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Under the Supervision of the Ministry of Higher Education, Scientific Research and Technology

The Centre of Biotechnology of Sfax

Organized the

International Symposium on Biotechnology k o o b s

g n i eed

c o r P

The International Centre For Genetic Engineering and Biotechnology

Institut de Recherche pour le Développement

The University of Sfax

Institut Français de Coopération

The Tunisian Union for Industry, Commerce and Handicrafts

ISB 2008, May 4th – 8th, 2008 Sfax - Tunisia

Foreword Dear Colleagues, This meeting coincides with the 25th anniversary of the CBS and with the completion of the new CBS building. We are extremely delighted and enthusiastic about these two events and hope this scientific manifestation will prepare the ground for more biotechnology glowing in Sfax, Tunisia, and all over world The increasing request for organic food products and a safe natural environment have given new insights in to biotechnology research and development. Genomics that entered its golden era in the 21th century has had a fabulous impact on the unraveling of the mystery of life and has provided us with innovative solutions related to health, environment, food sources and bioindustries. The main objective of this symposium is to promote the creation and dissemination of rapidly growing knowledge in the different biotechnology areas covered on this occasion. This event includes plenary lectures given by internationally known scientists and experts as well as oral communications given by senior and young researchers and poster and satellite sessions, including sessions on public health, water treatment, innovations and start-up and quality control. In a privileged location within approximately 270 kilometers from the capital Tunis on the south–eastern Mediterranean coast of Tunisia, Sfax is rich with its industries and its commercial port which is one of the most important commercial ones in Africa and which plays a major economic role through the export of olive oil, dried fish, phosphates and derivatives. The organizing committee welcomes contributions from all countries and hopes that this event will be a nice opportunity for senior and junior scientists from all over the world to meet in Tunisia, a modern safe country open to all civilizations and cultures and to exchange ideas and experiences in the vast domain of biotechnology.

Professor Hammadi Ayadi Chairman

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Acknowledgements The organizers of the symposium are very grateful to the honorable Minister of Higher Education, Scientific research and Technology and the State Secretary for the Minister of Higher Education, Scientific Research and Technology in charge of Scientific Research and Technology for their kind continuous support and encouragements during all the preparation of the Symposium. The organizers extend their gratitude to the regional authorities Governorate and Mayor of Sfax, for their enduring support devotion. The organizing committee addresses its grateful thanks to the USA and French embassies for accepting to participate in the organization and in the financial support of this event. The organizing committee is also grateful to all the institutions, the companies and the scientific associations that supported the organization of this manifestation whether morally or financially. The organizing committee addresses its grateful thanks to the exhibitors for their participation to this international event.

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Committees Honour Committee: Ellouze Radhouane, Emeritus Professor, CBS, Sfax, Tunisia Ben Dhia Hamed, President of the University of Sfax El Abed Amor, State Secretary for the Minister of Education and Formation charged by the Professional Formation. Hilal Nidal, Director o fthe Centre for Clean Water Technologies, beneficiary of the Nobel Prize 2006 in water Technology, UK. Thomas Daniel , University of Technology of Compiegne (UTC)

Organizing Committee: Ayadi Hammadi, Committee Chair Abdelhak Sonia, Pasteur Institute of Tunis Alimi Mohamed Adel, National Engineering School of Sfax Aouni Mahjoub, Faculty of Pharmacy of Monastir Bejar Samir, Centre of Biotechnology of Sfax Belghith Hafedh, Centre of Biotechnology of Sfax Ben Ali Mamdouh, Centre of Biotechnology of Sfax Ben Ayed Nathalie, Sfax Chamber of Commerce and Industry Ben Salah Abdelhamid, Faculty of Sciences of Sfax Bouhaouala Balkiss, Pasteur Institute of Tunis Fourati Amin, Tunisian Chemical Group Gabsi Slimane, Higher Institute of Biotechnolgy of Sfax Gargouri Raja, Centre of Biotechnology of Sfax Ghorbel Abdelfatteh,Technopark of Sfax Ghorbel Moncef, Tunisian Union of Industry Commerce and Handicraft Hammami Adnane, Faculty of Medicine of Sfax Jamoussi Ahmed, Tunisian Union of Industry Commerce and Handicraft Jaoua Samir, Centre of Biotechnology of Sfax Karray Boubaker, Olive Institute Khemiri Mongia, FIPA Martial Adele, French Institute of Cooperation Makni Ikram, Business Centre of Sfax Makni Zouheir, Regional Direction of Tourism (Sfax & Mahdia) Marrakchi Mohamed, Faculty of Sciences of Tunis Mitchell Beth, US Embassy Morio Takahiro, Japan International Cooperation Agency and the University of Tsukuba Nasri Hasen, University of Sfax Rachdi Ferid, National Center for Scientific Research CNRS Rekik Ahmed, Municipality of Sfax Sayadi Sami, Centre of Biotechnology of Sfax Smaili Abdelkerim, US Embassy Triki Nabil, Triki-Le Moulin confectionery Walper Beddies, German Company for Technical Cooperation in Sfax Zouari Nabil, Centre of Biotechnology of Sfax Zouari Wassim, Association of Sfax International Fair

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Scientific Committees: National: Ayadi Hammadi, Committee Chair, Sfax Barteji Aghleb, Higher Institute of Biotechnology of Monastir Bejar Samir, Centre of Biotechnology of Sfax Boudabbous Abdellatif, Faculty of Sciences of Tunis Drira Noureddine, Faculty of Sciences of Sfax El Ayeb Mohamed, Pasteur Institute of Tunis Fakhfakh Faiza, Faculty of Medicine of Sfax Gargouri Ali Faouzi, Centre of Biotechnology of Sfax Gargouri Raja, Centre of Biotechnology of Sfax Gargouri Youssef, National Engineering School of Sfax Ghorbel Abdelwahid, Centre of Biotechnology of Borj Cedria Hentati Faical, Institute of Neurology in Tunis Jaoua Samir, Centre of Biotechnology of Sfax Marzouki Najib, National Institute of Applied Sciences and Technology in Tunisia Masmoudi Khaled, Centre of Biotechnology of Sfax Rebai Ahmed, Centre of Biotechnology of Sfax Saad Ali, Faculty of Medicine of Sousse Sayadi Sami, Centre of Biotechnology of Sfax Zouari Nabil, Centre of Biotechnology of Sfax International: AlFadhli Suad, Faculty of Allied Health Sciences, Kuwait Chauhan Virander S., ICGEB, India Chouaieb Salem, INSERM, France Frutos Roger, CIRAD, France Giacca Mauro, ICGEB, Italy Haser Richard, CNRS, France Miyasaki Hitoshi, Arena, University of Tsukuba, Japan Molina Franck, CNRS, France Parry Martin, Centre For Crop Genetic Improvement, UK Potts Malcolm, Virginia Tech, USA and Qatar University Tholozan Jean Luc, IRD, France

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Scientific Programme

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Programme of the:

INTERNATIONAL SYMPOSIUM ON BIOTECHNOLOGY ISB2008 SFAX, TUNISIA 4 - 8 May, 2008

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PROGRAMME Sunday, May 4th 09:00-15:00: Registration 16:30-18:30: Opening ceremony chaired by: -the Secretary of State to the Minister of Higher Education, Scientific Research and Technology, in charge of Scientific Research and Technology : Mr. Ridha Ben Mosbah & -the Secretary of State to the Minister of Public Health, in charge of Public Hospitals : Mrs.

Najoua Miladi

18:30-19:30: Plenary Conference (PC1) Pr. Daniel Thomas, University of Technology of Compiègne, France Conference title: "BIOREFINERY- Biotechnology for the conversion of biomass into biomolecules, biomaterials and biofuels"

20:00: Buffet

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Monday, May 5th Session 1

Session 2

Biotechnology for animal and human health and biopharmaceuticals 9:00-10:00 : PC 2 10:00-10:30: TC1-S1 10:30-11:00: Coffee break 11:00-11:15: OC1S1 11:15-11:30 :OC2S1 11: 30-11: 45: OC3S1 11: 45-12:00: OC4 S1 12 :00-12:15: OC5 S1 12:15-12:30: OC6 S1 12:30-14:00: Lunch

Microbial and Environemental Biotechnology 9:00-10:00 : PC 2 10:00-10:30: TC1-S2 10:30-11:00: Coffee break 11:00-11:15: OC1S2 11:15-11:30 :OC2S2 11:30-11: 45: OC3S2 11:45-12:00: OC4 S2 12:00-12:15: OC5 S2 12:15-12:30: OC6 S2 12:30-14:00: Lunch

14:00-15:00: Poster session 15:00-16:00 : PC 3 16:00-16:30:TC2-S1 16:30-17:00: Coffee break 17:00-17:15: OC7 S1 17:15-17:30: OC8 S1 17:30-17:45: OC9 S1 17:45-18:00: OC10 S1

14:00-15:00: Poster session 15:00-16:00 : PC 3 16:00-16:30:TC2-S2 16:30-17:00: Coffee break 17: 00-17:15: OC7 S2 17:15-17:30: OC8 S2 17:30-17:45: OC9 S2 17:45-18:00: OC10 S2

Session 3

Satellite Session

Agricultural, Food and Marine biotechnology

Innovative Biotechnology

9:00-10:00 : PC 2 10:00-10:30: TC1-S3 10:30-11:00: Coffee break 11:00-11:30: TC2-S3 11:30-11:45 :OC1S3 11:45-12: 00: OC2S3 12:00-12:15: OC3S3 12:15-12:30: OC4 S3

- Promembrane International conf. -Food safety & traceability -Nanobiotechnology - Biotechnology and Society (Bioethics, education in biotechnology)

12:45-14 :00: Lunch 14 :00-15 :00: Poster session 15 :00-16 :00 : PC 3 16:00-16:30:TC3-S3 16:30-17 :00: Coffee break 17: 00-17:15: OC6 S3 17:15-17:30: OC7S3 17:30-17:45: OC8 S3 17:45-18:00: OC9 S3

- Principles of Quality Management -Incubating innovations (Start-ups, Spin-offs) -Financing young innovative companies -From patent to profit: commercialising your research

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Tuesday, May 6th Session 1

Session 2

Session 3

Satellite Session



Biotechnology for animal and human health and biopharmaceuticals 9:00-10:00 : PC 4 10:00-10:30: TC3-S1 10:30-11 :00: Coffee break 11:00-11:15: OC11S1 11:15-11:30 :OC12S1 11:30-11: 45: OC13S1 11:45-12:00: OC14 S1 12:00-12:15: OC15 S1 12:15-12:30: OC16 S1 12:30-14:00: Lunch

Microbial and environemental Biotechnology (M E B) 9:00-10:00 : PC 4 10:00-10:30: TC3-S2 10:30-11:00: Coffee break 11:00-11:15: OC11S2 11:15-11:30 :OC12S2 11:30-11: 45: OC13S2 11:45-12:00: OC14 S2 12:00-12:15: OC15 S2 12:15-12:30: OC16 S2 12:30-14:00: Lunch

9:00-10:00 : PC 4 10:00-10:30: TC4-S3 10:30-11:00: Coffee break 11:00-11:15: OC10S3 11:15-11:30 :OC11S3 11:30-11: 45: OC12S3 11:45-12:00: OC13S3 12:00-12:15: OC14 S3 12:15-12:30: OC15 S3 12:30-14:00: Lunch

14:00-15:00: Poster session 15:00-16:00 : PC 5 16:00-16:30:TC4-S1 16:30-17:00: Coffee break 17: 00-17:15: OC17 S1 17:15-17:30: OC18 S1 17:30-17:45: OC19 S1 17:45-18:00: OC20 S1

14:00-15:00: Poster session 15:00-16:00 : PC 5 16:00-16:30:TC4-S2 16:30-17:00: Coffee break 17:00-17:15: OC17 S2 17:15-17:30: OC18 S2 17:30-17:45: OC19 S2 17:45-18:00: OC20 S2

14:00-15:00: Poster session 15:00-16:00 : PC 5 16:00-16:30:TC5-S3 16:30-17:00: Coffee break 17: 00-17:15: OC16 S3 17:15-17:30: OC17S3 17:30-17:45: OC18 S3 17:45-18:00: OC19 S3

- Promembrane International conf. -Food safety & traceability -Nanobiotechnology - Biotechnology and Society (Bioethics, education in biotechnology) - Principles of Quality Management -Incubating innovations (Start-ups, Spin-offs) -Financing young innovative companies -From patent to profit: commercialising your research

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Wednesday, May 7th Session 1 Biotechnology for animal and human health and biopharmaceuticals 9:00-10 :00 : PC 6 10:00-10:30: TC5-S1 10:30-11:00: Coffee break 11:00-11:15: OC21S1 11:15-11:30 :OC22S1 11:30-11: 45: OC23S1 11:45-12:00: OC24 S1 12:00-12:15: OC25 S1 12:15-12:30: OC26 S1 12:30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : PC 7 16:00-16:30:TC6-S1 16:30-17:00: Coffee break 17: 00-17:15: OC27 S1 17:15-17:30: OC28 S1 17:30-17:45: OC29 S1 17:45-18:00: OC30 S1

Session 2 Microbial and environemental biotechnology (M E B) 9:00-10:00 : PC 6 10:00-10:30: TC5-S2 10:30-11:00: Coffee break 11:00-11:30: TC6-S2 11:30-11:45 :OC21 S2 11:45-12: 00: OC22 S2 12:00-12:15: OC23 S2 12:15-12:30: OC24 S2 12:30-12:45: OC25 S2 12:45-13:00: OC26 S2 13:00-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00: PC 7 16:00-16:30:TC7-S2 16:30-17:00: Coffee break 17: 00-17:30: TC8-S2 17:30-17:45: OC27 S2 17:45-18:00: OC28 S2 18:00-18:15: OC29 S2 18:15-18:30: OC30 S2

Session 3

Satellite Session



9:00-10:00 : PC 6 10:00-10:30: TC6-S3 10:30-11:00: Coffee break 11:00-11:15: OC20S3 11:15-11:30 :OC21S3 11:30-11: 45: OC22S3 11:45-12:00: OC23S3 12:00-12:15: OC24 S3 12:15-12:30: OC25 S3 12:30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : PC 7 16:00-16:30:TC7-S3 16:30-17:00: Coffee break 17: 00-17:15: OC26 S3 17:15-17:30: OC27S3 17:30-17:45: OC28 S3 17:45-18:00: OC29 S3


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Thursday, May 8th Session 1

Session 2

Session 3

Satellite Session

Biotechnology for animal and human health and biopharmaceuticals

Microbial and environemental biotechnology (M E B)



9:00-10:00 : PC8 10:00-10:30: TC7-S1 10:30-11:00: Coffee break 11:00-11:15: OC31 S1 11:15-11:30 :OC32 S1 11:30-11: 45: OC 33 S1 11:45-12:00: OC 34 S1 12:00-12:15: OC 35 S1 12:15-12:30: OC 36 S1

9:00-10:00 : PC8 10:00-10:30: TC9-S2 10:30-11:00: Coffee break 11:00-11:30: TC10-S2 11:30-11:45: OC 31 S2 11:45-12:00: OC 32 S2 12:00-12:15: OC 33 S2 12:15-12:30: OC 34 S2 12:30-12:45: OC 35 S2

9:00-10:00 : PC8 10:00-10:30: TC8-S3 10:30-11:00: Coffee break 11:00-11:30 : TC9-S3 11:30-11: 45: OC 30S3 11:45-12:00: OC 31S3 12:00-12:15: OC 32 S3 12:15-12:30: OC 33 S3

13:30: Closing ceremony chaired by the Minister of Higher Education, Scientific Research and Technology: Mr. Lazhar Bou Ouni



14:30: Lunch 15:15: Touristic trip 20:00: Dinner-Ceremony




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• PC: Plenary Conference • TC: Session Conference • OC: Oral Communication • Satellite Session: Economic and Biosafety aspects of Biotechnology: - Start-ups, Spin-offs - Biosafety - Principles of Quality Management - Biotechnology and Society - Education in Biotechnology

800 participants from 32 countries 107 Oral Communications 26 Session Conferences 08 Plenary Conferences 540 Posters

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ISB2008 SCIENTIFIC PROGRAMME Plenary Conferences: Sunday, May 4th 18:00-19:00: Chairpersons:

Pr. A. El Abed [Loaded of Mission to the Prime Minister] Pr. H. Ben Dhia [President of the University of Sfax] & Pr. R. Ellouz [Centre of Biotechnology of Sfax- CBS] PC1: Pr. Daniel THOMAS, University of Technology of Compiègne (UTC), France Title" BIOREFINERY- Biotechnology for the conversion of biomass into biomolecules, biomaterials and biofuels." Monday, May 5th 09:00-10:00: Chairperson: Pr. H. Ayadi [Director General of the CBS] PC2: Pr. Hechmi LOUZIR, Pasteur Institute of Tunis Title:" Immunology of human leishmaniasis: challenges for vaccine development” 15:00-16:00: Chairperson: Pr. S. Jaoua [CBS] PC3: Pr. Brian FEDERRICI, University of California, USA Title:” Basic Biology and Genetic Engineering of Bacillus thuringiensis” Tuesday, May 6th 9:00-10:00: Chairperson: Pr. S. Hamon [IRD Montpellier] PC4: Pr. Martin PARRY, Centre for Crop Genetic Improvement, UK Title:" Integrated Approaches to Wheat Improvement under drought." 15:00-16:00: Chairperson: Pr. S. Abdelhak [Pasteur Institute of Tunis] PC5: Pr. Hitoshi MIYAZAKI, ARENA, Tsukuba University, Japan Title:" Effects of Olive Leave Compounds on the Functions of Vasculature and Ovary. " Wednesday, May 7th 9:00-10:00: Chairperson: Pr. A. Gargouri [CBS] PC6: Pr. David HOPWOOD, John Innes Centre, UK Title:" Using Microbial Genetics to Find and Develop New Antibiotics." 15:00-16:00: Chairperson: Pr. S. Sayadi [CBS] PC7: Pr. Christine PETIT, Pasteur Institute, France Title:" Human Hereditary Deafness: from genes to pathogenis and beyond." Thursday, May 8th 09:00-10:00: Chairperson: Pr. H. Makni [Faculty of Medicine of Sfax] PC8: Pr. S. Chauhan VIRANDER., ICGEB, India Title: “Development of Malaria Vaccine: Current Scenario” 8

Session Conferences: Monday, May 5th

10:00-10:30: TC1-S1: Pr.Takashi KURIKI, Biochem.Res.Lab., Ezaki Glico Co., Japan

Title: “Relationship between Structure and Immunostimulating Activity of Enzymatically Synthesized Glycogen” Chairpersons: Pr. F. Fakhfakh [Faculty of Medecine of Sfax] & Pr. C. Granier [CNRS FRE Montpellier] 10:00-10:30 : TC1-S2: Pr. Hidetaka HORI , University of Niagata, Japan

Title: “Recycle of waste /sludge from food industry and live stock feces for compost” Chairpersons: Pr. S. Jaoua [CBS] & Pr. M. Potts [Virginia Tech.] 10:00-10:30: TC1-S3: Pr. Emmanuel GUIDERDONI , CIRAD, France Title: “Rice Mutant Resources for Deciphering Developmental and Adaptive Processes” 11:00-11:30: TC2-S3: Pr. Juan FERRE, University of Valencia,Spain Title: “Basis of resistance to Bacillus thuringiensis toxins in Lepidoptera ” Chairpersons: Pr. N. Marrakchi [Pasteur Institute of Tunis] & Pr. M. Mathlouthi [Faculty of Science, Reims] 16 :00-16:30: TC2-S1: Pr. Mauro GIACCA, ICGEB, Italy

Title:" Gene and cell therapy for cardiovascular disorders” Chairpersons: Pr. M. Frikha [Faculty of Medicine of Sfax] & Pr. T. Morio [University of Tsukuba, Japan] 16:00-16:30: TC2-S2: Pr. Malcolm POTTS, Virginia Tech, USA and Qatar University Title: “Roof Biofilm: A Unique Biotechnological Resource” Chairpersons: Pr. N. Zouari [CBS] & Pr. Y. T. Gargouri [ENIS] 16:00-16:30:TC3-S3: Pr. Gerald BERKOWITZ ., University of Connecticut, USA Title: “Mechanisms mediating Ca2+ involvement in pathogen perception, programmed cell death and plant innate immunity” Chairpersons: Pr. A. Ghorbel [CBBC] & Dr. E. Bonnin [INRA, France]

Tuesday, May 6th 10:00-10:30: TC3-S1: Dr. Balkis BOUHAOUALA, Pasteur Institute of tunis ,Tunisia Title: “From classical antibody based immunotherapy to engineered nanobodies” Chaipersons: Pr. R. Gargouri [CBS] & Pr. M. Aouni [ Faculty of Pharmacy of Monastir] 10:00-10:30 TC3-S2: Pr. Samir BEJAR , CBS, Tunisia

Title: “Two original isomerases involved in low caloric sugar production: Screen, characterization, cloning, study of structure function relationship, improvement and application.” Chairpersons: Pr. S. Ellouz-Chaâbouni [ENIS] & Pr. H. Mejdoub [ Faculty of Science of Sfax] 9

10:00-10:30 TC4-S3: Dr. Khaled MASMOUDI, CBS, Tunisia Title: “Functional characterization of genes involved in drought and salinity tolerance in plants” Chairpersons: Dr R. Gargouri Bouzid [ENIS] & Pr. J. Ferre [University of Valencia Spain] 16:00-16:30:TC4-S1: Pr. M. PARKER IQBAL, National Research Center, Egypt

Title: “Gene-Environment Interactions in Oesophageal Cancer in South Africa” Chairpersons: Pr F. Hentati [Institute of Neurology of Tunis] & Pr. A. Ghorbel [Faculty of Medicine of Sfax] 16:00-16:30:TC4-S2: Pr. Gerhard SCHORIES , TTZ, Germany

Title: “Lignocellulosic co-substrates for anaerobic digestion of agricultural wastes and energy crops” Chairpersons: Dr. S. Tounsi [CBS] & Dr. S. Aifa [CBS] 16:00-16:30:TC5-S3: Dr. Estelle BONNIN, INRA, France Title: “Extraction of “green labelled” pectin from plant by-products” Chairpersons: Pr S. Bejar [CBS] & Pr. C. Franche [IRD, Montpellier]

Wednesday, May 7th 10:00-10:30 TC5-S1: Pr. Salem CHOUAIB, INSERM, France

Title: “Tumor resistance to specific lysis : a major hurdle for successful immunotherapy of cancer” Chairpersons: Pr. M. ElAyeb [Pasteur Institute of Tunis] & Pr. A. Bartegi [Higher Institute of Biotechnology of Monastir] 10:00-10:30 TC5-S2: Pr. Abdellatif BOUDABBOUS , FST, Tunisia

Title: “Bacterial species, reality or mirage”? 11:00-11:30:TC6-S2: Pr J. A. C. ARCHER, Dep. Genetics. Univ. Cambridge , UK Title: “Building of Genomics led green chemistry for industrial biotechnology Chairpersons: Pr. M. Nasri [ENIS] & Dr. L. Mellouli [CBS] 10:00-10:30 TC6-S3: Pr. Claudine FRANCHE, IRD, France

Title: “Genetic transformation of forest trees” Chairpersons: Pr. S. Hamon [IRD, Montpellier] & Pr. F. Molina [CNRS, France] 16:00-16:30:TC6-S1: Pr. Suad AL FADHLI, Faculty of Allied Heath sciences, Kuwait Title: “Susceptibility genes for Systemic Lupus Erythematosus in the Kuwaiti population” Chairperson: Pr. J. Hachicha [Faculty of Medicine of Sfax] & Pr. L. Keskes [Faculty of Medicine of Sfax]

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16:00-16:30:TC7-S2: Pr. Nidhal HILAL , Centre for Clean Water Technologies, UK Title: “Quantification of (bio) interactive forces at the nano-scale” 17:00-17:30:TC8-S2: Pr. Jean Luc THOLOZAN Title: “Extremophylic anaerobic bacteria: Characterization, ecology and potential applications” Chairpersons: Dr. H. Belguith [CBS] & Pr. M. Labat [IRD Marseille] 16:00-16:30:TC7-S3: Pr. Frank MOLINA, CNRS, France

Title: “BaSysBio, a systems biology approach for Bacillus Subtilis modelling” Chairpersons: Pr. N. Drira [ Faculty of Science of Sfax] & Pr R. Frutos [CIRAD Montpellier]

Thursday, May 8th 10:00-10:30 TC7-S1: Pr. Marie-Paule LEFRANC, Université Montpellier 2, IMGT, CNRS, France

Title: “IMGT®: a paradigm for genetics, genome and 3D structure data integration towards Systems Biology” Chairpersons: Dr. S. Masmoudi [CBS] & Pr. M. Hachicha [Faculty of Medicine of Sfax] 10:00-10:30 TC9-S2: Pr. Richard HASER, CNRS, France

Title: “News from sugar-converting enzymes: from structures towards catalysis and synthesis of attractive oligosaccharides” 11:00-11:30 TC10- S2: Pr. Mohamed AMAR, CNRST, Morocco Title: "Biological Resource Centres for Bioeconomy in Developing Countries" Chairpersons: Pr. N. Gharsallah [ Faculty of Science of Sfax] & Dr. S. Maâlej [ Faculty of Science of Sfax] 10:00-10:30 TC8-S3: Pr. Noureddine DRIRA, FSS, Tunisia Title: "Biotechnologie et déterminisme du sexe chez les végétaux supérieurs" 11:00-11:30 TC9-S3: Pr. Mahmoud SAKER, National Research Center, Egypt Title: “Plant Biotechnology in Egypt: Current Status and Future Scenarios” Chairperson: Pr M. N. Marzouki [INSAT]& Dr. K. Masmoudi [CBS]

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SESSIONS PROGRAMME Sunday, May, 4th 2008 09:00-15:00: Registration 16:30-18:30: Opening ceremony chaired by: -the Secretary of State to the Minister of Higher Education, Scientific Research and Technology, in charge of Scientific Research and Technology : Mr. Ridha Ben Mosbah & -the Secretary of State to the Minister of Public Health, in charge of Public Hospitals : Mrs.

18:30-19:30: Chairpersons:

Najoua Miladi

Pr. A. El Abed [Loaded of Mission to the Prime Minister] Pr. H. Ben Dhia [President of the University of Sfax] & Pr. R. Ellouz [Centre of Biotechnology of Sfax- CBS]

Plenary Conference 1 (PC1): Pr. Daniel Thomas, University of Technology of Compiègne, France Conference title: "BIOREFINERY- Biotechnology for the conversion of biomass into biomolecules, biomaterials and biofuels"

20:00: Buffet

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SESSION 1: BIOTECHNOLOGY FOR HUMAN HEALTH Monday, May 5th 09:00-10:00: Chairperson: Pr. H. Ayadi [Director General of the CBS] PC 2: Pr. Hechmi LOUZIR “Immunology of human leishmaniasis: challenges for vaccine development” 10:00-12:30: Chairpersons: Pr. F. Fakhfakh [Faculty of Medecine of Sfax] & Pr. C. Granier [CNRS FRE Montpellier] 10 :00-10 :30: TC1-S1: Pr.Takashi KURIKI “Relationship between Structure and Immunostimulating Activity of Enzymatically Synthesized Glycogen” 10:30-11:00: Coffee break 11:00-11:15: OC1 S1 : M. SLIMANI “Effects of stress on brain inflammation and its Remediation by Moroccan Endemic Medicinal Plants Products (MEMPP)” 11:15-11:30: OC2 S1: Maria KABBAGE “HSP 27, PDI and PPI Are Dysregulated in both Female and Male Breast Infiltrating Ductal Carcinomas (IDCA): An IEF/NEPHGE-2-DE- MS investigation” 11:30-11: 45: OC3 S1: Jenan AL-MATOUQ “Pattern of Expression of Triiodothyronine , Somatostatin hormone Receptors, and 5` Deiodinase in different Breast Pathologies versus Normal and Lactational tissues” 11:45-12:00: OC4 S1: Jihène ELLOUMI “EGFR expression in E.coli: N and C-termini truncation” 12 :00-12:15: OC5 S1: Imen MILADI-ABDENNAD:ER “Aberrant CpG island hypermethylation of multiple genes in colorectal neoplasia” 12:15-12:30: OC6 S1: Sondes KARRAY-CHOUAYEKH “DNA hypermethylation in breast cancer and its association with clinicopathological features” 12:30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00: Chairperson: Pr. S. Jaoua [CBS] PC 3: Pr. Brian FEDERICI "Basic Biology and Genetic Engineering of Bacillus thuringiensis" 16:00-18:00: Chairpersons: Pr. M. Frikha [Faculty of Medicine of Sfax] & Pr. T. Morio [University of Tsukuba, Japan] 16:00-16:30:TC2-S1: Pr. Mauro GIACCA " Gene and cell therapy for cardiovascular disorder" 16:30-17:00: Coffee break 17: 00-17:15: OC7 S1: Olfa Siala-Sahnoun “First successful prenatal diagnoses of MDC1A and LGMD2C forms in Tunisia and in Africa” 17:15-17:30: OC8 S1: Sofiene BEZZINE “Overview on phospholipase A2 and involvement in inflammation” 17:30-17:45: OC9 S1: Rim CHATTER “Novel analgesic and anti-inflammatory bromineted diterpenoid from red algae genus Laurencia” 17:45-18:00: OC10 S1: Saloua LASSOUED “In vitro Analysis of the oxidative profile in Epstein-Barr virus target cells during the early stages of infection and after lytic cycle induction”

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Tuesday, May 6th 9:00-10 :00 : Chairperson: Pr. S. Hamon [IRD Montpellier] PC 4: Pr. Martin PARRY "Integrated Approaches to Wheat Improvement under drought” 10:00-12:30: Chaipersons: Pr. R. Gargouri [CBS] & Pr. M. Aouni [ Faculty of Pharmacy of Monastir] 10:00-10:30: TC3-S1: Dr. Balkis BOUHAOUALA “From classical antibody based immunotherapy to engineered nanobodies” 10:30-11:00: Coffee break 11:00-11:15: OC11 S1: Imen HADJI SFAXI “Characterization of Superoxide Dismutase Iso-enzymes From Allium sativum. Effects on Tumor Cell Lines” 11:15-11:30 :OC12 S1: Salma ABDELMOULA-SOUISSI “High-level expression of human tumour suppressor P53 in the methylotrophic yeast: Pichia pastoris” 11:30-11: 45: OC13 S1: Ines Zidi “Importance of HLA-G regulation by TNF- and glucocorticoids in treatment of cancer” 11:45-12:00: OC14 S1: Sarrah M’BAREK "Solid phase synthesis, a biotechnogical approach for structure-activity relationships of scorpion toxins : Innovative strategy of chemical synthesis of a Maurotoxin with Constrained Standard Disulfide Bridging” 12:00-12:15: OC15 S1: M’hamed GRATI “Molecular Analysis of USH genes in a large cohort of Usher type I, II and III patients shows the importance of MYO7A and USH2A implication” 12:15-12:30: OC16 S1: Imen BEN REBEH “Localization of candidate regions for a novel gene for Usher II syndrome” 12:30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : Chairperson: Pr. S. Abdelhak [Pasteur Institute of Tunis] PC5: Pr. Hitoshi MIYAZAKI " Effects of Olive Leave Compounds on the Functions of Vasculature and Ovary" 16:00-18:00: Chairpersons: Pr F. Hentati [Institute of Neurology of Tunis] & Pr. A. Ghorbel [Faculty of Medicine of Sfax] 16:00-16:30: TC4-S1: Pr. M. PARKER IQBAL “Gene-Environment Interactions in Oesophageal Cancer in South Africa” 16:30-17:00: Coffee break 17: 00-17:15: OC17 S1: Hassen HADJ KACEM “The PDS gene expression in different pathological thyroid tissues” 17:15-17:30: OC18 S1: Salah LAROUI “Genetic polymorphism of the Hyperhomocysteinemia with Valvulopatients in the region of Aures in the East of Algeria” 17:30-17:45: OC19 S1: Saima SADAF “High-level expression and production of bubaline somatotropin in Escherichia coli” 17:45-18:00: OC20 S1: Basma Hadj Kacem “A novel nonsense mutation (at Ser23) in GPIbβ gene affects GPIb-IX complex expression in two Tunisian Bernard-Soulier Syndrome unrelated families”

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Wednesday, May 7th 9:00-10:00: Chairperson: Pr. A. Gargouri [CBS] PC6: Pr. David HOPWOOD "Using Microbial Genetics to Find and Develop New Antibiotics" 10:00-12:15: Chairpersons: Pr. M. ElAyeb [Pasteur Institute of Tunis] & Pr. A. Bartegi [Higher Institute of Biotechnology of Monastir] 10:00-10:30: TC5-S1: Pr. Salem CHOUAIB “Tumor resistance to specific lysis : a major hurdle for successful immunotherapy of cancer” 10:30-11:00: Coffee break 11:00-11:15: OC21 S1: Laurence Molina “The inter-individual variability of the human urinary proteome assessed by 2D electrophoresis analysis” 11:15-11:30 :OC22 S1: Olfa Frikha-Gargouri “Comparison of an in house ELISA test and the pELISA MEDAC for the detection of Chlamydia trachomatis IgG antibodies in Danish woman seeking abortion” 11:30-11:45: OC23 S1: Riadh BEN MANSOUR “Oxidative stress in Systemic lupus erythematosus and rheumatoid arthritis and its relationship with autoantibodies production” 11:45-12:00: OC24 S1: Walid AL ACHKAR “CML Profile in Syria” 12:00-12:15: OC25 S1: Jos VAN PELT “the application of gene expression array and bioinformatics to investigate molecular mechanisms of liver disease” 12:30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : Chairperson: Pr. S. Sayadi [CBS] PC7: Pr. Christine PETIT " Human Hereditary Deafness : from genes to pathogenis and beyond" 16:00-18:00: Chairperson: Pr. J. Hachicha [Faculty of Medicine of Sfax] & Pr. L. Keskes [Faculty of Medicine of Sfax] 16:00-16:30: TC6-S1: Pr. Suad AL FADHLI: “Susceptibility genes for Systemic Lupus Erythematosus in the Kuwaiti population” 16 :30-17:00: Coffee break 17:00-17:15: OC27 S1: P. BILLIALD “Recombinant antibodies: Towards a new generation of antivenoms?” 17:15-17:30: OC28 S1: Rym BENKHALIFA “Electrophysiological Strategy for Ion Channel Drug Discovery” 17:30-17:45: OC29 S1: Shoukat PARVEZ “Co-culturing of Bifidobacterium bifidum Enhanced Bile Salt Hydrolase Activity in Media”17 h3017:45-18:00: OC30 S1: Olfa KALLECH-ZIRI “Chemical synthesis of lebestatin, an anti-angiogenic disintegrin, and its analogues. Structureactivity relationship”

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Thursday, May 8th: 09:00-10:00 : Chairperson: Pr. H. Makni [Faculty of Medicine of Sfax] PC8: Pr. S. CHAUHAN VIRANDER “Development of Malaria Vaccine: Current Scenario” 10:00-12:45: Chairpersons: Dr. S. Masmoudi [CBS] & Pr. M. Hachicha [Faculty of Medicine of Sfax] 10:00-10:30: TC7-S1: Pr. Marie-Paule LEFRANC “IMGT®: a paradigm for genetics, genome and 3D structure data integration towards Systems Biology” 10:30-11:00: Coffee break 11:00-11:15: OC31 S1: Ahmed REBAI “Computational biology approaches help in understanding cancer” 11:15-11:30: OC32 S1: Yaqoub ASHHAB “Developing a Powerful Bioinformatics Tool for Prediction of Caspase 3 Substrate: Preliminary Analysis of the Human Proteome” 11:30-11: 45: OC33 S1: Gregory NUEL “Taking into account missing genotypes and errors in Family Based Association Testing using an Expectation-Maximization framework” 11:45-12:00: OC34 S1: Mohamed Mahmoud SOWILEM “Utilization of GIS and Space data in assessment of victor borne diseases and appropriate bio-control measures” 12:00-12:15: OC35 S1: Imen KALLEL “Screening of pro-apoptotic activity of pure molecules and extract derived from medicinal plant on breast cancer cell lines” 12:15-12:30: OC36 S1: Dr Soroush SADARI “A New Network in the Eastern Mediterranean Region Spreads its Wings:DEVELOPMENT OF THE EASTERN MEDITERRANEAN HEALTH GENOMICS AND BIOTECHNOLOGY NETWORK (EMHGBN) » 13:30: Closing ceremony chaired by the Minister of Higher Education, Scientific Research and Technology: Mr. Lazhar Bou Ouni 14:30: Lunch 15:15: Touristic trip 20:00: Dinner-Ceremony

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SESSION 2: MICROBIAL AND ENVIRONMENTAL BIOTECHNOLOGY Monday, May 5th 9:00-10:00: Chairperson: Pr. H. Ayadi [Director General of the CBS] PC 2: Pr. Hechmi LOUZIR “Immunology of human leishmaniasis: challenges for vaccine development” 10:00-12:30: Chairpersons: Pr. S. Jaoua [CBS] & Pr. M. Potts [Virginia Tech.] 10:00-10:30: TC1-S2: Pr. Hidetaka HORI “Recycle of waste /sludge from food industry and live stock feces for compost” 10:30-11:00: Coffee break 11:00-11:15: OC1S2: Nushin AGHAJARI “Structural- and mutagenesis studies give insights into sucrose isomerization” 11:15-11:30 :OC2S2: Ena ALBA “treatment for bioremediation of olive oil mill wastewater” 11:30-11:45: OC3S2: Fatma DRISS “Enhancement of the insecticidal activity of a Bacillus thuringiensis subsp. kurstaki strain via the production of chitinase-containing crystals” 11:45-12:00: OC4 S2: Sonia JEMLI “The β-cyclodextrin glycosyltransferase of Paenibacillus pabuli US132: characterization, cloning and overproduction of the recombinant enzyme” 12:00-12:15: OC5 S2: Ines MAALEJ “Purification, biochemical characterisation and immobilization of a xylanase of a thermophilic fungus” 12:15-12:30: OC6 S2: Sami MNIF “Isolation and characterization of Halomonas sp. strain C2SS100, an hydrocarbon degrading bacterium under hypersaline conditions” 12:30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : Chairperson: Pr. S. Jaoua [CBS] PC3: Pr. Brian FEDERICI "Basic Biology and Genetic Engineering of Bacillus thuringiensis" 16:00-18:00: Chairpersons: Pr. N. Zouari [CBS] & Pr. Y. T. Gargouri [ENIS] 16:00-16:30: TC2-S2: Pr. Malcolm POTTS “Roof Biofilm: A Unique Biotechnological Resource” 16:30-17:00: Coffee break 17:00-17:15: OC7 S2: Hamed Mohammed EL-SHORA “Immobilization and modification of laccase from Bacillus thuringiensis is promising for enzymatic removal and decontamination of phenolic compounds from polluted water systems” 17:15-17:30: OC8 S2: El Akrem HAYOUNI “Multiparametric flow cytometry, a powerful tool for the assessment of the mechanism of action of Melaleuca armillaris (Sol. Ex Gaertu) Sm. essential oil, on six LAB strains” 17:30-17:45: OC9 S2: Abdelmalek BADIS “Characterization and biodegradation of humic acids extracted from different soil of Algeria: selection of performance microbial strains and optimisation of culture conditions” 17:45-18:00: OC10 S2: Wissem MNIF “Occurrence of endocrine disrupting activity in Tunisian sewage treatment plants”

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Tuesday, May 6th 9:00-10:00 : Chairperson: Pr. S. Hamon [IRD Montpellier] PC 4: Pr. Martin PARRY "Integrated Approaches to Wheat Improvement under drought” 10:00-12:30: Chairpersons: Pr. S. Ellouz-Chaâbouni [ENIS] & Pr. H. Mejdoub [ Faculty of Science of Sfax] 10:00-10:30: TC3-S2: Pr. Samir BEJAR “Two original isomerases involved in low caloric sugar production: Screen, characterization, cloning, study of structure function relationship, improvement and application.” 10:30-11:00: Coffee break 11:00-11:15: OC11 S2: Jean-Jacques GODON “Unstable environment enhanced ecological resilience in anaerobic digestors” 11:15-11:30:OC12 S2: José DUARTE “New bioreactor for ethanol production with flocullent yeast” 11:30-11:45: OC13 S2: Mireille KALLASSY-AWAD “Novel isolates of Bacillus thuringiensis strains presenting a polymorphism of cry1A genes with difference of in vivo activity” 11:45-12:00: OC14 S2: Samiha SIOUD MEDDEB “Detection of the biosynthesis pathway of a DKP active molecule produced by the new isolated Streptomyces sp.US24 strain” 12:00-12:15: OC15 S2: Walid SAIBI “ Purification and biochemical characterization of a transglucosilating β-glucosidase of Stachybotrys strain” 12:15-12:30: OC16 S2: Mohamed CHAMKHA “Biodiversity of aerobic bacteria from a Tunisian high-temperature oil field and biodegradation potential of some aromatic compounds and hydrocarbons” 12:30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : Chairperson: Pr. S. Abdelhak [Pasteur Institute of Tunis] PC 5: Pr. Hitoshi MIYAZAKI " Effects of Olive Leave Compounds on the Functions of Vasculature and Ovary" 16:00-18:30: Chairpersons: Dr. S. Tounsi [CBS] & Dr. S. Aifa [CBS] 16:00-16:30: TC4-S2: Pr. Gerhard SCHORIES “Lignocellulosic co-substrates for anaerobic digestion of agricultural wastes and energy crops ” 16:30-17:00: Coffee break 17:00-17:15: OC17 S2: Abdelkader BEKKI “Rhizobia-leguminous symbiosis: Diversity and biotechnology application” 17:15-17:30: OC18 S2: Layla BEN AYED “Detection of Cryptosporidium oocysts and Giardia cysts from sewage and sludge samples in Tunisia” 17:30-17:45: OC19 S2: Raja REZGUI “Phylogenetic caracterisation and metabolic potentialities of original bacteria isolated from a South Tunisian deep subterrestrial petroleum environment” 17:45-18:00: OC20 S2: Sabiha ACHAT “Antibactérial activity (in vitro) of phenolic extract of three medicinal plants sage, celery and laurel”

Wednesday, May 7th 18

09:00-10:00 : Chairperson: Pr. A. Gargouri [CBS] PC 6: Pr. David HOPWOOD "Using Microbial Genetics to Find and Develop New Antibiotics" 10:00-12:30:Chairpersons: Pr. M. Nasri [ENIS] & Dr. L. Mellouli [CBS] 10:00-10:30: TC5-S2: Pr. Abdellatif BOUDABBOUS “Bacterial species, reality or mirage”? 10:30-11:00: Coffee break 11:00-11:30: TC6-S2: Pr. J. A. C. ARCHER “Building of Genomics led green chemistry for industrial biotechnology 11:30-11:45: OC21 S2: Kais JAMOUSSI “Heterologous expression of the B. thuringiensis vegetative insecticidal protein Vip3LB in Photorhabdus temperata strain K122 and oral toxicity against the Lepidoptera Ephestia kuehniella and Spodoptera littoralis” 11:45-12:00 :OC22 S2: Fakher KAMOUN “Purification, Biochemical Characterization and Genes Identification of New Bacillus thuringiensis Bacteriocins” 12:00-12:15: OC23 S2: Bassem JAOUADI “Novel oxidant- surfactant- and bleach-stable serine alkaline protease from a newly isolated Bacillus pumilus CBS strain: biochemical and molecular characterization” 12:15-12:30: OC24 S2: Ameny FARHAT “Gene cloning and characterization and of a novel thermostable phytase from Bacillus subtilis US417” 12:30-12:45: OC25 S2: Rashed AL-SAÈD “Assessment of current conventional and membrane technologies for wastewater treatment and effluent reclamation in Palestine” 12 :45-13:00: OC26 S2: Zouhaier BOUALLAGUI “Hydroxytyrosol recovery through microbial transformation” 13:00-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : Chairperson: Pr. S. Sayadi [CBS] PC 7: Pr. Christine PETIT " Human Hereditary Deafness : from genes to pathogenis and beyond" 16:00-18:30: Chairpersons: Dr. H. Belguith [CBS] & Pr. M. Labat [IRD Marseille] 16:00-16:30: TC7-S2: Pr. Nidhal HILAL “Quantification of (bio) interactive forces at the nano-scale” 16:30-17:00: Coffee break 17: 00-17:30:TC8-S2: Pr. Jean Luc THOLOZAN “Extremophylic anaerobic bacteria: Characterization, ecology and potential applications” 17:30-17:45: OC27 S2: Sonia KHOUFI “Ultrafiltration for advanced olive mill wastewater treatment for ferti-irrigation” 17:45-18:00: OC28 S2: Olfa BEN SAID “study of microbial community diversity in superficial sediments of coastal anthropized bizerte lagoon (Tunisia)” 18:00-18:15: OC29 S2: Ines BORGI “A spontaneous direct repeat deletion in the Pgex fusion vector decreases the expression level of recombinant proteins in E. coli” 18:15-18:30: OC30 S2: Rima ALBESHARAT “Investigations on the influence of the fermentation conditions on the vitality of a probiotic culture after drying”

Thursday, May 8th : 19

9:00-10:00 : Chairperson: Pr. H. Makni [Faculty of Medicine of Sfax] PC8: Pr. S. Chauhan VIRANDER., “Development of Malaria Vaccine: Current Scenario” 10:00-12:45: Chairpersons: Pr. N. Gharsallah [ Faculty of Science of Sfax] & Dr. S. Maâlej [ Faculty of Science of Sfax] 11:00-11:30: TC9 S2: Pr. Richard HASER “News from sugar-converting enzymes: from structures towards catalysis and synthesis of attractive oligosaccharides” 10:30-11:00: Coffee break 10:00-10:30: TC10-S2: Pr. Mohamed AMAR "Biological Resource Centres for Bioeconomy in Developing Countries" 11:30-11:45: OC31 S2: Raida ZGHAL ZRIBI “Evidence of the importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa protein cytolytic activity” 11:45-12:00 :OC32 S2: Cherif BEN-YOUSSEF “Kinetic modelling of the benzene inhibitory effect on nitrifying batch cultures” 12:00-12:15: OC33 S2: Nour SHAFIK EL-GENDY “Assessment of biostimulation effect on a highly heavy metals and oil polluted soil” 12:15-12:30: OC34 S2: Maxime DUMONT “Automatic Control may Help to Investigate Microbial Ecological Questions” 12:30-12:45: OC35 S2: Ouardia BELOUANA BORN BENBELKACEM “Study of the nitrate pollution conversion to the gas nitrogen with a fixed biomass on consumable support (Alfa Stem)” 13:30: Closing ceremony chaired by the Minister of Higher Education, Scientific Research and Technology: Mr. Lazhar Bou Ouni 14:30: Lunch 15:15: Touristic trip 20:00: Dinner-Ceremony

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SESSION 3: AGRICULTURAL, FOOD AND MARINE BIOTECHNOLOGY Monday, May 5th 9:00-10:00: Chairperson: Pr. H. Ayadi [Director General of the CBS] PC 2: Pr. Hechmi LOUZIR “Immunology of human leishmaniasis: challenges for vaccine development” 10:00-12:45: Chairpersons: Pr. N. Marrakchi [Pasteur Institute of Tunis] & Pr. M. Mathlouthi [Faculty of Science, Reims] 10:00-10:30: TC1-S3: Pr. Emmanuel GUIDERDONI “Rice Mutant Resources for Deciphering Developmental and Adaptive Processes” 10:30-11:00: Coffee break 11:00-11:30: TC2-S3: Pr. Juan FERRE “Basis of resistance to Bacillus thuringiensis toxins in Lepidoptera ” 11:30-11:45: OC1S3: Karim MEZHOUD “Global quantitative analysis of protein expression and phosphorylation status in the liver Medaka exposed to microcystin-LR” 11:45-12:00 :OC2S3: Imen BEN SALAH “Comparative study of the response of growth, nitrogen fixation and carbohydrate metabolism in Medicago cilaris lines cultivated under salt stress” 12:00-12:15: OC3 S3: Rania BEN SAAD “Over expression of an alien ZnFAl gene from A. littoralis in tobacco enhances salt and drought stress tolerance” 12:15-12:30: OC4 S3: Slim TOUNSI “Improvement of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and Manduca sexta by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes” 12:45-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : Chairperson: Pr. S. Jaoua [CBS] PC 3: Pr. Brian FEDERICI "Basic Biology and Genetic Engineering of Bacillus thuringiensis" 16:00-18:15: Chairpersons: Pr. A. Ghorbel [CBBC] & Dr. E. Bonnin [INRA, France] 16:00-16:30: TC3-S3: Pr. Gerald BERKOWITZ “Mechanisms mediating Ca2+ involvement in pathogen perception, programmed cell death and plant innate immunity” 16:30-17:00: Coffee break 17: 00-17:15: OC6S3: Muhammad AASIM “Efficient, Simple and Rapid Transformation of two Turkish Cowpea (Vigna unguiculata L.) Cultivars by Agrobacterium tumefaciens” 17:15-17:30: OC7S3: Kakha NADIRADZE “In vitro and in vivo virus free potato seed production using Elisa Reader” 17:30-17:45: OC8 S3: Aymen EZZINE “Cloning and Expression of Human Papillomavirus type 16 Capsid Protein L1 in Arabidopsis thaliana” 17:45-18:00: OC9 S3: Essaid AIT BARKA “The induced state of resistance in inflorescence of Vitis vinifera L. by a Plant growth promoting bacteria, Burkholderia phytofirmans strain PsJN against Botrytis cinerea Pers” 21

Tuesday, May 6th 9:00-10:00 : Chairperson: Pr. S. Hamon [IRD, Montpellier] PC 4: Pr. Martin PARRY "Integrated Approaches to Wheat Improvement under drought” 10:00-12:30 : Chairpersons: Dr R. Gargouri Bouzid [ENIS] & Pr. J. Ferre [University of Valencia Spain] 10:00-10:30: TC4-S3: Dr. Khaled MASMOUDI “Functional characterization of genes involved in drought and salinity tolerance in plants” 10 :30-11:00: Coffee break 11:00-11:15: OC10 S3: Ikram ZAIDI “identification and molecular characterization of durum wheat map kinase phosphatase 1 (tmkp1)” 11:15-11:30 :OC11 S3: Orhan ARSLAN “Rapid and Highly efficient micropropagation of Two Turkish woads Isatis constricta Davis and I. aucheri Boiss. under in vitro conditions” 11:30-11:45: OC12 S3: Lina AL-AMIR “The effect of crude aqueous Juniperus excelsa leaf and fruit extracts on some human pathogenic bacteria” 11:45-12:00: OC13 S3: Mariam DAMMAK KARRAY “Evidence of the involvement of the C-terminal region of Bacillus thuringiensis Cry1Ac in delta-endotoxin crystallization” 12:00-12:15: OC14 S3: Imen SAADAOUI “Cloning, sequencing and expression of cry1A genes of a new Bacillus thuringiensis strain highly toxic to Lepidopteran larvae” 12:15-12:30: OC15 S3: Shahidul HAQUE MD “Biotechnological Approaches for Improvement of Garlic (Allium ativum L.)” 12 :30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : Chairperson: Pr. S. Abdelhak [Pasteur Institute of Tunis] PC 5: Pr. Hitoshi MIYAZAKI " Effects of Olive Leave Compounds on the Functions of Vasculature and Ovary" 16:00-18:00: Chairpersons: Pr S. Bejar [CBS] & Pr. C. Franche [IRD, Montpellier] 16:00-16:30: TC5-S3: Dr. Estelle BONNIN “Extraction of “green labelled” pectin from plant by-products” 16:30-17:00: Coffee break 17:00-17:15: OC16 S3: Fadila Maiza BENABDESSELAM “In vitro antioxidant studies on phenolic and alkaloid extracts of two Fumaria Algeria species” 17:15-17:30: OC17 S3: Lobna MESRATI-ABDELKEFI “Characterization of a novel Vip3-type toxin of Bacillus thuringiensis strain BUPM95” 17:30-17:45: OC18 S3: Mohamed TRIGUI “Seasonal variation in antimicrobial activity of crude extracts of the green alga Ulva rigida (Ulvaceae)” 17:45-18:00: OC19 S3: Hayat MAKEE “Effect of inherited sterility and Bacillus thuringiensis on mortality and reproduction of Phthorimaea opercullela Zeller (Lepidoptera: Gelechiidae)”

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Wednesday, May 7th 9:00-10:00 : Chairperson: Pr. A. Gargouri [CBS] PC 6: Pr. David HOPWOOD "Using Microbial Genetics to Find and Develop New Antibiotics" 10:00-12:30:Chairpersons: Pr. S. Hamon [IRD, Montpellier] & Pr. F. Molina [CNRS, France] 10:00-10:30: TC6-S3: Pr. Claudine FRANCHE “Genetic transformation of forest trees” 10:30-11:00: Coffee break 11:00-11:15: OC20S3: Mohamed Rabie EL-AKHAL “Genetic diversity of rhizobial isolates obtained from root nodules of Arachis hypogaea in northwestern Morocco” 11:15-11:30 :OC21S3: Wafa JALLOULI “Overcome of Photorhabdus limitations of production and overproduction of corresponding bioinsecticides by fermentation” 11:30-11:45: OC22S3: Saoussen BEN KHEDHER “Development of a new generation of Bacillus thuringiensis bioinsecticides based on asporogenic and oligosporogenic mutants” 11:45-12:00: OC23 S3: Nader Farid ABUSARA “Optimal Growth Conditions for the Production of β-Carotene and Glycerol from a Halophilic Microalga Dunaliella sp. Isolated from the Dead Sea, Jordan” 12:00-12:15: OC24 S3: Maher CHAOUACHI “A High-Throughput Multiplex Method adapted for GMO Detection” 12:15-12:30: OC25 S3: Houda HANANA “Primary cultures of heart cells from the clam Ruditapes decussatus as an alternative method for ecotoxicological studies” 12:30-14:00: Lunch 14:00-15:00: Poster session 15:00-16:00 : Chairperson: Pr. S. Sayadi [CBS] PC 7: Pr. Christine PETIT " Human Hereditary Deafness : from genes to pathogenis and beyond" 16:00-18:00: Chairpersons: Pr. N. Drira [ Faculty of Science of Sfax] & Pr. R. Frutos [CIRAD Montpellier] 16:00-16:30: TC7-S3: Pr. Frank MOLINA “BaSysBio, a systems biology approach for Bacillus Subtilis modelling” 16:30-17:00: Coffee break 17: 00-17:15: OC26 S3: Samia DALDOUL “Cloning of full length cDNA for Vitis vinifera SIP/raffinose synthase related to salt stress response” 17:15-17:30: OC27 S3: Khan GOHARTAJ “Alteration of ABA mediated resistance against Alternaria blight in Brassica through osmotin gene transformation” 17:30-17:45: OC28 S3: Bassam AL-SAFADI “Selection of potato mutants tolerant to salinity using in vitro and DNA marker techniques” 17:45-18:00: OC29S3: Sameh SELLAMI “Overexpression of the Vegetative Insecticidal Protein of Bacillus thuringiensis during vegetative growth and sporulation phases”

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Thursday, May 8th : 09:00-10:00 : Chairperson: Pr. H. Makni [Faculty of Medicine of Sfax] PC8: Pr. S. Chauhan VIRANDER., “Development of Malaria Vaccine: Current Scenario” 10:00-12:45: Chairperson: Pr M. N. Marzouki [INSAT] & Dr. K. Masmoudi [CBS] 10:00-10:30: TC8-S3: Pr. Noureddine DRIRA "Biotechnologie et déterminisme du sexe chez les végétaux supérieurs" 10:30-11:00: Coffee break 11:00-11:30: TC9-S3: Pr. Mahmoud SAKER “Plant Biotechnology in Egypt: Current Status and Future Scenarios” 11:30-11:45: OC30 S3: Rafiq MUHAMMAD “Production of azadirachtin through in vitro culture of neem (Azadirachta indica A. Juss)” 11:45-12:00 :OC31 S3: J. SATHEESH “microbial technology of Bacillus thuringiensis var. kurstaki (berliner) formulation against defoliator PODOPTERA LITURA (Fabricius) in groundnut (Arachis hypogea)” 12:00-12:15: OC32 S3: Mohammed Najib SAIDI “Molecular investigation of brittle leaf disease affected date palm (phoenix Dactylifera L) leaves” 12:15-12:30: OC 33 S3: Halima EL-HATMI “Protective whey proteins from camel (Camelus dromedarius) milk” 13:30: Closing ceremony chaired by the Minister of Higher Education, Scientific Research and Technology: Mr. Lazhar Bou Ouni 14:30: Lunch 15:15: Touristic trip 20:00: Dinner-Ceremony

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Satellite Sessions Monday, May 5th Satellite1 Innovative Biotechnology PROMEMBRANE International Conference: 09:00-10 :00 : PC 2 10 :00-10 :315: Opening ceremony and welcome to PROMEMBRANE International Conference: Prof. Sami Sayadi 10 :15-10 :30: Presentation of ProMembrane project : Ms. Marisol Oropeza 10 :30-11 :00: Coffee break 11 :00-11 :45: Key Note Lecture: Current water scarcity situation in MENA countries: an overview Mourad Bino (to be confirmed) 11 :45-12 :30: Key Note Lecture: Visualization and quantification of membrane properties at the nano-scale Prof. Nidal Hilal 12 :30-14 :00: Lunch 14 :00-15 :00: Opening Fair and visit to the stands 15 :00-15 :30 : Nanotechnologies and membrane engineering at the core of water and related questions in Mediterranean and MiddleEast countries : Prof. Gilbert M. Rios 15 :30-16 :00 : Water recovery, reuse and advanced products formulations with integrated membrane operations: Enrico Drioli 16 :00-16 :10 : Young Scientist Award: Results and Certificate deliverance 16 :10-16 :30 : Young Scientist Award North Africa: 1st place Anaerobic membrane bioreactor treatment of domestic and agro-industrial wastewaters in Tunisia Ahlem Saddoud (Tunisia) 16 :30-17 :00: Coffee break 17 :00-17 :20: MBR technology: towards the search of an innovative and suitable wastewater treatment solution for isolated areas Miguel Ángel Orbaneja Bioazul S.L. (Spain) 17 :20-17 :40: Young Scientist Award Middle East: 1st place Optimization of membrane bioreactor system integrated with cross flow membrane filtration 17 :40-18:00: Mohammad Issa (Syria): Membranes in the sanitation field Barreto Leonellha: ttz Bremerhaven (Germany) 18 :00-18:30: Panel discussion: Moderator: Dr Drioli

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Tuesday, May 6th Satellite1

Satellite 2

Satellite 3


- Biosafety and Quality Management in biotechnology laboratories 9:00-10 :00 : PC 4

Eurohear: Advances in hearing science: from functional genomics to therapies

9:00-10 :00 : Key Note Lecture: Membrane Bioreactor Technology: Design and Applications Dr Tom Arnot (to be confirmed) 9:30-9 :50 : Young Scientist Award North Africa: 2nd place: Sally Ibrahim (Egypt) 9 :50-10 :10: Young Scientist Award Middle East: 2nd place Membrane used for water treatment Daad Hassan (Syria) 10 :10-10 :30: Panel discussion Moderator: Dr Schories 10 :30-11 :00: Coffee break 11 :00-11 :20: Advancing membrane technologies for wastewater treatment and effluent reclamation in selected Arab Mediterranean and North African countries Dr. Rashed Al-Saèd: Birzeit University (Palestine) 11 :20-11 :40: Applying the MBR technology in wastewater treatment with reuse of wastewater on coastal areas in Croatia Alena Vlasic: Water Management Institute (Croatia) 11 :40-12 :00: Membrane operations for water purification Dr. Alberto Figoli: Institute on Membrane Technology (ITM-CNR),Italy 12 :00-12 :30: Current water scarcity situation in the Middle East Prof. Adnan J. Ghata and Prof. Fouad Atallah Director of Training &Development, Al-Baath University (Syria)

10 :00-10 :30: -Quality management: what for ?: Dr. Roger Frutos 10 :30-11 :00: Coffee break 11 :00-11:30: Quality assurance for biotechnology laboratories: Dr. Roger Frutos 11 :30-12 :30: Discussion: Chaired by Pr. Samir Jaoua 12 :30-14 :00: Lunch

16 :30-17 :00: Coffee break 17:00-17:05: Opening of the Eurohear satellite session 17:05-17:15: Tunisia: an incredible population and genetics journey: Pr. Hammadi Ayadi 17:15-17:25: OC1: GJB2 mutation 35delG: Prevalence in Algeria and further evidence for founder effect:Imen Amar 17:25-17:35: OC2: Mutational analysis of the mitochondrial 12s rRNA, the tRNASer(UCN) and the tRNALeu(UUR) genes in Tunisian patients with nonsyndromic inherited hearing loss: Emna Mkaouar-Rebai 17:35-17:45: OC3: Novel mutations in the TMPRSS3 gene in Spanish DFNB8/10 families with autosomal recessive nonsyndromic hearing impairment: Dr. Antonio VIÑUELA 17:45-17:55: OC4: Redefining the DFNB33 locus to the chromosome 10p11.23-q21.1: Hanen Belguith 17:55-18:15: Discussion

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12 :30-14 :00: Lunch 14 :00-14 :30: Potential and examples for applications of membrane technology in water treatment Dr. Gerhard Schories Tech. Dir. Environmental Institute, ttz Bremerhaven (Germany) 14 :30-14 :50: Membrane bioreactor for wastewater treatment: overview and application in Egypt Prof. Hussein Abdel-Shafy Water Research & Pollution Control Dept., National Research Centre (Egypt) 14 :50-15 :10: Membrane operations for water purification Dr. Alberto Figoli: Institute on Membrane Technology (ITM-CNR),Italy 15 :10-15 :30: PV-RO desalination stand-alone system in the village of Ksar Ghilène (Tunisia). Vanessa Milan (or co-author) Spain 15 :30-15 :50 : Young Scientist Award North Africa: 3rd place :Firas Feki (Tunisia) 15 :50-16 :10 : Young Scientist Award Middle East: 3rd place Treatment of water with high (TDS , hardness , SO4--,and H2S) by using Reverse Osmosis system and the problems (scaling salts) of membrane : Shadi Shaheen (Syria) 16 :10-16 :30 : Panel discussion Moderator: Dr Rashed Al-Sa’ed 16 :30-17 :00: Coffee break 17 :00-17 :30: Conclusions and Closing of the Conference

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Wednesday, May 7th Satellite 3

Satellite 4

Satellite 5

Eurohear: Advances in hearing science: from functional genomics to therapies

Seame, a treasor of healthy benefits

9:00-10 :00 : PC 6 10:00-10:30: TC1: Mammalian sound amplification, towards a molecular map of the auditory hair cell lateral wall: Dr. Aziz EL-Amraoui 10 :30-11 :00: Coffee break 11:00-11:15 : OC5: Large scale computational model of the cochlea of inner ear: predictions about the cochlear cellular amplifier and mutations in the potassium-transport related genes: Dr. Mistrík PAVEL 11:15-11:30: OC6: Interactions between myosin VI and interacting proteins in the sensory hair cell: Tamar Tenne 11:30-11:45: OC7: Effects of mobile Ca2+ buffer deficiency on the IHC synaptic transmission: Dr. Tina PANGRŠIČ1 11:45-12:00: OC8: Mutations in the human miR-96, a microRNA expressed in the inner ear, causes non-syndromic progressive hearing loss: Ángeles MENCÍA RODRÍGUEZ 12:00-12:15: OC9: Prevention of hair cell death in transcription factors Pou4f3 and Gfi1 knockout mice using anti-apoptotic factors: Orna ATAR 12:15-12:30: Discussion 12 :30-14:00: Lunch 14:30-14 :45: Dissemination within the EuroHear project / FP7 opportunities by Laurent CHARVIN 15:00-16 :00 : PC 7 16:15-17:00: TC2: Control of connexin gene expression in cochlear organotypic cultures: Pr. Fabio MAMMANO. 17: 00-18:00: Round table cheered by Pr. Christine PETIT “Eurohear Project and research on hearing: present and future”

9:00-10 :00 : PC 6 10:00 - 10:15: Sesame based foods: Technological processing: (Confiserie Triki) 10 :15 - 10:30: Policy related to sesame use in agro-industry: Mr Mohamed Ben Rejeb (CTAA)

Developments for biotechnological research: success stories and networking 14 h00-15 :00: Poster session 15 :00-16 :00 : PC 7 16 :00-16 :20: Analysis of water for pesticides....using 2D LCMSMS: Mr Loïc BEYET (Applied Biosystem) 16 :20-16 :40: The next generation sequencer for genomic analysis has arrived: SOLID System: Mr Marco Pirotta (Applied Biosystem) 16 h40-17 :00: Coffee break 17 :00-17 :30: Biotechnology in Malaysia: Pr. Abdellatif IBRAHIM, Malaysia 17 :30-17 :45: NEPAD/North Africa Biosciences Network (NABNet): Promoting Biotechnology projects: Pr. Mohamed Elarbi Aouani 17 :45-18 :00: Construction of a novel multifunctional extracellular matrix-bound epidermal growth factor for tissue engineering: Dr Imen Elloumi

10 :30-11 :00: Coffee break 10 :00 - 10:20: Lipides: nutrition et obésité Pr Robert Verger (France) 11 :20-11:35: Digestibilité et stabilité de l’huile de sésame: Pr Youssef Gargouri (ENIS) 11 :30-11 :40: Lipides et diabetes: Dr Yassine Ben Ali 11 :40-11 : 55: Composition nutritive du sésame: Mme Naila Abid 11 : 55-12 : 10: Effets du sésame sur la santé humaine: Dr Mouna Mnif 12 : 10-12 : 30: Discussion 12 :30-14 :00: Lunch 14 :00-15 :00: Réunion/débat spécifique: consommateurs et presse : Mr Nabil Triki

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Thursday May 8th: Satellite 6 Incubating innovations (Start-ups, Spin-offs): From opportunities to Innovative companies 9:00-10 :00 : PC 8 10:00 - 10:10: CBS-innovation : strategy and logistic : Pr Hammadi Ayadi (CBS) 10 :10 - 10:20: Success stories of biotechnology transfer from CBS: Pr Sami Sayadi (CBS) 10 :20 - 10:30: Spin-off stories from CBS incubator: Mr Ramzi Trigui, Melle Hajer Yaakoubi, Melle Sana Smaoui 10 :30 - 10:45: Panel discussion 10 :45-11 :00: Coffee break 11 :00-11:15: Incitations for innovating companies: Dr Ikram Makni ( Business Center of Sfax, Tunisia) 11 :15-11 :35: INSERM- transfert expertise in biotechnology: Mallory Wolff (INSERM-TRANSFERT, France) 11 :35-11 : 55: Technology transfer and spin off mechanism in biotechnology, current challenges and practical examples from Germany: Frank Graage (Germany) 11:55-12:30: Discussion: Good practices for innovation in CBS: perspectives and consequences: Chaired by Ing. Gerhard Ebert and Pr. Daniel Thomas 13:30: Closing ceremony chaired by Minister of Higher Education, Scientific Research and Technology: Mr. Lazhar Bou Ouni 14:30: Lunch 15:15: Touristic trip 20:00: Dinner-Ceremony

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Plenary Conferences

Plenary Conference

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ISB 2008, May 4 – 8 , 2008 Sfax - Tunisia

Plenary Conference

PC 1

BIOREFINERY: Biotechnology for the conversion of biomass into biomolecules, biomaterials and biofuels. Daniel Thomas Compiegne University of Technology, FRANCE [email protected]

Abstract: Fossil fuel reserves are running out, global warming is becoming a reality, waste recycling is becoming ever more costly and problematic, and unrelenting population growth will require more and more energy and consumer products. There is now an alternative to the 100% oil economy; it is a renewable resource called biomass - the whole plant. In a global context of fossil energy dependence linked to oil and gas prices, it is essential to promote and increase the part of biobased products. Production and development of these new products are based on biorefinery concept. Biorefinery uses agro-resources to realize a valorization of the whole plant. Like crude oil, plants are composed of a huge number of different molecules. Each constituent of the plant can be extracted and functionalized in order to produce non-food and food fractions, intermediate agro-industrial products and synthons, whose value is generally inversely proportional to their volume. These fractions are then used directly or formulated according to the end users’ needs. Three major industrial domains can be concerned: molecules, materials and energy. Molecules can be used as solvent surfactants or chemical intermediates in substitution of petrol derivatives. Fibers can be valorized in materials like composites. Sugars and oils are currently used to produce biofuels like bioethanol or biodiesel, but second generation biofuels will use lingo-cellulosic biomass as raw material. Industrial biorefinery will be linked to the creation of new processes based on the twelve principles of green chemistry. Biotechnology, especially white biotechnology, will take a major part into these new processes with biotransformations (enzymology, micro-organisms…) and fermentation. The replacement of oil products by biobased products will develop a new bioeconomy and new industrial processes respecting the sustainable development concept. Industrial biorefinery can be developed on the principle that any residues of one can then be exploited as raw material by others.

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Plenary Conference

PC 2

Immunology of human leishmaniasis: challenges for vaccine development Hechmi Louzir Institut Pasteur de Tunis. E-mail : [email protected]

Abstract: Leishmaniasis constitutes a group of neglected diseases of public health importance in many parts of the world. Anti-leishmanial drugs are difficult to use and current experimental vaccines have not proven effective in humans. In fact, our knowledge of the immune response to Leishmania infection mainly stems from studying Leishmania infection in various experimental models, of which the L major murine model has been the most dominant, and has been extensively reviewed. Studies of the cellular immune responses in human beings have mostly been descriptive, because of the difficulties in defining the immunopathological and protective mechanisms in Leishmania infections, the necessity to do longitudinal studies, and by the genetic heterogeneity of human and parasite populations. Although epidemiological data from surveys of patients seem to confirm the Th1/Th2 dichotomy shown in experimental animal models, other studies show that the human immunological response is not exclusively explained in terms of Th1/Th2 subsets. AntiLeishmania responses play an essential role in: • The pathogenesis of visceral and cutaneous leishmaniasis. • The elimination and/or the control of the parasite multiplication. • The resistance to re infection. The later is crucial for vaccine development. Studies on vaccine development against human leishmaniasis have unfortunately remained fruitless until now, largely because of the lack of a precise definition of the immune mechanisms of resistance. In this contribution, I will summarize the present knowledge, and our laboratory experience, on human immune response to the parasite oriented toward vaccine development. A particular attention will be devoted to discuss: • The effector immune mechanisms that are associated with resistance against the parasite and how to analyze them • How to identify the target antigens (candidate vaccine) • The limits of the experimental models (mouse) for vaccine evaluation Selected references:

Nylén S, Sacks D. Interleukin-10 and the pathogenesis of human visceral leishmaniasis. Trends Immunol. 2007;28(9):378-84. Reithinger R, Dujardin JC, Louzir H, Pirmez C, Alexander B, Brooker S. Cutaneous leishmaniasis. Lancet Infect Dis. 2007;7(9):581-96. Kedzierski L, Zhu Y, Handman E. Leishmania vaccines: progress and problems. Parasitology. 2006;133 Suppl:S87-112.

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Plenary Conference

PC 3

Genetic Engineering of Bacterial Insecticides for Improved Efficacy Brian A. Federici Department of Entomology and Interdepartmental Graduate Programs in Genetics and Microbiology University of California, Riverside Riverside, California 92521 Email: [email protected]

Abstract: The bacteria Bacillus thuringiensis (Bt) and Bacillus sphaericus (Bs) hold promise for increased use as insecticides, which is currently limited by high costs compared to synthetic chemical insecticides. The insecticidal activity of these bacteria is due to larvicidal endotoxin proteins, which in Bt’s toxic to lepidopteran insects is due to Cry proteins, and in Bt israelensis (Bti), toxic to mosquitoes, is due to Cry and Cyt1A proteins. Importantly, Cyt1A synergizes Cry toxicity, delays resistance to these, and can suppress resistance to Bs and expand its target spectrum. Bs toxicity is due to Bin, a binary toxin acting as a single toxin. We used cyt1A promoters and a 5’ mRNA stabilizing sequence (STAB) to increase Cry and Bs Bin yields significantly, in both cases about 10-fold, although for Cry2A and Cry11A yields averaged only 2-fold. Recently, cyt1Ap/STAB was used to synthesize high levels of Bs Bin in Bti. The Bti/BsB recombinant (LC50 = 0.37 ng/ml) was 21-fold as toxic as Bti (LC50= 8.1 ng/ml) against fourth instars of Culex quinquefasciatus, and 32-fold as toxic as Bs2362 (LC50 =11.9 ng/ml). Moreover, Bti/BsB suppressed high levels of Bs resistance (>50,000-fold) in Cx. quinquefasciatus. Against Aedes aegypti and Anopheles albimanus, Bti/BsB was only twice as toxic as Bti. However, against larvae of An. gambiae, activity was more than ten-fold higher than that obtained with Bti and Bs strains used in current commercial products, indicating these new recombinant bacteria have potential for use in Africa against the most important malaria vectors. Such new recombinant larvicides should be highly effective against mosquitoes and certain agricultural pests, and much less prone to resistance due to their high toxicity and endotoxin complexity. The much higher efficacy combined with resistancedelaying properties and a high degree of safety to non-target organisms should expand use of bacteria for mosquito vector control, as well as control of some agricultural pests. For example, the same techniques used to improve the efficacy of mosquitocidal proteins can be used to improve the activity of Cry3 proteins against coleopteran pests and Cry2 proteins against lepidopteran pests.

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Plenary Conference

PC 4

Integrated approaches to wheat improvement under drought Parry Maj, Baudo, M, Madgwick Pj, Phillips A and Habash D Centre for Crop Genetic Improvement, Department of Plant Science, , Rothamsted Research, Harpenden, Herts. AL5 2JQ, UK

Abstract: Water is essential to sustaining human and environmental health but is becoming scarce in many countries. The availability of water is a major determinant of world-wide crop yield. Wheat is the most widely grown crop both in the UK and worldwide. Even in the UK yield losses due to drought average 1-2 t ha-1 but the losses are much greater in other countries with higher temperatures and lower rainfall. The predicted changes in climate patterns are projected to increase the losses. Although there is genetic variability for drought tolerance and yield in wheat germplasm, drought tolerance is a complex and multigenic trait which makes it difficult to breed for. Nevertheless molecular plant breeding has the potential to deliver high and stable yields under drought once the component traits and genes underlying traits to achieve high stable yields under drought have been identified. These can then be incorporated into new cultivars using conventional or biotechnological tools. In order to better understand the relationship between genotype, component traits, and environment over time we are adopting a multidisciplinary approach. By integrating genetics, genomics, soil science, crop physiology, biochemistry and biomathematics and agronomy we aim to both understand the drought response and identify candidate genes, QTLs and traits that can be used to develop crops with high stable yield under drought. Candidate gene will be evaluated by TILLING or transformation. The research will develop new conceptual models that describe drought-induced changes in gene expression and metabolism which underlie the adaptation mechanisms.

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Plenary Conference

PC 5

From human hereditary deafness to the cellular and molecular mechanisms of hearing Christine Petit Professeure au Collège de France, Pr à l’Institut Pasteur Unité de Génétique et Physiologie de l'Audition, INSERM UMRS 587, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris cedex 15, France

Abstract: The molecular mechanisms underlying the development and the functioning of the cochlea have eluded characterisation for a long time owing to the very small number of cells in each cochlear cell type. The study of hereditary deafness in humans provides a unique approach to gaining relevant insights into the understanding of these processes. Today, more than 40 genes involved in the isolated forms of deafness have been identified. Using these genes, functional modules underlying the morphogenesis of the hair bundle, the mechanoreceptive structure to sound stimulation, and its ability to work as a single unit can be built. For instance, the genes defective in the various types of Usher syndrome have enlightened the function of the hair bundle links. As for the hereditary auditory neuropathies, a rare subclass of hearing impairment, they provide insight into the molecular mechanisms accounting for the morphofunctional specificity of the inner hair cell ribbon synapse and the auditory pathway as well. Moreover, basic knowledge obtained on underlying pathogenic processes involved in hereditary deafness may lead to reassess the meaning of some clinical explorations.

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Plenary Conference

PC 6

Using Microbial Genetics to Find and Develop New Antibiotics David A. Hopwood Department of Molecular Microbiology, John Innes Centre, Norwich, NR2 2AH, England ([email protected])

Abstract: There is an urgent need to develop new antibiotics to meet the worldwide threat of emerging antibiotic resistance. The traditional method of discovering antimicrobial agents by the isolation and screening of microbes from natural habitats has failed to produce useful antibiotics for several decades. One way to address this deficit is to harness the powers of genetic manipulation of antibiotic producers, especially actinomycetes. Novel compounds can be made by the recombination of parts of the gene clusters for antibiotic biosynthesis from more than one organism in the process of “combinatorial biosynthesis of unnatural natural products”. This can be augmented by the total synthesis of artificial genes, according to the rules of the polyketide code, and their expression in convenient hosts to make totally new chemical structures. Meanwhile, total genome sequencing has revealed large numbers of “cryptic” gene clusters for potentially valuable secondary metabolites in the genomes of actinomycetes. A current challenge is to find generic ways to achieve expression of these gene clusters in order to evaluate the resulting novel metabolites as antibiotics. Both these approaches to antibiotic discovery will be reviewed.

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Plenary Conference

PC 7

Effects of Olive Leave Compounds on the Functions of Vasculature and Ovary Hitoshi Miyazaki Graduate School of Life and Environmental Sciences, Alliance for Research on North Africa, University of Tsukuba, Tennohdai 1-1-1, Ibaraki 305-8572, Japan

Abstract: We have examined the effects of major compounds of olive leaves on the vasculature and ovarian functions to aim toward the application of these compounds for food and medical industries. One of the five compounds of olive leaves stimulated the wound healing of cultured vascular endothelial cells (VECs) by inducing cell proliferation. In reverse, this compound induced apoptosis of cultured vascular smooth muscle cells (VSMCs). These functions of the compound in both cells are mediated by the generation of nitric oxide, a well-known vasodilator. Oral administration of the precursor of this compound to spontaneously hypertensive rats (SHR) significantly reduced the blood pressure. Because cell death of VECs, proliferation and migration of VSMCs, and hypertension are closely related to the initiation and progression of arteriosclerosis, these data suggest that the compound prevents the pathogenesis of the disease. This compound prevented oxidative stress-dependent apoptosis of ovarian granulosa cells, which play a critical role in the fate of follicular growth. Oral administration of the precursor of this compound to female mice completely prevented a marked decrease in the number of ovulation induced by heat stress, which is converted to oxidative stress in vivo. Therefore, the compound precursor can be used as supplements for livestock feed since heat stress-induced reduction of ovulation in the summer is a big problem in the livestock industry.

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Plenary Conference

PC 8

Development of Malaria Vaccine: Current Scenario Virander S. Chauhan Director International Centre for Genetic Engineering and Biotechnology New Delhi – 110 067

Abstract: Vaccination is the most reliable and affordable way of making human population healthy and maintaining it that way. Vaccine against Malaria, a major killer of children in the tropical world, has remained a scientific challenge. With the growing realisation of potential value of vaccination as a strategy for malaria management and enhanced financial support made available by international agencies, malaria vaccine development programmes have received a new lease of life. The renewed interest in vaccine development is also fueled by a better understanding of the immune system, the effector mechanisms involved in protective immunity and by the availability of complete genome sequences of pathogens and their hosts. What then is the current and future status of malaria vaccine(s)? Most new researches in Vaccinology per se have found their way in malaria vaccine development. Synthetic peptide vaccines, DNA vaccines, viral vector based constructs, new adjuvants and new methods of vaccine delivery have all been applied in malaria vaccine development. Scientific challenges apart, real hurdles in the vaccine development are lack of laboratory correlates, a reliable animal model and difficulties in carrying out efficacy trials of candidate vaccines. However, sustained efforts in malaria vaccine development, has seen several vaccine trials in humans. At least one malaria vaccine (RTS,S) has already shown impressive results and is scheduled to go forward beyond phase II trials, in Africa. Most crucial needs of the hour for now and for future seem to be production of clinical grade vaccines for trials and development of potential vaccine trial sites at different geographical locations. Results of ongoing blood stage malaria vaccines for the human malaria, plasmodium P.falciparum and plasmodium vivax will be discussed.


Session 1

Session 1: Biotechnology for Human Health -

Infectious, Monogenic and complex diseases Functional genomics and stem cells Innovative therapies Computational modeling and simulation in life sciences


Thematic Conferences

Session 1



TC 1 S1

Relationship between Structure and Immunostimulating Activity of Enzymatically Synthesized Glycogen Ryo Kakutani1, Hideki Kajiura1,2, Takashi Furuyashiki1, Tsunehisa Akiyama1, Reiko Ueyama1, Hiroki Takata1, Yoshiyuki Adachi3, Naohito Ohno3 and Takashi Kuriki1 1

Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502 Japan, [email protected] ２ Food Material Department, Glico Foods Co., Ltd. 3 Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy & Life Science

Abstract: Glycogen is highly branched (1→4) (1→6)-linked α-D-glucan. Two methods have been known to prepare glycogen. First, glycogen can be extracted from natural resources such as shellfishes and animal tissues. Second, glycogen can be synthesized from glucose-1phosphate by using two enzymes, α-glucan phosphorylase (EC 2.4.1.1) and branching enzyme (EC 2.4.1.18) (1). We recently established a novel method for synthesis of glycogen from starch (2), combining reactions of isoamylase (EC 3.2.1.68), branching enzyme, and amylomaltase (EC 2.4.1.25). The new method is the most efficient among the three methods, and furthermore we can control the molecular mass of glycogen in a range of 3,000 to 30,000 kDa by adjusting some parameters of reaction. Glycogen is exclusively known as energy and carbon reserves in animal cells and microorganisms. However, immunological activity of glycogen has long been suggested, although strong scientific evidence has not been obtained. Indeed, many scientists have been annoyed by the lack of reproducibility of their experimental results. Because their samples of glycogen have been extracted from natural resources, therefore, i) the effect of trace amount of the other materials cannot be ruled out; ii) the important characteristics of each glycogen sample, such as the average molecular mass and the chain length, are quite different depending on the source and purification procedures. We have cleared up the immunological activity of glycogen by using completely pure one with very uniform characteristics. The results revealed that the molecular mass of glycogen strongly related to its immunostimulating activity (3). The enzymatically synthesized glycogen with molecular mass of 5,000 and 6,500 kDa strongly stimulated RAW264.7, a murine macrophage cell line, in the presence of interferon-γ , leading to augmented production of nitric oxide, tumor necrosis factor-α, and interleukin-6. These results strongly suggested that glycogen functions not only as fuel reservoir but also as a signaling factor in vivo. References 1) G. T. Cori and C. F. Cori. 1943. J. Biol. Chem. 151:57-63. 2) H. Kajiura, R. Kakutani, T. Akiyama, H. Takata, and T. Kuriki. 2008. Biocatal. Biotransform. In press. 3) R. Kakutani, Y. Adachi, H. Kajiura, H. Takata, T. Kuriki, N. Ohno. 2007. Carbohydr. Res. 342:2371-2379.



TC 2 S1

Gene and cell therapy for cardiovascular disorders Mauro Giacca International Centre for Genetic Engineering and Biotechnology (ICGEB) Trieste, Italy

Abstract: The potential of inducing therapeutic angiogenesis and myocardial cell regeneration through gene transfer has engendered much excitement for the possible treatment of ischemic tissues. However, a number of crucial issues still have to be solved prior to successful clinical application, including the understanding of whether functional blood vessels can arise as a result of the delivery of a single angiogenic factor, or which are the most suitable vectors for efficient, stable and controllable gene delivery to the heart. To induce myocardial angiogenesis, different genes that promote new blood vessel formation (among which different VEGF isoforms, angiopoietins, and FGF2) can be safely administered to the myocardium and show remarkable efficacy in different animal models of acute and chronic ischemia. To be successfully used in patients, however, the gene combination that promotes both the formation of new vessels and their maturation needs to be found. For example, the unregulated expression of VEGF, a powerful trigger of neoangiogenesis, induces the formation of vessels that are leaky, poorly organized and do not account for improved tissue perfusion. Strikingly, the functional competence of these vessels can be significantly improved by the simultaneous delivery of other factors, such as Ang1, which promotes vessel maturation. An intriguing possibility suggested by some studies is that circulating progenitors of bone marrow origin might participate in the process of new blood vessel formation through their specific differentiation into vascular cells. However, recent evidence has challenged this notion, indicating that the newly formed vasculature that is formed as a response to VEGF arises from the activation and proliferation of resident vascular cells. Nonetheless, the recruitment of bone marrow mononuclear cells to the sites of neoangiogenesis appears essential for the process of arteriogenesis, involving the maturation of a primitive capillary network into more structured vessels covered by smooth muscle cells. The possibility of inducing the formation of a stable vasculature in the ischemic heart most likely requires the simultaneous delivery of multiple genes and their persistent and regulated expression over time. This objective can be met by the use of viral vectors based on the adeno-associated virus (AAV), a defective parvovirus with a ssDNA genome, which is largely diffused in the general population in the absence of any apparent associated pathology. Since AAV vectors do not express any viral protein, they are not immunogenic nor do they elicit an inflammatory response. Additional appealing features of AAV vectors are the possibilities to control expression of the transferred gene from any desirable promoter and to obtain highly concentrated and purified viral preparations. For still uncharacterized reasons, these vectors show a specific tropism for skeletal muscle cells and cardiomyocytes, in which they drive expression of the therapeutic genes for indefinite periods of time.



TC 3 S1

From classical antibody based immunotherapy to engineered nanobodies Bouhaouala-Zahar Balkiss1, Muyldermans Serge2 & El Ayeb Mohamed1 1

Laboratoire de Venins et Toxines, Institut Pasteur de Tunis, 1002 Tunis-Tunisie

2

Department of cellular and molecular interactions, VIB Brussels

[email protected]

Abstract: Nanobodies are single domain antigen binding fragments derived from functional Heavy-chain antibodies occurring in camelids such as dromedary. Data is presented on the study with monovalent, bivalent Nanobodies and reconstituted chimeric Heavy-chain antibodies targeting two major scorpion toxins that selectively block mammal sodium channels and that are involved in noxious effects. Scorpion envenoming is a real health problem in many countries in the world. In Tunisia about 40 000 sting cases occur each year from which 20 to 30 mainly envenomed children die. Horse polyclonal immunotherapy is the specific and recognized treatment. A lot of data dealing with immune sera quality improvement stated that sodium channel specific noxious toxins have a fast toxicokinetic characteristic. Delay in the application of the immune sera following envenoming can compromise seriously the chances on success and there is a critical need for a sufficient amount of administrated therapeutic compound by i.v. route. Some of these crucial parameters have been verified in random selection of envenomed children. Clearly immunotherapy application is beneficial for treated patients. However, in some severe envenoming cases Fab’2 fragment application failed to rescue the patients. In a recent effort aimed to develop and produce monoclonal antibody engineered fragments with new neutralizing and pharmacokinetic characteristics, dromedary VHH Nanobodies have been selected by phage display technology from combinatorial libraries. First, the immune system of dromedaries is exploited to elicit an immune response against scorpion toxins. Secondly, libraries were constructed containing the variable region repertoire of Heavy-chain antibodies from blood lymphocytes of the immunized dromedary. The libraries were screened for binders against Androctonus australis scorpion toxins (AahI and AahII) by phage-display. The VHH genes of the clones that scored positive in ELISA were subcloned into an E.coli expression vector. Affinity measurements were assessed by surface plasmon resonance on a BIAcore 3000 instrument. Bivalent constructs were generated as well as a chimeric Heavy chain antibody composed of the Nanobody and the human Fc part. Swiss mice model were used throughout to evaluate the neutralizing activity of our anti-toxin Nanobody constructs. We will described the successful strategy of isolation, production and characterization of Nanobodies and derived products (bivalent mono or bispecific and Fc-Nanobodies) that binds tightly and neutralizes native scorpion toxins. The design and performance of highly potent neutralizing Nanobody with long human serum half life is reported. VHH fragments with specificity to, respectively, Androctonus australis scorpion toxin 1 and 2 interact with high affinity (in nanomolar range). Moreover, some of these are able to neutralize potently the cognate toxin either following i.c.v. or s.c. administrations. These examples of engineered Nanobodies highlight how the many benefits, provided by these discovered natural proteins, are translated into a development of therapeutics against envenomation. Work is in progress to evaluate comparative pharmacokinetic effects over Fab’2 fragments. Expected favourable results will be the basis of a novel therapy tailored to treat severe cases.



TC 4 S1

Gene-Environment Interactions in Oesophageal Cancer in South Africa M. Iqbal Parker. International Centre for Genetic Engineering and Biotechnology (Cape Town), Wernher and Beit Building (South), UCT Faculty of Health Sciences, Anzio Road, Observatory 7925, Cape Town, South Africa.

Abstract: The tremendous variation in the observed susceptibility to complex diseases such as cancer may result as a consequence of an imbalance in the activities of the enzymes involved in the metabolism, conjugation and transport of xenobiotics and environmental factors such as pollutants and habits. In this study we investigated the functional polymorphisms in some of the phase I enzymes (CYP3A5, CYP2E1, Alcohol Dehydrogenase 2 and Aldehyde Dehydrogenase.) and phase II enzymes (GSTM1, GSTT1, GST P1 and SULT1A1) in oesophageal cancer patients and controls. A variety of environmental and behavioural factors were identified that were significantly associated with increased risk of developing oesophageal cancer. These factors included the exposure to smoke during the burning of wood or charcoal for cooking and heating purposes (AOR 15.2; pA mutation in the foetus who was diagnosed as normal. Screening of c.8007delT mutation in F2 showed that the first foetus was a carrier, while the second was homozygous for the c.8007delT mutation. So, the first foetus was diagnosed as normal; while the second couple was counselled that the foetus would be affected. Mutation screening of the 521delT mutation in the foetus of F3 revealed that he was normal. All pregnancies continued; and mutations screening on blood extracted DNA of newborns, as well as the clinical follow-up were in concordance with prenatal diagnoses. The present study represents the first successful prenatal diagnoses of MDC1A and LGMD2C forms in Tunisia and in Africa, and demonstrates the reliability of mutational screening strategy for prenatal diagnosis for genetic counseling and patient management.

ISB 2008, May 4th – 8th, 2008 Sfax – Tunisia

Session 1

OC20 S1

A novel nonsense mutation (at Ser23) in GPIbβ gene affects GPIb-IX complex expression in two Tunisian Bernard-Soulier Syndrome unrelated Families 1

2

1

2

Hadj Kacem Basma , Elleuch Hinda , Trigui Ramzi , Gargouri Jalel and Gargouri Ali

1

1-Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, BP”K”3038, Sfax -Tunisia 2-Centre Régional de Transfusion Sanguine de Sfax-Tunisia

Abstract: Bernard-Soulier syndrome (BSS) is a rare autosomal recessive genetic disorder characterized by a clinical bleeding tendency, platelets morphologic abnormality, thrombocytopenia, absence of ristocetin induced platelets aggregation and defective adhesion of platelets to the sub endothelium. BSS is explained by a defect in primary haemostasis owing to quantitative or qualitative defect in the GPIb-IX-V complex, a membrane receptor which plays an essential role in the thrombogenic function of platelets by interacting with von Willebrand factor. This complex normally consists of four subunits, GPIbα, GPIbβ, GPIX and GPV. For two unrelated BSS Tunisian patients, their parents and siblings we have amplified by PCR and sequenced the three candidates’ genes (GPIbα, GPIbβ and GPIX). Platelets were investigated by flow cytometry, western-blot and confocal microscopy. We report a novel, homozygous, GPIbβ defect in two unrelated BSS patients, in which Ser 23 is substituted by a Stop codon causing a premature termination of translation. The pedigree was determined for both families and revealed heterozygosity of parents. We studied the effect of this mutation on the expression of the GPIb-IX complex in both patients’ platelets by western blot, flow cytometry and confocal microscopy: GPIbα and GPIX were absent on the surface of platelets, whereas they were present in the cytoplasm with tiny amounts of GPIbα. The novel Ser 23 Stop mutation in GPIbβ is responsible of BSS in two unrelated families and hampers the complex to form on the platelets surface.


Session 1

OC21 S1

The inter-individual variability of the human urinary proteome assessed by 2D electrophoresis analysis Molina Laurence, Salvetat N., Peres S., Benameur R., Molina F. and Granier C., Sysdiag, FRE3009 CNRS-BIORAD, Cap Delta/Parc Euromédecine, 1682 rue de la Valsière, CS 61003, 34184 Montpellier Cedex 4, France [email protected]

Abstract: The detection of disease biomarkers in urine could be advantageous for clinical diagnostic, since urinary proteins comprise proteins filtered from plasma and proteins released from the renal/urinary system. Urine can be collected noninvasively and in large amount. Urine is however a complex mixture. Thus the proteomic approach appears to be a suitable method to thoroughly identify urinary proteins and to search for potential biomarkers of kidney disease. The present study was devised to study the inter-individual variability of the human urinary proteome. To this aim, we separated by two-dimensional gel-electrophoresis, proteins from urinary samples of healthy volunteers and then compared 2D protein profiles using image analysis software. Twenty healthy volunteers were included in this study. Urinary samples were collected during the morning and stored at -80°C. Samples were treated and then analysed in 2D gels. The simultaneous analysis of the 20 protein profiles was realised with 2D gel analysis software (ProgenesisSameSpot, NonLinear). Statistical treatments of data were used to study the protein variability. 2D gel electrophoresis allowed us to observe a heterogeneous variability of urinary proteins in the studied population. Different protein categories were defined according to the variability of their occurence in normal urine. The more constant and the more variable proteins were further identified by mass spectrometry. We will take advantage of this analysis of the variability of the healthy human urinary proteome to look more confidently for biomarkers of kidney diseases.


Session 1

OC22 S1

Comparison of an in house ELISA test and the pELISA MEDAC for the detection of Chlamydia trachomatis IgG antibodies in Danish woman seeking abortion Frikha-Gargouri, Olfa1, Gdoura, R1, Znazen, A1, Birkelund, S2, Christiansen, G2, Hammami, A1. 1: Department of Microbiology and research laboratory "Microorganismes et Pathologie Humaine" Habib Bourguiba hospital of Sfax, Tunisia. 2- Institute of Medical Microbiology and Immunology, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark. [email protected]

Abstract: Chlamydia trachomatis (CT) is an obligate intracellular bacterium causing genital tract infections. Serology is used for the diagnosis of CT infections. In this study, we compared the performance of our in house ELISA test using the variable domain 4 (VD4) of major outer membrane protein to a commercial ELISA test (the pELISA (Medac)) using peptide from the VD4 for the detection of CT IgG antibodies. In our in house ELISA test, the full length sequence of VD4 was cloned in pGEX-6P1, expressed and purified by affinity chromatography. 105 sera from women seeking abortion were tested by these ELISA tests. After removal of reactivity due to the GST for each sera, a good correlation was found between the tests (R2=0.84). Subsequently, the GST was cleaved from the GST-VD4. The VD4 was purified, verified for it’s homogeneity by mass spectrometry, tested in ELISA and compared to the results of the pELISA. Cleaving the vector tag does not enhance the performance of the in house test since a lower correlation was found between the two tests (R2=0.63). In conclusion, the performance of our in house ELISA tests was better when using GST-DV4 and subtracting the reactivity due to the GST compared to pELISA.


Session 1

OC23 S1

Oxidative stress in Systemic lupus erythematosus and rheumatoid arthritis and its relationship with autoantibodies production Riadh Ben Mansour, Saloua Lassoued, Bochra Gargouri, Amel El Gaïd, Hamadi Attia, Faïza Fakhfakh Institut Supérieur de Biotechnologie de Sfax, Route Sokra Km 4 BP 261. 3038 Sfax Tunisie. [email protected]

Abstract: In the present study, we hypothesized that autoimmune background in Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) diseases induced anti-SOD and/or anti-CAT autoantibodies that inhibit SOD or CAT activities and thereby contributed to increasing ROS production, as previously described. The oxidative/antioxidative profile was tested among 40 SLE patients and 39 RA, and 50 controls by measuring serum malondialdehyde (MDA), conjugated dienes (CD), catalase (CAT) and superoxyde dismutase (SOD) activities. In all patients the lipid peroxidation was confirmed by high levels of MDA and conjugated dienes (p14775µg/ml. Key words: Enterobacteriaceae – Epidemiology – Extended spectrum B-lactamases – Gram (-) bacteria – Resistance to antibiotics – Seek plasmid – Thymus capitatus (L) Hoffm et Link – Ammoides verticillata – Antibacterial activity.


Session 1 /

PC93 S1

Antispasmodic activity of aqueous and organic extracts of the brown algae, Dictyopteris membranaceae Bouzgarou Olfa1, Mhadhebi Lamia1, Ben Aoun Zouheir2, Dellai Afef1, Farhat Farhat2 and Bouraoui Abderrahman1. 1 Unité URSAM, Laboratoire de Pharmacologie, Faculté de Pharmacie, 5000 Monastir (Tunisie). 2 Unité de chimie analytique, Faculté de Pharmacie-5000 Monastir (Tunisie).

Abstract: The role of marine organisms, essentially algae and invertebrates in drug discovery has been greatly enhanced the last few years. As part of our search for a new potential drug, two extracts of brown alga Dictyoptéris membranacea, collected from Tunisian coast were screened for their antispasmodic activity on rat isolated duodenum, in the presence of Acetylcholine (Ach) or in the presence of Barium chloride (BaCl2). Both extracts of Dictyopteris membranacea exhibited in a concentration and reversible manner, a significant inhibitory activity for the contractile reponse induced by Ach (5 µg/ml) and by BaCl2 (5 mg/ml). The percentages of inhibition of aqueous extract (2.5 mg/ml) and chloroformic extract (0.5 mg/ml) were respectively 45.5 % and 72 % in the presence of Acetylcholine. Whereas the percentages of inhibition in the presence of BaCl2 were respectively of 73% for aqueous extract at concentration of (1mg/ml) and 72 % for the chloroformic extract at (0.5 mg/ml).


Session 1 /

PC94 S1

Hormonal Theory Of Aging O. Chehab A,B, R. Hmizi B, M. Ouertani A, A. Bouraoui A, K. Mahdouani A,B a: Unite of research URSAM 03/UR/07-01, Faculty of Pharmacy of Monastir, street Ibn-Sina, 5000 Monastir, Tunisia, E-mail: [email protected] b: Laboratory of Biochemistry, Hospital Ibn El Jazzar of Kairouan, 3100 Kairouan, Tunisia Postal address: Laboratory of Biochemistry, Hospital Ibn El Jazzar of Kairouan, 3100 Kairouan, Tunisia

Abstract: The hormones are primarily used to coordinate the different functions of our bodies. Secreted by various glands, they are essential to a correct function of our organisms. The rate of certain hormones decreases with age. It is considered that this reduction is an aging marker; the rate hormonal measurement in the organism is regarded as a reliable means to evaluate our biological age, independently of the number of our years. During this study, we evaluate the hormonal status in healthy elderly Tunisian population; we subsequently carry out the proportioning of some hormones: DHEA-S, Cortisol, GH and Melatonin. The relations between the concentrations of these hormones and the various parameters of lipid, electrolytic and fertility assessment were studied. The relationships to age, sex, composition of the body, blood pressure and other biochemical parameters were also given. Aging process and endocrinal system were intensively reviewed during these last years and certain hormones merit serious consideration, in fact of their multiple effects which confer their fascinating potentialities. Consequently, the pharmacological reconstitution by using these hormones meeting a certain success with the reverse of functional decline associated with age.


Session 1 /

PC95 S1

Performance of CT694 based Enzyme Linked ImmunoSorbent Assay for detection of Chlamydia trachomatis antibodies Frikha-Gargouri O.1, Gdoura R1, Znazen A1, Birkelund S2, Christiansen G2, Hammami A1. 1: Department of Microbiology and research laboratory "Microorganismes et Pathologie Humaine" Habib Bourguiba hospital of Sfax, Tunisia. E-mail: [email protected] 2: institute of Medical Microbiology and Immunology, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark.

Abstract: Chlamydia trachomatis (CT) and Chlamydophila pneumoniae (CP) are obligate intracellular bacteria causing genital tract infections and respiratory tract infections, respectively. Serology is used for the diagnosis of CT infections. A newly hypothetic protein CT694 was described to be antigenic in CT. In this study, we aimed to evaluate the performance of an in house ELISA test using as antigen CT694 to the microimmunofluorecsence (MIF) test routinely used in our hospital. The sequence of CT694 was cloned in pGEX-6P-1, expressed in Escherichia coli and purified by affinity chromatography. 348 sera were tested by MIF and the in house ELISA tests. These sera were classified according to the results of the CT and CP MIF tests: 176 sera were CT and CP negative, 65 were CT negative CP positive, 48 were CT positive and CP negative and finally 59 were CT and CP positive. The new developed antigen was found to be specific for CT with a specificity of 95%. However, the in house ELISA test sensitivity (54%) was low compared to MIF test. CT694 appears to be specific, however it lacks sensitivity. Thus, this antigen could not be used alone for CT serology. Further studies would focus on its utility when associated with other antigens in ELISA tests for CT serology.


Session 1 /

PC96 S1

Evaluation of antimicrobial, antioxydant and cytotoxic activities of marine plant extracts Kesraoui, Ons1,3, Picot, L2., Maugard T. Marzouki, M.N.1 And Limam, F.3 1 : Lab Interactions Légumineuses-Microorganismes, CBBC, BP 901, 2050 Hammam-lif 2: Lab Biotechnologies et Chimie Bio-organique CNRS FRE2766, Av Michel Crépeau- 17042 La Rochelle 3 : Unité Génie Biologique 99 UR 09-26, Zone Industrielle Charguia1, INSAT-Tunisia. E-mail: [email protected]

Abstract: Methanolic and acetonic extracts of marine plant, collected from Tunisian coast, were analysed for their antimicrobial, antioxidant and cytotoxic activities. The antimicrobial property of marine plant was studied against ten human pathogenic bacteria and two yeast strains using disc diffusion method. Acetonic and methanolic extract showed similar antibacterial activity and were effective against Bacillus lichiniformis, Bacillus thuringensis and Staphylococcus aureus strains. The anti-oxidant activity was evaluated by DPPH free radical scavenging activity. The IC50 values obtained for mathanolic and acetonic extract were 200 and 150 µg/ml respectively. The antiproliferative activity of these extracts was examined in two human breast cancer cell lines MDA-MB-231 and MCF 7 using methyl tetrazolium (MTT) assay. After 48 h of exposure, methanolic extract inhibited the growth of the two cell lines in a dose dependant manner. Whereas, acetonic extract increased cell proliferation.


Session 1 /

PC97 S1

Redefining the genetic localisation and clinical aspect of Buschke-FischerBrauer Palmoplantar Keratoderma in a seven generation Tunisian family. Mamaï, O., Gribaa, M., Adala, L., Bouyacoub, Y., Ben Charfeddine, I., Belazreg, T ., Mili, A ., Kasem S., Saad, A. Cytogenetic, molecular genetics, and reproduction biology in human laboratory, CHU Farhat HACHED Sousse. Rue Ebn Eljazzar CHU Farhat HACHED, 4000, Sousse, Tunisia [email protected]

Abstract: Punctate palmoplantar kertoderma (PPK), named Buschke-Fischer-Brauer syndrome, is a rare autosomal dominant cutaneous disorder characterized by numerous hyperkratotic papules distributed on the palms and soles. Tow loci for this PPK were found to be located on chromosome 8 (8q24.13-8q24.21 (9.89 cM)) and chromosome 15 (15q22.2-15q22.31 (5.06 cM)). No gene for this disease has been indentified to date. We have to confirm linkage with one locus and to refine it, to identify the gene disease. We investigate a seven-generation KPPBFB Tunisian family to study the segregation of the two loci with STR marker. A clinical investigation was directed to find association between cancer and KPPBFB and other affection of the skin. No significant evidence for linkage was fined with the locus of chromosome 8. But a significant evidence for linkage was observed in the region of 15q22.215q22.31 with a maximum tow-point LOD score of 3.0184 at D15S108 (θ=0). Haplotype and recombination analysis showed a new locus for punctate KPP defined with D15S987 and D15S153 different to the previous loci. This locus overlaps 3.261 Mb (3.35 cM). An anomaly in the skin pigmentation is associated to this type of KPP. In this study we refine the Buschke-Fischer-Brauer locus to 3.261 Mb and we describe the most important candidate gene that can cause this disease, and that will be sequenced in the previous study.


Session 1 /

PC98 S1

Molecular investigation of genetic disorders in Tunisian consanguineous families. R. Kefi-Ben Atig, Diseases1 and Collaborators2 Research Unit on Molecular Investigation of Genetic Orphan Institut Pasteur de Tunis.

Abstract: Tunisian population, like other North African populations, is characterized by its heterogeneous ethnic background and a high rate of consanguinity. The rate of consanguineous mating is estimated to 33% in Northern Tunisia. Depending on the studied area, this rate reaches over 60%. This can be explained by the fact that endogamy is culturally favoured. In consequence of the high rate of inbreeding, there is an increase in the prevalence of recessive monogenic as well as multifactorial genetic disorders. With the availability of the human genomic sequence and of other model organisms genome sequences, identification of candidate genes for genetic disorders has been largely improved. We report here on molecular investigation of several rare disorders and particularly severe genodermatosis such as epidermolysis bullosa and xeroderma pigmentosum. Although different diseases are studied, a standardized strategy has been adopted. In a first step, patients and their families are typed with microsatellite markers flanking the already known genes to assess linkage to these candidates using homozygosity mapping. In a second step mutation screening is performed by direct sequencing of the coding region of the gene of interest. Despite the relatively small size of the Tunisian population, clinical and genetic heterogeneity is generally observed. Taking into account the historical, socio-cultural and economic context of southern Mediterranean countries, we propose a strategy for the investigation of genetic disorders as a time and cost effective tool for molecular diagnosis of these diseases in consanguineous populations.

1

equal contribution in alphabetical order: Sonia Abdelhak, Ahlem Amouri, Imène Arfa, Manel Bali, Mbarka Bchetnia, Slim Ben Ammar, Sabrine Ben Brik, Nizar Ben Halim, Mariam Ben Rekaya, Faten Ben Rhouma, Chérine Charfeddine, Sonia Chakroun, Faika Chérif, Wafa Chérif, Lamia Chérif-Ben Abdallah, Ibtissem Chouchane, Imen Dorboz, Amel Ghouila, Sana Hsouna, Haifa Jmel, Selma Kassar, Imène Kraiem, Khaled Lasram, Chokri Maktouf, Olfa Messaoud, Habib Messai, Sonia Nouira, Farah Ouechtati, Houyem Ouragini, Faten Talmoudi, Ahmed Turki, Mohamed Majdi Zorgati. 2 Mourad Mokni, Med Samir Boubaker, Neji Tebib, Marie-Françoise Ben Dridi, Jelel Chemli, Koussay Dellagi, Leila Elmatri, Fatma Warda, Kamel Monastiri, Héla Ben Abid, Ahmed Rebai, Najoua Miladi, Med Tahar Sfar, Mohamed Zghal, Fethi Amri.


Session 1 /

PC99 S1

Determination of polyphenolic composition and antioxidant activity of Crataegus azarolus var. aronia calli Bahri-sahloul R.a, Harzallah-Skhiri F.b, Saguem S.c, Grec S.d, Trotin F.e, Ammar S.a a

U.R. Plant Morphogenesis and Biotechnology, Faculty of Sciences of Tunis, Tunisia U.R. Agrobiodiversity, Institute Superior of Biotechnology of Monastir, Tunisia, E-mail: [email protected] ; Fax: + 216 73 465 405- Tel : +21673465404 c Metabolic Biophysics and Applied Pharmacology Laboratory, Department of Biophysics, Faculty of Medicine, University of Sousse, Tunisia d University of Sciences and Technology of Lille, "Abiotic Stress and cultivated plant differentiation" UMR1281 USTL, INRA, ERT1067, IFR147, SN2 Bulding, F-59650 Villeneuve d'ascq cedex, France. e Pharmacognosy laboratory, Faculty of Pharmacy, BP 83, 59006 Lille-France b

Abstract: The production of polyphenol by fourty-two-weeks old callus cultures from leaf and floral bud ovaries of Crataegus azarolus var. aronia has been studied in relation to growth variation within a fourty-days-subculture period. Fresh weight, dry weight, content in total phenols, flavonoids, proanthocyanidins were determined. Chlorogenic acid, hyperosid, rutin, isoquercitrosid, (-) epicatechin and procyanidin B2 were identified by HPLC. Antioxidant activity of calli extract was determined spectophotometrically with DPPH and ABTS radicals. An increasing of fresh and dry weights has been observed. The optimal contents of total phenols, flavonoids and proanthocyanidins were reported at the 24th day culture. They were respectively equal to 3500.7 ± 34.5; 1100.2 ± 23.5 and 1845.7 ± 25.8 mg/100g dry wt for floral bud ovaries calli and 4100.2 ± 26.2; 1145.8 ± 26.7 and 2500.7 ± 38.9 mg/100g dry wt for leaf calli. Optimal antioxidant activity (2810.7 ± 10.2 µM trolox /100g dry wt for floral bud ovaries calli and 2757.0 ± 14.4 µM trolox /100g dry wt for leaf calli) was attained when maximal growth is reached (at the 24th day culture). Strong correlation was found between antioxidant activity and polyphenol contents.


Session 1 /

PC100 S1

Technology Forsight in Stem Cell Sector – using patent information as a tool Guerrante, R. National Institute of Industrial Property – INPI, Praça Mauá 7 / sala 716, Centro, Rio de Janeiro – RJ, Brasil, CEP: 20.081-240, E [email protected]

Abstract: Considering the absence of studies showing the state of art of Brazilian research on stem cells, the present work intends to outline the present situation of protecting intellectual property rights on stem cells through patents in Brazil, as well as on their therapeutic applications and technologies related to isolating, purifying, cultivating and differentiating the above-mentioned cells. The present work makes a quantitative evaluation on patent protection in Brazil and also intends to identify the technological development of stem cell research along the years; the main stem cell patent applicants in Brazil; their nationality and nature (public/private universities or institutes, companies, etc.); the existence (or not) of cooperation among them to develop research on stem cells; the countries more interested in protecting stem cell property rights through patents in Brazil; the countries where stem cell research is in a more developed stage; and other scientific and technological areas of knowledge affecting or being affected by stem cell research. The results obtained from the present work are of great relevance to society (in particular, the Brazilian one), basing their right of choice upon truthful and exempt information; to the government, assisting it in setting forth Brazilian public policies on the matter; and also to academic researchers, showing them the importance of the patent system to protect intellectual property rights arising from their research.


Session 1 /

PC101 S1

Chemical sympathectomy induces atherosclerosis in abdominal aorta of hypercholesterolemic rats Hachani, R.1, Dab, H.1, Sercombe, R.2, Sakly, M.1, Vicaut, E.2, And Kacem, K. 1 1) Laboratoire de Physiologie intégrée, Unité de Pathologies Vasculaires, Faculté des Sciences de Bizerte. Jarzouna 7021 TUNISIE. 2) Laboratoire de Microcirculation EA 3509, Faculté de Médecine Lariboisière Saint-Louis, 75010 Paris. E-mails : [email protected] ; [email protected]; [email protected]

Abstract: This study was carried out to verify whether periarterial sympathetic denervation combined with cholesterol-supplemented diet, a high risk factor for atherosclerosis, are able to disrupt rat protection from atherosclerosis. Wistar rats were given 50mg/kg guanethidine subcutaneously. Control rats were treated with saline. Rats were fed standard rat chow supplemented with 1% cholesterol for three months. Cross sections of abdominal aorta were examined with a confocal microscope after immunolabelling for four markers of phenotypic modulation of Vascular Smooth Muscle Cells : vimentin (expressed in dedifferentiated cells, such a phenotype is more susceptible to migrate and proliferate during atherosclerosis) and desmin, α-actin and h-caldesmon (expressed in differentiated cells). Immunolabelling for markers were assessed by computer-assisted color image analysis. The same markers were studied by immunoblot. Our results indicated that sympathectomy combined with hypercholesterolemic diet was able to induce thickened intima in 5 out of 9 rats. The crosssectional area of the neointima was 558.60 ± 88.79 µm2. As showen by immunohistochemistry, medial SMCs were stained intensely with anti-vimentin in sympathectomized rat as compared with control. Quantitated by immunoblotting, vimentin relative expression increased by about 50%. However, α-actin and h-caldesmon expressions were significantly decreased in sympathectomized rat compared with control (-42.89 ± 11.5% and -39.83 ± 7.5% respectively, p 150 K UI / l) weaker than the homozygous patients Ser / Ser (p = 0. 0129; odds ratio: 0.295; 94% Reliable interval: 0.10-0.76). Furthermore, the combination IL-13 Gln130 + IL-4RSER / SER478 is found with a significantly frequency more raised with the patients whose rate of total IGE is superior of 150k UI / l (p = 0.0078; odds ratio: 2.38; 94%Reliable interval: 1.23 4.59). The other polymorphism or their association- two with two- are not correlated to the rate of the total IGE. Key words: genetic polymorphism, Interleukin, immunoglobulin E, hypersensitivity, Beta lactamin.


Session 1 /

PC129 S1

Decreased Fatty Acid And Glycerol Efflux From Adipose Tissue And Improved Glucose Tolerance In Triacylglycerol Hydrolase Deficient Mice Yassine Ben Ali Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS - Sfax, Tunisia

Abstract : Increased levels of plasma free fatty acids (FFA) have been identified as one of the key factors responsible for insulin resistance and type 2 diabetes. Reducing excessive free fatty efflux from adipose tissue is a target for the treatment of insulin resistance. Triacylglycerol hydrolase (TGH) is expressed in 3T3-L1 adipocytes and adipose tissue. Attenuation of TGH activity in 3T3-L1 adipocytes by chemical inhibition or RNA interference reduced FFA and glycerol release into culture media, suggesting TGH participation in the adipose tissue triacylglycerol (TG) turnover. We sought to assess whether ablation of TGH – mediated lipolysis affects fatty acid and glycerol efflux, as well as, glucose metabolism in mice. We show that TGH null mice have decreased levels of plasma free fatty acids (FFA) and glycerol in both fed and fasted states, accompanied by accumulation of triacylglycerol (TG) in white adipose tissue. In addition to ablation of TGH activity, TGH deficiency suppressed fasting-induced expression of hormone-sensitive lipase, adipose triglyceride lipase and diacylglycerol acyltransferase 1 and decreased diacylglycerol acyltransferase 2 expression. TGH null mice exhibited marked augmentation in glucose tolerance and insulin sensitivity. These results suggest an important role of TGH in adipose tissue triacylglycerol metabolism and may represent a suitable pharmacological target to alleviate the lipotoxicity associated with the pathogenesis of insulin resistance.


Session 1 /

PC130 S1

Y chromosome microdeletion in male infertility Kaabi Y 1, Abdelmoula NB 1, Louati R1, Kallebi F1, Amouri A2, Chrif M3, Rebai T 1 Laboratoire d'Histologie Faculté de Médecine de Sfax, Avenue Majida Boulila 3029 Sfax-Tunisie E-mail : [email protected]

Abstract: Microdeletions in Yq11 overlapping azoospermia factor’s (named AZFa, AZFb and AZFc for azoospermia factors a, b and c) are recurrently detected in about 10-15% of idiopathic azoospermia and severe oligozoospermia. Screening for Y chromosome microdeletions is often performed by analyzing the presence of Y chromosome-specific markers using multiplex PCR. The aim of this study was to detect frequency of microdeletions of Yq11 chromosome in male infertility from North Africa. Patients were subjected to detailed clinical, endocrinological and cytogenetic examinations. Fifty patients with normal cytogenetic findings, 40 with azoospermia and 10 with severe oligozoospermia, were included in the study. Detection of 20 Y-specific STS of AZF regions were conducted by means of 5 multiplex polymerase chain reactions. Four cases of Y microdeletion were found among the 50 infertile patients with a prevalence of 8%. All cases of Yq11 microdeletions were detected in patients with azoospermia. The first had a microdeletion of the three AZF regions, the second presented a large deletion involving AZFb and AZFc regions, the third had a deletion of the AZFc region involving the DAZ (deleted in azoospermia) gene and the fourth had a small deletion of the terminal part of AZFc region. Our finding supports that testing for Y chromosome microdeletions should be considered, besides cytogenetic analysis, as an important element in diagnosis and genetic counseling of infertile men with azoospermia and severe oligospermia, particularly before their enrolment in ICSI or TESE-ICSI program.


Session 1 /

PC131 S1

Molecular characterization of Candida species resistance associated to the hospital infections Souiden, Y., Eddouzi, J., Chaieb, K. and Mahdouani, K. Hospital Ibn El Jazzar of Kairouan, Laboratory of Biology, Street of Ibn El Jazzar, 3140 Kairouan-Tunisia

Abstract: Candida species are opportunistic pathogens which can cause a wide range of infections from mucocutaneous to systemic infections. These latter, are usually observed in immunocompromised and weakned individuals. The emergence of new pathogenic agents and the increase of the antifungal resistant streans incidence, require an assumption of these infectious agents responsibility in hospital medium. The aim of this study was to identify the different species implied in the infection and to seek the possible correlation between phenotypical resistance observed and its genetic support. In this study, the identification of twenty four isolates was carried out by Api ID32 C strips. The antifungal succeptibility testing determined by the ATB Fungus 2, show that an acquired resistance is observed for triazoles with percentages of 16% and 62% noted respectively for Fluconazole and Itraconazole. However, 5-Fluorocytosine and Amphotericin B were the most potent antifungal agents assayed (95.83 % of susceptible strains). Molecular mechanisms of the phenotypical resistance at the predominant two species (C albicans (n=10) and C glabrata (n=10)), was appreciate by RT-PCR method. The results prove that this resistance is correlated to the upexpression of the multidrug efflux transporter CDR1. At present, acquired antifungal resistance represents a common finding for the most Candida ssp circulating in hospital medium. In fact, a good identification as well as a better management of the results of the susceptibility patterns appreciated by the molecular study, makes it possible to adapt an effective treatment in front of the serious Candida systemic infections of which mortality can reach high percentages.


Session 1 /

PC132 S1

HLA allele Distribution in breast cancer female patients from Kingdom of Bahrain. Zainab Ashkanani, and Fadhel Saleh, MD, FRACP Centre of Biotechnology of Arabian Gulf University, Bahrain E-mail: [email protected]

Abstract: Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. Several HLA alleles have been linked with susceptibility or protection in breast cancer. The particular alleles vary depending on the geographical region and ethnic origin of subjects. Herein we present the first report on the correlation between breast cancer with HLA class I and class II markers in Kingdom of Bahrain. Serologic HLA typing for Class I and class II was performed for consecutive 60 Bahraini female patients with histology proven breast cancer. Comparison of allele and haplotype distribution between patients and 118 age-matched female control subjects is presented and results are discussed.


Session 1 /

PC133 S1

Facilities for the production and the experimentation in standardized conditions (SPF) Z. Benlasfar Service des Unités Animalières, Institut Pasteur de Tunis

Abstract: Animal experimentation still remains the least developed activity in Tunisia and most African countries. The controls on animals for biological products are being done in only few research institutions. In addition, the absence of experimental facilities hinders the development of such activities. Conventional facilities available now are not adapted to the ambitious programs of the scientists at Pasteur institute who have the good will to reach an international level in quality and to move on further in their research works and biological productions. For this, the Pasteur Institute has made big investment in building up a new and a reliable integrated experimental unit for the production of standardized laboratory animals (SPF), in controlled conditions known for level 2 security units. This unit fully responds to the European standards (Directive 86-685/CEE and European Convention STE 123) in regard to animal welfare. Aware of the importance of such unit for research activities, Pasteur institution would give the opportunity to all academic and research institutions biopharmaceutical, and biotechnological producers, from the public and the private sectors, to take advantage of this P2 Unit and its well defined environment. In fact, the main goals are: - To produce laboratory animals with sanitary and genetic status in conformity to international standards; - To invite experienced users to take advantage of such unit and do their research work in conformity with the applied procedures; - To propose protocols for expertise needs; - To train students and give them qualification; Hopefully, the unit will ready by the end of the year 2008 and we will be glad to answer demands.


Session 1 /

PC134 S1

Establishment of the karyotyping in the Ambiguous genitalia Daoui z, Benachour N, Brihmat A, Bouchereb M,Hannachi S, Satta D Laboratory of biology and molecular genetic, MONTOURI university route AIN EL BEY, Constantine, Algeria. E-mail: [email protected]

Abstract: Ambiguous genitalia is a medical and social problem. It can be detected in birth in front of an anomaly of the external genitals which presents a metabolic urgency associated with the syndrome salt loss within the framework with a congenital adrenal hyperplasia, causes more the frequency of the PHF. The diagnosis of ambiguous genitalia can also be late at the age of puberty, in front of a puberty delay where appearance of unmatched natures sexual with the assigned civil sex. The goal of this work consists to determine the sex of ambiguous genitalia by a cytogenetic study, and to establish the percentages of the various types of sexual ambiguity in our country. From 400 patients we listed 45 individuals with a ambiguous genitalia, with a frequency of 11,25 %, 24 among them were male, 17 of female and 4 of unspecified phenotype. The cytogenetic study revealed discordance between the phenotypic and genotypic sex, 3/24 of male are genetically XX, and 5/17 of female are genetically XY. The chromosomal anomalies of a number were observed for 4 cases. We distinguished 2 % true hermaphroditism, 40 % female pseudohermaphroditism, and 58 % male pseudohermaphroditism. Only the karyotyping chart would have made it possible to establish the real sex, while knowing that the standard chromosomic chart only is not enough with the complete orientation towards the phenotype and a complementary molecular study is recommended. Key words: Ambiguous genitalia, true hermaphroditism, female pseudohermaphroditism, male pseudohermaphroditism, karyotyping, cytogenetic


Session 1 /

PC135 S1

Effect of alcohol chronic dose on histopathological aspects of the heart, aorta and liver in male adult rats Zeineb Kamoun1, Ahmed Hakim2, Khaled Zeghal2 And Najiba Zeghal1 1: Laboratory of Animal Physiology. Faculty of Sciences.BP 802. 3018 Sfax. Tunisia 2: Service of Pharmacology. Faculty of Medicine. Sfax. Tunisia E-mail: [email protected]

Abstract: Alcoholic beverages are consumed by most of the human societies in the world. Chronic alcoholism, which is associated with hepatic, kidney and cardiovascular system, is one of the major health problems in the world with high morbidity and mortality. Cardiovascular system and liver are the important targets of excessive consumption of alcohol abuse. The purpose of this study was to investigate the effects of alcohol chronic dose administrated to male adult rats on histopathological aspects of the heart, aorta and liver. Twelve male Wistar rats were divided into two groups of six animals each: a control group (C), and a treated group (T) receiving for 15 days, by intraperitoneal way a single daily dose of ethanol (30%). Food and water were supplied ad libitum and measured daily during full period of treatment. Before sacrifice, blood samples were collected and centrifuged to determine activities of some inflammatory biomarkers in plasma such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT). For histopathological studies, some samples of liver, heart and aorta were drawn from control and alcohol treated rats, cleaned, weighed and fixed in formalin solution. Leukocytes infiltration was observed in cardiac muscle, aorta and liver indicating inflammation. Moroever compared to controls compared to controls an increase of AST and ALT activities in plasma by 43% and 38%, respectively confirmed inflammation in liver and its histopathological aspect in alcohol treated rats. Keys words: Ethanol, adult rat, heart, aorta, liver, AST, ALT.


Session 1 /

PC136 S1

An investigation of alkaline phosphatase and lactate dehydrogenase activities in some diseases, which affect zinc status in human Kechrid Z and Derouiche S Laboratory of Biochemistry and Microbiology Application, Department of Biochemistry, Faculty of Sciences, University of Annaba. Tel/Fax: 00213 38 87 57 01, E-mail: [email protected]

Abstract: The present investigation was undertaken to study the effect of zinc variation on some enzymes activities in patients suffering from certain diseases. The enzymes chosen due to their needs to zinc element as a catalytic or structural. In other words serum alkaline phosphatase and serum lactate dehydrogenase activities were measured in patients suffering from diabetes type I, diabetes type II, insufficient renal failure and gastrointestinal disorder which are usually affect zinc body status. Serum zinc concentration was also estimated in all different diseases. The results, which have been obtained, indicated that zinc level was decreased in all studied cases and there was no difference in zinc variation between both sexes. We found also that there was a diminution of alkaline phosphatase activity in most studied cases and there was a strong significant correlation between zinc concentration and the activity of this enzyme in both sex and each sex alone. However no changement of lactate dehydrogenase activity. In other words there was no effect of zinc diminution on LDH activity and a negative correlation between zinc and LDH activity in all studied diseases. In conclusion we can say that the present investigation result in that alkaline phosphatase activity is susceptible or sensitive to zinc variation as compared to LDH. Therefore we can probably take that alkaline phosphatase activity as a good factor (parameter) for the diagnosis of zinc status in the organism. Key words: Zinc, Enzymes, Diseases, Activity


Session 1 /

PC137 S1

In vitro sensitivity test of moulds with respect to Cistus ladanifere extracts Mohammedi Z. et Atik F. Natural product Laboratory, Department of Biology-Faculty of sciences, University Abou Bakr Belkaïd BP 119 Tlemcen (ALGERIE)

Abstract: Medicinal plants represent a very good natural source of bioactive molecules; these vegetable substances represent for the man a great interest. Cistus ladaniferus is a resinous aromatic plant, which believes spontaneously, rich in bioactive compounds. We have extracted essential oil from the leaves by hydrodistillation process with a Clevenger apparatus type. This natural aromatic mixture was subject to an antifungal test against seven moulds. A good fungistatic activity was expressed by essential oils on all strains whose most sensitive Mucor (CMI = 2µl/ml), and most resistant mould Aspergillus flavus (CMI = 7.5µl/ml). Key words: Cistus ladaniferus, essential oil, moulds, antifungal activity


Session 1 /

PC138 S1

Comparing methods for nuclear hormone response element prediction Ahmed Fourati, Mouna Choura, Sami Aifa, Ahmed Rebaï Bioinformatics Unit, Centre of Biotechnology of Sfax, Laboratory of Enzymes and Metabolites of Prokaryotes Road of Sidi Mansour Km 6, P. O. Box « K » 3038 Sfax-Tunisia

Abstract: One of the challenges of bioinformatics in genome analysis is to predict particular motifs within DNA sequences serving as trans-regulation sites. Nuclear Hormone Response Elements (NHRE) are among the most important response elements adopted by Eukaryotic regulation of genetic expression. An NHRE is a DNA sequence characterized by its palindromic structure with 3 specific features: the sequence of the base pairs in the half-site, the number of base pairs between the half-sites, the relative orientation of the two half-sites. These sequences can be predicted through different methods such as Weight Matrix (WM) and Bayesian Networks (BN). In this work, we first outline the performance of the three methods and how they can be adapted to NHRE prediction. Then we present two programs that implement WM and BN methods. We used these programs to perform a genome wide search of NHRE in the human genome. This allowed us to evaluate the performance of the two methods and to compare them to each other. We finally propose a combination of BN and WM to improve the precision of the prediction.


Session 1 /

PC139 S1

Using Bayesians networks in modeling the EGFR signal transduction pathway: comparison between Bayesian and implicit inference Bouchaala, L., Ben Hassen, H., Rebaï, A. Centre of Biotechnology of Sfax, Unit of Bioinformatics, Road of Sidi Mansour Km 6, P. O. Box « K » 3038 Sfax-Tunisia

Abstract: Since its introduction in the 1980s, the Bayesian Network has been applied in many fields including biology. In fact, the Bayesian networks constitute one of the most complete and coherent formalisms for the acquisition and the modeling of complex systems. Bayesian networks are directed acyclic graphs whose nodes represent variables, and whose arcs encode conditional independencies between the variables. Variables in a Bayesian network can be discrete or continuous. They can represent mRNA concentrations, protein concentrations, protein modifications or complexes, metabolites or other small molecules, experimental conditions, genotypic information. In this work we search to model the signaling pathway of the Epidermal Growth Factor Receptor (EGFR) wich is a receptor protein that is activated after fixing a ligand and activates, via a phosphorylation cascade, a large number of proteins leading to the regulation of the expression of target genes. To estimate the structure and the probabilities of the EGFR network we used the K2 algorithm and the maximum likelihood approach respectively. Data were simulated within Matlab and structure learning was carried using the K2 implementation in Matlab (BNT toolbox). Robustness of the K2 algorithm was evaluated. We estimated the parameters of the network (conditional probabilities of each node being in a given phosphorylation state) using two methods: the classical Bayesian estimation method using uniform priors and a new method called implicit inference. The implicit method does no need any priors to be provided by the user. We show that the implicit method gives more accurate and robust estimates of the parameters. We can thus combine Bayesian network approach and implicit inference to get an efficient method for the modelling of complex biomolecular networks.


Session 1 /

PC140 S1

Mutations in the human miR-96, a microRNA expressed in the inner ear, causes non-syndromic progressive hearing loss Mencía, A.1,2,, Modamio-Høybjør, S.1,2, Redshaw, N.3, Morín, M.1,2, Mayo, F.1,2, Olavarrieta, L.1,2, del Castillo, I.1,2, Dalmay, T.3, Moreno, F.1,2, Moreno-Pelayo, M.A.1,2. 1

Unidad de Genética Molecular, Hospital Ramón y Cajal, 28034 Madrid, Spain. E-mail: [email protected] 2 Centre for Biomedical Research on Rare Diseases (CIBERER), Madrid, Spain. 3 School of Biological Sciences University of East Anglia, NR4 7TJ Norwich, UK.

Abstract: We previously mapped a novel autosomal dominant (AD) deafness locus, DFNA50, in a Spanish family with postlingual, progressive, non-syndromic all-frequency hearing loss. DFNA50 is located on 7q32, within a 3.8 cM region in which more than 40 genes have been identified. Our initial sequencing analysis excluded ten genes as responsible for DFNA50 deafness. Recently, a set of three microRNAs with reported expression in the inner ear, MIRN96, MIRN182 and MIRN183, was positioned in the interval. No changes were found in MIRN182 and 183; however, a point mutation was found in MIRN96. This change segregated with the affected status in the family and it was not found in more than 100 normal-hearing controls. Interestingly, this mutation places within the conserved seed region of the ~22-nt mature miRNA sequence, which is involved in the mRNAs target-sites recognition for posttranscriptional repression. We extended the screening to our cohort of undiagnosed AD deafness families and found a novel point mutation in the seed region that co-segregated with the hearing loss in the family. We have verified that both mutations have no impact on the processing of hsa-miR-96 and do not prevent the expression of luciferase protein coupled to 3’UTR of human selected targets. The expression of hsa-mir-96 appears to be restricted to the hair cells, where it might facilitate their differentiation and function. On this basis, we hypothesise that the mutations identified in this study might be altering the role that hsa-miR96 may play on the maintenance of the expression profiles in hair cells.


Session 1 /

PC141 S1

Novel mutations in the TMPRSS3 gene in Spanish DFNB8/10 families with autosomal recessive non-syndromic hearing impairment Viñuela, A., del Castillo, F.J., Villamar, M., Moreno-Pelayo, M.A., Moreno, F., del Castillo, I.* Unidad de Genética Molecular, Hospital Ramón y Cajal, Madrid, Spain *E-mail: [email protected]

Abstract: Autosomal recessive non-syndromic hearing impairment (ARNSHI) is genetically very heterogeneous. To date, 53 loci for ARNSHI have been mapped, and 29 genes have been identified. Mutations in the TMPRSS3 gene, which encodes a transmembrane serine protease, cause ARNSHI with congenital onset (DFNB10) or with onset later on in childhood (DFNB8). We have investigated the contribution of TMPRSS3 mutations to ARNSHI in the Spanish population. Sixty unrelated families with at least two affected siblings were genotyped for four microsatellite markers close to TMPRSS3. In families in which haplotype analysis revealed compatibility with linkage, all the 13 exons and flanking intronic sequences of TMPRSS3 were sequenced. The affected subjects from three families carried two mutant alleles of this gene. In total, we identified three novel pathogenic mutations (p.Ser81X, p.Val116Met and p.Ala141Pro), and two mutations previously described (c.207delC, in two families, and p.Pro404Leu). We confirmed the cosegregation of the mutations with the hearing impairment in these families. All mutations were not found in 200 healthy controls. The TMPRSS3 protease contains a transmembrane domain, a low-density-lipoproteinreceptor A domain, a scavenger receptor cystein-rich (SRCR) domain, and a protease domain. The two novel missense mutations, p.Val116Met and p.Ala141Pro, affect highly conserved amino acid residues in the SRCR domain. The proteolytic activity of the TMPRSS3 proteins is being tested by using a yeast-based protease assay. Our data indicate that mutations in the TMPRSS3 gene may account for up to 5 % of all cases of ARNSHI in the Spanish population.


Session 1 /

PC142 S1

Interactions between myosin VI and interacting proteins in the sensory hair cell Tamar Tenne, Tama Sobe and Karen B. Avraham Department of Human Molecular Genetics & Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, E-mail: [email protected]

Abstract : Myosin VI, an unconventional myosin that travels along actin filaments toward the pointed end is responsible for deafness in humans and mice. In mammalian cells, myosin VI is involved in endocytosis, in maintenance of Golgi complex morphology and secretion, and in membrane ruffling. However, in the inner ear, myosin VI is expressed solely in the sensory hair cells. In the Snell's waltzer (sv) mouse, an intragenic deletion leads to truncation of the myosin VI gene. These mice exhibit circling, head-tossing, hyperactivity and they are deaf. In hair cells of normal mice, myosin VI is found in the pericuticular necklace and at the base of the stereocilia. Using the yeast-two hybrid system with the myosin VI tail as bait, we identified two putative binding partners: snapin and kaptin. Snapin has been implicated as a synaptic vesicle membrane protein in neuronal cells and as a soluble factor in non-neuronal cells. Kaptin is known as an actin-binding protein that may be expressed at the tips of hair cell stereocilia. The interaction of both proteins with myosin VI was verified by a GST pull-down assay, using GST-snapin and GST-kaptin incubated with total cochlear extracts. Immunoflourosence staining with specific antibodies against snapin and kaptin showed co-localization with myosin VI in the cuticular plate of the cochlear and vestibular sensory hair cells of wild type mice. In order to develop a model describing myosin VI and partners and their function in hair cells, we will further study the interactor's localization and function in the Snell's waltzer mice in comparison to wild type.


Session 1 /

PC143 S1

Molecular SMA technical Approaches: A comparative study Kallebi F.1, Abdelmoula NB.1, Amouri R.2, Rebai T.1 Laboratoire d'Histologie Faculté de Médecine de Sfax, Avenue Majida Boulila 3029 Sfax-Tunisie E-mail : [email protected]

Abstract: Spinal muscular atrophy (SMA) represents one of the most common autosomal recessive diseases often leading to death in early infancy. The SMA gene named survival motor neuron gene (SMN) is localised in 5q11.2 within a large inverted duplication of a 500 kb element. The SMN gene exists in two highly homologous copies, SMN1 and SMN2.The coding region of the SMN1 gene differs from that of the SMN2 gene by only a single nucleotide in exon 7. Both SMN copies are expressed, but the C→T transition in exon 7 of SMN2, although translationally silent, decreases the activity of an exonic splicing enhancer, so that truncated transcripts are generated and less full-length protein is expressed. Deletions of the SMN1 gene is involved in SMA since exon(s) 7 (and 8) of SMN1 are undetectable in over 95% of patients, irrespective of their clinical type and severity, either as a result of homozygous deletions, or because of conversion of sequences of SMN1 into those of the SMN2 gene. Homozygous deletions of exons 7 and 8 of SMN1 can be detected by several methods including PCR-RFLP, SSCP and sequencing. Quantitative assays based on competitive amplification of SMN1 and SMN2 exons 7 to determine their gene-copy number are used to identify patients with compound mutations and for carrier detection. We set up in the Laboratory of Histology of the Medical University of Sfax, these different molecular technical approaches. We try through this work to demonstrate usefulness of each technique. Our finding will be presented and discussed.


Session 1 /

PC144 S1

Design and humanization of an antithrombotic scFv directed against human platelets glycoprotein VI Muzard, J.1,2, Ollivier, V.2, Lacapère, J.J.3, Jandrot-Perrus, M.2, Billiald, P.1 1

Muséum national d'Histoire naturelle, EA4105, CP39, 12, rue Buffon, F75005 Paris. U698, Inserm, Hôpital Bichat, 46 rue Henri Huchard; Université Paris 7, Paris. 3 U773, Inserm, CRB3 Faculté Xavier Bichat, 16 rue Henri Huchard; Université Paris 7, Paris. E-mail: [email protected] 2

Abstract: Given the key role of platelets glycoprotein (GP) VI in hemostasis, recombinant antibody fragments that neutralize GP VI / collagen interaction at the site of vascular injury may have important clinical applications. Here, we present the design and humanization of an scFv with antithrombotic activity. We first engineered a murine scFv derived from the anti-GP VI hybridoma 9O12 of which Fab fragments have previously been shown to prevent thrombus formation under arterial flow conditions without significantly prolonging the bleeding time. The murine scFv not only binds recombinant GP VI but also completely blocks platelet aggregation induced by collagen under arterial flow conditions and inhibits the procoagulant activity of collagen-stimulated platelets. A humanized version of this scFv was also designed after CDR-grafting and structural refinements using homology-based modeling. The final product was produced in recombinant bacteria. It was found to retain the epitope specificity of the original antibody and to preserve a high affinity (KD = 3nM), which are the main parameters usually impaired by humanization procedures. The method we report here is a simple, efficient, and straightforward processing that could also be used for humanizing other antibodies with therapeutic potential.


Session 1 /

PC145 S1

A tentative a means of natural fight against the mite Varroa destructor (Anderson and Trueman) important pest of algrean honeybee: The sugar powdering Adjlane. N Département de Biologie, Faculté des sciences, Université M'hammed Bougara, Boumerdes, Algérie E-mail: [email protected]

Abstract : The bees, in addition to their of production of the honey, ensure the pollination of the fruit trees and other harvests flowers. Any threat on the bees, which it comes from weedkillers, pesticides, diseases or of parasites is thus heavy consequences not only for the bee-keeping but also for agriculture as a General thus, the varroase is one of the most frightening diseases, because of the considerable damage caused on the hives, also involving the total collapse of the colony. The causal agent of this disease is the acarina Varroa destructor which attacks with the larvae and the adult bees. Currently in Algeria, several products are used in the fight against this parasitosis (Fluvalinate, flumethrin, amitraz). These molecules presents disadvantages related to the appearance of the mites resistants at these products, and contamination of honey by the residues risks it. Therefore it seems to us interesting to be directed towards other means of natural fight. The sugar powdering belongs to the methods of fight which can present an alternative. The objective of this test of is tested for the first time in Algeria the effectiveness of this method of fight against the varroase. Two experimental batches were used in this test, each batch consists of 8 hives. A pilot batch and a batch treated with the sugar powdering. The method consists in pulverizing sugar industrial to crush directly between the executives of bees two made per week. The estimate of the effectiveness consists in comparing mortality varroa to find on the lange between the two batches (witness and treaty The results obtained highlight a more significant mortality of varroa of the batch treated compared to the pilot batch, which highlights the good effectiveness of this method. In addition, this method of fight does not have to cause side effects on the development of the hives. From these results, one can propose this technique like a method complementary to the chemical treatments, especially for the bee-keepers who have a number restricted of hive Finally of other tests are necessary to determine with precision the exact percentage of the effectiveness of this method, and to finish the effects of the use of sugar on the couvain and this to be able to integrate it in a strategy of alternative fight against the mite. Key words: varroa destructor, HoneyBee, sugar, effectiveness


Session 1 /

PC146 S1

Human Cytosolic 5’-nucleotidase: a target of therapeutic interest 1

1

2

2

Moez Rhimi , Richard Haser , Lars Jordheim , Charles Dumontet and Nushin Aghajari

1

1

Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, CNRS et Université de Lyon, UMR 5086, IFR 128 "BioSciences Lyon-Gerland", F-69367 Lyon, France 2

Laboratoire de Cytologie Analytique, Inserm U590, Université de Lyon, F-69008 Lyon, France

Abstract : Cytotoxic nucleoside analogs are small molecules widely used in the treatment of haematological malignancies and some solid tumors as well as in the treatment of certain viral diseases. However, resistance to these cytotoxic nucleoside analogs is a major problem in the treatment of cancers (see Jordheim & Dumontet [1] for an overview). The human cytosolic 5'-nucleotidase (cN-II) also known as high Km 5’-nucleotidase or purine 5‘-nucleotidase is a key enzyme for the regulation of intracellular concentrations of nucleotides required for anticancer activity. This enzyme which functions as a tetramer catalyses the dephosphorylation of nucleoside monophosphates. It furthermore posses’ phosphotransferase activity, and catalyses the phosphate transfer from the monophosphate substrate to the nucleoside acceptor. It represents a target of high therapeutic interest, especially for the treatment of leukemia since it has been reported that patients showing a poor outcome of treatment with nucleoside analogs displayed a higher cN-II expression level or enzyme activity than patients with good response ([2] and references herein). In order to contribute to optimizing the existing treatments and to the understanding of the mechanisms of recognition and catalysis, and to propose new types of biomolecules for the treatment of eg. leukemia, expression and purification protocols of this protein have been established. We have crystallized an N-terminal trunctated form of this enzyme in the presence of fludarabine monophosphate (F-ara-AMP), a drug used in the treatment of chronic lymphocytic leukemia, and collected X-ray diffraction data to 1.7Å resolution. The determination of the corresponding 3D structure using the “molecular replacement” method as well as a selenomethionylated form of the protein failed, and very recently the crystal structure of a C-terminal truncated form was solved by Nordlund and colleauges [3]. Nevertheless, this 3D structure does not include the 73 C-terminal residues of cN-II, as well as the strecth 401-417, and the C-terminal of this enzyme has been reported to be of high importance in the activation and the formation of multimers [4]. We therefore continue our studies based on the presently used N-terminal truncated form as well as newly planned constructions. The results reported from the solved 3D-structure [3] along with our ongoing studies should contribute to broaden our understanding of the regulation of nucleoside/tide pools in tumor cells and to design appropriate and new agents for the control of the enzyme activity. References [1] Jordheim, L.P. & Dumontet, C. (2007) Biochim. Biophys. Acta, 1776: 138-159. [2] Bretonnet, A.S., Jordheim, L.P., Dumontet, C. & Lancelin, J.M. (2005) FEBS. Lett., 579: 33633368. [3] Walldén, K., Stenmark, P., Nyman, T., Flodin, S., Gräslund, S., Loppnau, P., Bianchi, V. & Nordlund, P. (2007) J. Biol. Chem., 282: 17828-17836. [4] Spychala, J., Chen, V., Oka, J., & Mitchell, B.S. (1999) Eur. J. Biochem., 259: 851-858.


Session 1 /

PC147 S1

Comparison of efficacy of single antigen DNA vaccine with polytope DNA vaccine against Visceral Leishmaniasis in mouse model Rakhee Sachdeva, M.L. Dubey and Nancy Malla Department of Parasitology, Postgraduate Institute of Medical Education & Research, Chandigarh, India

Abstract: DNA vaccination holds great promise for the future of vaccine development against infectious diseases, especially in developing countries. We investigated the protective potential of single antigen and polytope DNA vaccines against Leishmania donovani infection. The coding region of gp63 gene of Leishmania donovani was used for the preparation of single antigen DNA vaccine. Polytope DNA vaccine was prepared using immunogenic T cell epitopes. pcDNA3.1 eukaryotic expression vector was used for the preparation of DNA vaccines. Balb/c mice were immunized with the vaccines at 4 weekly doses with and without Gp63 protein boost at the fourth dose. The vaccine showed high Immunogenicity as seen by Splenocyte proliferation, cytotoxic and cytokine responses. Protein boost enhanced Immunogenicity. The vaccine was potentially efficacious as determined by parasite load at 4 weeks of challenge infection with Leishmania donovani promastigotes in both spleen and liver. Protein boost did not affect the efficacy of the vaccines. The results show that polytope DNA vaccine can be a successful approach against potentially fatal Visceral Leishmaniasis.


Session 1 /

PC148 S1

Effectiveness Of Herbal Decoctions On Reduction Of Vibrio Cholerae On Chicoreus Ramosus Meat P. Ashok Kumar1 and Karpagam 2 and Jamila Patterson3 1

MVJ College of Engineering, Bangalore, Karnataka, India Sri Kaliswari College Sivakasi, Tamil Nadu, India 3 Suganthi Devadason Marine Research Institute, Tuticorin, Tamil Nadu, India E-mail: [email protected] 2

Abstract: Medicinal plants have been an integral part of human society since the start of civilization. Different national and international pharmaceutical companies are utilizing such plant-based formulations in treatment of various diseases and disorders world around. The World Health Organization has estimated that more than 80% of the world’s population in developing countries depends primarily on herbal medicine for basic healthcare needs. This study attempts to determine whether or not the empirical application of plant drugs could be supported by scientific examination. Aqueous extracts, the most frequent form of application in traditional medicine, were tested for their antibacterial activity. Forty-five higher plants which have been described in ancient herbal books and medicinal folklore reports as vulneraries were studied in an antibacterial screening. Aqueous extracts of different aerial parts (leaves, flowers, seeds, fruits, barks) and subterranean parts (roots, rhizomes) of the plants were investigated. The authenticated plant materials used in study were collected from Thiruvannamalai mountain hills. Preliminary studies were done to screen aqueous decoctions of herbs commonly used to soften meat for their ability to inhibit the growth of V. cholerae before doing immersion treatments of inoculated C. ramosus. The plant extracts were prepared using the modified method of Alade and Irobi (1993). The modified agar well diffusion method of Perez et al. (1990) was employed. Thirteen potent medicinal herbs were finally chosen from all the 45 medicinal plants for the preparation of decoctions. Studies were done to determine the effectiveness of herbal decoctions in killing V. cholerae inoculated onto raw C. ramosus meat. The efficacy of treatments in reducing populations of naturally occurring aerobic microorganisms on raw C. ramosus meat was evaluated. The inoculated raw C. ramosus meats stored in the refrigerator were analyzed for populations of V. cholerae and total aerobic microorganisms within 1 h of treatment (0 day storage) and after storage for 24, 48 and 96 h. Among the 45 medicinal herbs, only 30% (13 plants) of the medicinal herbal decoctions tested exhibited a pronounced antibacterial effect against V. cholerae. Cleom gynandra (leaf), Launaea sarmentosa (leaf), Cordia obliquawillel (leaf), Cassia angustifolia (leaf), Abrus precatorius (leaf), Smilax china (leaf), Sida cordifolia (leaf), Tinospora cordifolia (leaf), Moringa oleifera (leaf), Aegle marmelos (fruit), Alpinia galangal (leaf) and Lippia nodiflora (rhizome) produced outstanding antibacterial effects against V.cholerae. Alternanthera sessilis, Gymnema sylvestra, Pergularia extensa, Berberis aristata, Cadaba tarinosa, Terminalia belerica, Eclipta prostrate, Ipomoea obscura, Melothria maderaspatana, Phyllanthus niruri, Acalypha indica, Taraktogenous kurzii, Enicostimma littorale, Piper longum, Mucuma prurita, Metia azedarach, Cocculus hirsatus, Myristica fragrans, Morinda coreia, Trachyspermum ammi, and Centella asiatica showed no activity against V.cholerae. Aegle marmelos, Alpinia galangal, Cleom gynandra, Cordia obliquawillel, Moringa oleifera, Sida cordifolia and Tinospora cordifolia herbal decoctions inhibited completely the growth of V.cholerae on C.ramosus meat at 96h. However all the thirteen herbal decoctions experimented exhibited some reduction in the V.cholerae load on 48th hour. No complete inhibition rather than a reduction in the population of V.cholerae was noticed from the following herbal decoctions viz., Cassia angustifolia, Launaea sarmentosa, Lippia nodiflora, Marsilea quadrifolia and Smilax china.


Session 1 /

PC149 S1

Encapsulation By Solid Double Emulsion Wafa ESSAFI1 and Jérôme BIBETTE2 1

INRAP - Pôle technologique de Sidi Thabet -2020 sidi Thabet- TUNISIE, Unité d’études Physico-chimiques des Milieux et Substances Naturels, E-mail : [email protected] 2 Laboratoire Colloïdes et Matériaux Divisés – ESPCI, 10 rue Vauquelin-75231 Paris

Abstract : Encapsulation consists to incorporate an active molecule in solid or liquid state within a support colloidal material which isolates it from the media and leads to the release of the active species (either by rupture of the support material or by diffusion of the active species through the material). Any double (water/oil/water) emulsion can encapsulate hydrophilic species but the originality of our material stems from the use of a crystallisable oil, solid at room temperature, which constitutes a solid shell containing aqueous reservoirs able to encapsulate hydrophilic species and also to control their release. The encapsulation capacity of these matrixes was first studied on low molecular weight species. A passive leaking was observed, but it remained below 10% over 12 months. A complete release can be obtained by various means. One way is to create an osmotic shock, which leads to 100% release in 2 hours. Release mechanism and release control are currently investigated and we are interested in the future in the encapsulation by these materials, to create new drugs or food formulations.

Inner aqueous droplet stabilised by a lipophilic surfactant

Continuous phase : water

Solid oil

Double globule stabilised by an hydrophilic surfactant

Figure of a double emulsion


Session 2

Session 2: Microbial and Environmental Biotechnology -

Biocatalysts and enzyme engineering Geneomics and metabolites Bio refinery Wastewater treatment and reuse Biodepollution, biomass and renewable energy



Session 2



TC1 S2

Recycle of Waste Sludge from Food Industries and Live Stock for Compost -Exploitation of Various Gears for Greening of Arid LandTakateru Ishimori, Takako Kaneda, Saravanan Ayyakannu, Makoto Togashi, Rie Honma, Mizuho Miura, Tsuyoshi Okajima, Hideaki Taira, Tsohihiro Sanada And Hidetaka. Hori Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan E-mail: [email protected] Tel and Fax : +81-25-262-7637

Abstract: We succeeded to develop an ultra high performance composting system for food industrial sludge by employing paper mixing method. This was successfully adapted for the composting of pig feces. The sludge and feces were mixed with cut pieces (3 x 12 mm) of newspapers, in the range of 10-20 % (w/w) in an electric mixer to enhance the porosity and reduce water content of the materials. We performed conventional method of sawdust mixing as control. The mixture was subjected to aeration at room temperature with an electric blower at 80 L/min/m2 bottom area of bioreactor (1m h x 0.6m x 0.6m). The aeration was effective and temperature at three points, i.e., top, middle and lower points in the reactor, were almost equally fluctuate, and surprisingly, mature compost was resulted during 15 days in contrast to the conventional cases where it takes 60 to 90 days to complete composting. Chemical analyses of the compost showed concentration of nitrogen (N) 4%, phosphorus (P2O5) 4% and potassium (K2O) 0.5% while all heavy metal contents were below the standard required level. The compost showed pH 7 and carbon/nitrogen (C/N) ratio 8. Since the EC value of the compost was substantially low, it was capable of applying to the soil at the same time as seeding. Compost containing a large amount of cellulose fibrous materials can preserve moisture therefore it may have a good effect on growth of the plantlets in dried arid lands having poor nutrition. Therefore, we cast various gears with the compost and test the efficacy of those for enhance the growth of seedlings and plantlets under the controlled water supply. In this meeting, we like to show the wonderful effect of the compost to enhance the rooting of various plantlets. The gears made with the compost are also shown to support effectively the growth of plantlets under very limited water supply. We also like to show the usefulness of the compost for proliferation of cedar（Cryptomeria japonica D. Don）by cuttings, which does not make pollen.



TC2 S2

Roof Biofilm: A Unique Biotechnological Resource Potts, M. and Helm, R.F. Virginia Tech Center for Genomics and Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, USA

Abstract: Surfaces of roof shingle on virtually every dwelling and commercial property in the US, as well as aerophytic environments in temperate and tropical regions worldwide, support a unique, biofilm. Group I intron analyses identified a diverse community of cyanobacteria, Gram positive and negative forms, including actinomycetes and human pathogens, as well as fungi and eukaryotic algae. The biofilm, dominated typically by the extremophilic cyanobacterium Gloeocapsa cf sanguinea, withstands temperatures that exceed 100 °C in summer months and below -30 °C in winter; redox-active copper (and zinc), which roofing manufacturers incorporate in shingle to prevent growth; erratic cycles of desiccation and rehydration; intense photon fluxes of UV-A/B irradiation; hydrocarbons that leach from the asphalt-based shingle; paucity of nutrients, and acid rain. This biofilm community is a valuable biotechnological resource that can provide fundamental knowledge on the mechanisms for cell survival and macromolecule stability in extremophiles, the process of metabolic arrest (quiescence) and recovery, community signaling processes, biological and mechanical energy storage, novel bioelectrode designs based upon cell to surface interactions, electron and proton transfer (coupling of cells with heavy metals), reactions and kinetics of secondary products and enzymes in immobilized cells, and enzyme engineering for faster catalysis under normally prohibitive conditions.



TC3 S2

Two original isomerases involved in low caloric sugar production: Screen, characterization, cloning, study of structure function relationship, improvement and application. Samir Bejar, Moez Rhimi, Hichem Chouayekh, Mohamed Ali Borgi, Mamdouh Ben Ali Karima Belguith-Srih, Emmanuelle Maguin2, Nushin Aghajari3, Richard Haser3 1: LEMP-CBS; 2: Unité de GM, Centre INRA de Jouy-en-Josas, France; 3:LBC-IBCP Lyon-France, Tel/fax: + 216 74 870 451, E-mail: [email protected]

Abstract : Nowadays, marketing new low-caloric prebiotic products will undoubtedly find success especially for diabetes and low-caloric diet persons. In this goal, our group studied two original isomerases, a D-glucose isomerase and an L-arabinose isomerase involved in the production of low-caloric sweeteners. Glucose Isomerases (GI), are enzymes largely used at industrial level for the production of fructose containing syrup. The glucose isomerase of Streptomyces sp. SK (SKGI), displayed very attractive physico-chemical properties for an industrial exploitation. In effect, it exhibited maximal activity at pH 6.5 and temperature 90°C (Belguith et al. 1998, 2002). This enzyme shared large homology with GIs from other Streptomyces but, it presented some subtle amino acid substitutions including an Ala residue at position 103. The site directed mutagenesis technique was used to prove the implication of the Ala103 residue in the thermostabilily and the acidotolerance of the enzyme as well as in their interesting kinetic parameters. We have also demonstrated by molecular modelling, that Ala103 plays an important structural role by maintaining the 91-109 loops, that includes the important catalytic residue Phe94, via hydrophobic contacts, (Borgi et al. 2004, 2007). The L-arabinose isomerases (LAI) are also attractive enzymes for industrial production of D-tagatose; a sweet and low calorific ceto-hexose having a taste and physical properties similar to sucrose. The LAI from G. Stearothermophilus US100 (L-AI US100) is optimally active at pH 7.5 and 80 °C. It was distinguishable by its behaviour towards divalent ions. Indeed, the L-AI US100 activity and thermostability were totally independent toward metallic ions until 65 °C (Rhimi et al., 2006a, b). The homology modelling and site directed mutagenesis studies, allowed to identifying mains aa implicated on the catalytic isomerisation (Rhimi et al. 2007a). The development of novel applications using: (i) the L-AI US100, or the both enzymes, will be presented (Rhimi et al. 2007b). References.

Belguith and Bejar S. (1998). Biotechnology Letters 20: 553-556. Belguith.et al. (2002). Patent N° US 6,372,476 B1. Borgi et al. (2004). Biochimie, 86 : 561-568 Rhimi M. and Bejar S. (2006a). Biochimica and Biophysica Acta (BBA) 1760: 191-199. Rhimi, et al. (2006b) : Patent Application N° WO 2006/071203 A2. Borgi et al.. (2007). Biotechnology Journal. 2, 254-259. Rhimi M., et al. (2007a). Enzyme and Microbial Technology. 40, 1531-1537. Rhimi M., et al. (2007b). Journal of Bacteriology 189(9): 3556-3563.



TC4 S2

News from sugar-converting enzymes: from structures towards catalysis and synthesis of attractive oligosaccharides Richard Haser1, Hildegaard Watzlawick3, Xavier Robert1, Birte Svensson2, Ralf Mattes3 & Nushin Aghajari1 1

Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines,UMR 5086-CNRS/UCBL,

IFR128 « BioSciences Lyon-Gerland » (7 Passage du Vercors, F-69367 Lyon cedex 07, France) [email protected] http://www.ibcp.fr/rhaser/ 2

Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark Lyngby

3

Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany

Abstract: Recent results will be reported on enzymes from various origins (plants, bacteria…) involved in sugar recognition and processing, on the basis of their high resolution structures in the presence and absence of ligands of interest (natural substrates, inhibitors, protein partners..) and of appropriate mutants. In this context we are studying the structure/function relationships of two major α-amylase isozymes produced in the aleurone layer of barley seeds. These enzymes in combination with limit dextrinase, β-amylases, are of pivotal importance for starch degradation and embryo growth during seed germination. Coupled enzymatic and structural analysis using site directed mutagenesis, gene shuffling, and X-ray crystallography have provided the essential data that enables the fundamental understanding of the catalytic hydrolytic cleavage of α-1,4linked carbohydrates, in starch and related oligosaccharides. A number of bacterial amylases are also known in terms of detailed 3D architectures. Our contribution to the structure/function relationships of amylases from psychrophilic microorganisms led to clarify the features which control molecular adaptation, recognition of sugars and high catalytic efficiency at low temperatures, including the synthesis of novel oligosaccharides of high interest for biomedical applications. Characterization of highly efficient sucrose isomerases have also been reported from isomaltulose-producing bacteria. The first three-dimensional structures (native and complexes) of a sucrose isomerase producing predominantly trehalulose (a nutritional sugar with high health advantages for diabetics and nondiabetics) was recently established, and helps to elucidate the mechanism of isomerase action. Finally, a number of new and unexpected insights into the action of enzymes belonging to the αamylase and sucrose isomerase family at the molecular level will be presented. They contribute to the understanding on how these enzymes tackle the processing of the different substrates they act with in nature. Ultimately, technological processes as well may benefit from the improved insight into the conversion of starch and related sugars, and in the case of the isomerases into the industrial biosynthesis of sugars with significant health advantages.



TC5 S2

Extremophilic anaerolic bacteria : characterization, ecology and potential applications Pr. Jean Luc Tholozan Abstract not communicated



TC6 S2

Espèces Bactériennes, réalité ou mirage ? Boudabous Abdellatif, Cherif Ameur, Gtari Maher, Ben Slama Karim et Ouzari Hadda-Imène Laboratoire Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis.

Abstract : Trois siècles après les premières observations des microorganismes, la notion d’espèces bactériennes a effectuée un grand progrès ou même une révolution. De la notion d’animalcules ou microbes aux organismes eucaryotes et procaryotes. Ces derniers divisés en Bacteria et Archea sont considérés comme les parents ancestraux des cellules vivantes sur terre. Les bactéries sont actuellement divisées en classes, ordres, familles, genres et espèces avec un nom de genre et d’espèce comme les espèces animales ou végétales. Malgré la recherche continue d’une taxonomie claire, les limites et les frontières entre ces groupes, genres ou espèces sont troubles et même illusoires. Cette relative image des microorganismes provient : (i) d’une évolution de quatre milliards d’années, (ii) une diversité presque illimitée des espèces (iii) une multiplication rapide impliquant des mutations et une sélection – adaptation dans tous les milieux (iv) cette évolution est aussi accélérée par des transferts génétiques entre souches et espèces. (v) La relativité de la taxonomie provient aussi de notre connaissance limitée au 1/100 à 1/1000 des microorganismes actuels, (vi) et à notre observation du monde microbien à un moment ponctuel de l’échelle temps. Comme les croyants, les taxonomistes ont développé un débat passionnel sur la notion d’espèce bactérienne et mis au point plusieurs méthodes de description, caractérisation et classification des bactéries. A partir d’une taxonomie conventionnelle et dichotomique basée sur des critères phénotypiques ayant un poids arbitraire, ils ont développé une taxonomie numérique avec des critères à poids équivalents. La description des espèces ou taxons bactériens a été approfondie par la taxonomie polyphasique basée sur des critères morphologiques, métaboliques, physiologiques, ultrastructure, acides gras membranaires, GC%, et homologie DNA/DNA. Cependant, la taxonomie même avancée définit un système d’identification et de classification sans relation entre espèces ou genres. L’émergence de la génétique moléculaire a permis une évolution rapide de la phylogénie des espèces. L’espèce bactérienne est actuellement définie comme une population homogène de souches, ou espèce génomique (genospecies), partageant en commun un nombre élevé de critères biochimiques, phénotypiques et structuraux, confirmés par une homologie ADN/ADN supérieure à 70%, une similitude en GC%, en gènes ADNr, rpoB, recA. Une comparaison multi- génique des souches de la même espèce est souvent exigée. Bien que la majorité des espèces connues est caractérisée par ces critères biochimiques et génétiques, une nouvelle taxonomie – phylogénie plus précise a déjà émergé à partir de la comparaison des six cent génomes bactériens entiers disponibles. Les nouveaux regroupements sont plus cohérents que



jamais, cependant le mirage persiste avec de beaux horizons. De nouvelles questions se posent : (i) Qu’est que Escherichia coli, si les souches de cette espèce ont 25% de gènes variables ? (ii) Quels sont les gènes les plus importants pour l’évolution et la phylogénie, les gènes du moteur (ADN et ARN polymérase, les opérons ribosomaux) ou

les gènes de ménages et métaboliques ? (iii) Quels rythmes et quelles

importances des transferts génétiques horizontaux (intra - espèces et inter - spécifiques) dans l’évolution des espèces microbiennes ? (iv) Comment peut améliorer nos concepts de souches, variétés, espèces, genres, familles bactériennes ? La notion d’espèces bactériennes réelles ou illusoires, a permis une grande évolution de notre compréhension du monde microbien ancestral et actuel. Elle reste imparfaite et utile comme toute théorie enrichissante, elle est toujours passionnante avec des perspectives étonnantes pour la pensée humaine. Comme un concept, comme un rêve, elle permet toujours d’avancer.



Building of Genomics led green chemistry for industrial biotechnology Pr J. A. C. Archer, Dep. Genetics. Univ. Cambridge , UK

Abstract not communicated

TC7 S2



TC8 S2

Quantification of (bio) interactive forces at the nano-scale Professor Nidal Hilal Director of Centre for Clean Water Technologies The University of Nottingham United Kingdom

Abstract: Atomic Force Microscopy (AFM) is a new technology with a rapid growing range of applications. It is versatile technique to study (bio)engineering processes at the nanoscale level. This powerful technique may used to visualise both conductive and non-conductive surfaces in air and process relevant aqueous environments. Since 1994 the research of Professor Hilal has been focused on developing and applying atomic force microscopy in process engineering, mainly membrane separation, (bio)colloidal interactions and measurement of complex fluid properties. The major achievement of his recent research has been the development of the smallest colloid probe reported in the literature, coated colloid probe technique and cell probe technique techniques. The presentation will focus on the use of such probes to directly quantify the force of interaction of coated colloid or microbiological cell in a direction normal to the surface at which the interaction was taking place. The potential of the techniques was demonstrated when colloid probes, coated colloid probes and cell probes were used to assess rejection of colloids at the entrance of membrane pores and the adhesive characteristics (fouling) of synthetic ultrafiltration membranes used in industry. The approach correctly identified membranes with low fouling properties and introduced the concept to (bio)process engineering of using AFM in the development of novel surfaces prior to costly pilot plant procedures. The use of the Atomic Force Microscope as a nano-viscometer to measure extensional properties of complex fluid at the nanoscale level is a recent application was identified by the presenter. The presentation will show atomic force microscopy as a powerful asset in the understanding of existing processes and the development of new process.



TC9 S2

Biological Resource Centres for Bioeconomy in Developing Countries V. Storms1, J. Swings1 and M. Amar2 1

Ghent University, Belgium.

2

Centre National pour la Recherche Scientifique et Technique – CNRST, Rabat, Maroc

Abstract : The term bioeconomy clearly stresses the importance of biology and biotechnology in the global economy. Biotechnology is considered as an ‘enabling’ technology, supportive to many traditional economic sectors and leading to many new applications and products in a wide range of fields. For instance, industrial or white biotechnology mainly uses Microorganisms and their enzymes to make useful products, materials and processes, including fine and bulk chemicals, bio-plastics, vitamins and colorants, food ingredients, bio-flavours as well as biofuels. Within the bleu or marine biotechnology, micro-algal biotechnology has grown and diversified significantly during the last 30 years, to become a very promising field for a wide range of applications. Amongst the different cornerstones of the bioeconomy, Biological Resource Centres and Bio-informatics are of major importance. Biological Resource Centres (BRCs) are an essential part of the infrastructure underpinning life sciences and biotechnology. The OECD task force on BRCs has done a great effort of thought to define the new BRC and form the basis for the future development of the actual culture and reference collections. Specifically, a series of best practices for BRCs were developed in extensive consultation with the scientific community. These best practices were written down in a new report that was published recently (1). In general, for Culture Collections or BRC’s, important fundamentals can be distinguished. First, BRCs organize the professional conservation and distribution of biological materials and the data related to them. Second, BRC’s have to be linked to scientific centres in taxonomy, molecular genetics, biochemistry, cellular biology, biochemistry or genomics. Finally, common for all BRCs, is the valuation and thus demonstrable utilization of its holdings and its data, e.g. in biodiscovery. Culture Collections, in this purpose, form the backbone onto which the user community can rely. They provide the infrastructure, know how and legal framework and are the centers for professional conservation of biological material. At present we also witness another important ongoing development, which consists of the transition of the ‘computational science’ as a tool, into the ‘computer science’, including concepts, tools and theorems, as a science in its own right. It is postulated that this development represents the foundations of a new revolution in biology, biotechnology and medicine, and will



have profound implications for society and for life on earth. A prototype of these developments is found in the StrainInfo.net initiative (www.StrainInfo.net) For developing countries’ bioeconomy, many important challenges can be identified: the green, red and blue biotechnologies and the extended use of bioinformatics. It is clear that therefore, a durable and sustainable action is absolutely necessary to preserve biodiversity for future use in research and industry and that also all other ‘types’ of biotechnology will be needed. (1) OECD Best Practice Guidelines for Biological Resources Centres – OECD 2007



TC10 S2

lignocellulosic co-substrates for anaerobic digestion of agricultural wastes and energy crops (Project AGROBIOGAS) Pr. Gerhard Schories TTZ, Germany

Abstract not communicated


Oral Communications

Session 2


Session 2

OC1 S2

Structural- and mutagenesis studies give insights into sucrose isomerization 1

1

1

2

1

Stéphanie Ravaud , Moez Rhimi , Xavier Robert , Hildegard Watzlawick , Richard Haser , 2

1

Ralf Mattes and Nushin Aghajari 1

Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, CNRS et Université de Lyon, 2

UMR 5086, IFR 128 "BioSciences Lyon-Gerland", F-69367 Lyon Cedex 07, France Institut für Industrielle Genetik, Allmandring 31, D-70569 Stuttgart, Germany

Universität Stuttgart,

Abstract: The structural isomers of sucrose (α-D-glucosylpyranosyl-1,2-β-D-fructofuranoside), Trehalulose (α-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (α-D-glucosylpyranosyl-1,6-D-fructofuranose) are very attractive sugar substitutes as they have similar taste profiles and very similar physical and organoleptic properties to sucrose. Various diseases related to the over-consumption of sugar make a growing need for sugar substitutes. Sucrose is an inexpensive and readily available D-glucose donor, and the industrial potential for enzymatic synthesis of the sucrose isomers trehalulose and/or isomaltulose from sucrose is large. The product specificity of sucrose isomerases which catalyze this reaction depends essentially on the possibility for tautomerization of sucrose which is required for trehalulose formation. A number of three-dimensional structures of native and mutant complexes of a trehalulose synthase from Pseudomonas mesoacidophila MX-45 which mimic successive states of the enzyme reaction have been solved by x-ray crystallography methods. Combined with mutagenesis studies they give for the first time thorough insights into substrate recognition and processing, and reaction specificities of these enzymes. Amongst the important outcomes of this study is the discovery of an aromatic clamp defined by Phe256 and Phe280 playing an essential role in substrate recognition and in controlling the reaction specificity, which is further supported by mutagenesis studies. Furthermore, this study highlights essential residues for binding the glucosyl- and fructosyl-moieties. Ravaud S., Watzlawick H., Haser R., Mattes R. & Aghajari N. 2005. Acta Cryst F. 61, 100103. Ravaud S., Watzlawick H., Haser R., Mattes R. & Aghajari N. 2006. Acta Cryst F. 62, 74-76. Ravaud, S., Robert, X., Watzlawick, H., Haser, R., Mattes, R. & Aghajari, N. 2007. J. Biol. Chem. 282, 28126-28136.


Session 2

OC2 S2

Treatment for bioremediation of olive oil mill wastewater Ena Alba, Faraloni Cecilia Pintucci Cristina and Torzillo Giuseppe Institute of Ecosystem Study, National Research Council, Italy, Via Madonna del Piano 10 50019, Sesto Fiorentino, Florence, Italy

Abstract: Olive oil mill waste water (OMW) is the by-product of the olive oil production. It is characterizedby a dark brownish colour, and by a strong odour and it is considered one of the most pollutant waste produced in agricultural industry. Currently OMW is spread on cultivated lands under strictlaw control which strongly limits their use in agriculture (D.L. 574/96). In this work, an innovative procedure (1) for depuration of olive oil waste water is briefly described. Our procedure also make possible to recover valuable substances, such as phenols having important commercial applications. The treatment here suggested uses two different packed vegetablematrices which remove most of the pollutant substances by absorption. Following the filtration of olive oil waste water on the matrices, the pollutant load of the waste is greatly reduced, e.g. theorganic content is reduced by 80% while the phenol compounds are completely removed. From the exhausted matrices very important substances can be recovered such as phenols which have received an increasing interest in applied research in the last few years as they can be used (2),in the prevention of cardiovascular illness(3), as antiviral, as antioxidant, and for antitumor protection (4). Besides, they can be used for food, cosmetic and pharmaceutical applications. Moreover, the filtered water resulting from the treatment can be used to grow purple bacteria (e.g. Rhodopseudomonas palustris) for biohydrogen production. In the end, R. palustris when is under unbalanced growth conditions (5) can produce biopolymers (Polyhydroxyalkanoates) which have apotential applications in medicine and surgery as biodegradable plastic material (6). 1) Ena A., Pushparaj B., Carlozzi P., Sacchi A., Torzillo G., Peccianti F., Citernesi S., Pintucci C., Petrai I., Brevetto di invenzione industriale nr FI2006A00015 2) Visioli, F., Caruso, D., Plasmati, E., Patelli, R., Mulinacci, N., Romani, A., Galli, G., Galli, C.; Free Rad. Res., 2000, 1, 1-5. 3) Visioli, F.; Bellosta, S.; Galli, C. Atherosclerosis 1997,134, 336. 4) Ohno, T.; Inoue, M.; Ogihara, Y.; Saracoglu, I.; Biol. Pharm. Bull. 2002, 25, 666-668. 5) Vincenzini M., Marchini A., Ena A., De Philippis R.; Biotech. Lett., 1997, 19, 8: 759-762. 6)Yamamoto Y, Abe H, Doi Y. Abstracts of the Int. Symposium on bacterial Polyhydroxyalkanoates, 1996, Davos, Switzerland.


Session 2

OC3 S2

Enhancement of the insecticidal activity of a Bacillus thuringiensis subsp. kurstaki strain via the production of chitinase-containing crystals Driss, F., Baanannou, A., Azzouz, H., Zouari, N. and Jaoua, S.* Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box: K, 3038. Sfax, Tunisia *E. mail: [email protected]

Abstract: A new strategy to exploit the chitinase performances to enhance the insecticidal activity of Bacillus thuringiensis was adopted. It consists on the synthesis during sporulation of recombinant crystals that contain chitinases. A newly constructed vector pF harbouring a fusion of the chi255 gene (Driss et al., 2005; 2007) and the cry1Ac gene region responsible for delta-endotoxin Cry1Ac crystallization was transferred to the B. thuringiensis strain BNS3. The recombinant bacterium produced a fusion protein and showed an improvement in chitinolytic activity. Immunological methods using two polyclonal antibodies, Anti-Cry and Anti-Chi/Cry, showed the presence of the chitinase in the crystal and at the surface of the spores of the recombinant strain. The effect of these new locations of this protein on the insecticidal activity was studied. A significant increase in the toxicity of the recombinant bacterium toward the Lepidopteran larvae of Ephestia kuehniella and Spodoptera littoralis was detected. It was concluded that the co-expression of the chitinase Chi255 and the delta-endotoxins of the wild strain enhances the insecticidal activity of the recombinant strain. Moreover, the chitinase display at the spore surface might facilitate the toxin’s role to create pores into the insect midgut membrane. References: Driss, F., Baanannou, A., Rouis, S., Masmoudi, I., Zouari, N. and Jaoua, S. (2007) Effect of the chitin binding domain deletion from Bacillus thuringiensis subsp. kurstaki chitinase Chi255 on its stability in Escherichia coli. Mol Biotechnol, 36, 232-237. Driss, F., Kallassy-Awad, M., Zouari, N. and Jaoua, S. (2005) Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki. J Appl Microbiol, 99, 945-953.


Session 2

OC4 S2

The β-cyclodextrin glycosyltransferase of Paenibacillus pabuli US132: characterization, cloning and overproduction of the recombinant enzyme Sonia Jemli , Ezzedine Ben Messaoud, Sameh Ben Mabrouk, Ayadi-Zouari Dorra and Samir Bejar Laboratory of Prokaryotic Enzymes and Metabolites of the Centre of Biotechnology of Sfax, P.O. Box 'K', 3038, Sfax-Tunisia Tél. : 74 870 451, Fax : 74 870 451, e-mail : [email protected]

Abstract: Cyclodextrin glycosyltransferases (CGTases) are starch degrading enzymes which generate cyclodextrins (CDs). CDs are able to encapsulate various molecules, thereby modifying their physico-chemical properties. Consequently, CDs are widely used in pharmaceutical, chemical, cosmetic and food industries [1]. A Paenibacillus pabuli US132 strain, isolated from a Tunisian soil, was selected for its production of a potent CGTase activity. The US132 CGTase is able to produce a high level of CDs reaching 42 g/l with a β-CD ratio of 63%. Furthermore, the enzyme improved the loaf volume and decreased bread firmness during the storage [2]. The gene encoding this CGTase was cloned and its entire sequence was determined. The deduced amino acid sequence revealed that the mature enzyme of 684 aa was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity of 94% with the CGTase from Bacillus circulans no 8. The US132 CGTase production, in shake flasks by recombinant E. coli strains was very low (about 1 U/ml) suggesting the formation of inactive inclusion bodies. Interestingly, this production reached 22 U/ml after 22 h of induction by shifting the post-induction temperature from 37 to 19°C and using 2TY instead LB medium. High enzyme production (35 U/ml) was attained after 18 h of induction in fermentor using the same culture conditions as in shake flask. The purified recombinant US132 CGTase retained the same biochemical properties to those of the original one. The determination of the kinetic parameters suggested that this CGTase was more specific towards starch than the other reported CGTases. References: [1] Del Valle E.M.M. (2004) Process Biochem. 39: 1033-1046. [2] Jemli S., Ben Messaoud E., Ayadi-Zouari D., Naili B., Khemakhem B. and Bejar S. (2007). Biochem. Eng. J. 34: 44-50.


Session 2

OC5 S2

Purification, biochemical characterisation and immobilization of a xylanase of a thermophilic fungus Maalej Ines; Belhaj Ines, Guerfali Mohamed; Gargouri Ali and Belghith Hafedh Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, BP “K” 3038-Sfax, Tunisia

Abstract: In this study a certain number of thermophile alkaline fungi were isolated due to their capacities to produce extracellular alkalophilic xylanase at high temperature. One of these strains, Talaromyces thermophilus stolk produces a xylanase at high level in a modified Mandels medium pH 8 and 50°C as temperature of growth. This fungus is apparently non cellulolytic and could thus be a possible source of hemicellulases “cellulase-free” with a potential of application in paper and pulp industry. The purification to homogeneity of one endoxylanase of this fungus with a recovery yield of 17.5% was achieved through ammonium sulphate precipitation, DEAE–Cellulose, gel filtration and Mono Q chromatography. The molecular mass, as determined by gel filtration and SDS-PAGE, was about 25 kDa. The secreted cellulose-free xylanase presented interesting characteristics regarding its optimal temperature and heat stability; The enzyme activity was optimal at 75°C and pH 7-8. The enzyme retained 100% of its activity after 7 days at 50°C and could retain 50% of activity after 60 min at 100°C. These characteristics are required for various industries in order to reduce the process cost. Various immobilization methods of Talaromyces thermophilus xylanase were tested, such as the immobilization by inclusion, adsorption and covalent bond on various supports. The enzymatic activity of the membranes was evaluated and compared with the free enzyme. Immobilization on gelatin 10% with the bifunctional agent glutaraldehyde gave the best efficiency: xylanase activity recovery: 113.5%, immobilization yields: 98.8 %. By immobilization, a shift in the pH optima from 7 to 8 was observed but optimum temperature of activity broadened between 75 and 80°C as compared to 75°C in the case of the free enzymes. Immobilization increased enzyme stability mainly for extremes pH. We observed that the half-life time of the immobilized enzyme increased up to 3h instead of 1h at 100°C for the free enzyme. We also observed that certain additives have an activator effect on the immobilized enzyme 2+ 2+ 2+ such as Co and Fe . However others inhibited the immobilized enzyme as EDTA and Mn . The immobilized enzyme retained about 94.0% of the initial catalytic activity even after being used 13 cycles of hydrolysis at 50 °C. The main hydrolysis products yielded from xylan by Talaromyces thermophilus xylanase were xylobiose and others xylooligosaccharides such as the xylotriose. Consequently, the immobilized enzyme can be used for continues production of xylobiose in a large scale starting from xylan of hemicellulose of lignocellulosic materials waste. The co-immobilization of the β-xylosidase and the xylanase of Talaromyces thermophilus permits the increase of the efficiency of xylane saccharification by the liberation of xylose; such result proves the synergic action of both enzymes.


Session 2

OC6 S2

Isolation and characterization of Halomonas sp. strain C2SS100, an hydrocarbon degrading bacterium under hypersaline conditions Sami Mnif, Mohamed Chamkha and Sami Sayadi Laboratoire des bioprocédés, Centre de Biotechnologie de Sfax, BP « K » , 3038 Sfax, Tunisie. [email protected]

Abstract: Formation water collected from “Sercina” petroleum reservoir, located near the island of Kerkennah, Tunisia, was used for the screening of halotolerant or halophilic bacteria able to degrade crude oil. Bacterial strain C2SS100 was isolated from the petroleum reservoir after enrichment on crude oil, in the presence of 100 g/l NaCl. This stain was aerobic, Gram-, rod-shaped, motile, oxidase + and catalase +. Phenotypic characters and phylogenetic analysis based on the 16S rRNA gene of the isolate C2SS100 showed that the strain was related to Halomonas genus. Strain C2SS100 was grown on crude oil as sole carbon and energy source, in the minimal medium, without yeast extract added, in the presence of 100 g/l NaCl and at 37 °C. The degradation of several compounds present in crude oil was confirmed by GC-MS analysis. The use of refined petroleum products such as diesel fuel and lubricating oil as sole carbon source, under the same conditions of temperature and salinity, showed that significant amounts of these heterogenic compounds could be degraded. The potential of the strain C2SS100 to degrade individual hydrocarbons such as aliphatic, cyclic and aromatic hydrocarbons was also studied. Strain C2SS100 was able to degrade hexadecane (C16) via monoterminal oxidation. The proposed metabolism of hexadecane proceeded via hexadecanol, hexadecanaldehyde and hexadecanoique acid. During growth on hexadecane, cells surface hydrophobicity and emulsifying activity increased indicating the production of biosurfactant during the microbial degradation of hydrocarbons. Thus, this strain can be used in the bioremediation process in hydrocarbon contaminated marine and soil environments with high level of salinity.


Session 2

OC7 S2

Immobilization and modification of laccase from Bacillus thuringiensis is promising for enzymatic removal and decontamination of phenolic compounds from polluted water systems 1

2

Hamed M. El-Shora and Metwally, S.A. 1

2

Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt Botany Department, Faculty of Science, Tanta University, Tanta, Egypt

Abstract : Laccases (benzenediol oxygen oxidoreductase, E.C. 1.10.13.2) are phenol oxidases that catalyze the oxidation of various substituted phenolic compounds by using molecular oxygen as electron as the electron acceptor. Bacillus thuringiensis showed a remarkable capability to excrete th laccase to the growth medium. The maximum laccase activity was measured at the 5 day after inoculation. The productivity of laccase increased in presence of GA3, 2,4-dichlorophenoxyacetic acid (2,4-D), 6benzylaminopurine (6-BAP) and methyljasmonate (MJ). Laccase was purified from -1 Bacillus thuringiensis with specific activity of 280 Umg . The pure laccase was immobilized on kaolinite. The Km values for the free and immobilized laccases were 0.30 and 0.21 mM indicates a higher affinity for the substrate after immobilization. Vmax values were 6.5 and 4.5 U for free and immobilized laccases, respectively. The immobilization of laccase improved its stability at a wider pH range (3-7) compared with the free counterpart. The temperature-activity profiles of the enzymes indicate that the highest laccase activity occurred at 40 ºC and 60 ºC for free and immobilized laccases, respectively. After 4 months of storage at 4 ºC, no loss of activity was detected for immobilized laccase; by contrast, free laccase lost 95 % of its initial activity. The immobilized laccase showed higher efficiency than free laccase in oxidizing various phenolic substrates. The purified laccase was modified using polyalkyleneoxide-co-maleic anhydride (PAOMA). Chemical modification of laccase resulted in 30 to 15-fold increase in thermal stability at 40 ºC and 60 ºC, respectively. The modified enzyme expressed a remarkably improved efficiency over that of the native laccase in terms of removing various phenolic substrates including dimethylphenol, o-chlorophenol, 2,4 dichlorophenol, 2,6-dichlorophenol, 2,4,6trichlorophenol, xylenol and cresol. Native laccase was found to decrease in removal efficiency when treating a mixture of phenolics, in comparison with the case of decontamination in single component systems. In contrast, the modified enzyme retained its efficiency performance with respect to treating a phenolics mixture, as well as in treating a single substrate. The results presented indicate a new opportunity for application of immobilized laccase in bioremediation. Also, these results open promising perspectives for applying the PAOMA-modified laccase in a practical removal process of phenolics from polluted waters.


Session 2

OC8 S2

Multiparametric flow cytometry, a powerful tool for the assessment of the mechanism of action of Melaleuca armillaris (Sol. Ex Gaertu) Sm. essential oil, on six LAB strains. A

B

C

B

A

Hayouni, E.A. , Bouix, M. , Abedrabba, M. , Leveau, J-Y. , Hamdi, M. a

Laboratoire d’Ecologie et de Technologie Microbienne: Institut National des Sciences Appliquées et de la Technologie (INSAT), BP. 676, 1080 Tunis, Tunisia. ([email protected]) c

AgroParisTech, INRA, UMR 782 Génie et Microbiologie des Procédés Alimentaires, CBAI, 78850 c

Thiverval-Grignon, France. Unité de Recherche: Physico-chimie et Moléculaire: IPEST, La Marsa, Tunisia.

Abstract: Recently, there’s growing interest in the use of plants, herbs, spices and their derived essential oils (EOs) namely as flavourings in food industry. Nevertheless, little is known concerning the effects of EOs against lactic acid bacteria (LAB) either of the human gastrointestinal microflora or those involved in industrial processes. Additionally, since there are no reports dealing with the antibacterial activities of Melaleuca armillaris (Myrtaceae) against LAB; this work describes, for the first time, the use of a multiparametric flow cytometry technique to assess the viability of six LAB strains at the single cell level. GC and GC-MS analysis of M. armillaris EO resulted in the identification of 68 compounds amounting 99.63% of the oil. Eucalyptol (1,8-cineole) was the major compound (68.92%).Time-survival kinetics of each strain incubated with increasing concentrations of the EO (0.25; 2.5; 5 and 25 µg/ml) were established using an automated microtiter assay (Bioscreen C). Bacteriostatic or bactericidal effects were noticed depending on the studied strain and on the applied concentration of the EO. The mathematical modelling of the kinetics showed that in presence of increasing concentrations of M. armillaris EO, the lag phases of growth were extended (0.69% to 97.5%) and both the growth rate and final cell density were reduced. Variations depending on the strain were noticed. Live/dead assays of the multiparametric flow cytometry technique (combining cFDA and PI fluorescent probes) were done by dual staining of each sample culture to differentiate viable, dead and stressed cells. The behaviour of each strain, in presence of increasing concentrations of M. armillaris EO, was evaluated by quantifying the relative percentages of each subpopulation throughout 3 days of culture. Results displayed disparate patterns of subpopulations which reveal dynamic changes in cells behaviour. This is probably due to disparate influences of the EO components on cellular physiological properties throughout the incubation period. This study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of LAB, and was well correlated with plate count results. Such study could be useful to understand how to fully take advantage of LAB as probiotics or as potential candidates to improve food hygiene and to assure food quality; namely when they are associated with natural preservatives such as EOs. Keywords: Melaleuca armillaris, essential oil, LAB, antibacterial, automated microtiter assay, multiparametric flow cytometry, cFDA, PI.


Session 2

OC9 S2

Characterization and biodegradation of humic acids extracted from different soil ofAlgeria: selection of performance microbial strains and optimisation of culture conditions. Badis A., Ferradji F. Z Et Fodil D. Département de chimie industrielle. Faculté des sciences de l’ingénieur. Université Saàd DAHLAB de Blida. Algérie. E-mail : [email protected]

Abstract: Our present study focuses primarily on the characterization of humic acids (AHs) extracted from different types of soil in the Mitija region (Algiers). This very fertile region is known by rapid organic matter depletion phenomena. Therefore leaching of soil causes a high AHs concentration in surface waters that feed the largest population in Algeria. While the second part of this work is to test the biodegradability of AHs by microbial strains isolated locally. The morphological observation by scanning electron microscope and UV, visible spectra and infra-red analysis of AHs extracted by International Humic Substances Society method led to a preliminary characterization of these compounds. The adaptation method based on synthetic AHs medium has permeated to isolate 90 microbial strains (50 fungi and 40 actinomycetes), which showed a very important biodegradability. A flowing of biodegradation kinetics in batch by performance strains revealed the need of simple substrate (glucose) addition for starting growth. A total discoloration in additional medium (contained carbon and nitrogen sources + 0.5 g/l AHs) was obtained with the three selected fungi strains and to a lesser degree with the three actinomycetes strains. The uses of AHs as sole sources of C and/or N require a very long incubation period up to four weeks. In addition, a comparison was also made between the biodegradation of synthetic and natural AHs. The UV spectra and HPLC analyses showed the formation of intermediaries products who demonstrate the complexity of these humic substances extracted from soil. Key words: Humic acids – characterization – biodegradability – fungi – actinomycetes.


Session 2

OC10 S2

Occurrence of endocrine disrupting activity in tunisian sewage treatment plants a,*

b

c

c

b

Wissem Mnif , Arnaud Pillon , Elena Gomez , Claude Casellas , Marie-Josèphe Duchesne , b

a

Patrick Balaguer and Aghleb Bartegi a

Laboratoire de Biochimie, Unité de Recherche 02/UR/09-01, Institut Supérieur de Biotechnologie de Monastir, 5000, b

Tunisia. INSERM, U824 Signalisation Hormonale, Environnement et Cancer, Montpellier 34298, France. c

CNRS, UMR 5569, Montpellier, F-34293, France

Abstract: With reporter cell lines developed in the lab and allowing the detection of specific activities, we characterized the endocrine disrupting profile of waters and sediments from three Tunisian sewage treatment plants (STPs), which inputs were of domestic and touristic or domestic and industrial origins. We used chimeric cell lines to detect ligands of the estrogen, androgen, glucocorticoid, mineralocorticoid, progesterone, xenobiotic (pregnane X and dioxin) receptors. Treatment efficiency depended on the STP. All the STP waters exhibited a very high estrogenic activity whereas particulate matter and sediments showed a strong xenobiotic activity, mediated by pregnane X and dioxin receptors. A very weak androgenic activity was detected in STP waters. No antiandrogenic activity and no agonistic or antagonistic activity of glucocorticoid, mineralocorticoid and progesterone receptors were detected. By performing competition experiments, we demonstrated that the estrogenic activity present in water mainly contained compounds with a strong affinity for recombinant ERα. On the contrary, the low estrogenic activity present in sediments showed a weaker affinity for recombinant ERα. Kinetics studies allowed us to state that the compounds present in STP sediments and able to activate dioxin receptor behaved like benzo[a]pyrene or 3-methylcholanthrene rather than like dioxin.


Session 2

OC11 S2

Unstable environment enhanced ecological resilience in anaerobic digestors Zemb,O., Delgenes J.P. , Lebaron,P. and Godon J.J. INRA, UR050, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, Narbonne, F-11100, France. [email protected]

Abstract: Pollution removal microbial processes are subject to constant variation. To maintain functional stability microbial communities must be able to face with unstability. Thus, resilience and are a central interest in microbial processes. The role of small perturbation as driving force to increase ecological resilience was explored on lab scale anaerobic reactors. Structure and dynamic of microbial communities were followed by molecular fingerprint. Simple and complex substrates were tested, both based on a selection of community by various small disturbances during 90 days and followed by different major shocks. Compared to the control, small perturbation drove: (i) structure of the microbial community and ecological resilience to the major shock in complex substrate experiment, (ii) only ecological resilience to the major shock in simple substrate. In both cases, reactor functioning remained similar. However, population of duplicated controls and perturbed reactors had slightly different behaviors lead by in terms of community evolution and reaction to a major shock. This demonstrates that history has an effect on the evolution of the system, and that the systems tested have an internal dynamic that is decoupled from the external dynamic.


Session 2

OC12 S2

New Bioreactor For Ethanol Production With Flocullent Yeast JC Duartea, B Ribeiroa, MC Sáàguaa, L BAETA-Halla, V Lourençoa, A Ferreirab And A Fonsecab a

INETI, Department of Biotechnology, Lumiar, 1649-038 Lisboa, Portugal TECNIA-Polígono Industrial do Alto do Ameal, C6, 2560 Torres Vedras, Portugal

b

Abstract: Ethanol production via fermentation became an important issue during the last decades for economical and environmental reasons, and several works have been published in scientific literature since then. The production of ethanol by continuous fermentation is a more productive process, compared to batch ones, as higher productivities are achieved. However, industrial implementation of continuous processes requires a detailed understanding of the process behaviour, so control strategies may be efficiently developed and the process optimized. A high productivity keeping the substrate conversion and product concentration maximal can be accomplished by having high concentration of microorganisms in the bioreactor. Flocculation systems present low associated capital and operational costs and design simplicity. Continuous culture experiments were performed using a strain of Saccharomyces cerevisiae and cane molasses as feedstock to produce ethanol. First trials were carried out in a 1 L-glass tubular reactor, followed by 5 L and 20 L-reactors. In the 1 L and 5 L-reactors feeding sugar concentrations varied between 100 and 120 g/L. Biomass was retained within bioreactors by flocculation, without requiring any support. High cells densities were always achieved, with 80-100 % of viability, with a maximum attained at 120 g/L (1 L-reactor). Best ethanol productivities ranged between 14-16 g/Lh at dilution rates from 0.4 to 1.0 h-1. Nevertheless, during fermentations ethanol yields were not higher than 0.35, corresponding to ethanol concentrations of 20-50 g/L. The 20 L-reactor was used to perform a second step fermentation, to run out the residual sugar from the 5 L-reactor effluent. The reactor design was scaled-up for 500 and 1500 L respectively for trial and optimization experiments. Acknoledgements We thank DVT-Destilação Vinícola Torrejana and Dr. Klaus Bertsch for support and collaboration. This work was partially funded by the EU DG XII. (FERMATEC Project, ENK5-CT-2002-30029)


Session 2

OC13 S2

Novel isolates of Bacillus thuringiensis strains presenting a polymorphism of cry1A genes with difference of in vivo activity. Kallassy-Awad, M.,* Daou, N.,* Azzouz, H. ** and Jaoua, S.** * Faculté des Sciences, Université Saint-Joseph, Beirut, Lebanon, ** Centre of Biotechnology of Sfax, Tunisia E. mail: [email protected]

Abstract : Bacillus thuringiensis is a gram positive bacterium. It has the possibility of forming crystalline inclusions encoded by the cry genes against a broad spectrum of various insects’ larvae. In order to identify a new toxic Bacillus thuringiensis strains against Lepidoptera pests, we isolated different Bacillus thuringiensis strains from Lebanese soil samples. Five strains named D28, D33, B255, Lip, D6, were selected for the revealed presence of cry 1Aa, cry1Ab and cry1Ac genes by the Polymerase Chain Reactions (PCRs). The profile of the plasmid DNA showed a difference between these strains. Bioassays to determine the insecticidal activity of the five selected strains, D28, D33, B255, Lip, D6 was conducted against Ephestia kuehniella larvae, using spore-crystal mixtures. We noted that D6 (96%) and Lip (93%) have higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1) (73.3%) and BNS3 (70%). The Southern Blot analysis of the plasmid DNA after digestion with different restriction enzymes and hybridized with different cry1A probes, revealed a polymorphism among the cry1A genes. The cloning and sequencing of some of the genes from these strains showed a difference in the nucleotide sequences. Our results show that the B. thuringiensis isolates Lip and D6 may have applied value as a significant microbial control agent against lepidopteran pests.


Session 2

OC14 S2

Detection of the biosynthesis pathway of a DKP active molecule produced by the new isolated Streptomyces sp.US24 strain *

Samiha Sioud Meddeb, Ines Karray-Rebai, Samir Bejar, And Lotfi Mellouli

Laboratory of Prokaryotic Enzymes and Metabolites of Centre of Biotechnology of Sfax P.O. BOX ‘K’: 3038 Sfax-Tunisia. Tel and Fax : 00 216 74 870 451, E-mail: [email protected] *

Corresponding author:E-mail: [email protected]

Abstract: We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro) diketopiperazine (DKP) [1, 2]. The aim of this study was to detect the biosynthetic pathway of this DKP derivative. To reach this purpose, PCR amplification of a DNA fragment of 696 bp was carried out using the Streptomyces sp. US24 genomic DNA as template, and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS). The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach, showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro) biosynthesis in Streptomyces sp. US24 strain [3]. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide. Then a cosmid library of Streptomyces sp. US24 genomic DNA has been constructed and probed by the 696 bp PCR fragment. Two positive recombinant cosmids have been retained and the studies of the corresponding inserts are in progress. References: Mellouli L., Ben Ameur-Mehdi R., Sioud S., Salem M. and Bejar S. (2003). Isolation, purification and partial characterization of antibacterial activities produced by a new isolated Streptomyces sp. US24 strain. Research in Microbiology, 154: 345-352. Ben Ameur-Mehdi R., Mellouli L., Chabchoub F., Fotso S. and Bejar S. (2004). Purification and Structure Elucidation of two Biologically Active Molecules from a New Isolated Streptomyces sp. US24 Strain. Chem. Nat. Comp. 40: 510-513. 3. Sioud S., Karray-Rebai I., Aouissaoui H., Bertrand A, Samir B. and Lotfi M. (2007) Targeted gene disruption of the Cyclo (L-Phe, L-Pro) biosynthetic pathway in Streptomyces sp. US24 strain. J.Biom. Biotech, Volume 2007; Article ID 91409.


Session 2

OC15 S2

Purification and biochemical characterization of a transglucosilating β-glucosidase of stachybotrys strain W. Saibi. B. Amouri. and A. Gargouri Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de biotechnologie de Sfax, CBS, route Sidi Mansour, Sfax 3061, Tunisia e-mail: [email protected]

Abstract: The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C. The kinetic parameters, Km and Vmax, on para-nitro-phenyl-β-D-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846±0.11 mM and 211± 0.08 μmol min−1 ml−1. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase activity towards salicin, methylumbellypheryl-β-D-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.


Session 2

OC16 S2

Biodiversity of aerobic bacteria from a Tunisian high-temperature oil field and biodegradation potential of some aromatic compounds and hydrocarbons a

a

a

b

Mohamed Chamkha , Sami Mnif , Sarra Boujelbene , Marc Labat , b

b

Jean-Luc Tholozan & Sami Sayadi a

b

Laboratoire des bioprocédés, Centre de Biotechnologie de Sfax, BP « K » , 3038 Sfax, Tunisie. Laboratoire de Microbiologie IRD, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France. [email protected]

Abstract: An aerobic, thermophilic and halotolerant, bacterium, designated strain C5, was isolated from a high-temperature oil field, located in Sfax, Tunisia, after enrichment on tyrosol. Strain C5 was able to degrade tyrosol aerobically, in the presence of 30 g/l NaCl and at 55 °C. The metabolism of tyrosol proceeded via p-hydroxyphenylacetic acid and 3,4dihydroxyphenylacetic acid, before the aromatic ring degradation. Strain C5 was also found to degrade a wide range of other aromatic compounds. In addition, strain C5 was grown on diesel and crude oil as sole carbon and energy sources. Phenotypic characters and phylogenetic analysis of the 16S rRNA gene sequence of the isolate C5 revealed that it was related to members of the genus Geobacillus with Geobacillus pallidus being the most closely related (sequence similarity of 99 %). The diversity of aerobic bacteria was also studied. Sixty bacterial strains were isolated after enrichment in the presence of crude oil and at various temperatures and salinities. Sixteen thermophilic and twelve mesophilic strains were used after Amplified Ribosomal DNA Analysis. Based on the phylogenetic 16S rRNA analysis, the taxonomic affiliation of the isolates was established. The aerobic bacterial community belonged to the genera Bacillus (43 %), Geobacillus (29 %), Brevibacillus (11 %), Pseudomonas (7 %), Staphylococcus (7 %) and Micrococcus (3 %). 50 % of the isolates were able to grow in the presence of octanoic acid as carbon and energy source. Many strains were also found to degrade a range of aromatic compounds.


Session 2

OC17 S2

Rhizobia-leguminous symbiosis: diversity and biotechnology application Bekki, A., Merabet, C., and de Lajudie, Ph. Laboratoire de biotechnologie des rhizobium et amelioration des plantes. Département de biotechnologie. Université d’Oran, Es-Senia. Oran Algérie E-mail : [email protected]

Abstract: In the purpose of selecting nitrogen-fixation rhizobia/legumes couples well adapted to saline and arid areas in Algeria, we prospected the leguminous plants of ecological and agronomical interest present in these areas. A collection of 100 isolates were obtained from root nodules and identified by phenotypic and genotypic analyses. All strains were tested for nodulation with their host plant in standard tube conditions. A first screening of the bacterial collection, based on the 591 bp variable central part of the 16S rDNA sequencing, affiliated 88 strains to 11 groups in five genera, Ensifer, Rhizobium, Agrobacterium, Phyllobacterium and Bradyrhizobium. Surprisingly, we found that Medicago ciliaris is, in Algeria, associated to three rhizobial species, Ensifer, Rhizobium and phyllobacterium. The full length 16SrDNA sequencing of representative strains of each group revealed five new groups. Multilocus Sequence Analysis (MLSA) of five housekeeping gene (recA, atpD, glnA, thrC and gltA), auxanographic data and nodA gene sequencing confirmed that Ensifer and Medicago sativa, represent two new species, for which we propose the names Ensifer maghrebium sp. Nov. and Ensifer xericitae sp. Nov. respectively. A third group is close to Ensifer adhaerens gvA and nodulate their host plant of isolation, Lotus arabicus. We selected three Ensifer medicae and two Ensifer meliloti strains of the Algerian collection for greenhouse plant tests of their effectivity with two Medicago species (M. polymorpha and M. ciliaris). Their effectivity was estimated by ARA and plant aerial part dry weight measurement using statistical calculations of data. The most effective strains were tested in open field located in semi-arid zone of South-Western of Algeria. Key words: rhizobium, diversity, Ensifer, MLSA, salinity, inoculum.


Session 2

OC18 S2

Detection of cryptosporidium oocysts and giardia cysts from sewage and sludge samples in tunisia 1

2

3

Ben Ayed Layla , Alouini Zoubeir , Cama Vitaliano and Xiao Lihua

3

1

Laboratoire Sciences et Techniques de l’Eau. Institut National Agronomique de Tunisie. 2

Laboratoire de parasitologie des eaux usées et des boues résiduaires. INRGREF. Rue Hédi Karray, BP n°10. Ariana 2080. Tunis. Tunisie 3

Center for Disease Control and Prevention, Atlanta, Georgia Corresponding author: [email protected]

Abstract: Sewage and sludge used for agricultural, industrial and domestic purposes needs to have acceptable levels of chemical and microbiological contaminants so that it will not represent a threat to human and animal health. This study compared the recovery of Cryptosporidium oocysts and Giardia cysts from sewage and sludge samples using three different techniques: Bailanger method, Immunomagnetic separation (IMS) procedures and PCR. Samples collected from plants in Tunisia included raw wastewater, treated wastewater, and sludge. The Bailanger method detected a myriapod of helminths eggs and protozoa cysts. So, we detected the presence of cysts of Giardia sp., Entamoeba histolytica/dispar, Entamoeba coli, and eggs of Ascaris sp., Enterobius vermicularis and Taenia sp. Then, Cryptosporidium and Giardia were identified by immunofluorescent microscopy in 7 and 10 samples respectively. Additionally, community DNA was extracted from all samples and tested using specific molecular tools developed for parasite characterization: a SSU rRNA based molecular tool for Cryptosporidium and a triose phosphate isomerase (tpi) based tool for Giardia duodenalis. We identified several distinct genotypes for each parasite that were predominantly from human and cattle origins: Cryptosporidium hominis and Cryptosporidium parvum and assemblages A and B of Giardia duodenalis. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water and indicate the presence of Cryptosporidium spp. and G. duodenalis among people in Tunisia. Further studies are needed to determine the public health impact of these human pathogens in Tunisia.


Session 2

OC19 S2

Phylogenetic caracterisation and metabolic potentialities of original bacteria isolated from a South Tunisian deep subterrestrial petroleum environment. 1,2

1

1

2

2

Raja Rezgui , Abderrazak Maaroufi , Said Ben Hamed , Jean Luc Cayol , Jean-Luc Tholozan and 2 Marc Labat 1 Groupe des Bioprocédés, Laboratoire de Microbiologie, Institut Pasteur de Tunis (IPT), Tunisie. Bp 74, 13 place Pasteur, Belvédère –1002 Tunis. [email protected], [email protected] 2

Laboratoire de Microbiologie IRD (LMI), UMR_D180 Microbiologie et Biotechnologie des Environnements Chauds, Universités de la Méditerranée-ESIL, Marseille, France. [email protected]

Abstract: Thanks to traditional microbiological ways as enrichment and isolation cultures associated to molecular approaches (16S DNA- based phylogenetical identification), several active bacterial strains in hot and salted media were isolated from a South Tunisian deep subterrestrial petroleum environment and from its related soil. Amongst the isolated strains, three representatives belonging to three different bacterial genera were kept. A bacteria was affiliated to Streptococcus sanguins whom strain Sabriaf37 is its representative, was found as the only bacterium present in all circulatory elements of the petroleum system studied, which suggests an external contaminant adapted to this deep petroleum environment.Two strains with original properties, Sol2f37 and Sol3f37, were isolated from two different oilcontaminated soils. On one hand, Sol2f37, identified as Bacillus licheniformis, was able to grow on crude-oil, the only carbon source, in the presence of nitrate as electron acceptor. On the other hand, Sol3f37 could not be identified to any species. In fact, it was close to four different bacterial genera but even its closest species “Clostrisalibacter paucivorans” presented a phylogenetical difference of more than 7%. Other results will thus be necessary in order to classify this strain either as a novel species of the genus “Clostrisalibacter” or as belonging to a novel bacterial genus.


Session 2

OC20 S2

Antibactérial activity (in vitro) of phenolic extract of three medicinal plants sage, celery and laure Achat S, Madani, K and Chibane, M. *Corresponding author: [email protected] elle

M ACHAT Sabiha.Université Abderrahmane Mira de Bejaia, Faculté des Sciences de la Nature et de la Vie, Département des Sciences Alimentaires, Laboratoire de Biomathématiques, Biochimie, Biophysique et de Scientométrie. Béjaia 06000. Algérie. Tel/Fax: 034214762

Abstract:

The therapy by the medicinal plants (Herbal medicine), has always been a part of our environment. They have several thérapeutic virtues (anti-inflammatory, antibacterial and antioxidant). These properties are generally attributed to secondary metabolites such as phenolÿÿÿÿÿÿpounds (ÿÿlyphÿÿols), ÿÿich have multiple interests in the food and pharmaceutical industrieÿÿ The aim of this study is to contribute of the valorization of vegetable rein as a source of naturel substances bioactives. In this context we are interested in three médicinal plants, sage (Salvia officinalis), laurel (Laurus nobilis) and celery (Apium graveolens), which are used traditionally in the treatment of various diseases (digestive unrests and gynecological affections). This survey focused on extraction of polyphenols, phytochemical investigation and to evaluate their antibacterial activity on selected pathogenic microorganisms but also the interaction of these compounds with a target protein. The results of carried out work showed that the moisture content of the leaves of plants studied ranged from 47, 74 to 85.32% and the yield extraction of Laurus nobilis is 47.5%, which is higher than the value of Apium graveolens (27.5%) and Salvia officinalis (25%). The phytochemical analysis of plant extracts is characterized by the presence of different phenolic classes: total polyphenols (Laurel: 70.69±5.90 mg/g; Sage: 54±0.05 mg/g; Celery: 29.10± 0.92mg/g), flavonoids (Sage: 7.86±0.62 mg/g; Laurel: 5.09±0.06 mg/g; Celery: 4.49±0.20 mg/g) and tanins (Sage: 21.30±1.47 mg/g; Laurel: 2.21±0.72 mg/g; Celery: 2.06±0.26 mg/g). The test of antibacterial activity demonstrated that the methanolic extracts of laurel, celery and sage had an inhibitory effect against some bacterial strains: Escherichia coli, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Klebsiella pneumoniae, and Staphylococcus aureus. The alcoholic extract from Salvia officinalis precipitated the protein bovin serum albumin. This interaction is influenced by the concentration of the extract, like by the ionic strength. All these results suggest the presence of a positive correlation between biological activities of three plants studied and their content in polyphenols. Keywords: Salvia officinalis, Laurus nobilis, Apium graveolens, Methanolic extract Polyphénols, Antibacterial activity, Interaction.


Session 2

OC21 S2

Heterologous expression of the B. thuringiensis vegetative insecticidal protein Vip3LB in Photorhabdus temperata strain K122 and oral toxicity against the Lepidoptera Ephestia kuehniella and Spodoptera littoralis 1

1

1

2

Jamoussi, K. , Sellami, S. , Mesrati, L.A. , Givaudain, A. and Jaoua, S.

1

1

Centre of Biotechnology of Sfax, Laboratory of Biopesticidess, P.O. Box «K» 3038 Sfax, Tunisia. 2

Laboratoire Ecologie Microbienne des Insectes et Interactions Hôte-Pathogène UMR 1133 INRA-UMII, Université de Montpellier 2, Montpellier, France. E-mail: [email protected]

Abstract: Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB (1) is a B. thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we studied for the first time the heterologous expression of vip3LB gene by Photorhabdus temperata strain K122. Firstly, polyclonal antibodies against Vip3LB have been produced, then Western blot analysis of whole cultures of recombinant P. temperata showed that Vip3LB was produced and appeared lightly proteolyzed. Cellular fractionation showed that in vitro-cultured recombinant P. temperata K122 accumulates Vip3LB in the cytoplasm and the periplasm and appears to not release this protein into the culture medium. Oral toxicity of whole cultures of recombinant P. temperata K122 strains has been assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Contrarily to the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth depending on the amounts added to diet. These results demonstrated that heterologous expression of B. thuringiensis toxins in Photorhabdus is an excellent tool for improving Photorhabdus insecticidal activities (2). References (1) Mesrati, AL., Tounsi, S. and Jaoua, S. (2005) FEMS Microbiol Lett. 244:353-358. (2) Tounsi, S., Ebn Aoun, A., Blight, M., Rebaî, A. and Jaoua, S. (2006) J. Invert Pathol 91: 131–135.


Session 2

OC22 S2

Purification, Biochemical Characterization and Genes Identification of New Bacillus thuringiensis Bacteriocins Kamoun, F., Ben Fguira, I. and Jaoua, S* Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box: K, 3038. Sfax, Tunisia *E. mail: [email protected]

Abstract: A comparative study of the bacteriocins of five different Bacillus thuringiensis strains revealed that the highest activity was obtained at the late exponential growth phase and early stationary one, except for one strain named BUPM103 whose bacteriocin was produced at mid-exponential growth phase. These bacteriocins revealed several differences in their activities and resistance to pH and temperature. Bactericidal effects were observed especially against Bacillus species like B. cereus. Except for the strain BUPM103, whose bacteriocin has a novel apparent molecular mass of about 11 kDa, all the four others were of 3 kDa. Although, the latter have the same molecular mass, the differences observed in their biochemical properties and the existence of a cross-activity among their hosts, suggest differences between them. One 3 kDa bacteriocin produced by BUPM4 was purified by a two-step procedure: ammonium sulphate precipitation of protein from culture supernatant followed by a reverse phase chromatography. Upon purification, the specific activity was increased 100-fold. Its molecular mass, determined by mass spectrometry was 3160.05 Da. Direct N-terminal sequencing of BUPM4 bacteriocin revealed the following sequence: DWTXWSXL [Kamoun et al. 2005]. BUPM103 bacteriocin was purified using a combination of chromatographic methods based on high performance liquid chromatography and anion exchange principles. Its N-terminal sequencing revealed the following sequence: AVLPYDDVNITNT. In order to identify genes associated with BUPM4 bacteriocin synthesis, insertional mini-Tn10 transposon mutagenesis approach was used. One transposant, containing a Mini-Tn10 single-copy insertion, has lost the bacteriocin activity but maintained its immunity. The analysis of the mini-Tn10 insertion flanking sequences revealed that a secretion system involving an ABC type transporter was required for the bacteriocin export. Reference: F. Kamoun, H. Mejdoub, H. Aouissaoui, J. Reinbolt, A. Hammami and S. Jaoua (2005). Purification, Amino Acid Sequence and Characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis. J. Appl. Microbiol. 98, 881-888


Session 2

OC23 S2

Novel oxidant- surfactant- and bleach-stable serine alkaline protease from a newly isolated Bacillus pumilus CBS strain: biochemical and molecular characterization a

b

a,

Jaouadi, B., Ellouz-Chaâbouni, S. and Bejar, S. * a

Centre of Biotechnology of Sfax, Laboratory of Enzymes and Metabolites of Prokaryotes Road of Sidi Mansour Km 6, b

P. O. Box « K » 3038 Sfax-Tunisia National Engineering of Sfax, Unit of Enzymes and BioConversion Road of Sokra Km 3, P. O. Box « W » 3038 Sfax-Tunisia *Corresponding author. Tel/Fax: +216 74 8704 51. E-mail address: [email protected] (S. Bejar).

Abstract: A novel detergent-stable serine alkaline protease (termed SAPB) was studied from a newly isolated bacterium from Tunisian soil. Based on API 50 CHB system and 16S rRNA gene sequencing, the isolated bacterium was classified as a new Bacillus pumilus CBS strain. The enzyme was purified to homogeneity and a 12% recovery of enzyme with 38–fold purification was recorded. Pure enzyme was found to be monomeric protein with a molecular mass of 34598.19 Da. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine protease family. The optima of pH and temperature were 10.6 and 65 °C, respectively. Its 2+ 2+ 2+ activity showed total inhibition with Hg ions, while Ca and Mg enhance thermal stability. Its -1 -1 catalytic efficiency (kcat/Km) calculated to be 45265 min mM using casein as substrate which is, in our knowledge, the highest among others already reported Subtilisins. In addition, SAPB showed remarkable stability in presence of 5% Tween 80, 1% SDS, 15% Urea and 10% H2O2. This enzyme removed human blood or chocolate stain completely. Furthermore, xylitol at 1% enhanced the stability of this enzyme during spraydrying and lyophilizing process. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in E. coli.Purified recombinant SAPB has the same biochemical properties than the native one. It had an ORF of 1149-bp encoding a protein of 383 aa. The deduced aa sequence inspection displays an important homology with other subtilisins.


Session 2

OC24 S2

Gene cloning and characterization and of a novel thermostable phytase from Bacillus subtilis US417 Ameny Farhat, Hichem Chouayekh, Kameleddine Bouchaala & Samir Bejar Laboratoire d’Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax, Route de Sidi Mansour Km6, BP “K” 3038 Sfax, Tunisie. E-mail: hichem.chouayekh @cbs.rnrt.tn Tel.: +216 74 870451; fax: +216 74 870451;

Abstract: Phytases can increase feed nutritional value by releasing phosphate from phytate, the major storage form of phosphorus in plants. A novel extracellular phytase (Phy) was purified from the newly isolated Bacillus subtilis US417 strain. The purified enzyme of 41 kDa, was calcium-dependent and optimally active at pH 7.5 and 55 °C. Phy was found to be highly specific for phytate and exhibited good pH stability (from 3 to 9). This enzyme showed also high thermostability as it was able to recover 90% of its original activity after denaturation at 70 °C for 10 min in the 2+ presence of 5 mM Ca . The phytase gene was cloned by PCR from Bacillus subtilis US417. Sequence analysis of the encoded polypeptide revealed high similarity to the calcium-dependent phytases from Bacillus characterized by the absence of the active site motif RHGXRXP which is highly conserved among the phytases belonging to the sub-family of histidine acid phosphatases. With its high substrate specificity, neutral pH optimum as well as its great pH and thermal, this phytase could be an interesting candidate for animal feed application.


Session 2

OC25 S2

A spontaneous direct repeat deletion in the pGEX fusion vector decreases the expression level of recombinant proteins in E. coli Ines Borgi and Ali Gargouri Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, BP”K”3038, Sfax –Tunisie E-mail address: [email protected]

Abstract: pGEX vectors are widely used for GST-fusion protein expression in Escherichia coli under the control of a strong IPTG inducible tac promoter. While using pGEX-4T-2 vector in heterologous protein expression we noticed that the GST or GST fusion protein were expressed at a very low levels. Interestingly, we found a spontaneous deletion of 701 bp DNA fragment harbouring the tac promoter in both, native and recombinant pGEX-4T-2 vectors. This deletion took place between two direct repeats of 43 bases and led to the loss of a 701 bp DNA fragment. This explained the decrease in GST or GST-fused protein level since the tac promoter, that directs transcription was deleted. The lacZ promoter, located upstream of the deleted fragment, replaced tac promoter but was less efficient. The deleted DNA also specifies part of the lacZ gene coding for the N-terminal end of the beta galactosidase (the alpha-peptide), which is slightly functional. Consequently, bacterial cells transformed with the original pGEX are of a faint blue colour while those bearing the deleted ones are white, when plated on X-Gal containing medium. The deletion, didn’t affect neither the sequence nor the molecular weight of GST and fusion protein since it took place just before the GST start codon. It occurred in E. coli TOP10 cells which are recA-, suggesting that the deletion didn’t require the RecA recombination system.


Session 2

OC26 S2

Hydroxytyrosol recovery through microbial transformation Zouhaier Bouallagui* And Sami Sayadi Laboratoire des Bioprocédés, Centre de Biotechnologie de Sfax: B.P “K”. 3038, Sfax, Tunisia.

, B.P “K”. 3038, Sfax, Tunisia. * Corresponding author: Tel: +216 22 94 78 94; Fax: +216 74 44 04 52; e-mail: [email protected] Abstract: Microorganisms have been successfully employed to produce high regio- and enantioselectively transformed organic compounds. More interestingly immobilization in alginate beads is being widely used to overcome limitations imposed by substrate excess or toxicity. Owing its ease, immobilization can be applied to viable cells, purified enzymes or crude extracts. In this contribution, Hydroxytyrosol, an ortho-diphenolic compound known as antioxidant, anti-atherosclerotic and cancer preventive agent, was produced after bacterial catalysed hydroxylation of tyrosol. Biocatalysts were immobilized in alginate beads and their efficiency to transform tyrosol into Hydroxytyrosol was compared to free catalysts. Immobilized cells were revealed to tolerate more tyrosol concentration (5 g/l) than free cells (4 g/l) resulting in high bioconversion molecular yield exceeding 86%. Cells immobilization was also advantageous for stabilising the enzymatic activity after reuse of alginate beads in repeated batches since their reusability was extended for at least four batches with almost same original yield. Enzymatic parameters have shown that immobilization has no effect on the catalytic efficiency expressed as Kcat/Km. Consequently, numerous advantages of immobilized cells are to be noted. The most important are the easy separation of the cells, their reusability and their improved stability. These pointed advantages have encouraged the exploitation of immobilized cells for industrial application in agricultural, pharmaceutical and cosmetic fields.


Session 2

OC27 S2

Ultrafiltration for advanced olive mill wastewater treatment for ferti-irrigation Khoufi, S., Aloui, F. and Sayadi, S. Laboratoire des Bioprocédés, Pôle d’Excellence Régional AUF (PER- LBP), Centre de Biotechnologie de Sfax, BP: « K », 3038, Sfax, Tunisie, E-mail : [email protected]

Abstract: Treated olive mill wastewater (OMW) by electro-Fenton-methanisation process is a valuable water source that can be reused for diverse agricultural purposes. However, in order to minimize environmental risks and to maintain adequate levels of sustainable agriculture production, advanced treatment is required. In this study, we investigated the improvement of the effluent quality by membrane separation technology. For this purpose, membranes (Mb) having 2, 25 and 100 kDa molecular weight cut-offs were tested at ambient temperature. The characteristics of the ultrafiltered anaerobic effluent show that the 25 kDa cut-off membrane was able to remove 100% of total suspended solids (TSS), 95% of the color and 45% of the chemical oxygen demand (COD). Moreover, the results show that high molecular mass polyphenolic compounds, as well as a high percentage of residual monoaromatic compounds were retained by the Mb 25 kDa. On the basis of results, Mb 25 kDa was selected as the best membrane for the improvement of effluent quality. Microbial toxicity tests using the bioluminescent bacteria, Vibrio fisheri, showed that compared to 100% inhibition of untreated OMW, the ultrafiltration (UF) post-treatment seems to be a good option since it completely detoxifies the wastewater. Phytotoxicity experiments showed that the application of UF increased the germination index percentage of Lepidium sativum up to 124% compared to 100% for control (water). On the other hand, untreated OMW led to a decrease of the GI to 12%. This proves that treated OMW could be used for ferti-irrigation without negative effects.


Session 2

OC28 S2

Study Of Microbial Community Diversity In Superficial Sediments Of Coastal Anthropized Bizerte Lagoon (Tunisia). Olfa Ben Said, Maria Soledad Goñi, Monia El Bour, Patricia Aissa And Robert Duran Abstract: In order to estimate how pollution affects the bacterial community structure and composition of sediments, chemical and molecular approaches were combined to investigate 8 stations around the Bizerte lagoon. Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA genes revealed that each station was characterized by a specific bacterial community. The combination of this data with those of chemical analysis showed a correlation between the bacterial fingerprint and the pollutant content. The analysis of the bacterial community composition revealed a higher diversity in the most heavy-metal-contaminated station than in the most PAH-contaminated station. Sequences affiliated to α, β, γ, δ, ε, subclass of the Proteobacteria, Actinobacteria and Acidobacteria were detected in both selected stations although in different extent. In contrast, sequences affiliated to Nitrospirae were detected only in the most heavy-metal-contaminated station. In the most PAH-contaminated station, phylotypes affiliated to γ-Proteobacteria were the most abundant while the most heavy-metals-contaminated station was dominated by phylotypes affiliated to δ-Proteobacteria main of which closely related to sulphate-reducing bacteria (SRB).


Session 2

OC29 S2

Assessment of current conventional and membrane technologies for wastewater treatment and effluent reclamation in Palestine Rashed Al-Saèd*,1, M. Khamis2, A. Manassra2 1

Institute of Environmental and Water Studies, Birzeit University, P. O. Box 14, Birzeit, Palestine Tel. +972 (2) 298-20 70; Fax +972 (2) 298-21 20; E-mail: [email protected] 2 Center for Chemical and Biological Analysis, Al-Quds University, Jerusalem, Palestine E-mail: [email protected]

Abstract: Palestine shared other arid and semi-arid Mediterranean countries with a looming water shortage issues but exacerbated with sanitation crises. This calls for endorsement of sustainable sanitation facilities with adequate effluent quality useful for different utilization purposes. Treated effluent of existing wastewater treatment systems is a valuable water source after adequate reclamation stages. Using field results (6 and 12 months) and compiled literature data, this study presents a comparative technical and financial analysis between two activated sludge systems (ASS) with different advanced reclamation stages. One ASS plant followed by slow sand filter (SSF) and another by two membrane technologies (MT); an ultrafiltartion (UF) and a reverse osmosis (RO) stage. Results obtained on effluent quality of both systems revealed that MT produced high quality water source suitable for unrestricted irrigation. The SSF showed removal efficiencies for TDS, COD, NH4-N and FC (9.9%, 82.6%, 64.8% and 99.999%, respectively) compared with MT (99.5%, 96.4%, 93.7%, and 100%, respectively). The annual capital and running costs for both reclamation options were calculated at 0.35 US$/m3 (ASS) and 0.50 US$/m3 (MT). This study pointed that MT can be a viable reclamation option with additional efforts to improve membrane performance and efficacy resulting in treatment costs reduction. Keywords: Effluent reclamation; Agricultural reuse; Membrane technology, Effluent quality


Session 2

OC30 S2

Investigations on the influence of the fermentation conditions on the vitality of a probiotic culture after drying Albesharat, Rima(1), FöRST Petra (2) 1-National Commission of Biotechnology – Ministry of High Education - Syria 2-Inestitute of Food Process Engineering at Technical University in Munich - Germany

Abstract: The industrial exploitation of Lactic acid bacteria (LAB) as starter cultures and probiotics is dependent on concentration and preservation technologies, which can guarantee the delivery of stable cultures in terms of viability and bacteria functionality. Vacuum drying is one of the promising preservation processes. In this work we explored first the growth and metabolism of the probiotic strain F19 (Lactobacillus Paracasei ssp. paracasei) under different fermentation conditions (pH, neutralizing agent and temperature) and different fermentation media. In the second step, vitality after drying (CFU + metabolic activity) was studied under selected fermentation conditions and medium composition. Moreover, to explore the effect of added protectants prior to drying on the survival after drying, a concur test was carried on with two different sugars (Sorbitol and Lactose) added as protectants. Our results showed: A higher dehydration resistance in bacteria harvested earlier during the stationary growth phase. pH stress has positive influence on cells resistance to the dehydration inactivation. The preferred neutralizing agents for cells growth and viability are Ca (OH)2 and Na(OH) compared to NH4(OH), but for the survival after drying the most effective neutralizing agent was NH4(OH). Medium composition and glucose \ lactose concentration had significant influence on the survival after drying. In general, our results demonstrated that survival of L. paracasei following vacuum drying could be improved by the addition of sorbitol and optimization of fermentation conditions


Session 2

OC31 S2

Evidence of the importance of the met115 for Bacillus thuringiensis subsp. israelensis cyt1aa protein cytolytic activity Zghal Zribi, R., Trigui, H., Azzouz, H., Jemaâ, M., Ben Ali, M. and Jaoua, S.* Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box: K, 3038. Sfax, Tunisia *E. mail: [email protected]

Abstract: B. thuringiensis israelensis is the mosquitocidal subspecies most thoroughly studied (Zghal et al., 2006). One of the major components of its toxins is the cytolytic endotoxin Cyt1Aa. Cyt1Aa was demonstrated to increase Cry protein toxicity when expressed simultaneously, through synergism. Cyt synergizes or overcomes resistance to mosquitocidal-Cry proteins by functioning as a Cry membrane bound receptor. Moreover, expression of cloned Cyt1Aa is lethal to E. coli and to B. thuringiensis kurstaki cells. Current evidence suggests that the helical hairpin, containing αC, is the most likely machinery for membrane insertion and channel formation. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1A97 gene has been isolated from a B. thuringiensis israelensis strain named BUPM97 (Zghal and Jaoua, 2006). Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix αC. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in E. coli. It revealed that the mutated Cyt1A97 protein was overproduced showing a very weak toxicity to E. coli contrarily to Cyt1A98. This mutation should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa (Zghal et al., 2007). Other investigations are being undertaken to clarify the role of the observed mutation in the Cyt1A synergistic activity with other Cry proteins. References Zghal, R.Z., Tounsi, S. and Jaoua, S. (2006) Biotechnol. Appl. Biochim. 44, 19-25. Zghal, R.Z. and Jaoua, S. (2006) Mol. Biotechnol. 33, 191-198. Zghal, R.Z., Trigui, H., Ben Ali, M. and Jaoua, S. (2007) Mol. Biotechnol. (in press)


Session 2

OC32 S2

Kinetic modelling of the benzene inhibitory effect on nitrifying batch cultures 1, 2*

Chérif Ben-Youssef 1

2

3

3

, Alejandro Zepeda , Anne-Claire Texier and Jorge Gomez 2

Instituto Tecnológico de Cancún, Av. Kabah km 3, 77500 Cancún, Q. Roo, México Universidad Politécnica de Pachuca, Departamento de Biotecnología, Carr. Pachuca-Cd. Sahagún, km 20, Zempoala, 43084 Pachuca, Hgo., 3

México Universidad Autónoma Metropolitana-Iztapalapa, Div. CBS, Departamento de Biotecnología, Av. San Rafael Atlixco 186, 09340 México D. F., México Email: [email protected]

Abstract: The inhibitory effect of benzene on the nitrifying activity of a sludge produced in steady state nitrification was evaluated and modelled in batch cultures. The proposed model structure is based on the sequential oxidation of ammonia to nitrite and nitrite to nitrate by two distinct bacterial groups, the ammonia-oxidizing biomass and the nitrite-oxidizing biomass. Since benzene concentration was monitored during the experiments, a dynamical equation describing benzene transformation was incorporated to complete the two-step model. Reliable predictions of ammonia oxidation, nitrite oxidation, nitrate accumulation and benzene transformation with one single set of kinetic parameters were obtained. Model validation was performed using the experimental data obtained during the different batch cultures at different initial concentration of benzene (from 0 to -1

10 mg benzene-C l ).


Session 2

OC33 S2

Assessment of biostimulation effect on a highly heavy metals and oil polluted soil Nour Sh. El-Gendy*, A. A. El-Feky, Yasser M. Moustafa and Raed M. Hegazi Egyptian Petroleum Research Institute (EPRI), Nasr City, Cairo, Egypt *Corresponding author. Tel: +202 274 7917; Fax: +202 274 7433. E-mail address: [email protected] (N. Sh. El-Gendy).

Abstract: Seven heavy metals tolerable bacteria were isolated from highly heavy metals (HM) and petroleum contaminated soil (PCS) collected from an Egyptian oil field. The isolated strains were identified using the Biolog/Microlog 3420 program. Six of them were Gram positive Brevibacterium otitidis N1, Cellulomonas hominis N2, Clavibacter agropyri N3, Geobacillus thermoglucosidasius N4, Micrococcus luteus N5 and Pediococcus pentosaceus N6 and only one Gram negative bacterium, Sphingomonas macrogoltabidus N7. The most HM tolerable bacterial strain was Cellulomonas hominis N2, tolerated up to 500mg/l heavy metals mixture (Cu, Mn, Ni and o Zn). After 7 days of incubation at 30 C, pH 7 and 150rpm; the seven bacterial strains showed different biodegradation potentials on 500mg/l phenanthrene (9.5-98%). N2 showed the highest biodegradation potential (98%) while N4 showed no capability of degradation. Biostimulation treatment of the highly HM and PC soil through nutrients addition showed immediate increase in the indigenous bacterial populations with enhanced degradation of most hydrocarbons present in the oil o pollutant up to 80% of its content after 35 days of incubation at 30 C. Key words: Biostimulation, soil, heavy metals, oil pollution


Session 2

OC34 S2

Automatic Controlmay HelptoInvestigate Microbial Ecological Questions M. Dumont, A. Rapaport, B. Benyahia, J-J. Godon and J. Harmand Abstract: In the actual context of the study of the consequences of anthropic actions on the biodiversity, it is important to establish links between the functions of an ecosystem and its biodiversity. More precisely, it is crucial to assign to each species a particular function. This "assignation problem" is related to the question of "who does what?" in a complex ecosystem (here, "complex means that we consider ecosystems involving several interacting species, each of them possibly realizing different functions). Molecular fingerprint techniques offer a way of monitoring microbial ecosystems by providing new pictures of microbial ecosystems. In a recent work, it has been shown, under appropriate conditions (and in particular for ecosystems with relatively low diversity), that the relative abundances of species could be monitored with this technique while it is not possible if the ecosystem under interest is too diverse, Loisel etal., 2006. These techniques allow us to monitor the concentrations of the species that are present in the ecosystem. Assuming a functional model is available (typically a mass-balance type model describing both biomass and substrates/products dynamics), we propose an "indirect" approach based on specific tools of automatic control theory to solve this problem. In a first step, functional biomass trajectories which best explain the substrate/products dynamics are generated in using optimal control theory while, in a second step, the combination of individual species concentrations which best approximate these functional biomass trajectories are searched for. Such an indirect approach is compared to recent proposed strategies based on nonlinear observers. In this paper, the approach is proposed for the large class of biosystems involvingPbioreactions in cascade realized byPmicrobial functional consortia in which the product th

st

of the i reaction is the substrate of the i+1 reaction. In addition, preliminary experimental results are presented. Loisel, P., J. Harmand, O. Zemb, E. Latrille, C. Lobry, J. P; Delgenes and J. J. Godon (2006) "DGE and SSCP molecular fingerprintings revisited by simulation and used as a tool to measure microbial diversity, Environmental Microbiology, Vol. 8, No. 4, pp. 720-731. J. Harmand, J. J. Godon and M. Dumont are with the National Institute of Agronomic research, LBE-INRA, Avenue des étangs, 11100 Narbonne, France (J. Harmand is the corresponding author, phone: (+33) 468-425-159; fax: (+33) 468-425-160; e-mails: [harmand, godon, Dumont]@supagro.inra.fr). A. Rapaport is with the INRA, UMR-ASB, 2 place Viala, 34000 Montpellier, France (e-mail: [email protected]). B. Benyahia is with the automatic control lab. At the University Abou Bekr Belkaid, BP 230 Tlemcen, 13000, Algeria (e-mail: [email protected]). J. Harmand and A. Rapaport are also with the INRA-INRIA MERE Research Team, 2, place Viala, 34000 Montpellier, France.


Session 2

OC35 S2

Study of the nitrate pollution conversion to the gas nitrogen with a fixed biomass on consumable support (Alfa Stem) O. Belouanas -Benbelkacem1 and. K. Benrachedi2. 1. Department of Chemistry, Faculty of Sciences, University of Boumerdes, [email protected]. 2. Laboratory research of alimentary Technology and Environment, University of Boumerdes, [email protected]

Abstract: This work presents the results optimisation of the biological process to convert the nitrate to the gas nitrogen with fixed biomass using a (consumable support Alfa Stem). The objective is to study the kinetics of denitrification on hand to bring to the fore the fundamental parameters intervening in denitrification bacteria activity. In the first part, we have analysed the influence of hydrolyc and volumic load to value the capacity of nitrate purification in a down flow submerged bio filter. Then with an experimental design approach, we have analysed the qualitative and quantitative aspects of the effects of some factors: concentration of nitrate, temperature and velocity on different responses like the apparent rate of denitrification, as well as concentration of nitrite and nitrate in the reactor outlet. In the second part, we have analysed the behaviour of an up flow bio filter constituted of (Alfa Stem) realizing the denitrification. This was realized with action of some factors (pH, temperature, and dissolved oxygen). Under these conditions, we have established the kinetics of denitrification into account the conditioned or not of nitrite. Key words: Biological treatment, denitrification, "Alfa Stem", "consumable support", biological reactor, denitrifying bacteria.


Poster Communications

Session 2


Session 2 /

PC1 S2

Production of Aspartyl protease by the Penicillium camemberti on curdwhey and its action on cow milk 1,4

2,4

3,4

4

A. Mokhtari , A. Benlounissi , M. Benkahoul , H. Boukhalfa , and A. Mechakra

4

th

(1) 8 May 1945 University, B.P. 401, 24000 Guelma, Algeria, E-mail: [email protected] th

(2) 20 August 1955 University, Route d'El-Hadaiek BP 26, 21000 Skikda, Algeria (3) Larbi Ben M’hidi University Center, B.P 358, 40000 Oum-El Bouaghi, Algeria. (4) Laboratory of Microbiological Engineering and Application, Enzymal Engineering Unit, Life Sciences Department, Mentouri University, B.P. 325 Route Ain El Bey, 25017 Constantine, Algeria.

Abstract : The production of Aspartyl protease (acid protease) is obtained by fermentation of a growth medium based on a curd-whey with a cheese mould (Penicillium camemberti). The culture media are enriched by three nutriment factors, which are lactose, trypticase, and minerals (FeSo4, MgCl2), already selected by a previous study (Mechakra, 1999). The determination of the concentration optima of these factors are carried out with the help of the centred composite plan of Box and Wilson. The following values are found: 56.94 g/l for lactose, 10 g/l for trypticase, 0.0149 g/l for FeSo4 and 1.368 g/l for MgCl2. This optimization allowed us to obtain a maximal proteolytic activity of 4253 Proteolitic Units. The tridimensional representation of the response surface equation for the three studied factors reveals that the stationary points (optima) found ensure a maximum production of acid protease for lactose and trypticase. But they ensure a minimum production for the minerals. The separation scheme of protease consists of a divided precipitation by acetone at 30% and 50%, followed by a dialysis. The yield of the recovered activity is 91.45% with a purification level equal to 2. The coagulation test of the cow milk by the enzymatic extract shows that the coagulation time is 2h 45mn, which is too long compared to 22 min for the rennet. Respective contents of sugars and proteins in the curd-whey given by the action of our extract are 28.3 g/l and 5.3 g/l. For the rennet they are 45.3 g/l and 3.2 g/l. These results show that curd-whey is a cheap medium that is suited for the production of Penicillium camemberti acid protease, even if it requires a nutritional supplementation in order to increase the enzymatic production. The coagulation tests show a weak coagulating activity (coagulation force) of the aspartyl protease produced by Penicillium camemberti.


Session 2 /

PC2 S2

Occurrence of CTX-M-type β-lactamases among clinical isolates of Enterobacteriaceae in Bejaia, Algeria 1

2

1

2

1

Abdelaziz Touati , Lucien Brasme , Said Benallaoua , Janick Madoux , Alima Gharout , and 2 Christophe De Champs 1

Laboratoire de microbiologie appliquée, FSNV, Université A/MIRA de Béjaia, 06000, Algérie, Laboratoire de Bactériologie-Virologie-Hygiène Hospitalière, CHU Reims, Hôpital Robert DEBRE, Avenue du 2

Général Koenig, 51092 Reims Cedex, France. E-mail: [email protected]

Abstract : We screened 443 clinical Enterobacteriaceae isolates recovered from 4 hospitals in Bejaia, Algeria, from 2004 to 2006 for ESBL production. Among theme, 13 isolates including 5 K. pneumoniae, 4 E. coli, 2 E. cloacae, 1 K. oxytoca and 1 S. marcescens were found to produce an Extended-Spectrum β-lactamase. Isoelectric focusing, PCR analysis and sequencing demonstrated the presence of 2 different ESBL. CTX-M-15 produced by 4 E. coli and 3 K. pneumoniae isolates and CTX-M-3 in 2 E. cloacae, 2 K. pneumoniae, 1 K. oxytoca and 1 S. marcescens isolates. All blaCTX-M-15 genes, except in 1 isolate of K. pneumoniae, were transferred in E. coli C600 by mating out assays, while transfer of blaCTX-M-3 genes was obtained only for the 2 K. pneumoniae isolates. Key words: Enterobacteriaceae, CTX-M-15, CTX-M-3, Algerian Hospitals


Session 2 /

PC3 S2

Contribution to the biological indicator of pollution search: Cases of the influence of a chronic toxicity by a xenobiotic (Cu) on the morphology, response of catalase activity and energy metabolites of freshwater fish: The Tilapia (Oreochromis niloticus) 1, 2

1

2

Meknachi A , Djillali M and Badis A . 1-URDPA. Bou-Ismail. Tipaza. Algérie. E-mail : [email protected] 2-Faculté des sciences de l’ingénieur. Université Saad Dahlab de Blida. Algérie.

Abstract: The pollution of water by toxic substances is not easy to identify. If the dramatic events (fish deaths) spill affecting public opinion, impregnation gradual chronic rejection due to pass mostly unnoticed, although it can have serious consequences on the functioning of ecosystems. In the identification of pollution situation, the use of media concentres, including sediment, living beings (fish, mussels, etc.)., Certainly allows the drawing up of a presence, especially for heavy metals. Our objective is to determine the influence of a xenobiotic (Cu) ecophysiological parameters (morphometric and energy metabolites) and Catalase activity in liver, gill and intestine of fish. The results showed that the study of metabolism through the measurement of +

ammonium NH4 as a function of time has proved very interesting. The allure of curves shows that there has been a slowdown in metabolism in terms of body weight and an increase in the production of ammonium depending on the concentration of copper . Also copper (1mg/l) , caused significant decreases in catalase activity in the liver and gill , so catalase may be considered as a sensitive biomarker for biomonitoring the aquatic environment This experience using organisms or bioindicators tests can be assessed so as early as possible the impact of water pollution. Key words: biological indicator-Oreochromis niloticus - xenobiotic (Cu)-Catalase.


Session 2 /

PC4 S2

Study of four strains of bacillus genera for their performances in hydrocarbon rejection biodegradation with pure and mixed culture. 1

2

1

Benhamed Said , Marc Labat and Maaroufi Abderrazek . 1-Laboratoire de Microbiologie, groupe de Bioprocédé, Institut Pasteur de Tunis. 2-laboratoire de Microbiologie IRD (LMI), UMR_D180 Microbiologie et Biotechnologie des Environnements Chauds, Université de la Mediternnée-ESIL, Marseille, France. Abstract: The oil which was the promoter of immense progress in the world economic activity but it generate in the same time many problems of pollution due to the toxic and bioaccumulable hydrocarbons substances that contains. Several studies related to the treatment of these hydrocarbon rejections but the limited output of biological treatment depend often on the polluting load, the physicochemical conditions and especially on competences of the implied micro-organisms. It is within this framework that we have isolated 121 bacterial strains from polluted sites. Thereafter we have studied the performances of these strains to product a biosurfactant and hydrocarbons degradation. The screening of biosurfactant producer’s strains was carried out according to two methods: drop collaps method and agar plate method (Nawart 2004).whereas the degrading capacity of hydrocarbons for each strain was studied according to the method of Van Hamme using the oil rejection as sole carbon source. (Van Hamme 2000). Among the strains having shown a significant activity for the secretion of the biosurfactants and the degradation of hydrocarbons we selected 4 strains of the genera Bacillus whose performances are remarkable at the same time for the production of biosurfactants and the decomposition of hydrocarbons on solid medium. The performance of these 4 strains to biodegradation of hydrocarbons in liquid medium was studied in a bioreactor of 20 L in batch system at the same time in pure culture and mixed culture. The follow-up of the kinetic of biological breakdown by the measurement of the total hydrocarbons, the DCO, SSM as well as the biomass formed along time, shows that the 4 selected strains (B.licheniformis, B.firmus B.sphaericus and B.stearothermophilus) are also powerful for the degradation of this hydrocarbon rejection in liquid medium. However in pures cultures the two strains of B.licheniformis and B.firmus degrade hydrocarbons with an output better than the two other strains (B.sphaericus and B.stearothermophilus.) In the mixed culture we notes a light improvement of the output of degradation compared with the pure cultures of two strains B.sphaericus and B.stearothermophilus. In the perspective to exploit these strains in the treatment process of the hydrocarbon pollutants, we have studied their competences under extremophiles conditions of temperature and salinity. Results obtained show that strains of B.licheniformis and B.stearothermophilus are quite active on temperatures which can reach 55°C and salinities going up to 120 g/l (almost four times the salinity of the marine environments) these results prove the capacity of these strains to eliminate this type of pollutant under extreme conditions of temperature and salinity.


Session 2 /

PC5 S2

Effectiveness of Pseudomonas syringae to control the Lesser grain borer in semi arid zone of Algeria . 1

1

Mebarkia, A., , Guechi, A. and Dehbi, F.

2

1

Laboratory of Microbiology and Phytopathology, Setif University –Algeria, E-mail: [email protected] 2

National Institutes for Agronomic Research, Setif Unit –Algeria, *Corresponding author. Tel: +213.06.63.27.59.49; Fax: 213.036.92.48.50,

Abstract: In this study we determined with appropriate method of control, the real impact of the antagonist of stored cereal grains, by assessing the degree of attack, which is about 15.02%. Without denigrating the advantage chemical methods that have allowed and even allow saving a significant amount of food, various alternative methods should be considered, particularly biological methods. Nevertheless, some antagonists of micro-organisms entomopathogens, Pseudomonas syringae against Rhyzopertha dominica (F.), as a very promising alternative for a mortality of the species after 48 hours of exposition to different temperatures 5

6

7

(0, 5 and 10 °C) and bacterial concentrations (2.10 ; 2.10 and 4.10 colonies/ml). The different mortality rates observed in the samples containing no bacteria showed that Rhyzopertha dominica (F.) is sensitive to low temperatures. The effect of gel-nucleant bacteria entomo-pathogenic manifests itself very well on this case at 0 °C when the dose of 5

2.10 colonies/ml with a mortality rate of 73.6% over a total mortality was observed at the 7

higher rate of 4.10 colonies/ml. The latter reached a mortality rate of 84.8% to 5 °C. However at 10 °C, the effect is very low not exceeding the 20% mortality. It should be noted that the mortality rate increases with the doses used and temperatures employed. This can be explained by the release of toxins produced by P. syringae and freezing of the various functions of the body of the insect at low temperatures thus reducing cold tolerance by increasing their point of under cooling. Key words: Stored wheat, insect, Pseudomonas syringae, biological control.


Session 2 /

PC6 S2

Optimization of cultural conditions for Biosynthesis of Extracellular Protease by Bacillus subtilis Qureshi, A.Sattar and Dahot, M. Umar Institute of Biotechnology and Genetic Engineering, University of Sindh Jamshoro, Pakistan. Email: [email protected]

Abstract: Bacillus subtilis isolated from soil is capable to produce extracellular protease. The production of proteases by Bacillus subtilis was studied by varying the age of inoculum, incubation time and carbon sources with different concentrations (0.5 and 1.0%). In this study pure carbon sources, agricultural sugarcane bagasse, rice husk and date syrup and industrial waste molasses were also utilized as substrate. The maximum enzyme yield was achieved using 1.0 % molasses was used as substrate. The maximum production was found after 8 hours at 45°C and pH 8.5 by using 21 hours old culture. Key words: Bacillus subtilis, agricultural waste and protease


Session 2 /

PC7 S2

Application of acidogenic fixed-bed reactor prior to anaerobic membrane bioreactor for sustainable slaughterhouse wastewater treatment Ahlem Saddoud & Sami Sayadi* Centre of Biotechnology of Sfax, Laboratory of Bioprocesses: Road of Sidi Mansour Km 6, P. O. Box « K » 3038 Sfax-Tunisia Tel/Fax: +216 74 440 452; E-mail: [email protected]

Abstract: The high rate anaerobic treatment systems such as anaerobic membrane bioreactors (AMBR) are less popular for slaughterhouse wastewater due to the presence of high fat oil and suspended matters in the effluent. This affects the performance and efficiency of the treatment system. In this work, AMBR has been tried for slaughterhouse wastewater treatment. After the start up period, the reactor was operated with an average organic loading rate (OLR) of 4.37 kg TCOD m-3d-1 with gradual increase to an average of 13.27 kg TCOD m-3d-1. At stable conditions, the treatment efficiency was high with an average COD and BOD5 reduction of 93.7 % and 93.96 %, respectively. However, a reduction in the AMBR performance was shown with the increase of the OLR to 16.32 kg TCOD m-3d-1. The removal efficiencies of SCOD and BOD5 were drastically decreased to below 53.6 % and 73.3 %, respectively. The decrease of the AMBR performance was due to the accumulation of VFAs. Thus, a new integrated system composed of a FBR for the acidogenesis step followed by the AMBR for methanogenesis step was developed. At high ORL, the integrated system improved the performance of the anaerobic digestion and it successfully overcame the VFA accumulation problem in the AMBR. The anaerobic treatment let to a total removal of all tested pathogens. Thus, the microbiological quality of treated wastewater fits largely with WHO guidelines.


Session 2 /

PC8 S2

The soil nitrogen cycle is it disturbed by olive mill wastewaters spreading? Ali Mekki*, Abdelhafidh Dhouib and Sami Sayadi Laboratoire des bioprocédés, Pôle d’Excellence Régionale AUF (PER-LBP), Centre de Biotechnologie de Sfax, BP: « K », 3038 Sfax, Tunisie. Tel/Fax: +216 74 440 452; E-mail: [email protected]

Abstract : Extremely high organic load and the toxic nature of olive mill wastewaters (OMW) prevent their direct discharge into domestic wastewaters treatment systems. To solve the problems associated with these wastewaters, different disposal methods have been proposed. The most frequently used methods nowadays are the direct application to agricultural soils as organic fertilizers. The present work was aimed at evaluating the occurrence of crude and biological treated OMW amendment on the soil nitrogen cycle. The OMW nitrogen content remained negligible owing the other elements as the carbon and the potassium. Compared to the control, soil amended with crude OMW showed low levels of total and inorganic nitrogen (38 and 8 mg g-1 dry soil) and the organic nitrogen brought by the untreated OMW presents a -1 slow mineralization. Biological treated OMW had a low content of pollutants (COD = 4 g l ; -1 phenolic compounds = 0.6 g l ) and nitrogen brought by these residues was rapidly mineralized in the soil. This phenomenon was accompanied by an increase of nitrifiers number and urease and ammonium oxydases activities. Key words: olive mill wastewaters; soil; nitrogen; nitrifiers; mineralization.


Session 2 /

PC9 S2

Purification of a bacteriocin produced by Lactobacillus acidophilus isolated from naturally fermented milk. A. Doumandji* et A. Hellal**. * Université Saâd DAHLEB, faculté des sciences agro-vétérinaire, département agronomie, route de Soumaâ, BP 270 – 9000 Blida, Algérie. ** Ecole Nationale Polytechnique, laboratoire des Sciences et Techniques de l’Environnement, 16 rue des frères Ouadek, Hassen Badi 16.200 Alger, Algérie. E-mail: [email protected]. *Corresponding author: A. Doumandji, Tel: 00213 93 27 27 01; Fax 00213 25 43 38 64.

Abstract : Lactobacillus acidophilus strain was isolated starting from a naturally fermented dairy product from the area from Blida. This species produces a bacteriocin presenting an antimicrobic effect towards a great number of bacteria. This bacteriocin inhibits some positive Gram species such as Staphylococcus aureus, Campylobacter jejuni and Bacillus subtilis. But it does not represent any effect towards the negative Gram species. Two species of bifidobacteries are inhibited by this bacteriocin (Bifidobacterium infantis and Bifidobacterium breve), on the other hand it does not represent any activity opposite Bifidobacterium bifidum. A maximum production of bacteriocin is obtained on MRS during the stationary phase with pH 6.5. Pepsin, trypsin, the pronase and the proteinase K completely deteriorate the antagonistic activity of the bacteriocin. This substance remains stable during 30 min of 70°C. until 90°C. It is stable also during 15 min with 100°C.The latter decrease quickly at the time of a treatment of 30 min or 45 min with 100°C. and a treatment of 121°C. during 30 min. this activity is not affected by the variation of the pH (from 4 to 10). The stages of purification are as follows: - Passage of the supernatant gross active in exchanger chromatography of cations - Passage of the fraction activates proceeding in chromatography in reversed phase. The study of the antimicrobic activity of the active compound starting from a gel of SDS PAGE, reveals a molecular lowweight protein. Key words: Lactobacillus acidophilus, bacteriocine, purification, characterization.


Session 2 /

PC10 S2

Biocontrol ability and future marketing of Pseudomonas fluorescens inoculant of the in the area of Mascara (Northern West of Algeria) 2

Meliani A., and Bensoltane A. 1

Laboratoire de Recherches sur les Systèmes Biologiques et Geomatiques ( LRSBG), Centre Universitaire Mustapha Stambouli de Mascara . 2 Laboratoire de microbiologie, Département de Biologie, Faculté des Sciences , Université d’Oran.

Abstract: There is increasing public concern regarding the continued use of agrichemicals that are damaging our ecosystems. So, it is necessary to search for more environmental friendly methods to control plant disease that will contribute to the goal of sustainability development. Within this period, and particularly within the past five years, intense scientific research has given rise to several well-characterised Pseudomonas BCAs (biocontrol agents) that have now become model strains for understanding regulatory mechanisms in Gramnegative bacteria. The soil-borne fluorescent Pseudomonas has received particular attention because of their aptitude to reduce the incidence of the root soil born phytopathogenes, Pseudomonas fluorescens that colonize rhizosphere and produce a wide range of active metabolites to prevent plant diseases present a realistic alternative to chemical pesticides. The aim of this work was to test the biocontrol ability of fluorescent Pseudomonas isolates from the area of Mascara (Northern West of Algeria) and their exploitation. In vitro, screening tests revealed an antifungal and antibacterial activity estimated 15 % to 75 %. The various tests showed an effective action of P. fluorescens strains with a rate of 75% for F. oxysporum, Pythium spp., 60 % for Alternaria spp., and fongistase action estimated at 15% in the case of Aspergillus spp.. Although, this antagonistic action had no effect on Verticillium spp. and Penicillium sp.. Also, a positive action against Erwinia sp. and Xanthomonas sp was estimated at 45 %. These results are very promising for biological prospects by using P.fluorescens as a biocontrol agent. In addition, future marketing of Pseudomonas inoculant products as environmentally friendly alternatives to chemical fungicides will depend on the generation of biosafety data required for the registration of biocontrol agents with the biotechnology potential that contribute enormously to this goal. Key words: Sustainability Development, Model, Pseudomonas Fluorescens, Biocontrol Agent, Marketing, Biotechnology Potenti


Session 2 /

PC11 S2

The Impact of Thermotolerant Micro-organisms for Biofertilizer Preparation in Agricultural Production Ajuo, A., Tinto, F., Ayuk, SN., Amanfred, T. and Wumbi, SE. Institute of Food Science and Nutrition, P. O. Box 590 Buea, Cameroon.

Abstract: Compost application is a popular means of improving soil physical properties and supplying appropriate plant nutrients in Cameroon. This study evaluated the impact of inoculation with thermotolerant micro-organisms to shorten the maturity and improve biofertilizer quality prepared by composting. Isolated thermotolerant micro-organisms from compost were reinoculated for biofertilizer preparation. During composting, the physical, biological and chemical properties of the biofertilizer were assessed. The effects of biofertilizer application on the growth and yield of tomato were studied. Of 4512 colonies of thermotolerant micro-organisms, Streptomyces thermonitrificans NTU88, Streptococcus sp. NTU-130 and Aspergillus fumigatus NTU-132 showed high growth rates, proteolytic and cellulolytic activities. A mixture of swine manure and rice straw were inoculated with these isolates and composted for 2 months, substrate temperature increased and later decreased gradually. Substrate pH also increased from 7.2 to 8.7. Microbial inoculation enhanced the maturity rate, and increased the ash content and total and immobilized nitrogen, improved rate of alfalfa seed germination, and decreased the total organic carbon content and the carbon/nitrogen ratio. The biofertilizer application increased the growth and yield of tomato. The inoculation of thermotolerant and thermophilic micro-organisms to agricultural waste for biofertilizer preparation therefore enhances the maturity rate and improves the resulting biofertilizer quality. Appropriate micro-organisms inoculation in biofertilizer preparation could be usefully applied to improve agricultural production.


Session 2 /

PC12 S2

Denitrification water by a advanced technique using an membrane bioreactor Cheikh, A. Chaib, N. Abdi, H. Grib and N. Mameri Ecole Nationale Polytechnique, Laboratoire des Biotechnologie Environnementales et Génie des Procédés BIOGEP, 10 avenue Pasteur, BP 182, El Harrach, Alger 16200, E-mail : [email protected]

Abstract : In recent years, water pollution, especially that caused by nitrates, is on the agenda. It goes back to a massive use of fertilizers and additives salting especially in countries with high industrialization. Indeed, nitrates are undesirable in drinking water because they can cause methemoglobinemia especially among infants and pregnant women and have a carcinogenic effect. In addition, they participate in the phenomenon that affects eutrophication in lakes and rivers causing a deterioration in their aesthetics and organoleptic characteristics. As a result, the European Community has decided that the distributed water must contain less than 50 mg / l of nitrate and therefore the presence of nitrates in water requires a very thorough treatment. There are diverse solution to improve water quality; immediate action can be taken: the change to the levels of pumping, dilution with water whose nitrate is low or even changing the resource used. Given the time required for the implementation of these measures and the load already present in the soil, it is essential to consider the use of technology for denitrification of the water, they are grouped into two classes: - Physico-chemical processes, -Biological processes. The objective of our study is to present denitrification by electrochemical bioreactor, that the coupling of a electrodialisys technique to a biological process, while focusing on the parameters that can influence this match. We are interested in understanding the effects of the initial concentration of nitrates in the coupling system. The results are very promising because we have a very good coupling with denitrification and especially for the low levels of nitrates; the performance of denitrification is of the order of 90% at the exit of the column for an initial -2 concentration not exceeding 10 M.


Session 2 /

PC13 S2

Impact of water pollution of surfaces on the environment:study of the north-east region of algeria (oued seybouse) A.hammadi*,A.louhi*, N.hezil* andY.Aitammar *Laboratoire de traitement des eaux et valorisation des déchets industriels. Université Badji –Mokhtar Annaba.Algerie. E-mail : ha_atika@ yahoo.fr

Abstract: Algeria one of the large countries in the process of development, is characterized by its natural richness, among most important of these water resource, one finds the Seybouse river which occupies the third place in Algeria, by its flow, its richness animal in cash and vegetable, it is regarded as a vital source for the North-eastern area of Algeria, but it is confronted of large problems of pollution confirmed by the scientific studies and research.. The taking away was carried out for the dry and wet period in various intake points of the Seybouse River and its Meboudja affluent. The proportioning of the metals at summer carried out by atomic absorption spectrometry and the majority of the contents found are higher than the standards: 15.5mg/l, 0.27mg/l, 5.5mg/l, 5.5mg/l, and 1.75mg/l respectively for Fe, Cu, Zn, Pb and Ni. The development of a way of pretreatment by clarification by using sulphate alumina like coagulant and cation silica like flocculating agent for each taken sample showed a reduction of the contents of metals reaches up to 96% for Pb. The sludge recovered after treatment was mineralized by aqua regia, the results found (115ppm, 2000ppm, 300ppm, and 400 ppm for Cu, Zn, Pb, Ni successively) show the existence of a important metallic pollution of this Oued. Key words: Water, Seybouse River, Pollution, Metals, Clarification


Session 2 /

PC14 S2

Antibacterial activity of the essential oil of Cymbopogon schoenanthus L. from Tunisia a,c*

Ayda Khadri

b

b

c

, M. Luisa M. Serralheiro , J. Manuel. F. Nogueira , Mohamed Neffati , M. b

a

Eduarda. M. Araújo and Samira Smiti , (a) University of El-Manar II, Faculty of Sciences, Unity of Research of Vegetal Ecology, Campus Academia, 2092 Tunis, Tunisia, E-mail : [email protected] (b) Center of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Campo Grande Ed. C8, 1749-016 Lisbon, Portugal (c) Institute of the Arid Areas, Head of Range Ecology Laboratory, 4119 Medenine, Tunisia

Abstract: The genus Cymbopogon belongs to the grass family of poaceae and comprises 56 species. Among them, Cymbopogon schoenanthus L. Spreng is known for their aromatic and medicinal properties. In the South of Tunisia, this plant is used for the treatment of rheumatism, and to diminish fever. Besides its use in culinary, C. schoenanthus is also used in folk medicine. The chemical composition and antibacterial activity of the essential oils dried leaves collected from the southern Tunisia, were evaluated. Essential oils were analyzed by GC13

Mass Spectrometry and C-NMR. More than thirty constituents were identified; the major components were limonene (24.6 %), β-phellandrene (15.7 %), δ-terpinene (8.9 %) and αterpineol (9.6 %). Furthermore, the volatile oil of C. schoenanthus was tested against seven bacteria at different concentrations. Discs diffusion method is used for antibacterial testing. Results showed that the oils of C. schoenanthus exhibited a strong antibacterial activity against: Escherichia coli, Staphylococcus aureus, Staphylococcus saprophyticus, Klebsiella pneumonia, Proteus mirabilis, Pseudomonas aeruginosa and Salmonella paratyphi. 13

Key words: Cymbopogon schoenanthus, essential oils, activity.

C-NMR, GC-MS, antimicrobial


Session 2 /

PC15 S2

Etude de l’évolution Lactobacillus bulgaricus et Streptococcus thermophilus au cours de la conservation du yaourt Bachir raho ghalem Département de biologie faculté des sciences Université Djilalli liabès de Sidi bel abbès, Tél : 0021370600017 E-mail : [email protected]

Résumé : Le yaourt, produit de la fermentation du lait. Le yaourt, si courant aujourd’hui dans notre alimentation, est connu et apprécié depuis fort longtemps : les premières références à ce produit datent de 3200 ans avant J.C. dans des écrits découverts en Irak sur des tablettes d’argile. Il est issu de la fermentation du lait par deux bactéries dites « lactiques » : Lactobacillus bulgaricus qui apporte au yaourt son acidité et Streptococcus thermophilus qui développe les arômes. Les ferments se développent au profit du lactose (glucide du lait) et ils provoquent ainsi la formation d'acide lactique qui fait lentement coaguler la caséine (protéine du lait). Les ferments lactiques apportent des effets bénéfiques sur la valeur nutritive et sensorielle du yaourt. Notre travail a pour objectif d’étudier la qualité microbiologique d’un ferment lactique et surtout l’évolution de ses deux espèces (Lactobacillus bulgaricus et Streptococcus thermophilus ) au cours de la conservation du yaourt. Les analyses bactériologiques du ferment lactique montrent l’absence totale des germes pathogènes conforment à la norme ce qui prouve la consommabilité du yaourt. Comme on a observé que la flore lactique acidifiante (Streptococcus thermophilus et Lactobacille bulgaricus), qui a servi à la fabrication est apportée majoritairement par les levains du yaourt, est largement majoritaire en début de la conservation ou on compte un nombre de 13.107 pour le premier jour et régresse à 96.103 pour la 22éme journée pour Lactobacillus bulgaricus alors que pour les Streptococcus thermophilus sont incomptable le premier jour mais régresse au 4.103 pour le 22éme jour. La différence entre le nombre de Streptococcus thermophilus et Lactobacillus bulgaricus dans le 1er jour de la conservation de yaourt paraisse acceptable puisque l’industrie de fabrication du yaourt la demande, et qui doit être fourni de cette manière. Les mots clés : Yaourt, ferment lactique, qualité microbiologique, Streptococcus thermophilus et Lactobacille bulgaricus, conservation.


Session 2 /

PC16 S2

Catalyzed reaction in microemulsions: the case of isomerases Bachra Khettal Enzymology laboratory, Department of Biological Physical-Chemistry, Faculty of Natural and live Sciences University A. Mira of Bejaia, Targa-Ouzemour, Bejaia 06 000, Algeria, *Email: [email protected]

Abstract: Water-in-oil microemulsions, or reverse micelles, are being evaluated as a reaction medium for a variety of enzymatic reactions. These systems have many potential biotechnological applications. This work illustrates the biotechnological applications of microemulsions as media for catalyzed reactions and focus on isomerases catalyzed processes. Key words: Microemulsions; Lipases; Reverse micelles; Isomerase; Biotechnological applications


Session 2 /

PC17 S2

Effect of marine spray and air pollution on the radial growth of Pinus halepensis Mill. In the Rimel forest: Dendroecological approach Beya Bachtobji1, Ali Aloui2 , Olfa Shaiek1 and Med H. El Aouni1 1- Laboratoire d’Ecologie Végétal Faculté des Sciences de Bizerte 2- lnstitut sylvo-pastoral de Tabarka

Abstract: Ring-width analysis has been used to assess the impact of the marine spray and the air pollution on radial growth of Pinus halepensis Mill. located in the Rimel coastal forest land. Ten samples trees have been selected in each of three sites: station I protected and stations II and III exposed respectively to marine spray or air pollution. The trees were sampled, taking into account exposure to the prevailing winds and their distance from the pollution source. Three cores were extracted at breast height with an increment borer from each sample tree on the north, south-west and south-east direction. The statistical analysis carried out on the whole of sampling cores show an absence of orientation effect on the radial growth within the unpolluted station. A significant decrease of tree ring widths on the sides exposed to prevailing winds from the north-west charged in marine spray and atmospheric pollutants in the stations II and III. In this particular study, a negative exponential curve: Yt= ae-kt + b was fit successfully to all averaged ring-width series. The decreasing of tree ring width was estimated depending on increasing of tree age. The mean sensitivity coefficients established from synthesis chronology in a 57 yearsold chronosequence (1948-2004) showed a low sensitivity of pine radial growth to the high frequency climatic variation particularly in the unpolluted station (21.7%). However, an increase of this sensitivity was seen in the station II (30.2%) due to the effect of marine spray and in the station III (32.9%) due to air pollution. Key words: Pinus halepensis Mill. radial growth, air pollution, marine spray, Rimel forest.


Session 2 /

PC18 S2

Comparison between Saccharomyces cerevisiae and Candida diddensiaeBehaviours on Ethanol Production by Batch Fermentation of Tunisian Carob Pod 1

1

2

2

1

Hadrich, B. , Boudhrioua, N. , Ahansal, L. , Boussaid, A. and Kechaou, N. Groupe de Génie des Procédés Agro-alimentaires - Unité de Recherche en Mécanique des Fluides Appliquée et 2

Modélisation - Ecole Nationale d’Ingénieurs de Sfax, BP ‘W’ 3038, Sfax, Tunisie – Fax: 0021674275595 Unité de Génie des Bioprocédés - Département de Biologie, Faculté des Sciences et Techniques, Géliz, Marrakech Avenue Abdelkrim Khattabi, BP 549, Marrakech – Tel: 00212 44433818 E-mails: [email protected], [email protected], [email protected], [email protected],

Abstract: The ethanol is widely-used as fuel in many countries such as France, Brazil and USA. Because of its high sugar concentration (≈ 40-50%), the carob kibble could be used as a source of ethanol production by fermentation. The purpose of this work is to compare the behaviour of Saccharomyces cerevisiae (ref: CBS 1171) to that of the Candida diddensiae (ref: 98 in laboratory of “Unité de Génie des Bioprocédés”) in batch fermentation of Tunisian carob pod with the aim of producing ethanol. The product was purchased from a local market in Sfax (Tunisia). After removing the seeds, kibbles were chopped into small particles and pulverized. The fermentation of pod powder was performed in batch without sugar extraction. Two concentrations of sugar (25g/l and 50g/l) for each yeast were tested. One solution of malt extract agar was used for each yeast as fermentation control solution. The gas chromatography was used to measure the variation of sugar and ethanol contents versus time of operation. At initial time, 100g of dried carob kibbles contain 75g of total sugar. The sugar composition was 9% of fructose, 6% of glucose and 85% of sucrose. After 80 hours, only the control solution and the carob solution were fermented with Candida diddensiae and produced small ethanol quantities: 5.4 g/l and 2.6 g/l, respectively. It was obtained with consumption of all types of sugar: 31% of fructose, 45% of glucose and 42% of sucrose.


Session 2 /

PC19 S2

Biodegradation pattern of hydrocarbon-contaminated soils by an acclimatized consortium Boutheina Gargouri, Fathi Aloui and Sami Sayadi Laboratoire des Bioprocédés, Pôle d’Excellence Régional AUF-LBP, Centre de Biotechnologie de Sfax, BP «K» 3038 Sfax, Tunisia Email : [email protected]

Abstract: This study was performed for the treatment of a hydrocarbon contaminated soil by bioremediation. This soil was characterised by a high total petroleum hydrocarbon (TPH) content reaching 6%. In order to study the effect addition of bacterial consortium to the hydrocarboncontaminated soil on biodegradation kinetics, the mass of n-alkanes were assessed by gas chromatography mass spectroscopie (GC-MS). The field trial has clearly demonstrated enhanced bioremediation when bacterial consortium was added and also significant hydrocarbon losses. The bioaugmentation using the acclimatized consortium was shown to be highly effective in decontaminating the soil, achieving hydrocarbons removal rates of around 92%. The bioaugmentation resulted in enhanced degradation of short and middle-chain aliphatic compounds in the soil compared to the soil in the control plot. Key words: Bioremediation; Hydrocarbon; Soil.


Session 2 /

PC20 S2

Analysis of phosphatase and phytase activities in nodules of common bean (Phaseolus vulgaris) inoculated with rhizobia strain Ciat 899 under phosphorus deficiency 1,2,3

1

2

1,3

1,3

Mandri, B. ; Oufdou, K ; Drevon, JJ ; Bargaz, A .; Faghire, M. and Ghoulam, C.

3

1

Laboratory of Biology and Biotechnology of Microorganisms, Faculty of Sciences-Semlalia P.O. Box 2390, 2

3

Marrakesh; UMR Rhizosphère & Symbiose, INRA, Montpellier, France, Unit of Plant Biotechnology and Symbiosis Agrophysiology , Faculty of Sciences and Techniques, P.O. Box 549, Gueliz, Marrakesh, Morocco E-mail: [email protected]

Abstract: The Common bean-rizobia symbiosis is an effective tool to improve Common bean yield through the biological nitrogen fixation in the plant. This phenomenon is limited by the low phosphorus (P) availability of some Mediterranean soils. Phosphatase enzymes improve plant P acquisition but a little information is available about the metabolic role of phosphatases and phytases. The objective of this work is to analyze the activities of these enzymes in common bean nodules under two P levels, and to localize the phosphatase activity by in situ RT-PCR. The interaction between Common bean genotypes and rhizobia strain ciat 899 was studied under controlled conditions in Hydroaeroponical culture using two phosphorus levels, 250µmole/plant/week as control treatment and 75µmole/plant/week as phosphorus deficiency treatment. Nodule samples were ground in acetate buffer at pH 5, and enzyme activities were estimated using p-NPP and phytate as substrates. The nodules were fixed and cut into 50 µm sections, and primers of acid phosphatases were used with in situ reverse transcription and PCR amplification. Bean plants cultivated under P deficiency showed an increase of phytase and phosphatase activities in nodules. The phosphatase activity is higher than phytase activity. The acid phosphatases transcripts were localized mainly in cell layers surrounding the infected zone, particularly in the nodule inner cortex. The phosphatase and phytase activities increase in nodules could be a strategy for bean plants to tolerate P deficiency through P availability increase. Key words: Common bean, ciat 899, phosphorus deficiency, symbiotic interactions, phosphatase and phytase activities, RT-PCR


Session 2 /

PC21 S2

Chemical composition and antibacteriial activity of tymus algeriensis essential oil 1

2

C.Chelghoum and T. Dob 1 – Laboratoire de chromatographie, Faculté de Chimie, USTHB , BP 32 ELALIA BAB EZ ZOUAR , ALGER , ALGERIE , Email [email protected] 2- Laboratoire de molécules bio-ctives et de la valorisation de la biomasse , ENS , KOUBA , ALGER, ALGERIE

Abstract : Essential Oil of tymus algeriensis was isolated from fresh vegetative aerial parts of the plants by hydrodistillation using a Clevenger – type apparatus and analyzed by GC / MS and GC / FID. A total yeld of 0.98 g of essential oil per 100 g of plant weight was obtenaid and more 80 coumpounds were identified. This oil shown a high antibacterialial activity. Key words: tymus algeriensis, essential oil, GC/MS, GC/FID, aintibacterial activity Characterization and Expression of Silks Proteins from the Brazilian spider Nephilengys cruentata


Session 2 /

PC22 S2

Characterization and Expression of Silks Proteins from the Brazilian spider Nephilengys cruentata 1

2,3

3,5

3

4

Bittencourt, D. ; Souto, B.M. ; Oliveira, P.E.F. ; Verza, N.C. ; Lewis, R.V. and Rech, 3

E.L. 1-Laboratório de Morfogênese e Biologia Molecular, EMBRAPA ACRE, Brazil; 2-Departamento de Biologia Celular, Universidade de Brasília, Brazil; 3-Núcleo de Biotecnologia, EMBRAPA CENARGEN, Brazil; 4-Department of Molecular Biology, University of Wyoming, USA; 5-Departamento de Ciências Genômicas e Biotecnologia Molecular, Universidade Católica de Brasília, Brazil.

Abstract: Spiders are able to produce up to seven different kinds of silk, each one for a specific biological function. Spiders’ silks are also known for their unique mechanical properties. The possibility to produce new materials with similar properties led to an advance in the studies about the silks’ proteins (spidroins). Using expression sequence tags from major ampullate, minor ampullate, flagelliform and tubuliform silk glands from the Brazilian spiders Nephilengys cruentata and Parawixia bistriata, we were able to identify a number of silk related proteins, including three distinct cDNAs encoding proteins similar to major ampullate spidroin 1 and 2 (MaSp1 and MaSp2), and flagelliform spidroin (Flag) from Nephila clavipes, a common garden spider. The major ampullate fibers of orb weavers are known to have strength on the same order of magnitude as that of the high performance fiber Kevlar associated with a reasonable elasticity, in the other hand the silk produced by Flagelliform gland is very elastic and is responsible for the formation of the capture spiral in the orb-web. Using modular engineering approaches, we cloned repetitive modules of spider silk MaSp2 and Flag genes in tandem into two distinct pET19b expression vectors and obtained recombinant E. coli BL21(DE3)pLysS expressing the engineered PbMasp2 and NCFlag. We proceeded the purification using affinity chromatography columns and the purified proteins were polymerized and a recombinant spider silk was produced in vitro.


Session 2 /

PC23 S2

Qualitative and Quantitative detection of Sinorhizobium meliloti in soil by T-RFLP and real-time PCR 1

2

1

2

Darine Trabelsi , Marco Bazzicalupo , Ridha Mhamdi , Emanuele G. Biondi , Alessio 2

1

Mengoni and Mohammed E. Aouani 1

Laboratoire des Interactions Légumineuses-Microorganismes, Centre de Biotechnologie de Borj-Cedria, BP 901, Hammam-Lif 2050, Tunisia; E-mail: [email protected] 2

Department of Animal Biology and Genetics, University of Firenze, via Romana 17, I-50125 Firenze, Italy.

Abstract: Here we report the development of a specific T-RFLP ribosomal intergenic spacer marker for the analysis of the genetic polymorphism of Sinorhizobium meliloti populations independently from nodule isolation and cultivation. We present also a qPCR protocol based on the S. meliloti chromosomal gene rpoE1 for estimation of S. meliloti cell number in soil which could represent a valid advantage over the current labour-intensive and timeconsuming nodulation-based protocol. The first protocol is based on a highly selective twostep PCR amplification of the 16S-23S IGS of S. meliloti followed by a T-RFLP analysis. This marker was tested on DNA extracted directly from soil and results were compared with those of cultivated populations isolated from nodules. The number of TRFs detected on pooled DNA of 25 S. meliloti isolates from each soil was lower than that of total DNA from each soil sample (about 30-40%). These data indicate that a fraction of the population was actually not sampled by plant trapping. Forward primer (IGS-mel-57f, HEX) was the most informative and it allowed the recognition of a total number of 108 TRFs, while the reverse primer (IGS-mel-1392r, FEM) produced 68 TRFs. The Real-Time PCR tested on soil samples containing natural populations of S. meliloti in comparison with the current nodulation-based protocol (MPN). Results obtained with qPCR were higher than those found with the plant trapping method which is very plausible since non-nodulating S. meliloti cells could be also detected by the PCR method. The qPCR protocol developed could be used as a fast and complementary tool for the estimation of the number of S. meliloti cells in soil. The polymorphism analysis of S. meliloti in soil could help to shed some light on a still obscure side of the ecological problem: what is the relationship between nodule-trapped strains and free-living strains in soil?


Session 2 /

PC24 S2

Performances of membrane bioreactor in conditioning sea – products effluents Treatment Dhouha Cherif, Sana Chaari and Raja Ben Amar* Laboratory of Material Sciences and Environnent, Faculté des sciences de Sfax, Université de Sfax, Route de Soukra Km4, 3038 Sfax, Tunisia. *Corresponding author: Tel. 216 21 603 013, Fax: 216 74 274 437, E-mail address: [email protected]

Abstract: From an environmental point of view, Tunisia is considered as one of the leading developing countries in the field of water treatment and reuse regarding discharge into the environment and management of water losses. Membrane bioreactors have been developed in the last two decades for municipal and industrial wastewater treatment in order to produce high quality water, to reduce reactor sizes and to minimise sludge production. Such process combines a biological reactor with a separation technique using microfiltration or ultrafiltration membrane. This paper aims at evaluating the performances of the membrane bioreactor during the treatment of the conditioning sea – products effluents. An activated sludge reactor of an 8litre volume, operating with an acclimated biomass and a microfiltration process with a ceramic tubular membrane of 0.1 µm were used. It was found that the used membrane bioreactor achieved very good organic removal efficiencies based on COD load. Effect of sludge load (MLSS of 4 g/l and 8 g/l, in MBR conditions) have been investigated experimentally. The high load concentration was found to be the most efficient to degrade pollutants: 90% against 82%. The contribution of the membrane in the increase in performance was due to the total suspended solid retention resulting in a very low value of treated effluent turbidity (inferior to 2 NTU). The MBR was performed at a constant 2 2 permeate flux of 8 l/h.m for low sludge load and 4 l/h.m at high sludge load under a transmembrane pressure of 0.3 bar. These conditions caused a variation in transmembrane pressure ranging from 0 to 0.4 bar for a period of 30 days. Key words: Conditioning sea – products effluents, Membrane bioreactor (MBR); Activated sludge, ceramic microfiltration membrane.


Session 2 /

PC25 S2

Rhamnolipids produced by Pseudomonas aeruginosa grown on Molasses 1

Djaber Tazdait and R.Bakour

2

1

Départment of Biotechnoloy, University of Mostaganem, Algeria, E-mail: [email protected] 2

Laboratory of Molecular and cellular Biology, Faculty of Biology, U.S.T.H.B., Algeria,

Abstract: A rhamnolipid producing bacterium, Pseudomonas aeruginosa was previously isolated from Hassi Messaoud soil contaminated with crude oil and was able to produce two types of rhamnolipids (RL1 and RL4). Several carbon sources such as ethanol, glucose, vegetable oil and hydrocarbon have been used to produce rhamnolipid. In this study we are trying to use sugar cane molasses as a carbon and energy source to produce rhamnolipid. Biosurfactant production was quantified by Emulsifying Index (E24). Results showed that the growth of bacteria on M1 media containing glucose as carbon source gives an emulsification index of 56% after 96hours culture. Whereas, the growth of bacteria on optimized M3 medium containing 31g/l molasses, gives an emulsification index of 71% after 76hours culture. The kinetic of rhamnolipid production on fermenter showed that production reached its maximum rate (E24=71%) after 26hours culture. Key words: Rhamnolipid, Pseudomonas, cane molasses, fermenter.


Session 2 /

PC26 S2

Production of lysine by a auxotroph mutant of corynebacterium glutamicum in medium containing acetic acid Trad Khodja d*. and Hellal A.** *INATAA. Université MENTOURI, Route de Ain El Bey, Constantine. Algérie. **Ecole Nationale Polytechnique, Département du Génie de l’Environnement, Alger. Algeria.

Abstract: Food security remains the main goal of third world peoples whose deficit in food resources is both quantitatively and qualitatively. The amino acids produced by microbial can play an important rule to fill protein gap mainly by the Corynebacterium glutamicum, that are the best producer of amino acid. The production of amino acids depends on several factors, including the environment and the conditions for crops that must be favourable for either the growth and production. Our research work is to produce lysine on a medium-based and acetic acid using a auxotrophe strain of Corynebacterium glutamicum while controlling differents parameters, pH, turbidimetry, and the concentration of lysine during fermentation. The culture passed by many steps needed the pH regulation and temperature at the optimal values for the growth of the mutants and the lysine production. The addition of new medium during the fermentation allow to maintain maximum growth. After 74 hours of fermentation, we get a yield of lysine, in the order of 20.66 g/l. Key words: Corynebacterium glutamicum, auxotroph, fermentation, acetic acid, lysine, turbidimetry, culture parameters.


Session 2 /

PC27 S2

Synthetics and Naturels Humics Acids biodegradation by Actinomycets Strains Isolated from surface soil. Fodil D., Ferradji F. Z and Badis A. Chemical Departement .Engineer sciences Faculty. SAAD Dahlab University . 09000 Blida. Algeria. E-mail : [email protected]

Abstract : The use of biosorption and biodegradation methods for the total Humics acids (HA) elimination and by-products results of the intensive physico–chemical methods use for the water treatment destined to human consumption, constitute actually a first choice in the environment bioprocess field. This study is a contribution for the actinomycets strains isolation from the surface soil, these strains able to degrade synthetics and three HA extrated from differents soil. The isolation using the adaptation method on solid ISP9 medium with 0,5g/l of HA allowed to get 19 actinomycets strains which are retained according to their importance biodegradation halo. The required assimilated substrat like glucose is proving indispensable to accelerate the biodegradation and to make easier in such a way the better strains selection. A discoloration of the medium tested for three efficients strains was obtained just after some days of incubation. However, the only use of HA as sources for C and/or N needs a long incubation time until 4 weeks and differently under agitation. In addition, a comparaison between synthetics HA biodegradation and naturels HA extracted from soil, had performed. Spectrals tests by UV visible (254 – 350 nm) and HPLC showed the intermediates products formation from the biodegradation process and give an idea about the complexity of humics substances and the environment influence on the biodegradation mechanism. Key words: Humics Acids, Caracterisation , Biodegradabiliy , Actinomycets.


Session 2 /

PC28 S2

Heterologous expression, secretion and characterization of the Geobacillus thermoleovorans US105 type I pullulanase Zouari Ayadi Dorra, Ben Ali Mamdouh, Jemli Sonia, Ben Mabrouk Sameh, Ben Messaoud Ezzedine, and Bejar Samir Laboratory of Prokaryotic Enzymes and Metabolites of the Centre of Biotechnology of Sfax, P.O. Box 'K', 3038, Sfax-Tunisia E-mail: [email protected]

Abstract: Pullulanase type I (PUL US105) of Geobacillus thermoleovorans US105 strain was already cloned and expressed as an intra-cytoplasmic protein (Ben Messaoud et al., 2002). In this work we described the heterologous expression and secretion of the PUL US105 recombinant protein as well as the purification and characterization of this protein. The PUL US105 was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the Open Reading Frame was connected downstream of the α-amylase signal sequence of the Bacillus stearothermophilus US100 strain (Ben Ali et al., 2001). The monitoring of the pullulanase activity and western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. By another way, pul US105 was fused to the Lipase A signal peptide (Brockmeier et al., 2006) of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction (80 %). The PUL US105 was purified to homogeneity from the periplasmic fraction, using a heat treatment, size exclusion and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2. References: * Dorra Zouari Ayadi, Mamdouh Ben Ali, Sonia Jemli, Sameh Ben Mabrouk, Monia Mezghani, Ezzedine Ben Messaoud and Samir Bejar (2007). Sous presse dans Applied Microbiology and Biotechnology. * Ben messaoud E, Ben Ammar Y, Mellouli L and Bejar S (2002). Enzyme and Microbial Technology 31: 827-832. * Ben Ali M, Mhiri S, Mezghani M and Bejar S (2001). Enzyme and Microbial Technology 28: 537542. * Brockmeier U, Wendorff M, Eggert T (2006). Current Microbiol 52: 143-148.


Session 2 /

PC29 S2

Investigation of extracts from the aerial parts of Peganum harmala as antibacterial antiviral and radical scavengers 1*

2

4

3

3

Edziri Hayet , Mastouri Maha , Ammar Samia , Matieu Mata , Patrich Gros , Mahjoub Mohamed Ali4, Ali Si Mohamed3, Laurent Gutmann3, Zine Mighri4, Aouni Mahjoub 1

1-Laboratoire des substances biologiquement actives et des maladies transmissibles Faculté de Pharmacie5000-Monastir-Tunisie 2-Laboratoire de Microbiologie C H U Fattouma BOURGUIBA -5000- Monastir-Tunisie 3-Laboratoire de Microbiologie et de virologie, Hôpital Européen George Pompidou, APHP, 20, Rue Leblanc 75905 Paris cedex 15 4-Laboratoire de chimie des substances naturelles et de synthèse organique 99/UR/12-26 Faculté des sciences de Monastir, 5000 Monastir, Tunisie Corresponding author : E-mail: [email protected]

Abstract: The aim of this study was designed to examine the in vitro antibacterial, antiviral and antioxidant activities of extracts issued from Peganumharmala growing in Tunisia. The antibacterial activity was evaluated by determination of MIC using the broth microdilution methods. The antiviral activity was determined against Human cytomegalovirus (HCMV) strain AD-169 (ATCC Ref. VR 538) and coxsakie virus type B3 by using Human diploid embryonic lung fibroblasts (MRC-5). The antioxidant activity was tested using the DPPH assay method. Amoug tested extracts; the chloroformic extract displayed a higher antibacterial activity against Gram positive bacteria than Gram negative bacteria. The methanolic extract showed the best antioxidant and antiviral activities with IC50 of 600.3 and 10µg/ml, respectively.


Session 2 /

PC30 S2

Transaldolase: purification and characterization of the enzyme involved in xylose fermentation to ethanol by the fungus Fusarium oxysporum Elisavet Kourtoglou, Diomi Mamma, Evangelos Topakas & Paul Christakopoulos* Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 157 80, Zografou, Attica, GREECE *

Correspondence to: Paul Christakopoulos, E-mail: [email protected]

Abstract: Ethanol produced from lignocellulose is an environment-friendly alternative to fossil fuels. Microbial species such as Neurospora crassa and Fusarium oxysporum have been reported to ferment cellulose directly to ethanol. If F. oxysporum is used as the fermentation organism, it is not necessary to perform a separate enzymic hydrolysis of the lignocellulosic material, as this microorganism produces the necessary enzymes. Since a substantial fraction of lignocellulose material may consist of pentoses, it is necessary to efficiently ferment the pentoses to ethanol to make the process cost-effective. During xylose fermentation an accumulation of sedoheptulose-7-P was observed, which might indicate a limitation in the transaldolase reaction or a competition of glyceraldehyde-3-phosphate between pentose phosphate pathway and glycolysis. This work describes the purification and characterization of the main aldolase activity from the fungus F. oxysporum involved in xylose metabolism, in order to be homologous overexpressed to improve this strain for ethanol production by hemicellulosic substrates. Transaldolase (FoTal) revealed a monomeric structure with molecular mass of 36 kDa. Transaldolase depicted an optimal pH of 7.5 using imidazole buffer, while loss of o activity was observed with Tris buffer. The optimal temperature was between 40 and 45 C and o transaldolase became unstable at temperatures above 50 C. The pI of the enzyme was 4.5. The kinetics of FoTal is consistent with a Ping Pong mechanism. The Km values for erythrose-4phosphate and fructose-6-phosphate were found 0.49 and 6.66 mM respectively, while the kcat -1 value was estimated at 4150 min .


Session 2 /

PC31 S2

Treatment of wastewater by electrocoagulation using pacticales electrodes. Farouk Bouhezila, Amel Bakalem, Hakim Lounici and Nabil Mameri. Laboratoire des Biotechnologies Environnementales et Génie des Procédés, Département de Génie de l’Environnement, Ecole Nationale Polytechnique 10 Avenue Hassen Badi B.P. 182- 16200 El Harrach Alger, Algerie, E-mail: [email protected]

Abstract : One of the major challenges facing mankind today is to provide clean water to a vast majority of the population around the world. The reuse of wastewater has become an absolute necessity. There is, therefore, an urgent need to develop innovative, more effective and inexpensive techniques for treatment of wastewater. Electrocoagulation is an electrochemical method of treating polluted water whereby sacrificial anodes dissolve due to a potential difference, producing active coagulant precursors (usually aluminum or iron ions). In the present study the effective performance of electrocoagulation technique in the treatment of synthetic colored wastewater has been investigated using particles electrodes. Several working parameters, such as aeration, rotating speed, and pH were studied in an attempt to achieve a higher removal capacity. The process produces a removal capacity of 95% of blue color just after 5min. the method was found to be highly efficient and relatively fast. Key words: Electrocoagulation, particles electrodes, wastewater treatment, electrochemical reactor.


Session 2 /

PC32 S2

Influence of enzymatic hydrolysis on the extraction of the R-phycoerythrin from the red algae Gracilaria verrucosa F. Mensi*, J. Ksouri, J. Fleurence and Y. Jobert *INSTM, centre Kheireddine, 29 Rue General Kheireddine 2015 le Kram – Tunis, E-mail: [email protected]

Abstract: Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe and analytical reagent. This protein possesses colouring properties and biologic activities susceptible to drive to new outlets in the agro-food domains. In red algae membrane contain polysaccharides, like alginates, carraghenanes, agars and cellulose. To facilitate the extraction of R-phycoerythrin from these algae, B-glucanaseses and cellulases were used. However, coupled action between the two types of enzymes has been tested. The action of two B-glucanases, Ultraflows (U) and Finizim (F) with a cellulase, celluclaste (C) on Gracilaria verrucosa has been tested. The concentrations used, expressed in µl, are the following: 50F/50C, 100F/100C, 150F/150C; 50U/50C, 100U/100C, 150U/150C. The results show that for Gracilaria verrucosa from Bizerte lagoon, the association 100F/100C gives the best output, in proteins and in R-phycoerythrine.


Session 2 /

PC33 S2

Treatment of landfill leachates by Aerobic Membrane Biological Reactor Fathi Aloui, Slim Loukil and Sami Sayadi Laboratoire des Bioprocédés, Pôle d’Excellence Régional AUF (PER- LBP), Centre de Biotechnologie de Sfax BP: «K» 3038 Sfax Tunisie, E-mail : [email protected]

Abstract: Landfill leachate (LFL) is a very complex wastewater and it posses potential hazard to the local communities and to the environment. This study was undertaken to investigate the performance of aerobic membrane bioreactor treating raw LFL from Djebel Chekir (Tunisia) discharge. LFL samples collected from this site were found to be highly loaded with organic matter, ammonia, salts, greases, phenols and hydrocarbons. A consortium acclimatized at the laboratory was used in biological LFL treatment carried out in a stir tank reactor (STR), coupled with a membrane. Important removals of chemical oxygen demand (COD) and + NH4 N were attained after 44 days of treatment at optimum conditions for the membrane and with organic loading rates (OLR) of 1.9 and 2.7 grams COD per litter and day. This treatment also allowed an important detoxification of the landfill leachates and an elimination of the microorganisms. Key words: Landfill leachates, Membrane Biological Reactor, Detoxification, Acclimatizing consortium


Session 2 /

PC34 S2

Effets des eaux usées sur quelques paramètres physiologiques et biochimiques du blé dur (Triticum durum Desf.) dans la région de ouargla Fatiha Bekhouche Laboratoire de Biologie Végétale et Environnement, Département de Biologie, Faculté des sciences, Université Badji Mokhtar BP 12, Annaba 23000, Algérie. Tel : 00213 74 30 66 65, E-mail : [email protected]

Abstract: Un essai sur trois variétés de blé dur (Triticum durum Desf.) à savoir Waha, Vitron et Gtax a été conduit afin de vérifier les effets des eaux usées et ce, sur quelques paramètres physiologiques et biochimiques. Pour cela un un semis a été réalisé pendant la campagne 2 2003-2004, sur une parcelle élémentaire de 240 m au niveau d’une station de refoulement des eaux usées (non opérationnelle) qui se situe vers le Nord à l’extérieur de la ville de Ouargla. Et qui transfère toutes les eaux usées vers un point de rejet qui est la Sebkha d’Oum Errane (zone d’éxutoire) sans aucun traitement préalable. Deux traitements ont été choisis, irrigation par les eaux usées, comparés à un témoin.. Un premier prélèvement a été effectué au stade tallage. .Les tests ont portés sur les teneurs en chlorophylles au niveau des feuilles et les protéines totales au niveau des racines et des feuilles. Les résultats obtenus ont montré, sur le plan énergétique les chlorophylles totales sont les plus élevées pour les plantes irriguées par les eaux usées, démontrant ainsi l’aptitude des cultivars à réagir favorablement sous les conditions du traitement aux eaux usées. les trois variétés de blé dur affichent une teneur en protéines totales également plus grande sous irrigation avec les eaux usées à ce stade physiologique des plantes. .


Session 2 /

PC35 S2

Physiological and molecular studies on strains from the qatar university culture collection of cyanobacteria Al-Emadi, M.*, Al-Saadi. F.* and Al-Thani, R.F. Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, POB 2713, Doha, Qatar *These authors contributed equally to this work.

Abstract: Cyanobacterium strain QU002 of the LPP group (Lyngbya-Plectonema-Phormidium) was obtained from the Qatar University Culture Collection of Cyanobacteria. Prolonged cultivation of the strain identified two associated heterotrophic, microaerophilic Grampositive eubacteria, with white and pink pigmentation, respectively. The cyanobacterium, and both eubacteria, were brought into axenic culture. The LPP-cyanobacterium produces two types of motile filaments, the smaller of which bores actively through 1.5% w/v agar. The eubacteria have extreme growth optima (>pH 11). Small-scale alkaline lysis and boiling techniques identified plasmids in these microorganisms. Purification and sequence analysis of the plasmid and chromosomal DNAs is in progress, in collaboration with the Virginia Tech Center for Genomics (USA). In mixed culture, on solid media, the eubacteria associate exclusively with filaments of the cyanobacterium. Detailed microscopic examination of strain QU002 at the Environmental Sciences Center of Qatar University identified an unusual distribution of extracellular polysaccharide along the apices of the larger class of filaments. The physiological and biochemical basis for the association between these microorganisms, including the potential for lateral gene transfer, is under investigation. Acknowledgements. Supported by a Qatar University award to Professor Malcolm Potts and Dr Roda Al-Thani. The authors are grateful to Hareb Al-Jabri for advice and technical support.


Session 2 /

PC36 S2

Heterologous expression of Penicillium occitanis cellobiohydrolase I gene in Escherichia coli Bhiri, F., Chaari, F., Gargouri, A. And Ellouz Chaabouni, S. Unité “Enzymes et Bioconversion”, Ecole Nationale d’Ingénieurs de Sfax, Route de Soukra, Km 3.5. BP W. 3038. SFAX- TUNISIA E-mail address: [email protected]

Abstract: The Pol6 mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases production with high cellulose degradation efficiency. The cellobiohydrolase I (CBHI) is the most abundant cellulolytic enzyme produced by this micro-organism, accounting for 40 to 60% of its extracellular proteins. We report in this work, the expression of the cbh1 gene in a heterologous host, Escherichia coli. For this purpose, the cellobiohydrolase I cDNA has been subcloned into the expression system pGEX-6P-1, in frame with a sequence encoding an N-terminal gluthatione Stransferase and under the control of tac promoter. The heterologous fusion protein was successfully produced but aggregated intracellularly in an insoluble and inactive form. Optimisation of the culture conditions (choice of medium, host cells, temperature, inducer concentration ….) need to be done in order to enhance the solubility of the expressed protein.


Session 2 /

PC37 S2

Purification and characterization of endo-β-1,3-1,4-D-glucanase activity from a new isolated Bacillus CF1 Chaâri, F. and Ellouz Chaabouni, S. Unité Enzymes et Bioconversion, Ecole Nationale d’Ingénieurs de Sfax, BP : W.3038 Sfax Tunisia , E-mail : [email protected]

Abstract: A new isolated thermophilic Bacillus CF1 produces constitutively at least four extracellular β1-3,1-4 glucanases upon induction with glucose. One of these extracellular enzymes EG1 was purified to homogeneity by concentration and ion-exchange chromatography procedures. The molecular weight of EG1 was estimated to be 30 kDa as determined by SDS-PAGE. EG1 was optimally active at 70°C and pH 5.0. The enzyme showed a high pH stability within the range of pH 4.0-10.0, and thermostability up to 60°C for 30 min, and it lost 20% of its activity at 50°C after1h of incubation. Furthermore, EG1 was highly active against barley β-glucan and lichenan respectively 18.75 U/mg and 15.62 U/mg, and it was not active against cellulose (CMC) and laminarin. The purified enzyme had a Km of 2.1 mg/ml and Vmax 21.25 µmoles/min/mg when lichenan was used as substrate. 2+ 2+ 2+ The enzyme activity was strongly inhibited by the divalent cations Hg , Zn and Fe . The N-terminal sequence of EG1 showed no significant homology to others known βglucanases.


Session 2 /

PC38 S2

Identification of Listeria species isolated in Tunisia by Microarray based assay: results of a preliminary study. 1

1

2

3

1

2

Hmaïed F. , Helel S ., Leberre V. , Kechrid A. , Barkallah I. and François J.M. Unité de Microbiologie, Centre National des Sciences et Technologies Nucléaires, Technopôle de Sidi Thabet, 1

2020 Sidi Thabet, Tunisia Plateforme Biopuces du Genopole, INSA Toulouse, Avenue de Rangueil 135, F-31077 Toulouse cedex 04, 2

France 3

Laboratoire de Bactériologie de l’hôpital d’enfants de Tunis

Abstract: Microarray-based assay is a new molecular approach for genetic screening and identification of microorganisms. We have developed a rapid microarray-based assay for the reliable detection and discrimination of Listeria spp. in food and clinical isolates from Tunisia. The method used in the present study is based on the PCR amplification of a virulence factor gene (iap gene). The PCR mixture contained cyanine Cy5-labeled dCTP. Therefore, the PCR products were fluorescently labeled. The presence of multiple species-specific sequences within the iap gene enabled us to design different oligoprobes per species. The species-specific sequences of the iap gene used in this study were obtained from GenBank and then aligned for phylogenetic analysis in order to identify and retrieve the sequences of homologues of the amplified iap gene analyzed. 20 probes were used for detection and identification of 22 food isolates and clinical isolates of Listeria spp (L. monocytogenes, L. ivanovi), L. innocua, L. welshimeri, L. seeligeri, and L. grayi). Each bacterial gene was identified by hybridization to oligoprobes specific for each Listeria species and immobilized on a glass surface. The microarray analysis showed that 5 clinical isolates and 2 food isolates were identified listeria monocytogenes. Concerning the remaining 15 food isolates; 13were identified listeria innocua and 2 isolates could not be identified by microarray based assay. Further phylogenetic and molecular analysis are required to design more species-specific probes for the identification of Listeria spp. Microarray-based assay is a simple and rapid method used for Listeria species discrimination.


Session 2 /

PC39 S2

Agronomic quality of composted organic wastes used as substrates for tomato seedling and culture: Effects on plant growth and soils fertility 1

2

2

3

Fatma Sellami , Hafedh Rigane , Imen Ben Mahmoud , Khaled Medhioub , 1 Emna Ammar 1

National Engineering School in Sfax, P.O. "W", 3038 Sfax - Tunisia, E-mail: [email protected] 2

Faculty of Sciences in Sfax, P.O. Box "802", 3018 Sfax - Tunisia, 3

Institute of Preparatory Engineering Studies; P.O. Box 805, 3018 Sfax - Tunisia UR: Etude et Gestion des Environnements Urbains et Côtiers, LARSEN

Abstract: A set of composted materials from industrial agro-alimentary wastes (sesame bark, animals’ waste, exhausted olive mill cake), trimmings yard waste and decomposed algae, was evaluated as substrate component for tomato seedling in nurseries and culture in different farms located in the South and the North in Tunisia (Sfax and Nabeul agricultural areas). The experiments in different tomato nurseries were conducted during a one-month period with a hundred of growing media tested. These included composted material mixed with different proportions of fresh coco nuts fibers as well as white and black peat. These new mixed substrates didn’t exhibit any phytotoxic problem for the plant growth and its performance. At the end of the nursery seedling growth period, the rates of emergence in the experimented media were measured. The biometric parameters such as seedling height, stem diameter, number of leaves and leaf area were determined during growth period. Plant growth on commercial products (peat-based substrate), used as a control, were compared to those on compost-based substrates. The seedling height tested with composts showed better stem elongation compared to that of the control products. The impact of the substratum used on root was investigated and confirmed the beneficial effect of composted materials use on tomato seedlings. The composted materials quality was confirmed on second step on tomato culture trials in different farms located in Sfax and Nabeul. The pedological study revealed positive effects of composts on the soil which was enriched in minerals and humic fractions. Furthermore, soil microstructure study exhibited an important aggregation and structural stability improvement. The different compost-amended parcels insured good stem elongation, compared to that of the control (amended with manure). These results predict that the soils amended with composts could present a higher productivity than those amended with manure. Indeed, at the biological end-cycle, the tomato -1

culture trials showed a yield on farm manure of 70 tons ha while on the other experimented -1

parcels it reached 85 tons ha .


Session 2 /

PC40 S2

Chemical fractionation and plant uptake of heavy metals in soil amended with urban compost (1)

(1)

Ayari Fethia *, Jedidi Naceur and Kosai Ridha

(2)

(1) Centre des Recherches et de Technologies des Eaux , Technopole Borj Cedria, Route touristique Borj cedria Soliman, B.P. 273, Soliman 8020 TUNISIE , E-mail : [email protected] (2) Université de Tunis- IPEIT. Laboratoire de Chimie de la Matière Condensée 2, rue Jawaher Lel Nehru, Monfleury-Tunis-Tunisie.

Abstract: Heavy metals are associated with different soil components in different ways, and these associations determine their mobility and availability. Water soluble and exchangeable forms are considered readily mobile and available to plants, while metals incorporated into the crystalline lattices of clays appears relatively inactive. The other forms precipitated as carbonate, occurred in Fe, Mn and Al oxides or complexed with organic matter-could be considered relatively active or firmly bound, depending upon the actual combination of physical properties of soil. Thus, soil texture (clay content) pH, organic matter, and Fe-Mn oxides have been found to be the most important soil properties influencing the lability and biological uptake of heavy metals. Sequential chemical extraction schemes are considered to be a great value than single extractant in the determining metal distribution in compost amended soil. Reagents utilized were chosen on the basis of their selectivity and specificity towards particular physicochemical form, although variations in reagent strength, volume and extraction time between the schemes are apparent. The major aim of extracting trace metals with various reagents from composts is to estimate the relative distribution and consequently identify which form(s) has high potential for plant uptake (bioavailability). Trace metal in the soluble, exchangeable and organically bound forms are believed to be highly available for plant uptake. Key words: Speciation, heavy metal, bioavailability, compost-amended soils.


Session 2 /

PC41 S2

Scale up for membrane bioreactor technology on treatment of industrial anionic surfactants rich-wastewaters Firas Feki, Abdelhafidh Dhouib, Sami Sayadi Centre of Biotechnology of Sfax, Laboratory of Bioprocess, Road of Sidi Mansour Km 6, P. O. Box « K » 3038 Sfax-Tunisia

Abstract: Every year Tunisian detergent and cosmetic industries produce more than 100 000 −1

metric tons of wastewater containing more than 1 g l −1

of surfactant, while the Tunisian

standards of wastewater allow only 5 mg l . Among these industries JASMINAL wich is part 3

from HENKEL multinational company situated in Sfax-Tunisia, produce daily 5 m of anionic surfactants rich-wastewaters. The objective of this work was to develop an effective membrane bioreactor process (MBR) for this industrial wastewaters treatment. In the laboratory level, the effluent treatment was conducted in a 10 litter stirred tank reactor 2

integrated with a cross-flow microfiltration membrane unit 0.04 m . The reactor was inoculated with an adapted consortium possessing high catabolic ability for degrading −1

−1

surfactant. The COD loading rates was increased from 0.5 to 4.2 g l d The MBR-pilot showed excellent pollutant removal efficiency with 99.3% of anionic surfactant and 92.9% of the influent COD at a HRT of 2.8 days. This pilot was scaled up to industrial station installed 3

2

in the manufacturer with reactor volume of 15 m coupled to a 15 m membrane surface. -1

This system worked under moderate filtration conditions of 2.3 m s of circulating -1

-2

fluid velocity and 1.5 bar of TMP, which allowed an average permeate flux of 14 l h m . 3

Currently the station treats 3 m /d and HRT is 5 days. An elimination of more than 99.6 % of the active anionic and 95% of the carbon pollution is observed. The rejection is achieved with a fully clarified elimination of microorganism and 100% of the suspended matter. The final treated wastewater respects the public discharge influents and could be used for washing equipment or soil, it could even be used for irrigation of green space in the plant, allowing us to restrict and save consumption drinking water.


Session 2 /

PC42 S2

Isolation and characterization phenotypic of rhizobia isolated from Genista saharae nodule *

Ghadbane Mouloud and Daoud Harzallah. * Laboratoire de microbiologe, université de Setif, Algeria

Abstract : Thirty rhizobia strains isolated from root nodules of Genista saharae and growing in a region of south Algeria (indigenous of Sahara) corresponding to the arid climatic zone were compared with 2 representatives of the recognized rhizobial species. G. saharae formed nodules with diverse rhizobia in Algeria soils. The presented results, suggest the relationship of G. saharae to Bradyrhizobium species. All microsymbionts were slow-growing Bradyrhizobium with generation time of 1012 h, acid tolerant, salt sensitive, and antibiotic resistant. Nodulation capabilities of rhizobia are diverse. We evidenced a high degree of the host specificity for G. saharae microsymbionts. Key words: Genista saharae, Nodulation, Algeria, Bradyrhizobium, Arid.


Session 2 /

PC43 S2

Chiken droppings recycling and use by earth worm composting Ouahrani G*., Gheribi-Aoulmi Z**. ElGaci M.* and Merdjeraoui M*. *Lab. Ecologie Fac. des Sci. de la Nat. Et de la Vie. Univ. Mentouri. Constantine. DZ. ** Lab. Maths. Appli. et Modélisation. Fac. des Sci.. Univ. Mentouri. Constantine DZ.

Abstract : Our study is a contribution to the recycling and use of chicken droppings by wormcomposting, the species used is Eisenia fetida. The biophysicochemical Analyses (earthworms’ number and biomass, pH, T ° C, C%; N%) as well as the statistical analysis (analysis of the variance ANOVA), reveal that some parameters are favourable both during the course of the worm composting and for earthworms growth. Thus earthworms accelerate the transformation of organic residues by contributing to the degradation of organic matter (C / N = 13). The development of earthworms (number and biomass) during the test show that the mixture of chicken droppings added to the sawdust is a high quality substrate for optimal development of earthworms. In addition, the results given by the growth test on tomato seeds (Riogradi M.) show that the wormcomposting allowed to obtain a high quality compost with an agronomical value. Thus, the wormcomposting would be an interesting alternative in the management and exploitation of droppings. Key words: Biotechnology, wormcomposting, Eisenia fetida, droppings, management, contribution, organic waste, experiment plan, ANOVA


Session 2 /

PC44 S2

Valorisation of mycelium biomass in waste water contaminated by metribuzin Behloul, M., Lounici, H., Abdi, N., and Mameri, N. Ecole Nationale Polytechnique ENP- 10 avenue Hacen Badi – El Harrach – ALGER E-mail : [email protected]

Abstract: Water consumed in agricultural or industrial fields is rejected at 80% on average as effluents charged in noxious substances for environment, and for all human beings. Among those pollutants, pesticides are highlighted because of the threat they represent. In order to face the risks that pollutants could represent, the strategy of the current scientific research moves towards the development of more elaborated and more adapted processes for the depollution of the highly concentrated effluents. In this context, we used a biomaterial: Pleurotus mutilus which is a fungus from industrial fermentation, mainly made up of mycelium cultivated for antibiotics production. The aim of this work was the valorisation of the biomass pleurotus mutilus in the biosorption of Metribuzin. This work is composed of two principal parts, in the first one; we were interested in the physical pre-treatment and the structural characterization of biomass. Second, we have studied the different parameters likely to have an influence on the biosorption capacity of Metribuzin such as biomass granulometry and Metribuzin concentration. The results of adsorption experiments obtained for synthetic water are convincing, we could reach an adsorption rate of Metribuzin of approximately 53%.


Session 2 /

PC45 S2

Use of Trametes trogii Laccases produced on olive mill wastewaters for industrial textile dyes decolourization Chakroun, H., Dhouib, A., Mechichi, T. and Sayadi, S. Centre de Biotechnologie de Sfax, Laboratoire des Bioprocédés, Pôle d’Excellence Régionale AUF, (PER-LBP) Route Sidi Mansour Km 6, BP: «K», 3038 Sfax, Tunisie.

Abstract: Olive-mill wastewaters (OMW) as a serious environmental polluting effluent and rich in phenolic compounds could be used as carbon source and inducer for laccases production by the white-rot fungus Trametes trogii DSM 17786. Sterilised OMW was supplemented by nitrogen sources and used for Trametes trogii growth medium and extracellular laccases production. Following optimisation of different nitrogen sources and percentage of OMW -1

dilutions, maximum of laccases production of 23 000 U l was obtained on OMW/water 80/20 (v/v) supplemented with 2 g/l of urea. Glucose supplementation to the growth medium didn’t increase laccases yield. The partially purified laccases obtained from the OMW cultures was able to totally decolourize textile industrial dyes as blue tubantin GLL 300 and black tubantin VSF 600. However, other dyes as blue solophenyl, yellow solophenyl and red BLL were partially or not decolourized.


Session 2 /

PC46 S2

Improvement of biogas yield by anaerobic co-digestion of solid, semi-solid and liquid organic wastes Bouallagui. H*, Lahdheb. H, Rachdi. B, Ben Romdan E and Hamdi. M Laboratory of Microbial Ecology and Technology, National Institute of Applied Sciences and Technology, Tunisia * Corresponding author. E-mail : [email protected] Tel: +216 22 524 406. Fax: +216 71 704329

Abstract: Analysis of some organic wastes indicated that they were deficient or access in certain nutrients such as carbon, nitrogen and phosphorus. The anaerobic co-digestion of different materials for achieving good carbon to nutrient balance for micro-organisms has been a well accepted approach of operating anaerobic digesters. Co-digestion of organic wastes is a technology that is increasingly being applied for simultaneous treatment of several solid, semi solid and liquid organic wastes. The main advantages of this technology are improved methane yield because of the supply of additional nutrients from co-digestates and more efficient use of equipment and cost-sharing by processing multiple waste streams in a single facility. The aim of this work was to present the research conducted in the laboratory scale on anaerobic co-digestion to identify applications of co-digestion of solid, semi-solid and liquid organic wastes. The organic fraction of municipal solid waste, municipal wastewater sludge and slaughterhouse wastewater are the main wastes used in these co-digestion processes. Biogas production, methane content, total organic matter, pH, volatile fatty acids (VFAs) and alkalinity were monitored to find the better conditions for the enhanced performance of co-digestion. The mesophilic and thermophilic anaerobic digestions of fruit and vegetabe waste (FVW) and slaughterhouse wastewater (SW) co-substrates were investigated. The mixture of wastes was prepared with a FVW content of 25% by volume. Under mesophilic condition, the results for co-digestion of FVW-SW mixture are better than those obtained -1

-1

from digestion of AW and FVW on their own. The gas production rates are 0.6 l d , l.5 l d and 2.6 l -1

d for SW, FVW and FVW-SW, respectively. Under thermophilic condition, the co-digestion process showed a decrease of biogas production rate due to the high amount of free ammonia in the digester. The effect of fish waste (FW) and waste activated sludge (WAS) addition as co-substrates on FVW anaerobic digestion performance was also investigated in mesophilic condition. It was observed that WAS addition with a ratio of 10% VS led to the enhanced biogas yield by 43.8% and total volatile solid removal by 11.7%. However FW addition led to improve the process stability proved by low VFAs/Alkalinity ratio of 0.28 and slightly improves the gas production yield (8.1%) compared to anaerobic digestion of FVW alone. The co-digestion of primary sludge and activated sludge was investigated. Results indicated that the ratio of primary sludge to waste activated sludge and the organic loading rate (OLR) are the main variables determining the methane production efficiency. The best results were obtained for a -3

-1

ratio (PS/WAS) higher than 50% together with an OLR up to 3.2 kg VS m d , with a biogas yield 3

-1

higher than 0.45 m kg VS removed and a methane content of 70%. Several authors have proposed co-digestion of the activated sludge, either with organic fraction. The benefits of co-digestion include: improved balance of nutrients, dilution of potential toxic compounds, synergistic effects of microorganisms, increased load of biodegradable organic matter and better biogas yield. The most significant factor for enhanced organic waste anaerobic digestion performance was the improved Carbon/Nitrogen ration provided by additional wastes.

Key words: Anaerobic co-digestion; Sequencing batch reactor; Fruit and vegetable waste; Abattoir wastewater; Waste activated sludge; Fish waste


Session 2 /

PC47 S2

Purification and characterization of chitinases crude extract obtained from the scorpion fish: Scorpeana scrofa 1

Laribi Hassiba and Mameri Nabil

1

Laboratoire des Biotechnologies Environnement et Génie des Procédés (BIOGEP), Ecole Nationale Polytechnique, El Harrach, Alger 1 : Ecole Nationale Polytechnique, El Harrach, Alger , E-mail : [email protected]

Abstract: Oceans are composed of a set a organismes and ecosystems characterized by a long evolution and original functioning. The implementation of these special features in the profit of human industry lead to the development of specific marine biotechnologies in the aim to develop the marine biomasse and produce polymers which is the case of the chitinases. In our study, in the occasion of an approach a development process of the marine biomasse had been undertaken to extract chitinases from scorpion fishes offals and determine optimal physicochemical conditions of their enzymatic activities. An attempt of purification by physicochemical and chromatographic methods had been done too. The obtained results allowed us to notice that the optimal conditions of the enzyme’s activity were of 45°C for the temperature, 4 for the pH and the incubation time was of 2 hours. Chitinases’ precipitation with the ammonium sulphate at different percentages of saturation revealed that it’s was total at 80%. The fragmentation of the crude extract by chromatography of gel filtration on G-75 revealed two peaks the second had chitinasic activity Key words: chitinases, purification, scorpion fish, characterization


Session 2 /

PC48 S2

Cloning, genomic organisation and mRNA expression of pectine lyase and polygalacturonase genes from Penicillium occitanis Hela Trigui-Lahiani and Ali Gargouri Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, BPK 3038, Sfax – Tunisie

Abstract: Penicillium occitanis has been studied, by our group, as a model system for pectinolytic genes organization and regulation in filamentous fungi. In fact we have already isolated, by classical nitrous acid mutagenesis, an exceptional mutant which is at the same time hyperpectinolytic and constitutive (Hadj-Taieb et al., 2002). We suppose that this mutation affect a trans-acting regulatory factor since several pectinases mRNAs are over expressed in the CT1 mutant. Therefore the study of the regulatory CT1 mutant should give us some clues in the field of pectinase gene expression. As the first step to understand the pectinolytic genes regulation in P. occitanis we had constructed a genomic library of the CT1 mutant. This bank was screened by previously isolated cDNA probes of a pectin lyase and a polygalacturonase. From several isolated clones, the nucleotide sequence of both genes was completed, led to the identification of introns and promoter-terminator regions and compared to other fungal pectin lyase and polygalacturonase genes. Inverse PCR was also performed in order to complete this strategy. In addition to the determination of transcription start site, the promoter sequence from both genes was analysed. It showed the conservation of known consensus sequences (CreA, Hap2-3-4 and PacC) and the existence of a particular sequence, CCTGA, which is similar to that already found to be specific of pectinolytic gene in Aspergillus (CCCTGA). Southern blot analysis of total DNA from P. occitanis revealed a single band for both genes. Northern analysis of both mRNAs were also performed and showed that these RNAs are clearly present from the third to the sixth day of culture, which means that both genes are expressed during all growth phases and not only at the stationary phase.


Session 2 /

PC49 S2

Isolation and put in culture of the Microcystis sp. which populates the dam Cheffia ( Algeria) Nasri H.(1,2,3), Bouaicha N.(3) and Kaid Harch M. (1) Institute of Biology, El Taref university, El taref 36 000. (2) The National look-out post of the Environment and the Durable Development O.N.E.D.D ) ., Algiers, Algeria. (3) Public Laboratory of health and environment, Faculty of Pharmacy, Paris University - the South, Paris, France. (4) Department of Biotechnology, Faculty of the Science, University of the Sciences and the technology of Oran, Oran, Algeria.

Abstract : At the moment, the availability in good quality water is indispensable for the good of the man. In Algeria we do not find the water which answers the qualitative requirements required for the drinking water, among these requirements the water has to be poor in microbial germs and in toxin. Indeed, the dam Cheffia, present for some years an increasing eutrophisation which is translated by more and more worrisome phenomena of proliferation of algae because of multiple bound problems has the potential toxicity of certain kinds of Cyanobacteria. Cyanobacteria possesses two characteristics deserving of attention: a photosynthetic function of type eucaryote; the capacity to fix the atmospheric nitrogen according to a process enzymatique classic, but often associated to a mechanism of protection against the original oxygen (certain tree stump filamentous). The capacity of genetic transformation (sure unicellular tree stumps), the presence of plasmides of coli E., make it potential, additional candidates or replacement, bodies at present used in vegetable Biotechnology. Our results of in vitro culture of the Microcystis sp. reveal that the growth of this sort is favored by a high temperature of the water, an optimal luminous intensity, and the abundance of nitrates and phosphates. Key words: vegetable Biotechnology, Microcystis sp ., culture, dam Cheffia.


Session 2 /

PC50 S2

A novel antifungal peptide from Aspergillus clavatus displaying a potent antifugal activity against F.oxysporum Houda Skouri-Gargouri and Ali Gargouri Laboratoire de Génétique Moléculaire des Eucaryotes-Centre de Biotechnologie de Sfax

Abstract : The mould Aspergillus clavatus, locally isolated in the laboratory secretes a protein of biotechnological interest, namely the A. clavatus Antifungal Peptide (AcAFP). AcAFP is a small and basic polypeptide of 51 amino acid residues. The cDNA and genomic sequences were determined and revealed a high content of cystein, tyrosines and lysines (8, 6 and 12 residues, respectively). The AcAFP was purified and has been tested, at several concentrations up to 0,3 mg/ml, against a wide variety of zoo and phytopathogenic fungi. Interestingly, our AcAFP displayed a potent antifungal activity against the phytopathogenic fungus Fusarium oxysporum, causing the “bayoudh” disease in date palm. The minimal protein concentration for total inhibition of F.oxysporum was 130µg/ml; however growth reduction could be seen starting from 34µg/ml. In order to elucidate the action mode of the antifungal peptide AcAFP, we were interested in the morphological changes induced by this peptide in target fungi. In this purpose we noted an abnormal cellular morphology of fungal hyhae pretreated with 50µg/ml AcAFP. Cell wall had lost its architecture and confers to the cells a protoplast aspect. Further preliminary investigations using AnnexinV staining are in progress in order to highlight the exposure of the phosphatidylserine residues (PS) on the surface of cytoplasmic membrane of F.oxysporum. These observations led us to the assumption that AcAFP may have a major role in the degradation of the cell wall of the target fungus, in the permeabilization of the cytoplasmic membrane and finally leads to a PS exposure on the surface of cells, which is a characteristic phenotype during apoptosis occurring in most cell types and organisms in response to diverse cell death signals.


Session 2 /

PC51 S2

Effet de la toxicité du bifenazate ( acaricide) sur un unicellulaire d’eau douce: Paramecium sp. Sbartai Ibtissem, Sbartai Hana, Berrebbah Houria, Djebar Med Réda Laboratoire de toxicologie cellulaire, Université de Badji Mokhtar, Tél : 00 213 72 98 91 34, E-mail : [email protected]

Résumé : L’espèce humaine n’a jamais été aussi vulnérable par rapport à son environnement, et particulièrement par rapport aux perturbations qu’elle même y introduit. Le problème est avant tout celui de l’environnement urbain où bientôt 90% de la population vivra, alors que les sources d’exposition aux toxiques et aux champs électromagnétiques ne cessent de s’y multiplier. Actuellement, en toxicologie, l’utilisation de modèles cellulaires permet de comprendre les mécanismes d'actions des toxiques, à différents niveaux d'organisation de la cellule. Le principal objectif de notre travail est d’étudier et de tester l’effet du Bifenazate qui est un acaricide sur un unicellulaire qui est protiste cilié d’eau douce : Paramecium sp . Le paramètre physiologique étudié est la cinétique de croissance des paramécies qui s’effectue par la mesure de la densité optique DO ( Lavergne, 1986 ; Sauvant et al., 1999), quant aux paramètres biochimiques nous avons opter pour le dosage des protéines totales (Bradford, 1976) , le Glutathion et la mesure de la respiration (Djebar et Djebar, 2000). Enfin une analyse statistique pour effectuer des tests de comparaisons multiples afin d’expliquer les différences entres les moyennes. Les résultats obtenus montrent que les paramécies sont sensibles à cet acaricide et présentent des modifications physiologiques et métaboliques assez intéressantes. Mots clés : Pollution, Acaricides, Bifenazate, Tests de cytotoxicité, métabolisme respiratoire, métabolisme biochimique, Glutathion.


Session 2 /

PC52 S2

Effect of the waste waters treated by infiltration percolation on the bacteriological and mineralogical quality of some market cultures a

a

b

d

Thabet I., Mkaddem M., Boukchina R. and EL Ferjani E . a

Laboratory of Modelling Analyzes and Orders Systems, National school of Engineering of Gabes, Omar Ebn El Kattab 6029 Gabes,Tunisia b

Institute of the arid Regions. Regional Direction of Gabes. d

Laboratory of Cell bio-Physiologie (LBPC), Bizerte. E-Mail address: [email protected]

Abstract: The reuse of the waste waters treated in agriculture comes up against sanitary obstacles. The risks of contamination are essentially bound to the pathogenic micro-organisms transported by these waters. One of the solutions recommended to incite the agriculturists to an adequate use of this resource, is the tertiary treatment of these waters. The infiltration percolation is a technical of purification aerobe to fixed biomass is recognized as efficient process concerning elimination of the pathogenic germs. The objective of this work is to study the bacteriological quality of some market cultures (Tomato, Potato, Pepper and Lettuce) irrigated by the secondary waste waters produced by the station of purification of the city of Gabès after have undergo a treatment by infiltration percolation. The follow-up of the bacteriological quality of the cultures irrigated showed that the source of irrigation water has an impact on the bacteriological load. The irrigation in gully by the secondary waters, balance himself by a very elevated bacterial contamination to the level of the fruits of the studied cultures: Tomato (FC: 103 germs/g), Potato (FC: 8.8 102 germs/g), Lettuce (FC: 3.2103 germs/g). When the irrigation drip with the infiltrated waters, data show that the bacteriological quality of the fruits doesn't present a meaningful difference in relation to the fruits irrigated with the conventional water. An evaluation of the quantities of nitrogen, phosphor and of potassium mobilized by the irrigated market cultures, according to the source of water has been done. The statistical analyses of the results of the contents in phosphor show the existence of a meaningful difference between the fruits irrigated by the tertiary waters (infiltration percolation) and the fruits irrigated by the secondary waters. The results gotten during this work are promoters and demonstrate a clear way the capacity of the infiltration percolation process to improve the physico-chemical and bacteriological quality of the waste waters to a compatible level to a non restraining reuse and permits to have the agricultural produce presenting a bacteriological quality comparable to those of the products marketed. Key words: Irrigation / Waste waters /Infiltration Percolation / bacteriological Quality


Session 2 /

PC53 S2

Phosphate solubilisation by soil microorganisms 1,2

2

1

Ilham Mardad , Emna Ammar and Abdelaziz Soukri 1

Laboratory of Physiology and Molecular Genetic (LPGM), University Hassan II, Faculty of Sciences Ain 2

Chock, Casablanca, Morocco National Engineering School in Sfax, BP « W », 3038 Sfax, Tunisia, E-mail: [email protected]

Abstract: For the plants, the phosphorus is an essential system for energy transfer. This mineral is abundant in soil but because of its intense reactivity, the phosphorus available amount for plants is a growth limiting factor. Phosphorus is sequestered through precipitation and adsorption phenomena. It is able to be adsorbed on soil particle surface and reacts with cations, especially iron, aluminium and calcium as well as with insoluble compounds, hindering its plant use. Microorganisms are able to solubilise phosphorus and may biotransform the insoluble phosphate into soluble form through acidification, chelating and exchange reactions. In this study, bacterial strains able to solubilise phosphorus were isolated from natural environment. The characterization of these strains was made by screening tests based on solid media including phosphates (NBRIP) and bromophenol blue. The culture of the efficient strains in liquid media was performed and showed that the liquid medium exhibited a better bacterial growth and solubilisation of the insoluble phosphate. This activity decreased the medium pH and was demonstrated to be dependent on the initial phosphorus concentration used. This solubilisating activity was related to peptides evidenced in the cultural media. These were partially purified.


Session 2 /

PC54 S2

Optimal conditions of levan and fructooligosaccharides production by a newly isolated thermophilic bacterium a

a

Imen Dahech* , Karima S. Belghith* , Jawhar Fakhfakh , Mohamed Damak, Hafedh a

c

Mejdoub and Hafedh Belghith ** Faculty of Sciences of Sfax, Department of Life’s Sciences, Road of Soukra km 4 , P. O. B. 802. 3018 SfaxTunisia

Abstract: Levan and fructooligosaccharides had several applications in the domain of medicine, cosmetic, agroalimentary, pharmacia. In this work we investigate the production of levan and fructooligosaccharides by a newly isolated thermophilic bacterium and the synthesis of levan by levansucrase at high temperature. This bacterium was identified as being Bacillus sp. The best nitrogen and carbon source that permitted good enzyme production were sodium nitrate with yeast extract and sucrose correspondingly. The optimal temperature and pH of culture were 50°C and 6.5 respectively. The levansucrase activity was very thermostable since it lost only 30% after one hour of incubation at 90°C. Oligosaccharides (fructooligosaccharides) were obtained by acidic hydrolysis of levan produced by this bacterium or by enzymatic way, while acting on times of synthesis and/on sucrose concentrations used, finally by hydrolysis of levan. Determination of the polymer content was done through RMN analysis, which indicates that we have a fructose polymer. This result was confirmed by CCM analysis after a total hydrolysis of the polysaccharide.


Session 2 /

PC55 S2

Occurrence of resistance to mercury, antibiotics and plasmids in environmental bacteria I. Daly, N. Saidi, L. Jaoua and A. Hassen Centre des Recherches et des Technologies des Eaux CERTE (ex INRST) , Laboratoire Traitement et Recyclage des Eaux E-mail : [email protected]

Abstract: Mercury and its compounds are distributed widely across the earth. Many of the chemical forms are toxic to all living organisms. The major sources of environmental contamination of mercury arise from human activities, such as burning coal and petroleum products, use of mercurial fungicides in papermaking and agriculture and mercury catalysts in industry. Bacteria in contaminated environments, which have developed a resistance to mercury, play a major role in decontamination. Mercury resistant bacteria were isolated from waste water, lixiviat, compost, sludge, and hydrocarbon basin. After identification by classic biochemical tests, the minimal inhibitory concentration (MIC) of mercury for each isolate was determined using tube dilution method. The tolerance of resistant bacteria to a range of antibiotics, were tested by disc diffusion method performed in Muller-Hinton medium. Results showed that the majority of bacteria are resistant both to mercury and broad antibiotic range. These isolates were screening for circular plasmids and some strains are chosen for curing experiments to detect if the resistance is located in plasmid. Key words: Mercury resistance, Antibiotic resistance, Plasmids, environmental bacteria


Session 2 /

PC56 S2

Production and molecular characterization of chitinases from Bacillus licheniformis S213 and Burkholderia cepacia S417 strains isolated from Tunisian soils 1

1

2

1

Debez-Benslimen, I.,1Tabbene,O., Karkouch, I., Urdaci, M. and Limam, F.

1

Laboratoire Interactions Légumineuses-Microorganismes, Centre de Biotechnologie, Technopole Borj-Cedria, 2

BP 901, 2050 Hammam-Lif, Tunisia. Laboratoire de Microbiologie et Biologie moléculaire (LMBA), Ecole Nationale d’ingénieurs des travaux agricoles de Bordeaux (ENITAB), Bordeaux, France.

Abstract: Chitin is an important constituent of the cell walls of most fungi. Chitinolytic bacteria have received considerable attention as potential biocontrol agents due to their ability to lose hyphae of fungal crop-pathogens. In the present study, two chitinase producing bacteria were screened and isolated from Tunisian soils. The gram positive bacterium was identified as Bacillus licheniformis and the gram negative one as Burkholderia cepacia, through sequence analysis of the 16S DNA gene. Both strains were grown in a medium containing 0.5% colloidal chitin for 14 days at 30°C. Enzymes were produced in culture supernatants were shown to be active against phytopathogen fungi. Chitinases produced by both bacteria were fractionated by ammonium sulphate precipitation, and their chitinolytic activity was determined using colloidal chitin as substrate. The activities showed levels of 54.99 and 94.39 chitinase Unit/mg protein respectively. Partially purified enzymes produced by Bacillus licheniformis S213and Burkholderia cepacia S417 were also analysed by polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS-MS). Results showed molecular weight of 90.135 and 65.769 kDa respectively. 1032pb from chitinase gene of Bacillus licheniformis S213 was sequenced and showed high homology with chitinase produced by Bacillus licheniformis strain F5.


Session 2 /

PC57 S2

+

Dynamics of (NO3 NH4 ) in an irrigated soil by treated Waste-water : Case of soil in the Nabeul region, NE Tunisia 1

2

1

Jemai.I , Ben Aissa. N and Gallali. T 1

: Faculté des sciences de Tunis 2

: Institut national d’agronomie de Tunis

Abstract : This study discusses and compares the effects of treated urban wastewater (TW) and Groundwater (GW) on the distribution of mineral nitrogen on soil. The study was based on two irrigation methods (sprinkler, S and integrated Gouttor, IG) and was carried out at the experimental station of Oued-Souhil in Nabeul Governorate, NE Tunisia. Water irrigations were undertaken simultaneously by TW and GW through five periods. In each period, soil samples were collected from various depths, down to 1m. The vertical evolution of the mineral nitrogen contents shows a consistent enrichment of nitrate nitrogen compared to ammonium independently of the irrigation water origin. In addition, nitrates seem to be rather associated to the surface horizons than the deeper horizons, regardless of the sampling period. Furthermore, this disparity is more notable within soil irrigated with treated water. The use of the two different irrigation systems (Sprinler, S and Goutter, IG) shows no significant effect on mineral nitrogen distribution. However, after adding the same amount of 3

nitrate nitrogen within either treated water irrigation or groundwater irrigation (967,5 g/m of 3

Groundwater and 200,7 g/m of treated wastewater), the accumulated quantity of nitrates in deep horizons seems to be more important using the irrigation system Springer than that of Goutter. This suggests that the Springer irrigation system may induce a higher nitrate lixiviation, compared to the Goutter system, and by consequence, may lead to an additional and aggravated water table contamination. Key words: soil, groundwater, treated wastewater, nitrate, ammonium.


Session 2 /

PC58 S2

Molecular and biochemical study of the thermophilic fungus Thalaromyces thermophilus lipase Ines Belhaj-Ben Romdhane, Ines Maalej, Ahmed Fendri*, Youssef Talèl Gargouri*, Ali Gargouri and Hafedh Belghith Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, BP “K” 3038-Sfax, Tunisia. Fax: 216-74 440 818 * : Laboratoire de Biochimie et de Génie Enzimatique des Lipases (LBGEL)

Abstract : The thermophilic fungus Talaromyces thermophilus, isolated from a soil sample of the south of Tunisia, has a lipase activity at high temperature. We have determined the lipase activity in the juice from different culture conditions by varying the carbon source. T. thermophilus produced 21 U/ml of lipase activity when grown on the Mandels media containing wheat bran as carbon source, after 4 days of fermentation at 50°C. This activity was determined at a temperature of 50°C and pH 8, in the presence of an emulsion of olive oil as a substrate. The lipase activity was as low as 1U/ml using Tributyrine (TC4) as a substrate and under the same conditions. The effects of pH and temperature on lipase activity are under further investigation. A Genomic library covering statistically the entire genome of Talaromyces thermophilus, was constructed and used for the cloning of lipase genes. We were able to isolate a clone containing a major portion of a lipase gene along with its promoter sequence. To clone the entire lipase gene, a fragment of this gene was used as a probe for the screening of the total genomic library. Several positive clones were identified by colony hybridization and confirmed by sequencing. The Southern blot analysis of genomic DNA and transcriptional study by Northern blot, using the same lipase probe, are underway.


Session 2 /

PC59 S2

DNA sequence analysis and identification of 21.3 kb plasmid involved in bacteriocin synthesis from Bacillus thuringiensis strain BUPM4 Ben Fguira, I., Kamoun, F., Tounsi, A. and Jaoua, S.* Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box: K, 3038. Sfax, Tunisia *E. mail: [email protected]

Abstract: Since their discovery, bacteriocins have been a subject of intense research. In this work, we were interested in the investigation of genes involved in the synthesis of a 3.1 kDa bacteriocin (BF4) produced by Bacillus thuringiensis strain BUPM4 (Kamoun et al. 2005) and the search for new ones. With this aim, two mini-Tn10 transposon insertion mutants affected in their bacteriocin activity were subjected to DNA sequencing of the mini-Tn10 target region. DNA sequence determination revealed that the insertion of the mini-Tn10 resulted in the duplication of a perfect 8 bp target site which was suggested to be original according to what is known about the usual mini-Tn10 transposition target site. This insertion site was mapped in an open-reading frame (ORF) sharing high nucleotide sequence homology (92%) with a gene implicated in bacitracin transport. The predicted amino acid sequence of the latter showed 35% identity with an ATP-binding cassette (ABC) transporter, a system involved in the transport of bacteriocins of many bacteria. These results suggest strongly the implication of this DNA sequence in the transport of the bacteriocin BF4. As for the other ORF(s), the highest similarities were identified with proteins closely related to phage tail-components, thus indicating that the BF4 is probably a phage tail-like one or that this structure is required for its export. Moreover, a plasmid of 21.3 kb harbouring the genetic determinants responsible of the BF4 activity, was evidenced for the first time. All these results were extremely useful for us to search and identify new bacteriocins of Bacillus thuringiensis completely different from BF4. Reference: F. Kamoun, H. Mejdoub, H. Aouissaoui, J. Reinbolt, A. Hammami and S. Jaoua (2005) J. Appl. Microbiol. 98, 881-888


Session 2 /

PC60 S2

Phenotypic, genotypic, and phylogenetic discrepancies to differenciate Pseudomonas species 1

1

1

1

1

Mehri Inès , Gtari Maher , Ouzari Imen , Turki Yousra , Jaoua Leila , Meyer 2

1

Jean-Marie , and Hassen Adennaceur . (1) Centre de Recherche et Technologie des Eaux : CRTE (ex- INRST), B.P 95, 2050 hammam-lif. (2) Laboratoire de Microbiologie et Génétique, Université Louis-Pasteur, CNRS FRE-2326, Stasbourg, France. E-mail: [email protected]

Abstract: The genus of Pseudomonas includes species with ecological, economic and healthrelated importance. The fluorescent pseudomonads form a major group within the rRNA homology group I of Pseudomonas ‘sensu lato’ and are characterised by the production of fluorescent pyoverdin pigments which are produced abundantly in the media of low iron content. The group includes species important for the biodegradation of a diverse range of compounds and for the production of useful metabolites and biotechnologically important substances. Some species are also agents of animal, human and plant disease while others can promote plant growth and control soil-borne diseases. A total of 160 environmental and clinical isolates were confirmed as Pseudomonas sensu stricto by fluorescent pigment production. These isolates were analyzed by biochemical (Api 20NE), analytical (Isoelectrofocalisation: IEF) and three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA) and 16S-23S rDNA intergenic spaceranalysis. So the aim of this investigation was to evaluate and compare the DNA-based typing techniques and the phenotypic fingerprinting for the purpose of assessing the inter- and intraspecific diversity of an identified collection of fluorescent pseudomonads. The reproducibility, typeability and discriminatory power of the methods were also compared to evaluate their application. Key Words: Pseudomonas, pyoverdin, ITS, ARDRA, IEF.


Session 2 /

PC61 S2

Optimization of cellulases and hemicellulases production by Neurospora crassa grown under SSF for the bioconversion of sweet sorghum to ethanol Dogaris I., Vakontios G., Mamma D., Kalogeris E. and Kekos D.* National Technical University of Athens, Department of Chemical Engineering, Biotechnology Laboratory, Zografou Campus, 15780 Ζografou, Αttica, GREECE, * E-mail: [email protected]

Abstract: Extensive research has been conducted in the last two decades for replacement of the limited fuel fossils by renewable energy sources. The conversion of lignocellulosic biomass to ethanol is a promising alternative, where hydrolysis of cellulose and hemicellulose is followed by fermentation of sugars to bioethanol. A few microorganisms, such as the fungi Neurospora crassa and Fusarium oxysporum, have the ability to carry out simultaneous hydrolysis and fermentation, thus combining these two steps into a cost-effective direct process. In the present work the production of cellulolytic and hemicellulolytic enzymes by the fungus Neurospora crassa, grown on agricultural wastes under solid state fermentation (SSF), was studied. The effect of important parameters, such as carbon source, nitrogen source, initial culture pH and initial moisture on enzyme production was investigated. The maximum activities of 492.8, 1.08, 26.7, 238.3 and 0.13 (U/g carbon source) of endoglucanase, exoglucanase, b-glucosidase, endoxylanase and b-xylosidase respectively, were obtained when the fungus was grown on a combination of wheat straw and wheat bran (5 to 1, w/w) supplemented with inorganic nitrogen (ammonium sulphate) and adjusting culture medium initial pH to 5 and initial moisture to 70%. Following media optimization, bioconversion of soluble and insoluble carbohydrates from sweet sorghum to ethanol was achieved by Neurospora crassa monocultures or mixed cultures with the yeast Saccharomyces cerevisiae. The addition of yeast resulted in 25% increase in maximum ethanol yield and reduced the time of maximum ethanol production by 4 days.


Session 2 /

PC62 S2

Establishing base-line data through measuring the concentration of Escherichia coli,fecal coliforms, total coliforms, and fecal streptococci in Zarqa River-Jordan prior to evaluation of the riverbank filtration (RBF) technology 1

2

3

4

Ismail Saadoun* , Ziad Al-Ghazawi , Mo,ayyad Shawaqfeh , Jamal AL-Rashdan , William 5

6

7

1

Blandford , Thomas Boving , Jack Schijven and Qutaiba Ababneh 1

Dept. of Applied Biological Sciences, College of Science & Arts, 2

Dept. of Civil Engineering, College of Engineering, Jordan University of Science and Technology, Irbid 22110, Jordan, 3

Dept. of Civil and Environmental Engineering, Mu’tah University, Karak 61710, Jordan, 4

Water Re-Use Division, Water Authority of Jordan, Ministry of Water and Irrigation, Amman-Jordan, 5

Dept.of Geology, Louisiana State Univ., Baton Rouge, LA-USA, 6

7

University of Rhode Island, Kingston, RI 02881, USA, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Abstract: The Zarqa River within Jordan, is heavily contaminated with treated domestic and industrial wastewater principally from the city of Amman, but still serves as the source for irrigation water. For the purposes of evaluation of riverbank filtration (RBF) for protection of Jordanian surface and ground water resources project, and to achieve its objectives throughestablishing base-line data on Zarqa river water quality prior to evaluation of the RBF technology, surface water samples were taken for analysis of microbial pathogen contamination through measuring the concentration of Escherichia coli (E. coli), fecalcoliforms, total coliforms, and fecal streptococci or enterococci. These were taken every sex months (June/2006 to December/2007) from station I (2 km from the main wastewater treatment plant in Jordan, Kherbit Al-Samra) and station II (the RBF site which isdownstream and 30 km from station I) along the river as well as from the RBF extraction well. Following 3

the membrane filtration (MF) technique, number of E. coli of June/2006samples was 9.1 x 10 3

and 20.0 x 10 cfu/100 ml at station I and II, respectively. 5

In January/2007 these numbers were increased by > 85% with 1.57 x 10 and 1.35 x 5

10 cfu/100ml at the above stations, respectively. For the intestinal enterococci bacteria, 2

2

June/2006 analysis from station I and II revealed values of 16.0 x 10 and 2.5 x 10 cfu/100 ml, respectively. By Jan./2007 and at station II, these bacteria increased to > 90% with a 3

concentration of 5.0 x 10 cfu/100 ml. By the multiple tube fermentation (MPN) technique,numbers of fecal coliforms, E. coli and intestinal enterococci of Jan./2007 at station 3

3

3

II were 18.8 x 10 , 12.3 x 10 and 2.4 x 10 , respectively. However, the numbers after one 5

4

3

year (Dec./2007) increased with concentrations of 4.6 x 10 , 9.3 x 10 and 2.7 x 10 , respectively. 5

6

As far as the bacteriophages are concerned, high concentrations (10 – 10 /100 ml) werefound in the river. For the chemical parameters at station I/II, COD, BOD, TSS and TDS analysis indicated values of 265/95, 48/216, 2024/1987 and 1691/1662 mg/L, respectively. Analysis of Vibrio and Clostridium perfringens indicated the presence of both pathogens at station I and II.


Session 2 /

PC63 S2

The solation of a pesticide-degrading bacteria from activated sludge. Iyad Ghanem*, Malek Orfi and Motassim Shamma Abstract: When an activated sludge sample was incubated in the presence of chlorpyrifos (13.9 -1

mg ml sludge), 46% of added chlorpyrifos were degraded within four days. Selection pressure resulted in the isolation of chlorpyrifos-degrading bacteria. The bacteria was identified as Klebsiella sp. It survived on poor culture medium with chlorpyrifos as the sole source of carbon, tolerated up to 17.28 g of chlorpyrifos in the poor medium, and was capable of degrading more than 90% of the insecticides.


Session 2 /

PC64 S2

Molecular cloning, sequence analysis of C-type lectin proteins from Macrovipera lebetina and expression of a recombinant, lebecetin in HEK cells 1

3

1

1,2

Jed Jebali , Assou Elbattari , Amine Bazaa , Sameh Sarray 3

5

1

1

, Raoudha Zouari , Sylvie 1,6

Matieu , Ali Gargouri , Mohomed El Ayeb and Naziha Marrakchi . 1: Laboratoire des venins et toxines, Institut Pasteur de Tunis, Tunisie 2: Faculté des Sciences de Tunis, Tunisie 3:Laboratoire des Glycociologies de la cellule tumoral digestive, faculté de Médecine Marseille, France 4: Laboratoire de Biochimie Cellulaire, Faculté de Pharmacie, Marseille, France 5: Laboratoire de génétique moléculaire des eucaryotes, centre de la biotechnologie de Sfax (CBS) 6: Faculté de Médecine de Tunis, Tunisie

Abstract: Tunisian viper Macrovipera lebetina venom contains a variety of C-type lectin proteins (CTL), causing platelet aggregation and consumptive thrombocytopenia in victims, we have characterised tow CTL: Lebecetin and lebectin. In addition to inhibiting platelets aggregation, Lebecetin and lebectin interact with cancer cells by inhibiting the adhesion, migration, invasion and proliferation. Using RNA extracted from Macrovipera lebetina and primers designed according to the conservative regions based either on published sequences of highly conserved 5’ and 3’ regions or translated consensus regions of various venoms CTL. Extensive clones screening by classical plasmid DNA analysis CTL were followed by sequencing several cDNA. More than one isoform CTL has been found in individual gland from adult snake. Variations in snake gland composition would be associated with factors occurring through “domains shuffling” from former genes, alternative splicing of pre-mRNA and trans-splicing or editing. The full-length cDNA clones containing encoding lectin precursor of 130 amino acids coding sequences for lebecetin α and β subunits were isolated. The cDNA of two subunits were transfected and expressed in human embryonic kidney separately or together by a novel strategy using two vectors with different selectable Tags. Analysis of transfect cells expression after two days followed by immunofluresence test revealed signification expression level lebecetin related protein.


Session 2 /

PC65 S2

Interactive MLST database management for Campylobacter species (incl. PubMLST data) using the BioNumerics® plugin. Dombrecht, J., Janssens, K., Vauterin, P., Vauterin, L. and Pot, B. Applied Maths NV, Keistraat 120, B-9830 Sint Martens Latem, Belgium Tel: +3292222100 / Fax: +3292222102 / www.applied-maths.com / [email protected]

Abstract: Multi Locus Sequence Typing (MLST) is a method to discriminate microbial isolates through the partial sequencing of selected housekeeping genes. BioNumerics® is widely used for the storage and analysis of MLST sequences. With the use of a plugin tool, BioNumerics automatically assembles and processes sequence trace files, connects to the online MLST databases, and retrieves allele numbers, sequence types as well as available clonal complex information. When installing a new database in BioNumerics, the organism available on the PubMLST site needs to be selected. During the import of MLST trace files, BioNumerics connects to the PubMLST website, retrieves the names of the housekeeping genes, their start and stop positions and creates the database environment to store this information. BioNumerics assembles the sequences into a consensus sequence, and automatically screens this sequence for start and stop positions. The sequence is stored in the database, and errors are reported. Consequently, BioNumerics compares assembled sequences to the online PubMLST allele libraries. This information is stored in the database. If available, MLST sequence type and clonal complex information is also retrieved and stored in BioNumerics. The data flow will be illustrated through the processing of MLST trace files from Campylobacter isolates. An UPGMA dendrogram will be compared to a Minimum Spanning tree. BioNumerics can type hundreds of isolates in only seconds. Results are stored in a database and are available for statistical and population analysis.


Session 2 /

PC66 S2

Study of the interaction of Nigella sp. alkaloids with serum albumin Hammam K.∱*, Khettal B.∱, Sobhi W.∱and Sadoun T

$

∱

Enzymology laboratory, Department of Physico-Chemical Biology, Faculty of Nature and Life Science, *

Abderrahmane Mira University, Bejaia , E-mail : [email protected] $

Chemistry of Polymers laboratory, Department of Process Engineering, Faculty of Engineer Science, Abderrahmane Mira University, Bejaia

Abstract: One of the aspects that modern biotechnology treats is extraction and study of new natural active molecules that have therapeutic effect. Actually, many of bioactive substances which come from secondary metabolism of plants have showed their pharmacologic effect in vitro. However, it has been shown, in vivo, that the distribution of these molecules and their metabolism can be changed significantly by their biding to serum proteins. The study of these aspects can give important information about structural characteristics that control therapeutic efficiency of these molecules. This study was designed to examine the kinetic of binding of Nigella sp. alkaloids to the serum albumin, protein of aspecific blood transport. Differential spectroscopy was used to determine the drug-binding mode and have shown that alkaloids bind specifically to serum albumin using a cooperative spontaneous process, mainly by hydrophobic and electrostatic interactions. Keywords: alkaloid, Nigella sp., interaction, human serum albumin, differential spectroscopy.


Session 2 /

PC67 S2

Isolation and characterization of cellulolytic yeasts from surrounding soil of thermal springs Labbani K F-Z, Benaouida K, Meraihi Z, Leghlimi H, Djekrif-Dakhmouche S, Bennamoun L and Meziani Z Laboratory of Microbiologic Geny and Applications Department of Biochemy and Microbiology, Mentouri University, Road of Ain-El Bey 25000, Constantine-ALGERIA, E-mail : [email protected]

Abstract : Cellulases are distributed throughout the biosphere such as plants, animals and microorganisms. However, microorganisms are considered to be the main source for cellulases with novel and high specific activities. Cellulases from fungi and bacteria have been studied extensively, but little attention has been given to cellulases from yeasts. Cellulases yeasts proved to work at a broad range of both pH and temperature. Also, they have a reasonable degree of pH and thermal stability. These properties make them suitable for biotechnological processes. Accordingly, the main goal of this study is to isolate and select yeast strains producing thermostable cellulase, from surrounding soil of thermal springs (Hammam Dbegh-Guelma and Hammam Teleghma-Mila, Algeria), with an aim of an industrial application. Five yeast strains were isolated. Their selection on a CMC-YP agar medium to 2% CMC with pH 5 and a temperature of incubation of 30°C showed that only the strain F has a cellulolytic activity. Tests of identification show that the strain F probably seems to belong to the Sporobolomyces genus. Key words: Yeasts, isolation, cellulases, thermal springs


Session 2 /

PC68 S2

Contribution to the study of microbial biomass in the alluvial soil of a Daya located in the region Guerrerra (Wilaya of Ghardaia) Mergoud L.* and Hamdi Aissa B. ** * Assistant lecturer in the Department of Biology, Faculty of Science and Engineering, University Kasdi Merbah of Ouargla ** Lecturer at the Department of Agronomy, Faculty of Science and Engineering, University Kasdi Merbah of Ouargla

Abstract: The objective of our work is the quantitative study of microbial biomass (bacteria, fungi, actinomycetes and algae) living in an alluvial soil Daya Ben Feïlah located in the region of Guerrerra. The results show that the alluvial soils of the daya studied, have different microorganisms capable of adapting to the conditions of such environments. The enumeration of different microbial groups reveals that the most dominant group blight than other microorganisms seen their great power multiplication especially in the basic soil (dry soil). Regarding the distribution of microorganisms in the profile studied, we found that the microbial biomass and the number of microorganisms reach their maximum level of moderately deep horizons. Key words: Microbial Biomass, alluvial soils, Daya, Guerrerra.


Session 2 /

PC69 S2

Analysis of water for pesticides at low parts per trillons (ppt) levels using two dimensional lcmsms without any sample pre-treatement. L. Beyet Applied Biosystems, 25 Avenue de la Baltique, 91943 Courtaboeuf, France. S. Lock, I. Gibb & N. Anderson, Applied Biosystems, 120 Birchwood Boulevard, Warrington, WA3 7QH, United Kingdom. Dave Evans, ALcontrol Laboratories, Rotherham S60 1BZ, Yorks, U.K.

Abstract: The provision of clean, uncontaminated drinking water is of paramount importance to the water industry. In recent times the requested limits of detection for pesticides have been decreasing as methodologies improve. Typically water companies need to be able to have limits of quantitation for pesticides between 0.02 – 1 µg/L (20 - 1000 ppt) which often means that methods should have limits of detection for certain pesticides at 10 – 50 ppt. These low levels often mean that water samples have to be extracted either using liquid/liquid or solid phase extraction in order to concentrate these contaminants to such a level where they can be detected. Sample pre-treatment can often be time consuming and add an additional cost to the analyses. We will present data acquired on the 3200QTRAP® LCMSMS system where pesticides have been detected in the low ppt range with no sample pre-treatment. High injection volumes were used with on-line solid phase extraction to pre-concentrate the pesticides before separation by reverse phase HPLC and detection. Data displayed in the poster indicated CV from spiked samples, were less than 15% at low ppt levels. Calibration lines over the range 20 – 1000 ppt were observed to be linear. For confirmation full scan enhanced product ion data was also acquired at low ppt levels using an MRM trigger to start the acquisition of an the enhanced product ion scan were a collision energy spread was used to enhance the spectral quality. Cycle times for a 50 pesticide screen were less than 1s allowing quantitative and qualitative data to be acquired in one run.


Session 2 /

PC70 S2

Study of the contribution of both soluble and insoluble fractions to ruminal methanogenesis using an in vitro gas production model. Boultifat L., Rira M., Laadjimi K., Driss D., Arhab R., Boufennara S.& Bousseboua H. Abstract: Methane (CH4) is the most important factor involved in the greenhouse effect after carbon dioxyde (CO2), respectively 16% and 30% of global green house effect. Only 30% of total methane emission originates from natural sources, whereas 70% is linked to human activities including livestock production. Generally, methane production by ruminants is estimated to be responsible for 22% of the anthropogenic sources. The aim of this study is to evaluate the contribution of cellulose degradation to methane production. So two natural substrates, originated from arid zones (dates residues and racemes), and two pure substrates (Cellulose type 101, starch) were used. At the same time, the contribution of both soluble and insoluble fractions (cellular content, cell wall) was evaluated. After incubation in miniaturized fermentors, CH4 production was qualitatively and quantitatively measured. The results shows that the total gas production, from substrates fermentation, vary significantly between substrates and their fractions (ppH 11). Small-scale alkaline lysis and boiling techniques identified plasmids in these microorganisms. Purification and sequence analysis of the plasmid and chromosomal DNAs is in progress, in collaboration with the Virginia Tech Center for Genomics (USA). In mixed culture, on solid media, the eubacteria associate exclusively with filaments of the cyanobacterium. Detailed microscopic examination of strain QU002 at the Environmental Sciences Center of Qatar University, identified an unusual distribution of extracellular polysaccharide along the apices of the larger class of filaments. The physiological and biochemical basis for the association between these microorganisms, including the potential for lateral gene transfer, is under investigation. Acknowledgements. Supported by a Qatar University award to Professor Malcolm Potts and Dr Roda Al-Thani. The authors are grateful to Hareb Al-Jabri for advice and technical support.


Session 2 /

PC79 S2

Prevalence and characteristics of Verotoxigenic producing Escherichia coli O157:H7 isolated from goats and cattle carcasses in Tanzania. Raji, M.A*, Minga, U.M and Machangu R.S Dept of Veterinary Microbiology and Parasitology, Sokoine University of Agriculture, P. O. Box 3019, Morogoro, Tanzania. Corresponding Address: *Dr Mashood Abiola Raji, Dept of Veterinary Pathology and Microbiology,Ahmadu Bello University, P. O. Box 1044, Samaru-Zaria,Kaduna State-Nigeria. Email: [email protected]

Abstract: The prevalence of Verotoxigenic producing Escherichia coli (VTEC) in cattle and goats carcasses were investigated between September 2002 and December to June 2003 by cultural and immunomagnetic separation technique. A total of 167 Escherichia coli colonies from carcasses of cattle (300), and goat (263), from Morogoro and Dar-es-salaam were isolated in this study. TEC O157 strains were recovered from 17 (5.67%) cattle carcasses and none from goats. Of 167 E.coli strains, 17 were grouped into sorbitol non-fermenting and glucuronide negative and 29 strains were sorbitol positive and glucuronide positive. The remaining 38 were sorbitol negative and glucuronide positive. Using Reversed passive latex agglutination kit from Denka Japan indicated that all isolates produced verotoxin. Further characterization of the VTEC isolates showed that 1(4%) of the bovine VTEC strains was positive only for stx1. Stx2 gene alone was detected in 4(20%) of bovine isolate. Both stx1 and stx2 gene were present in one (4%) of bovine isolates. Eae A was detected in 4 (20) of bovine isolates. Stx 1, stx2 eae A and Ehly A were present in one (4%) bovine isolates. Other bacterial agents such as Pseudomonas spp, Proteus spp and coliforms were also isolated. The VTEC O157 isolates were resistance to gentamicine, chloramphenicol, streptomycin, and amoxsylav. This study is the first attempt to investigate the prevalence of VTEC O157 in goats and cattle carcarsses in Tanzania. Key words: Carcasses, cattle, goats, Tanzania, PCR, Verotoxigenic producing Escherichia coli O157.


Session 2 /

PC80 S2

Talaromyces thermophilus β-D- xylosidase: purification, immobilisation and xylooligosaccharides synthesis Guerfali Mohamed, Maalej Ines, Gargouri Ali and Belghith Hafedh Laboratoire de Génétique Moléculaire des Eucaryotes du Centre de biotechnologie de Sfax BP”k” 3038 Sfax Tunisie

Abstract: In the industrial domain, theβ-xylosidases have an importance due to a duality in their action: β-xylosidases are enzymes catalysing hydrolysis (conversion and plant garbage valorisation) and synthesis reactions (synthesis of oligosaccharides and alkyl-xylosides). Recently, enzymes immobilization of hydrolase and glucosyltransferase activity became a very useful technique in the synthesis reaction; it offers an advantage for industrial applications. We purified and immobilized the β-xylosidase of a thermophile fungus Talaromyces thermophilus by different methods and using different supports of immobilization in order to improve the biochemical characteristics of this enzyme. The influence of enzyme immobilization on kinetic parameters Km and Vmax as well as the biochemical characteristics of Talaromyces thermophilus β-xylosidases were investigated. The chemical method of immobilization using chitosan as support in presence of glutaraldehyde permitted to get the best yield of immobilization with a high stability of the enzyme (until 25 cycles without loss of enzyme activity). We observed that temperature doesn't have an effect on immobilized β-xylosidases, since the activity profile for free and immobilized enzyme is nearly the same. On the other hand, we observed a displacement of the optimum pH (7.0 for the free enzyme to 8.0 for the immobilized enzyme). The immobilized β-xylosidases kept its activity at basic pH (pH=11), the loss of activity is less important for the immobilized enzyme (50%) in comparison to the free enzyme (90%). The immobilization enhanced also the enzyme stability in relation to thermal denaturation. Immobilization modified enzyme kinetic parameters, the enzyme affinity to its substrate (pNPX) decreased. Some ions (CaCl2, MnSO4, CoCl2 and KCl) showed a beneficial effect on βxylosidase activity either at immobilized or free enzyme state. This improvement is more important in conditions where the enzyme is in Free State. On the other hand, ZnCl2, CuSO4 and the FeSO4 ions as well as EDTA and SDS inhibited β-xylosidase activity. At the Free State and in presence of a saturating xylose concentration the β-xylosidase of Talaromyces thermophilus catalyses condensation reactions and allowed the synthesis of a high level of xylobiose at a yield of conversion of 11.3%. On the other hand, immobilized βxylosidase synthesized different xylooligosaccharides (xylotriose and xylotetraose…) at variable concentrations. The co-immobilization of the β-xylosidase and the xylanase of Talaromyces thermophilus permitted to increase the efficiency of xylane saccharification by the liberation of xylose, proving by the way the synergic action of the two enzymes.


Session 2 /

PC81 S2

Optimization of laccase production by the white-rot fungus Fomes fomentarius in solid-state fermentation using response surface methodology Neifar, M., Jaouani A. and Ellouz Chaabouni, S. Unité Enzymes et Bioconversion, Ecole Nationale d’Ingénieurs de Sfax, BP : W.3038 Sfax, Tunisia E-Mail : [email protected]

Abstract: Response surface methodology was employed for the optimization of different cultural and nutritional parameters for the production of laccase by the white rot fungus Fomes fomentarius MUCL 35117 in wheat bran-based solid medium. Initial screening of production parameters was performed using an asymmetrical design and the variables with statistically significant effects on laccase production were identified. Inoculum size, CaCl27H2O and CuSO45H2O were found to influence laccase production. These variables were selected for further optimization studies using a Hoke design. The statistical optimization by response surface methodology resulted in a three-fold increase in enzyme production as compared to the un-optimized medium. Key words: asymmetrical screening design; Hoke design; solid-state fermentation; Fomes fomentarius; laccase.


Session 2 /

PC82 S2

Characteristics OF chitinase extracted from new strain of Aeromonas hydrophila fn100 Mohammad B. Habibi Najafi, Fathi, M., and Forghani B. Ferdowsi University of Mashhad, Faculty of Agriculture, Department of Food Science & Technology, P. O. Box 91775-1163, Mashhad, I. R. Iran, [email protected],ir ; [email protected]

Abstract: Several organisms like bacteria, fungus, and plants produce chitinase for different purposes such as protection against pathogens, growth and nutritional requirement. Nowadays, chitinase application is entering our life in many ways. It is a good substitute for chloride and sulfate glucoseamine (effective in osteoarthritistropy). Due to the ability of Bifidobacterium in consuming N-acetylglucosamine, it is believed that chitinase activity can lead to increasing the Bifidobacteria population in the intestinal microflora, so they can produce enough lactase for hydrolysis of milk sugar. It is also used in formulation of environmentally friendly pesticides. In this study, chitinolytic bacteria were screened through sampling from different environments and their chitinolytic activity was determined after cultivation on chitin rich medium. The higher chitinase activity was then further identified using biochemical tests and 16s rDNA. Results showed that the isolated strain is a new strain from specie of Aeromonas hydrophila which was assigned by Genebank as FN100. Extracted chitinase from this novel strain was purified by chromatographic methods to 1.5 purification fold and 51% yield. The main characteristics of purified enzyme were as follow: optimum temperature 40°C, optimum pH 6, and optimum colloidal chitin for enzyme activity 3% w/v. It was proved by EDTA that the enzyme is not metalloenzyme. The purified enzyme retains 68% of its activity after 2h at 50°C and 85% of its activity at pH 7.


Session 2 /

PC83 S2

Potential for potable water savings by treatment and recycling the greywater M. Lamine, L. Bousselmi, and A. Ghrabi Laboratory of wastewater treatment and recycling, Center of researches and technologies of water BP 273 Slimane 8020, Tunisia

Abstract: Greywater reuse will play an important role in the sustainable water management approach. The main objective of this paper is to evaluate the potential water savings by using grey water and to plan a technical feasibility system to recycling water in El Manzeh students' house in Tunis. Studies of consumption reveal that more than 45% of the water used in the students' house is for showers. This may be reused for other purposes especially toilet flushing (30% of water used) and landscape irrigation. Greywater from showers has been examined for physical, chemical and microbiological parameters to determine the potential health and environmental risks associated with reuse. For its more-cost efficient removal, the sequential biological reactor (SBR) has been chosen to treat greywater. Several processing operations were carried out in laboratory reactor. Operational parameters have been optimized to produce an effluent with the quality in conformity with the standards recycling toilet flushing. But to guarantee zero faecal coliform levels at all times, disinfection of the effluent could be necessary. A pilot system of greywater recycling to flush toilets in students' house based on SBR treatment coupled with UV disinfection is installed.


Session 2 /

PC84 S2

Removal of phenolic compounds from olive mill wastewater by low cost bio sorbent “banana peel” a

b

a

c

a

M. Achak , A. Hafidi , N. Ouazzani , S. Sayadi and L. Mandi a Laboratoire d’Hydrobiologie, d’Ecotoxicologie et d’Assainissment, Département de Biologie, Faculté des Sciences Semlalia b

Laboratoire Sciences des Aliments, Département de Biologie, Faculté des Sciences Semlalia, Universitie Cadi Ayyad, Faculté des Sciences Semlalia, Département de Biologie, boulevard, Prince Moulay-Abdelah, BP 2390, Marrakech, Maroc c

Laboratoire des Bioprocédés, Centre de Biotechnologie de Sfax, Route Sidi Mansour BP: “K” 3038 Sfax, Tunisie

Abstract: Olive mill wastewater (OMW) are a significant source of environmental pollution especially in some important olive oil producing countries such as Spain, Italy, Greec, Tunisia, Morocco, Turkey, Lebanon, Syria and Portugal. When disposed into the environmental, OMW create serious deterioration such as coloring of natural waters, alteration of soil quality, phytotoxicity and odor nuisance. Several methods were reported for the removal of pollutants from these effluents. These technologies can be divided into three categories: biological, chemical and physical. All of them have advantages and drawbacks. Because of the high cost and disposal problems, many of these conventional methods for treating OMW have not been widely applied at large scale in the olive oil mill. The purpose of this work is to investigate the efficiency of banana peel as a biosorbent for removal of phenolic compounds from OMW. The effects of various operating parameters on biosorption such as initial pH, sorbent dosage and contact time were monitored and optimal experimental conditions were decided. Batch adsorption experiments were carried -1

out using a rotary shaker at 200 rpm.min and 30 ± 2°C. Different doses (1-5 g) were tested at various pHs (2-11). The changes of absorbance were determined at certain time intervals (124h) during the adsorption process. The obtained results show that the equilibrium solid-phase concentration of phenols decreased with increasing adsorbent (banana peel) concentration, an increase the pH of solutions leads to a significant increase in the adsorption capacities of phenols on the banana peel and the adsorbed amounts of phenols increased with increase in contact time and reached the equilibrium after 3h.


Session 2 /

PC85 S2

Production of Prebiotic Sugars by The Hydrolysis of Galactomannan Extracted from Carob and Tomato Seeds Blibech, M., Daoued, I., Ellouze Chaabouni, S. and Ghorbel, R. Unité Enzymes et Bioconversion à l’Ecole Nationale d’Ingénieurs de Sfax. Route de Soukra, Km 3.5 B.P.W 3038. Sfax, Tunisie. E-mail : [email protected]

Abstract: Seed galactomannans are vegetable, heterogeneous polysaccharides widely attracted considerable attention for various industrial applications. So, galactomannans from the flour of carob and tomato seed were extracted. The gum yield reached 12.3% and 2.46% from carob and tomato seed respectively. The chemical composition (moisture, ash, protein, fat…) of purified galactomannan from carob was similar to the commercial Locust Bean Gum (LBG) but the tomato galactomannan presented a difference. Results showed that the addition of galactomannans (from carob and tomato seeds) to the culture media as sole carbon source induced the formation of mannanases by Penicilluim occitanis (Pol6). Mannose and other sugars have been reported to be excellent prebiotic stimulating the growth of beneficial intestinal microorganisms. Then, an hydrolysis of both purified gums for 24h at 50°C by culture filtrate of P.occitanis containing mannanase activity (200U/g substrate) was performed. The yield of hydrolysis of commercial LBG, carob and tomato gum was 21.19±0.59, 12.02±2.1 and 11.27±1.13 respectively. HPLC analysis showed that galactose, mannose and glucose were the final hydrolysis products which stimulated the growth of Lactobacillus bulgaricus and thus attempt to improve the host health.


Session 2 /

PC86 S2

Characterization of sludge derived from waste water treatment electromigration: technology for the treatment of sludges polluted with metals Cherifi .M, Hazourli. S and Ziati. M Laboratory water treatment and valorization of the industrial waste, Department of Chemistry, Faculty of Science, University of-Badji Mokhtar, Annaba 23000. E.mail : [email protected]

Abstract : The urban sludge are produced at several stages of wastewater treatment, they are also called fertilizing residual matter or biosolids. According to their compositions, the sludges may have several final destinations. They can be valued in agriculture or be incinerated to produce energy. The presence of metal elements in the sludges, including heavy metals may be prohibitive for certain agricultural uses, the treatment of the sludge is then necessary. Techniques for preventing the production of sludge in wastewater treatment plants already exist; they use chemical or thermal voices destruction of a portion of the biomass produced in excess. These techniques require in many cases technical and financial resource very important Electromigration is an innovative technology which faces pollution metallic sludge, Soil and groundwater. It is an in or ex-situ method of treatment, it need a simple equipment and can treat a wide variety of pollutants (Metals, organic compounds, radionuclide). The first part of this paper is to characterize sludge derived from a drinking water treatment. The analyses show the presence of heavy metals in very low quantity with remarkable presence of aluminum. Electromigration tests at the laboratory level were performed to see the feasibility of this technique to decontaminate sludge from its aluminium. The experimental protocol used is simple. It is a glass cell containing sludge studied; the potential is applied to the sludge by the insertion of two electrodes in the middle. Phenomena such as the electromigration, electroosmosis and electrophoresis are responsible for the displacement of the aluminium from anode to the cathode. At the end of manipulation (10 days) a clear migration of aluminium to the cathodic region has been observed, but is influenced by the combination of several factors As the time of manipulation, the water saturation of sludge, as well as the acidity of the medium. Key words: Characterization, sludge, depollution, metals, aluminium


Session 2 /

PC87 S2

Isolation of soil bacteria with high ability for solubilizing insoluble inorganic phosphates Mounira Ben Farhat, Kameleddine Bouchaala, Ameny Farhat, Samir Bejar & Hichem Chouayekh Laboratoire d’Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax BP “K” 3038 Sfax, Tunisie. E-mail: hichem.chouayekh @cbs.rnrt.tn Tel./Fax: +216 74 870451

Abstract : Traditional phosphore (P) fertilizer production is based on chemical processing of insoluble mineral rock phosphate, which includes an energy intensive treatment with sulphuric acid at high temperature. This process is environmentally undesirable because of the release of contaminants with the main product, gas streams and by products. Furthermore, use of P fertilizers has becomes a costly affair and there is a need for alternative sources. To develop environmental-friendly biofertilizer solubilizing insoluble phosphates, five novel bacterial strains exhibiting strong ability to solubilize various insoluble phosphates (rock phosphate, hydroxyapatite, tricalcium phosphate, calcium phosphate) in the external culture medium, were newly isolated from tunisian soils. The solubilization of insoluble phosphates was associated with a drop in the pH of the culture medium. Soluble phosphate production under optimal conditions varies from 200 to 1100 mg/l depending on the nature of the inorganic phosphate source. HPLC analysis performed for one of these isolates revealed that the solubilization of hydroxyapatite was mainly caused by secretion of gluconic acid resulting from glucose oxidation. With their market inorganic phosphates solubilizing capacity, these bacterial isolates can be used as strong phosphate solubilizers in agriculture environments to increase plant-available phosphate.


Session 2 /

PC88 S2

Removal of zinc from aqueous solution by living duckweed (Lemna gibba). Khellaf N. and Zerdaoui M. Laboratoire du génie de l’environnement, faculté des sciences de l’ingénieur Université Badji Mokhtar, B. P 12, El Hadjar, Annaba 23000, Algérie.

Abstract: The ability of the duckweed Lemna gibba to remove zinc from water was investigated 2+

in a quarter coïc solution containing 18 mg/L of Zn supplied such as zinc sulphate (ZnSO4). The experiments were performed in plastic vessels with a 1 litre capacity along 7 days. The results showed that under experimental conditions (pH = 6 ± 0.1, T = 21 ± 1° C, photoperiod = 10h/j), 1,5 g (fresh weight) of Lemna fronds removed 80% of zinc from the nutrient solution. Elevated temperatures were more efficient in the metal uptake; at 29 ± 1° C, L. gibba reduced Zn concentration to 0.3 mg/L which is below the safety limit set by the US Environmental Protection Agency (EPA). The results obtained from this study, proved that the duckweed species could be used in clean up technologies for the removal of toxic elements from contaminated waters. The metal could then be extracted for industrial uses. Key words: L. gibba, Phytofiltration, Removal efficiency, Zinc.


Session 2 /

PC89 S2

Correlation de la biotypie, l’antibiotypie et la serotypie pour differentier des souches de p.aeruginosa isolees dans l’eau potable au chu dorban (1)

Bourafa N

(1)

, Ouanas S , Chéraiti N

(1)

(1)

et Boutefnouchet N

(1) : Laboratoire de Microbiologie, Département de Biochemie, Université Badji Mokhtar. Faculté des Science. BP. 12. 23000 Annaba. E-mail : [email protected]

Résumé : La corrélation entre les trois marqueurs phénotypiques : la biotypie, l’antibiotypie et la sérotypie pour différentier des souches de P.aeruginosa à un intérêt seulement en cas d’absence des souches non agglutinables ou auto-agglutinables. De ce fait nous avons combiné les résultats de ces marqueurs phénotypiques déjà cités pour différencier 18 souches de P.aeruginosa isolées dans l’eau potable dans deux services hospitalier ciblés au sein de CHU IBN-SINA (service de dermatologie : 8 souches, service de pneumologie : 10 souches). L’identification biochimique des souches collectées a été effectuer par la galerie miniaturisé API 20NE. La Gamme d’antibiotiques testée compte : les β-lactamines, les aminosides, les quinolones, la fosfomycine et la colistine. Le sérotypage a été réalisé par le test d’agglutination sur lame. Les résultats ont montré que les souches isolées dans le service de dermatologie présentant le même sérotype (O : 1) et les même profils biochimique et de résistance ; en revanche les souches isolées dans le service de pneumologie appartenant au même sérotype (O : 12) et présentant le même profil biochimique par contre elles montrant des profils de résistance différents (Antibiotype A : 7 souches, Antibiotype B : 3 souches) , ce résultat ne confirme pas la non appartenance de ces souche, car il ne faut pas négliger les inconvénients de l’antibiotypie qui sont l’acquisition possible de plasmide ou les réarrangements moléculaires au niveau du chromosome entraînant une modification de la sensibilité des souches. D’autre part, la similitude des résultats de l’antibiotypie et de la biotypie est un argument permettant de suspecter l’identité de plusieurs souches appartenant au même sérotype. Cependant, un approfondissement de notre travail en combinant des marqueurs moléculaires afin de confirmer l’identité de ces souches pour connaître si elles sont issues d’une même unité ancestrale. Mots clés : biotypie, antibiotypie, sérotypie, P.aeruginosa, différentiation.


Session 2 /

PC90 S2

Purification and biochemical characterization of a fungal pectate lyase 1

1

1

2

Naourez Damak , Rihab Aydi , Noomen HadjTaïeb , Estelle Bonnin , Jean François 2

1

Thibault and Ali Gargouri 1-Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, 2- Unité Biopolymères, Interactions, Assemblages, INRA Nantes France.

Abstract: The pectinolytic system is composed of several enzymes distributed in three main groups: pectinesterases which catalyse the de-esterification of methoxyl groups, pectin depolymerases that cleave α-(1-4) glycosidic linkages by hydrolysis or trans-elimination reactions (pectin and pectate-lyases) and disbranching class which catalyses the cleavage of hairy regions. Pectate lyases are widely used in industry considering their optimum pH of activity which is between 8 and 10. Considering the importance of these pectinases, we have purified a pectate lyase from the CT1 mutant strain of Penicillium occitanis and studied its biochemical characteristics and its mode of action by HPAEC. The CT1 mutant produces high amounts of pectate lyase (PAL) when grown on apple pectin. Using ion-exchange and gel filtration chromatography, one PAL enzyme was purified and found to have a molecular weight of 38 kDa. The purified enzyme exhibited its highest activity at pH 9 and showed another peak at pH 7. The maximal enzyme activity was obtained at a temperature of 60°C. The presence of 2mM calcium enhanced pectate lyase activity. The activity was strongly inhibited by EDTA. This Pectate lyase of the CT1 mutant strain has been applied on polygalacturonic acid and the products were analyzed by HPAEC: the highest level of tri-galacturonic acid was obtained after 30 min and the di-galacturonic acid after 1h of reaction. These oligosaccharides with high added value could be used in various biotechnological fields.


Session 2 /

PC91 S2

Reduction of surfactants by adsorption on hydrothermal kaolin N. Hezil and K. Guerfi Laboratory of water treatment and recycling of industrial waste, Department of Chemistry, University:-Badji Mokhtar, Annaba, 23000.Algérie, E-mail: [email protected]

Abstract: Surfactants are chemicals that are found in the effluents of several industries (textiles, paper, detergents,…). A current regulation in the field of environment requires a reduction of their residual concentration. The degradation of ionic surfactants in water has been the subject of several works. Thus, the biodegradability of anionic surfactants has been shown by aerobic microorganisms. But little work has been devoted to the separation of this type of molecules of water. Therefore, in our work we studied the adsorption in aqueous phase of ionic surfactants (BDDAB, SDS) and non-ionic surfactant (Tween20) onto the hydrothermal Algerian kaolin. The results show a good adsorption capacity of the solid. Key words: pollution, kaolin,, adsorption, surfactants.


Session 2 /

PC92 S2

Production and purification of xylanase from Jonesia sp. Isolated in Algerian soil Nabti-Boucherba N1., Benallaoua S1., Hebal H1. and Duchiron F2. 1-

Laboratoire de Microbiologie Appliquée/ Biochimie Microbienne, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia Route de Targa ouzemmour 06000, Algérie. 2

-Laboratoire de Microbiologie Industrielle, UFR Sciences, Université de Reims Champagne-Ardenne, Moulin de la Housse, BP 1039, 51687 Reims, France. Email : [email protected]

Abstract : Xylanase in synergism with several enzymes can be used for the generation of biological fuels, such as ethanol and xylitol from lignocellulosic biomass. Jonesia denitrificans BN-13 was isolated from a sample collected from Algerian soil. This culture produced extracellular xylanase, the maximum of xylanolytic activity (2.55U/ml) was observed at the beginning of the stationary phase of the bacterial growth. The best xylanolytic activity is obtained in a medium made up of xylan (7mg/ml), extract of yeast (2,5mg/ml), NaCl (3mg/ml), NH4Cl (6mg/ml), and of MgSO4 (0,3mg/ml). It also proved that the best xylanase production (0.6/ml) was obtained in growth medium containing Birchwood xylan at 37°C and pH7, xylanase activity was also observed when wheat bran (0.23U/ml) and wheat arabinoxylan (0.46U/ml) were used as substrate. In the presence of metal ions such as Mn2+, the activity of the enzyme increased, whereas inhibition of enzyme activity was observed in the presence of Hg2+. EDTA did not significantly affect these activities. The enzyme was purified to homogeneity using anion exchange on Q sepharose and gel filtration on sephacryl S200. At the best of our knowledge, this is the first report on the production and purification of xylanase by this bacterium and especially by the genus Jonesia.


Session 2 /

PC93 S2

Contaminated Sites: A Source of Biodegradable Plastic Producing Bacterial Strains 1

2

3

4

Nazia Jamil , Nuzhat Ahmed , David H. Edwards and Shahida Hasnain 1,4

Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore-54590, Pakistan. 2

Centre for Molecular Genetics, University of Karachi, Karachi-75270, Pakistan. 3

Department of Molecular and Cellular Medicine, University of Dundee, Dundee DD19SY, Scotland, UK.

Abstract: Biodegradable plastic was extracted from 70 bacterial strains, which were isolated from different contaminated environments of Pakistan. All the strains were analyzed for antibiotic and heavy metal resistant markers. Extraction of PHA was done by SDS and solvent methods. CMG607w bacterial strain isolated from sediment of Layari River out fall to Arabian Sea. PHA synthesis was substrate depended in CMG607w. In presence of sodium gluconate mcl-Pha was synthesized at the rate of 42% cell dry mass. Under highly enrich conditions, co production of polysaccharide and blends of PHB/PHA were observed. PCR base strategy was used to amplify Pha biosynthesis operon. In CMG607w Pha biosynthsis operon has PhaC1ZC2D (polymerase1, depolymerase, polymerase2 and hypothetical protein) genes orientation. Conserved sequences were observed in polymerase C1 and C2. All gene of Pha operon was cloned and sequenced. Pha biosynthesis operon of CMG607w has 98% homology to Pseudomonas aeruginosa PAO1 (AE004919). All gene sequences were submitted to GenBank. Key words: contaminated sites; biodegradable plastic, extraction, Pha synthase operon


Session 2 /

PC94 S2

Decolourisation of recalcitrant phenolic compounds of fresh and stored olive mill wastewaters by Geotrichum candidum 1,2

Nedra Asses

1

and Moktar Hamdi

1 : Laboratoire d’Ecologie et de Technologie Microbienne, Institut National des Sciences Appliquées et de Technologie (INSAT), 2 Boulevard de la terre, B.P. 676, Tunis1080, Tunisia 2 : Institut Supérieur des Sciences et Technologies de l’Environnement de Borj-Cedria (ISSTE), B.P. 1003 Hammam-Lif 2050, Tunisia. E-mail: [email protected]

Abstract: Treatment of olive mill wastewaters by different biological process induced an important decrease of COD but the black coloration of this effluent persists. Decolourisation of phenolic compounds contained in fresh and stored-black olive mill wastewaters by Geotrichum candidum was investigated. During storage of OMW, autooxidation and subsequent polymerisation of phenolic compounds and tannins, gives rise to darkly coloured phenolic compounds which are not easily biodegradable. G. candidum growth on fresh OMW decreased pH and reduced COD and colour by 75 % and 65 % respectively after 6 days of incubation. G. candidum hydrolysed phenolic compounds with high molecular weight and removed many simple phenolic compounds. G. candidum growth on the stored-black OMW was rapidly inhibited resulting in low reduction COD (15 %) with no decolourisation because phenol polymerisation was amplified by the increased pH and oxygen. The addition of oxygen limit biodegradation of phenolic compounds is critical in order to avoid the polymerisation of phenolic compounds and tannins. G. candidum growth on the stored-black OMW is enhanced by the addition of a carbon source easily metabolised, 10g / litre of glucose, leading an improvement of the COD reduction and decolourisation that reached 58 % and 48 % respectively. Decolourisation is the result of depolymerisation of high molecular mass polyphenols combined with mineralization of wide range of aromatic compounds. This depolymerisation is correlated with enzymes secretion of the lignin peroxidase and the manganese peroxidase in a static culture in presence of a substrate easily metabolised and in limited conditions of nitrogen.


Session 2 /

PC95 S2

Isolation and partial characterization of antimicrobial compounds from a new strain Streptomyces sp. CN207. 1

1

1

2

Nedra Slama , Hadeer Lazim , Insaf Barkallah and Fèrid Limam (1) Unité de Microbiologie et biologie moléculaire. Centre National des Sciences et Technologies Nucléaires de Sidi Thabet, 2020 Technopôle de Sidi Thabet, Tunisie. (2) Laboratoire Interactions LégumineusesMicroorganismes, Centre de Biotechnologie de Borj Cedria, BP910-2050 Hammam-Lif, Tunisie. E-mail: [email protected]

Abstract: A distinct Streptomycetes strains were isolated from Tunisian soil. The isolate designated CN207, was assigned to the genus Streptomyces on the basis of morphological and chemotaxonomic criteria. A 16S rDNA sequence of the isolate was determined. Streptomyces sp CN207 secreted large amount antibiotic against Gram positive bacteria, gram negative bacteria, yeast and fungi on his barley (HB) medium. HB medium was found to be suitable substrate of the medium for CN207 production. Maximum yield of CN207 product (700 mg/ml) after optimize fermentation process. Bioactive molecules from strain CN207 were extracted with ethyl acetate and analyzed by PTLC using silica gel plates. The separated compounds were visualized under UV at 254 nm and the active spots were detected by bioautography on silica gel plates using Salmonella thyphimurium NRRL B4420 and Staphylococcus aureus CDC 103 as indicator microorganisms. The crude extract (8.36 g) was fractionated on Sep-pack® column (C18 cartridge) and elution was performed using a discontinue gradient of methanol-water. Two active fractions eluted by 20% and 40% of methanol were obtained. The bioactive compounds were separated by preparative high performance liquid chromatography (HPLC) on a C18 reversed phase column and eluted with a linear gradient of acetonitrile-water in presence of 0.1% formic acid. The peaks were collected separately, concentrated and bioassayed against the routine indicator microorganisms. The absorption spectrum of the active molecules was determined with a Shimadzu UV-160 A spectrophotometer. Determination of the chemical structure of these compounds 1

13

on the basis on their IR, COSY and H/ C NMR is in progress.


Session 2 /

PC96 S2

Effect of the use of by-Product Resulting from some Agricultural Industries on Synthesizing Single – cell Proteins through the use of Strains from Candida Utilis Yeasts Nisrine Nakshoo Abstract: During the experiments on batch methods, fermentation using molasses as a medium a different pH, temperature, and levels of aeration . the highest productivity of 33.258% was at pH = 4.48 , temperature 33ºC level of aeration 6% , the productivity reached the maximum value of 2.6274g/l.h.under the same conditions. Therefore the above mentioned condition can be considered as optimal ones for the growth of Candida utilis yeast. The continuous fermentations method, has been applied under the same conditions of temperature, pH and -1

-1

aeration, the result show that the yield at (D1=0.142 h ) was 37.232% and at D2=0.206h ) -1

37.0118% it is also shown that the relative growth speed reaches the heist value (0.49 h ), while the value of Ks was 0.919 g/l. Another experiment of batch fermentation methods using a mixture of molasses and hydrolyzed starch as medium (50% of each), at different levels of aerations indicate that at 14% aeration , the yield was maximum 30.37% and the value of highest productivity is 1.51g/l.h, at the same level aeration for the growth of candida utilis is 14% in a mixture medium. The same level of aeration was used in the continuous fermentation method, and the -1

optimal results were; the yield of 37.47% obtained at (D1=0.081h ) and 36.322% at -1

(D2=0.139h ) which are higher than the batch method. -1

The relative growth speed reaches the maximum value (0.325h ), which the Ks value amount to 1.6965 g/l. It appears that, the increase of glucose in the medium affected negatively the use of soluble oxygen; it is also shown that during batch methods, the fermentation processes has been stopped when the sugar concentration reaches 22g/l, that because of the absences of other essential nutrient for the yeast growth normally found in molasses.


Session 2 /

PC97 S2

Recyclage des boues residuaires en agriculture et etude de leur impacte sur la fertilite chimique du sol Boulahbal O et Bourennane N. Laboratoire de Science du sol (INRAA)

Résumé : En Algérie, la restitution des sols en matière organique et en éléments nutritifs est insuffisante en raison d’une fertilisation minérale faible et d’apport organique très limité, provenant essentiellement des résidus de récolte. De ce fait les sols s’appauvrissent de manière accrue et voient 0,8 à 1,2 % de leur humus se détruit annuellement. Devant cette situation, l’amendement organique devient très impératif. En dehors des résidus agricoles couramment utilisés, il existe des déchets urbains solides et liquides (boues résiduaires, ordures ménagères, eaux usées traitées...), des déchets industriels (le bois, les textiles, les papiers, les huiles brûlées ...), et des sous produits agricoles et agro-alimentaires (marcs de raisin, sang d’abattoir, fiente et litière de volailles...) qui peuvent être valorisés comme amendements. Dans ce travail, il sera question de valoriser les boues issues de la station d’épuration de Reghaia, en les caractérisant et en étudiant l’évolution de leur matière organique dans le sol et son impact sur la fertilité chimique du sol. Mots clés : Boues résiduaires, amendement organique, sol, fertilité chimique


Session 2 /

PC98 S2

A new isolated Burkholderia cepacia strain having interesting antifungal activity against many phytopathogenic fungi Kilani Feki, O., Zouari, N. and Jaoua, S.* Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box: K, 3038. Sfax, Tunisia, *E. mail: [email protected]

Abstract: In the course of screening for antifungal compounds from microbial origins, various strains were isolated from environment using standard dilution agar plate method. One bacterial isolate having interesting antifungal activities was selected for further studies. It was designated Cs5 and identified as Burkholderia cepacia by many biochemical and molecular identification tests and particularly by 16S rDNA and recA sequence homology search. This bacterium exhibited an amazing activity against phytopathogenic fungi such as Aseargillus niger, Rhizoctonia solani, Botrytis cinerea, Alternaria alternata, Fusarium oxysporum Fusarium graminearum and Fusarium culmorum. Minimal inhibition concentrations (MIC) of the supernatant for each fungus were determined using serial broth dilution method. The data demonstrated a MIC ranging from 1 to 3 %. With optical and confocal microscopy, profound morphological alterations of fungal cells were observed after exposures to the supernatant of Cs5 because of the changes of cell permeability. The in vivo application of the bacterium showed that Cs5 is an endophytic bacterium and was able to protect in vitro cultivated vine totally against Botrytis cinerea in growth room conditions. To investigate antagonistic principle, antifungal compound was extracted and purified using organic solvent and column chromatography. The mass spectral analyses of the pure antifungal compound showed that this molecule have a mass of 414 Da.


Session 2 /

PC99 S2

Effects of antimicrobial activities produced by Bacillus subtilis sp. B38 on the viability of bacterial strains 1

1

2

1

Tabbene O ., Ben slimene I ., Mangoni M. L . and Limam F 1

Laboratoire Interactions Légumineuses-Microorganismes, Centre de Biotechnologie, Technopole Borj-Cedria, BP-901, 2050 Hammam-Lif cedex, Tunisia. 2

Unità di Diagnostica Molecolare Avanzata, II Facoltà di Medicina e Chirurgia, Azienda Ospedaliera S. Andrea Roma, Italy

Abstract: The Gram-positive bacterium Bacillus subtilis produces a large number of antibiotics with different mecanism of action. Bacillus subtilis sp. B38, isolated from soil, produces antimicrobial activities with both bactericidal and bacteriostatic effects against a Grampositive Bacillus thuringiensis sp. 15 and a Gram-negative E-coli D21 bacterial strains. The corresponding antimicrobial agents were extracted with methanol solvent and were partially purified by C18 reverse phase chromatography. Three active fractions were eluted with 20, 40 and 60% acetonitrile and were designated as fraction A, B and C respectively. The fraction A showed activity only against the Gram-positive bacterium Bacillus thuringiensis sp. 15. No activity was observed against E-coli D21. The MIC value on Bacillus thuringiensis sp. 15, determined in liquid media was about 213.75µg/ml. This fraction exhibited bacteriostatic effect even at 4MIC concentration. However, active fractions B and C were bactericidal to both Gram-positive and Gram-negative bacterial strains. Their MIC values on Bacillus thuringiensis sp. 15 were about 45 and 58,12µg/ml respectively and were about 150 and 186µg/ml on E-coli D21 respectively. In order to investigate the mecanism of action of these two fractions, we examined their effect on the integrity of the E-coli D21 inner membrane by monitoring the leakage of the cytoplasmic ßgalactosidase. Both fractions B and C increase the permeability of the E-coli D21 inner membrane. The enzymatic activity measured in the supernatant was approximately 65.5 and 80% respectively at bactericidal concentrations (600 and 744µg/ml respectively). To confirm results obtained with ßgalactosidase activity, we measured the influx of the vital dye TM

SYTOX green probe. A significant increase of the fluorescence was observed for both fractions at bactericidal concentrations. These results suggest that bacterial inner membrane might be the site of action for both fractions B and C.


Session 2 /

PC100 S2

Remediation of contaminated water by Cr (IV) using a sample clay sodium. O. Tobbi; K. Guerfi and N Rebbani. Laboratory water treatment and recycling of industrial waste, PO Box 12, Department of Chemistry, University Mokhttar Badji, Annaba.

Abstact: Clay is natural materials that contain clay minerals, compounds which do no plasticity (eg quartz) and sometimes organic matter. The determination of physical and chemical properties and the identification of phases constitute a prerequisite for studying the mechanisms of interaction between these materials and natural minerals from aqueous solution. This determination requires the combination of several techniques. The characterization techniques used in this work are: BET, RX, INFTR, ATG and ATD. The second part is the use of sodium bentonite for the remediation of contaminated water by Cr (IV). The results show that the sodium bentonite has a capacity sorptionnelle importantly; this capability could be interpreted in terms of adsorption isotherm exploiting the Langmuir model. Key words: environment, water, pollution, heavy metals.


Session 2 /

PC101 S2

Biotransformation of two polycyclic aromatic hydrocarbons (PAHs) by Absidiafusca Linnemann. Bordjiba Ouahiba Université Badji Mokhtar, Faculté des Sciences, Département de Biologie, Laboratoire de Biologie Végétale et Environnement Annaba. E-mail : [email protected]

Abstract: A strain of Absidia fusca was isolated from a pesticide-contaminated soil (Annaba, Algeria). The biotransformation capability of this strain towards two polycyclic aromatic hydrocarbons (PAHs): anthracene and fluoranthene was compared to that exhibited by another strain of A. fusca isolated from a non-contaminated milieu and considered as a control. The results obtained were statistically analyzed and showed that the strain isolated from the contaminated soil was more efficient than the control to remove anthracene from the medium, during all the kinetics (90% removed versus 45% after 24 hrs). Concerning fluoranthene, the amount removed by both strains was very high during the first 24 hrs however the control strain was slightly more efficient (94% versus 89%) while the results were similar for the two strains during the rest of the kinetics. This study reveals for the first time the potential interest of the species A. fusca for the bioremediation of PAHs. Key words: PolycyclicAromatic hydrocarbons, Fluoranthène, Anthracene, Absidia fusca Biodegradation, Kinetics


Session 2 /

PC102 S2

Isolation, identification and susceptibility testing of lactic acid bacteria associated with fish Ouissal Chahad Bourouni, Monia El Bour, Radhia Mraouna and Abdellatif Boudabouss Abstract: In order to isolate lactic acid bacteria from the fish in aquaculture (Sea bream and Sea bass), we have made several samples from the skin and the intestinal tract of fish. Thus, 58 lactic acid bacteria strains were isolated, purified and identified on the basis of the morphological characteristics, mobility and research of respiratory enzymes and then using API systems 50 CHL. Eight species were identified: Leuconostoc mesenteroides 6.9%, Lactococcus lactis 34.5%, Lactobacillus paracasei 15.5%, Lactobacillus plantarum 15.5%, Carnobacterium piscicola 13.8%, Lactobacillus brevis 6.9%, Lactobacillus pentosus 3.4% et Lactobacillus acidophilus 3.4%. All these species were found both on the skin of fish in their intestinal tract. The study of the sensitivity of these strains to 15 different antibiotics belonging to different families revealed a multiresistance of these strains to 4-10 antibiotics. The study also showed the effectiveness of penicillin, triméthoprim-sulfamids and furan to inhibit the growth of all strains.


Session 2 /

PC103 S2

Water treatment contaminated with aromatic hydrocarbons: Biodegradation of phenol by Pseudomonas aeruginosa in batch O. Ali, A. Namane and A. Hellal Ecole Nationale Polytechnique d’Alger, Laboratoire des Sciences et Technique de l’Environnement LSTE, 10 Avenue Pasteur, BP.182, El-Harrach, 16200, Alger, Algérie. E-mail : [email protected]

Abstract: Our common responsibility is to preserve the health and bequeath to our children a world in which resources and ecosystems are intact. It is in this vein that our work is directed towards the problems of water treatment contaminated by hydrocarbons, especially phenol. To do so and to avoid the toxic effects of phenolic substances discharged into the water on the environment, the World Health Organisation (WHO) as giving desirable maximum concentration of phenolic compound in the waters of beverages 1 μg/l as well as it is necessary to carry out appropriate treatment of these substances prior to discharge into the environment. Water pollution by phenolic compounds, whether chronic or accidental, is a big problem in terms of degradation. While the elimination of phenol, this toxic substance, uses various physical and chemical treatments, which are limited by their cost or their secondary impacts on the environment, and often these degradation pathways are merely transferring pollution from one medium another in concentrating, which encourages us to use biological treatments that are increasingly taking place mostly they are not expensive, and they allow mineralization total phenolic compounds. The degradation of phenol by the pure and mixed cultures has been a subject of many studies. A number of micro-organisms, including Pseudomonas (P.aeruginosa, P.putida) are able to grow using phenol as a source of carbon and energy. The present study focuses on the study of the influence of the composition of the middle of phenol degradation by Pseudomonas aeruginosa in batch.


Session 2 /

PC104 S2

Treatment of rinsing water containing chromic acid by electrodialysis Nabila Boutemine, Zahia Benredjem, Azzeddine Grid and Rachid Delimi Laboratoire de Traitement des Eaux et Valorisation de Déchets Industriels, Dépt. De Chimie, Faculté des Sciences, Université de Badji Mokhtar Annaba, BP12, 23000 Algérie Tel/Fax : 231 (0) 38 87 65 67, E-mail : [email protected]

Abstract: When passing from chromic to rinse bath, the treated spares draw important quantities of chromic acid in rinsing water. Due to the high toxicity of chromic acid, this contaminated water constitutes an important source of pollution. Indeed, there is a double interest of treatment: to avoid pollution and recuperate chromic acid relatively expensive. Also, the heavy metals are separated from the acid and confined into a small volume. The old techniques are either destructive (hydroxide, carbonate precipitation, with the inconvenient of metal and water loss) or recuperative, such as evaporation and recently ion exchange. Among the possible alternatives, electromembrane techniques, namely the electrodialysis, provide a lot of advantages that are integrated into the world tendency toward matter and energy management. The influence of certain parameters (current density, circulation speed and membrane nature) was studied. The results show that the elimination rate of impurities increases versus current density. Among the two ion exchange membranes CMV and CDS, the membrane CMV gives a higher elimination rate. Furthermore, the efficiency of impurities elimination grows strongly versus circulation speed. The developed speed can be considered as the appropriate technique for chromic acid recovery starting from rinsing water treatment of plated parts. Key words: water treatment, chromic acid, pollution, electrodialysis.


Session 2 /

PC105 S2

Actinomycetes strains isolation for biotechnological interest: resistance to heavy metals and antimicrobial activity. Mimouni*, R. and Khnifir, S. Ibn Zohr University, Faculty of Science, Laboratory of Aquatic Systems: Marine and Continental Environment, Agadir, Morocco. *E-mail: [email protected]

Abstract: The aim of this study was to isolate micro-organisms with biotechnological interest that can be exploited in the field of bioremediation of polluted soils. We isolated and studied bacteria belonging to the family of actinomycetes. We have searched for these microorganisms in sites polluted by waste water (Wastewater treatment plants, mounths,…). Thirteen isolates of actinomycetes were isolated and selected, on the basis of the aspect of the morphology aspect of their clones and their mutual antagonism. these isolates were studied to determine i) their resistance to heavy metals: cadmium (Cd (II)), Chromium (Cr (VI)) and Mercury (Hg (II)), by using qualitative Screening tests, semi quantitative and toxicity tests ii) their antimicrobial activity towards a set of bacteria and of yeasts known in human pathology. The results show that the majority of resistant strains isolated were tested for heavy metals and\or present an antimicrobial activity for one or more Gram negative or Gram positive bacteria. This preliminary study yielded some interesting results which are worth pursuing in order to improve the performance of these strains. Key words: Actinomycetes, antimicrobial Activity, Bioremediation, Waste water, heavy Metals.


Session 2 /

PC106 S2

Use of Phragmites australis as biological model of phytoremediation (1)

Rahmoune, C. , Lemzeri, H.

(1, 2)

, Maalem S.

(3)

(4)

and Ben Naceur M

(1) Laboratory of Ecotoxicology and Abiotic Stress, Biology and ecology, Faculty of SNV, University MentouriConstantine, Algeria.(2 Department of Biology, Jijel University, Algeria) E.Mail : [email protected] (3) Department of Biology, University Center of Sheik Larbi Tbéssi – Tébessa, Algeria (4) Laboratory of Plant Physiology and Biotechnology, INRAT, Hédi Karray Street, 2049 Ariana-Tunis, Tunisia.

Abstract: The pollution of the environement is a complex phenomenonthat should not pe perceived only through the vision of the polluents poured in streams or other receivers. In most of the cases, the polutants are spread far fromthe point of rejection by the water system circulation. Pollutants do not stay in earth and waters, but after wards, they pass to living organisms that they will contaminate. This leads to the use of biological models for the phytoremédiation of surroundings and to the the control of the urban and industrial wastes. In the framwork of the study of the impact of urban and industrial rejections, paticularly leather factory rejections, we have been interessed by the specificity of the bioaccumulatin of 6 heavy metals (Hg, Pb, Zn, Cu, Cr, Fe) by reed plants (Phragmites australis L.) and its photoremadiation power. The first results show that there is a best oppotunity for the use of this plant for a drastic reduction of water pollution bt heavy metals. Key words: Phragmites australis, Heavy matals, aquatic pollution, Phytoremediation


Session 2 /

PC107 S2

Contribution a une classification mycologique phylogenetique Djelloul R. Centre Universitaire d’El Tarf -AlgérieAdresse professionnelle : C.U.E.T. B.P. 78 W EL TARF 36000, E.mail : [email protected]

Résumé : Les systèmes de classification sont en perpétuelle évolution : l’introduction de caractères moléculaires et de la méthode phylogénétique a modifié l’approche actuelle de la classification des champignons, vaste groupe formé de plusieurs lignées évolutives indépendantes. Cette communication, augmenté d’un glossaire, propose une mise au point illustrée de la classification phylogénétique des champignons au sens large, jusqu’au niveau ordinal, avec un rappel des principales apomorphies morphologies et une discussion des termes obsolètes ou à surveiller. Mots clés : amibes, apomorphies, champignons, classification, phylogénie, hétérocontes.


Session 2 /

PC108 S2

Upgrading and pollution reduction of by-products generated by the seafood conditioning industry using enzymatic hydrolysis coupled to membranes processes: application to cuttlefish Sepia officinalis Emna – Soufi Kéchaou*, Abderrahman Bouain*, Pascal Jaouen**, Jean Pascal Berger*** and Raja Ben Amar* * Laboratoire Sciences des Matériaux et Environnement, Faculté des Sciences de Sfax, ** GEPEA/UMR CNRS 6144 , université de Nantes, *** IFREMER, Nantes

Abstract: It is well known that the seafood conditioning and transformation activity generates large quantities of solid by-products. Because environmental preservation has become a major problem, there is an urgent need to have recourse to different means of treatments in order to valorise these by-products, among these means, enzymatic hydrolysis and membrane processes. The aim of this work is to study the coupling enzymatic bioreactor and membrane in order to obtain interesting molecular fractions (peptides) for the cuttlefish by-products obtained from the cuttlefish conditioning and freezing industry. Some bioactivities like antimicrobial and antioxidant were be characterized in the different molecular cuts obtained after viscera hydrolysis, including the ink gland. The experimental work is composed of a specific part dedicated to enzymatic hydrolysis using commercial proteases and then fractioning by membranes techniques. The first chemical analysis carried on the supernatant on the ink showed a high content of proteins. The pH-stat enzymatic hydrolysis (24 h, pH=8, 50°C) led on the viscera without the ink gland, using alcalase, protamax and flavourzyme showed an important recovery of lipids, phospholipids and proteins. The highest degree of hydrolysis (DH) was obtained with alcalase (7%). The analysis of the molecular profiles by size exclusion chromatography done on the raw material (non hydrolysed) and the three supernatants shows a difference in the molecular weights. The 6 hours enzymatic hydrolysis without pH regulation was carried out with protamex, devolase and pepsin in their optimal conditions. The highest yield in proteins was obtained by protamex using two kinds of substrates: viscera with and without ink gland. The expecting results of this work concern not only the developing of new technologies and methodologies useful in the valorisation of seafood industry, but also the recovery of bioactive peptides, potentially interesting in animal nutrition.


Session 2 /

PC109 S2

Kinetic and interfacial properties of a novel Rhizopus oryzae lipase. 1

1

1,2

Ben Salah R , Gargouri Y and Mejdoub H . 1

Laboratoire de Biochimie, ENIS, Route de Soukra, “BPW”, 3038 Sfax –Tunisia . Unité de service commun pour la recherche scientifique, FSS, Route de Soukra, Km 3.5; BP 802, 3038 Sfax2

Tunisia .

Abstract: We compared several kinetic and interfacial properties of a lipase from a novel strain of Rhizopus oryzae (ROLw) with ROL29 lipase. In contrast to ROL29, ROLw was able to hydrolyze triolein emulsion in the absence of any additive, like bovine serum albumin (BSA). Furthermore, unlike Rhizopus oryzae lipase (ROL29), kinetic study of ROLw lipase when using tributyrin emulsion as substrate, shows linear dependency. ROLw can tolerate, more efficiently than ROL29, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate. The critical surface pressure πc of penetration into phosphatidyl choline from egg yolk films was found to be 23 mN/m with ROLw, in contrast to a value of 10 mN/m obtained with ROL29. The effect of calcium ion and synthetic detergent on the two lipases was studied. In contrast to ROL29, ROLw was activated in the presence of 100 μmoles TX-100. No significant difference on the two lipase activity was observed in presence or absence of calcium ion.


Session 2 /

PC110 S2

Trametes trogii crude laccase stability Rim Khlifi, Tahar Mechichi* and Sami Sayadi Ecole Nationale des Ingénieurs de Sfax, Route de Sokra Km 4.5, Sfax-Tunisia, Phone: (+216) 74 274 088 *E-mail : [email protected]

Abstract: Trametes trogii, isolated from decayed acacia wood (from Northewest of Tunisia) and identified, was selected in a broad plate screening because its ability to degrade textile wastewater and commercial dyes. The purpose of the present study was to determinate the effect of several naturally and synthetic occurring phenolic compounds to the stability of the T. trogii crude laccase in the process of effluent textile decolourization. We tested the effect of the textile wastewater to the reaction mixture on the textile effluent decolourization process. For this, the effluent decolourization was investigated by subsequent addition of the textile wastewater (5%). In the other hand, we characterized in terms of its catalytic stability laccase in the absence or presence of four common redox mediators and textile wastewater. The enzyme keeps 100 % of its activity in the presence of 20 % of textile wastewater, but loses from 10 % to 20 % of its initial activity in the presence of effluent in 40 %, 60 % and 100 % concentrations. Laccase was inactivated when incubated in the presence of mediators including syringaldehyde, acetosyringone, 1-hydroxybenzotriazole (HBT) and vanillin at pH 5 and 30 °C. The laccase is more stable in the presence of the vanillin (it lost 50 % of its activity in the presence of 3 mM of vanillin ), but with 3 Mm of syringaldehyde and acetosyringone the laccase lost 100% and 75% respectively of its activity after 3 days.


Session 2 /

PC111 S2

Effectiveness of an electrocoagulation of reactor in pretreatment of wastewater of a dairy industry Hazourli, S., Belkadi, W., Boudiba, L. Laboratory water treatment and valorization of the industrial waste, Department of Chemistry, Faculty of Science, University of-Badji Mokhtar, Annaba 23000. E-mail: [email protected]

Abstract: The wastewater from the dairy-Edough Annaba have shown significant pollution load mainly by the parameters of MES, BOD, COD, the report COD / BOD was 1.81; it is significant for a major organic pollution . In this case the treatment of these waters is necessary. Many authors agree to purify dairy discharges by biological treatment because the nature of the spill is mainly organic. In this study, it is attempted the physico-chemical treatment using the process of electrocoagulation, this process is simple and easy to use even in rural areas. The test of electrocoagulation of the wastewater is being conducted in a laboratory reactor of 1litre. The potential imposed is 10 V; the water temperature is 20 ° C with and without electrolyte support at different concentrations of NaCl. Two inter-electrode distances are tested 6 and 2 cm. Preliminary results showed that the clarification of water is done after 5h of electrocoagulation, the introduction of a concentration 0.01 M of NaCl with an interelectrode distance of 2 cm considerably reduces the time it takes to clarification less than 5 min. The turnover rate of turbidity is significant, it is about 99%. Key words: Clarification, waste water, dairy, Electrocoagulation [1] Donini, J.C., Kan, J., Szynkarczuk, J., Hassan, T.A., Kar, K.L., 1994. Operating cost of electrocoagulation. Can. J. Chem. Eng. 72, 1007–1012. [2] Ivanishvili, A.I., Przhegorlinskii, V.I., Kalinichenko, T.D., 1987. Comparative evaluation of the efficiency of electrocoagulation and reagent methods of clarifying waste water. Sov. J. Water Chem. Technol. 9, 468–469. [3] Chen, X., Chen, G., Yue, P.L., 2000. Separation of pollutants from restaurant wastewater by electrocoagulation. Sep. Purif. Technol. 19, 65–76.


Session 2 /

PC112 S2

Calibration and validation of oxidation ditch model process Ben Alaya, S., Haouech, L., Cherif, H. and Shayeb, H. ENIT, Laboratory of Hydraulics and Environment Modeling; B.P 37, Le Belvédère, 1002 Tunis E-mail: [email protected]

Abstract: Oxydation ditchs were heterogeneous reactor in whitch aeration system managment was controlled on local dissolved oxygen measurement. One of the methods, taken a possibility to related local oxygen measurement by the concentration in the different ditch zones, is simulation. Oxygen concentration distribution is one of the control functions in wastewater treatment systems. The alternations of aerobic/anoxic zones make a possibility to improve nitrogen and organic matter wastewater removal. The intensity of aeration should be adjusted to accommodate the variation of influent carbon/nitrogen loading conditions. If the influent load level is high, the intensity should be increased to complete nitrification. On the other hand, if the influent load is low, the intensity needs to be decreased to save unnecessary aeration energy. Implimentation an aeration managment control method basis in oxygen uptake rate prediction of the biomass according to the wastewater load is a best alternative to optimize aeration. Thus using a simulation tools have for best using equipement capacity and a better managment process. In this work, ASM1 model was used for modelling the biological process of mahres WWTP, infact to manage aeration system. COD fractions were estimated basis in literature results (Stricker, 2000; Ekama et al., 1986; Alper et al., 2005; Sperandio, 1998; Roeleved, 2002; Kappeler, 1992, Cherif et al., 2007). The COD fractions estimated were; Ss = 34% and Si=6 %, Xs =56% and Xi = 4% of total COD. The stoechiometric and kinetic parameters were adjusted in hope of improving the -1 fit, using the GPS-X (version 5.0) optimizer module (YH = 0.38 gCOD/gCOD, bH = 1.35 d , 3

-1

Ks = 12 gCOD/m and μH = 7.36 d ). Model parameters and COD fractions compared with those of other Europeans and Tunisians WWTP show the specificity of Mahres wastewater. Calibration model was used to reproduce oxygen profile and to identify the alternative anoxic and aerobic zones wich limit nitification and denitification process.


Session 2 /

PC113 S2

Application des electrodes ioniques (eis) pour le controle de l’environnement aquatique S. Alimokhnache, K. Mokrani djelloul, N. Djamil Ammouchi et Djelloul Messadi Université BADJI Mokhtar -Annaba- 23000- ALGERIE Pays : ALGERIE Fax : 038 87 65 67 Email : [email protected]

Résumé : Les électrodes ions-sélectivites (EIS) qui permettent la détermination précise, sensible, rapide et non destructive d’une trentaine d’ions dans les milieux divers constituent un puissant outil d’analyse. Si les EIS produites par « Orion Research » sont universellement reconnues, il existe sur le marché des EIS à des prix plus bas et d’une plus grande facilitée d’approvisionnement ; certaines électrodes peuvent etre préparées au laboratoire. Ces électrodes doivent etre testées selon des critères définis. La dispersion des résultats dans la littérature scientifique et la variabilité des conditions d’analyses dans chaque cas d’espèce rendent difficile l’exploitation directe des résultasaccumulés pas d’autres chercheurs. Nous nous sommes proposés d’étudier systématiquement certaines EIS, en vue de leur application ultérieure au contrôle de l’environnement aquatique. Cette étude tient compte de leur plus ou moins grande facilité d’utilisation, des limites de sensibilité, des interférénces trouvées et décrit les protocoles d’analyses développés. En outre, des méthodes d’optimisation ont été appliquées pour le calcul à l’avance des coefficients de sélectivité, et pour l’étude de l’influence de l’interaction de facteurs chimiques (Concentration, PH, force ionique) sur la valeur du potentiel de l’électrode.


Session 2 /

PC114 S2

Antibacterial activity of Scorpion venoms and their fractions Daoud , S., Ghrib, M., Ben Hadj Ameur, J., Ghram, A. and Elayeb, M. Laboratory of venoms and Toxins and Laboratory of Veterinary Microbiology. Institut Pasteur de Tunis.

Abstract: One of the most important and sustained driving forces for antibiotic discoveries over the past half century has been antibiotic resistance. Therefore, it is of considerable interest to develop antimicrobials with new mechanisms of action which can potentially evade the emergence of new bacterial resistances. In search for such new molecules, we have looked for effective antimicrobial peptides which are ubiquitous in nature as part of the innate immune system and the host defence mechanisms. They have been increasingly recognized as a critical first line of defence against many pathogens isolated from various sources. Scorpions like insects, are particularly resistant to bacterial aggressions, and it appears of great interest to analyse molecules responsible for this resistance. We investigated the antimicrobial and the hemolytic effect of Scorpion venoms and semi purified fractions of these venoms and demonstrated that the venom of Androctonus australis hector showed an interesting antibacterial activity against Escherichia coli and Staphylococcus aureus. Other venom fractions from scorpions Buthus and Maurus are being studied to find new effective activities.


Session 2 /

PC115 S2

Effect of a range of composts on palm dates in vitro plant survival during acclimatization phase 1

2

1

3

2*

Salma Hachicha , Walid Kriaa , Emna Ammar , Khaled Medhioub and Noureddine Drira 1

National Engineering School in Sfax, P.O. "W", 3038 Sfax – Tunisia, 2

3

Faculty of Sciences in Sfax, P.O. 802, 3018 Sfax – Tunisia, Institute of Preparatory Engineering Studies; P.O. 805, 3018 Sfax - Tunisia UR Etude et Gestion des Environnements Urbains et Côtiers, LARSEN * Laboratory of Applied Vegetal Biotechnologies on Culture Improvement E-mail: [email protected]

Abstract: In oasis culture, date-trees cultivation is an important activity based on the ecological and socioeconomical aspects. The development of such culture requires the use of in vitro techniques allowing the plant growth in a relatively short time. Moreover, such propagation technique improves selected varieties diffusion without any contamination risk, as frequently occur while using conventional methods in the case of date-plants dissemination. The in vitro propagation requires an acclimatization phase which may generates a noticeable lost of vitro plants when conducted in the winter. Indeed, some white rot fungi contaminating the neck will occur depending on external factors (green house environment, physical, chemical and biological acclimatization substrates) as well as internal factors (vitro plants, physiological state). In this study, the effect of different substrates including conditioned compost was investigated. Composted olive-by products mixed with peat, perlites, coco nut fibres and chemical fertilizer were used as substrate and death rate of vitro plant of Medjhoul variety was recorded during the acclimatization phase. The results exhibited the efficiency of the used support for vitro plants growth. The vitro-plants grown on the mixed composts compared to a control substrate performed by grape marc mixed with peat and manure, exhibited a lower plant death percentage. The survival was improved especially in winter period. The control used an optimized substrate allowing a survival rate of 60% during winter-period and a higher percentage of 80% in summer. The olive wastes composted mixed with the experimented materials showed a survival percentage of 87% in winter and 95% in summer, exceeding all the rate previously established in the case of palm-date propagation.


Session 2 /

PC116 S2

Effect of olive mill waste water on soil chemical characteristics a, b

Salwa Magdich

b

a

, Béchir Ben Rouina and Makki Boukhris

a

Laboratoire d’Ecologie Végétale, Département de Biologie, Faculté des Sciences de Sfax 3018 Sfax, Tunisie b

Laboratoire d’Amélioration de la productivité Oléicole et des Arbres Fruitiers, Institut de L’olivier de Sfax 3000 Sfax, Tunisie E-mail : [email protected], E-mail : [email protected]

Abstract: The objective of this work is to investigate the influence of olive mill waste water (OMW) application on the evolution of soil chemical characteristics. For this reason, four 3

treatments were applied (0, 50,100 and 200 m of OMW / hectare / year) in the experimental site «Taous» of the Olive Tree Institute of Sfax (Tunisia). Results obtained showed that an increase of the different nutrient contents (organic matter, nitrogen, potassium, phosphorus, calcium and magnesium), until an optimum reached after 2 months of application, was recorded. After that, a decrease of all the nutrients was noticed and differences were not statistically significant, in comparison to the control treatment with the except for potassium content which remained at high level. 3

The higher potassium content observed particularly in soil amended with 200 m of OMW reflected the important role of this amendment in soil enrichment especially in potassium.


Session 2 /

PC117 S2

Molecular and enzymatic characterization of maltogenic amylase from Bacillus sp US149 Sameh Ben Mabrouk, Ezzedine Ben Messaoud, Dorra Ayadi-Zouari, Sonia Jemli and Samir Bejar Centre de Biotechnologie de SfaxLaboratoire d’Enzymes et de Métabolites des Procaryotes BP « K » 3038 Sfax, TunisieTél. : 74 870 816, Fax : 74 870 818, E-mail : [email protected]

Abstract: Maltogenic amylases (EC.3.2.1.133) are members of α-amylase family (family 13 of glycosyl hydrolase) that exhibited specific characteristics and have multifunctional activities [1]. These enzymes have many attractive properties that make them useful for a variety of applications in several industries. Baker industry, however, seems to be one of the most promising industrial applications of this kind of amylases. Accordingly, our laboratory was interested by the selection and the isolation of strains that produced this enzyme. A gene encoding maltogenic amylase from acidic Bacillus sp.US149 (maUS149) was cloned, sequenced and over-expressed in Escherichia coli [2]. The nucleotide sequence analysis revealed an open reading frame (ORF) of 1749 bp encoding a protein of 582 residues. Amino acid sequence alignment revealed an identity of 60 % of with the maltogenic amylase of Thermus sp.IM6501 (THMA). The recombinant enzyme (MAUS149) was found to be intracellular and was purified. The recombinant enzyme has an apparent molecular weight of 135 kDa and is composed of two identical subunits of 67.5 kDa each. The Maximum activity was obtained at 40°C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose from starch, maltose and glucose from β-cyclodextrin, and panose from pullulan. The studies on structure-function relation ship, by the mean of site directed mutagenesis and directed evolution, are in progress in order to improve the thermal activity and stability of the enzyme. References [1] Kim T. J., Kim M. J., Kim B. C., Kim J. C., Cheong T. K., Kim J. W. and Park K. H. (1999). Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable Maltogenic Amylase, the Gene for which was cloned from a Thermus Strain. App. Env. Microbiol. 65; 1644-1651. [2] Ben Mabrouk S., Ben Messaoud E., Ayedi D., Jemli S., Roy A., Mezghani M. and Bejar S. (2007). Cloning and sequencing of an original gene encoding a maltogenic amylase from Bacillus sp.US149 strain and characterization of the recombinant activity. Mol. Biotech. To be published


Session 2 /

PC118 S2

Purification and characterization of an amylase activity from a new isolated Strain of Bacillus SBG Maktouf Sameh and Ellouz Chaabouni, S. Unité Enzymes et Bioconversion, Ecole Nationale d’Ingénieurs de Sfax, BP : W.3038 Sfax Tunisia E-mail : [email protected]

Abstract: A new thermophilic strain of Bacillus was isolated from the Tunisian Soil. It produces many hydrolase activities as amylases, xylanases and endoglucanases. This strain produced two amylase activities (Amy1and Amy2). The second (Amy2) seems to be the product of the proteolysis of the first (Amy1). Amy2 was purified to homogeneity by a combinaison of gel filtration and anion-exchange chromatography. The molecular weight of this protein was estimated to be 100 kDa as determined by SDS-PAGE. Amy2 was optimally active at 70°C and pH 5.0. The enzyme showed high pH stability within the range of pH 5.0-10.0, and thermostability up to 50°C for 60 min.The purified enzyme had a Km of 5.0 mg/ml and Vmax 1600 µmoles/min/mg when starch was 2+

used as substrate. The enzyme activity was strongly inhibited by the divalent cations Hg , 2+

and Fe . The N-terminal sequence of Amy 2 showed no significant homology to others known amylases.


Session 2 /

PC119 S2

Prevalence of Ambler class A and B β-lactamases among carbapenem resistant Pseudomonas aeruginosa in a Tunisian hospital 1

1

1

2

1

Hammami, S ., Ghozzi, R ., Mamlouk. K ., Arlet, G . and Ben Redjeb, S . 1

Laboratoire de Recherche « Résistance aux Antimicrobiens » Faculté de Médecine de Tunis 2

Laboratoire de Bactériologie Faculté de Médecine Pierre et Marie Curie, Paris, France Boulevard 9 Avril 1006 Tunis, Tunisia

Abstract: Resistance to extended spectrum β-lactams in P. aeruginosa is mostly associated with the overproduction of chromosomal AmpC β-lactamase, or with non enzymatic mechanisms. Recently, several class A, B and D extended-spectrum-β-lactamases have been reported in P.aeruginosa. In this study, clinical isolates of carbapenem resistant P.aeruginosa producing class a β-lactamases and /or metallo- β-lactamase (MBL) were reported. From 2000 to 2006, 115 non duplicate P.aeruginosa resistant to carbapenem were isolated at Charles Nicolle hospital of Tunis. Antibiotic susceptibility testing, Etest-MBL, PCR amplification and sequencing of genes encoding class A (blaTEM, blaSHV, blaCTX-M) and class B (blaIMP, blaVIM) β-lactamases were performed. PFGE was used to study the relatedness of isolates harbouring MBL. The isolates were particularly recovered from urology (n=44) and collected from urines (n=52). The isolates were highly resistant to β-lactams and showed increased MICs of imipenem (MIC range 8->2048 µg/ml; MIC50 >2048µg/ml) and meropenem (MIC range 82048 µg/ml; MIC50 =32µg/ml). The Etest MBL was positive in 44 strains. PCR amplification with specifically designed primers showed that VIM-2, CTX-M-9 group, TEM, and SHV were carried by 38.3%, 45.2%, 1.7% and 41.7% isolates, respectively. 20.9% carried more than two types of β-lactamase genes. PFGE analysis identified one major profile (A) and 2 others (B and C). Sequence analysis revealed that one isolate contained 4 different β-lactamases (VIM-2, SHV2a, TEM-1 and CTX-M-14). This study showed that carbapenem resistant P.aeruginosa may be harbour several types of β-lactamases at the same time.


Session 2 /

PC120 S2

Optimization of growth conditions and characterization of a biosurfactant produced by Pseudomonas fluorescens for an environmental application. 1

1

1

2

Ferhat S ., Abou-Saoud M ., Moulai-Mostefa n and Badis A . YAHYA Fares University Centre at Médéa. 026000 Médéa. Algeria. Department of Industrial Chemistry. Engineering Faculty. SAAD Dahlab University at Blida. 09000. Blida. Algeria. E-mail: [email protected]

Abstract: Our study focuses on the biosynthesis and characterization of a biosurfactant produced by Pseudomonas fluorescens strain in batch culture by the use of two types of cells free and immobilized. The flowing of kinetic production by the surface tension and the index emulsification measurements permeated to get the optimal conditions: olive oil/NH4NO3 with C/N concentration equal 10. The maximum biosurfactant production, equivalent to a minimum surface tension (30 dyn/cm) and emulsification index about 50%, was obtained after 40 hours at 25 °C and under agitation (1200 rpm). In this case, a marked improvement in production was observed by the immobilized cells (0,148 gr biomass / 100 ml sodium alginate at 4% + calcium chloride 0.05N). HPLC and IR characterization revealed the effectiveness of acetonitrile and KBr for proper extraction of biosurfactant. Decontamination tests of soil polluted by the Hexadecane and Naphthalene gave very encouraging results which will be very useful for a future approach of an environmental application. Key words: Biosurfactant -Pseudomonas fluorescens - bioremediation.


Session 2 /

PC121 S2

Delineation of Pseudomonas savastanoi pv savastanoi strains by random amplified polymorphic DNA analysis a

a

b

b

c

Samira Krid , Ali Rhouma , José M. Quesada , Ramón Penyalver , Ali Gargouri and María b

M. López a

Unité de Recherche Protection des Plantes Cultivées et Environnement, Institut de l’olivier, Route de Soukra Km 1.5, 3003 Sfax, Tunisia. b

Centro de Protección Vegetal y Biotecnología. Instituto Valenciano de Investigaciones Agrarias (IVIA). Apartado Oficial, 46113 Moncada, Valencia, Spain. c

Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax B.P. K3038 Sfax, Tunisia.

Abstract: Random amplification of polymorphic DNA method (RAPDs) was used for discrimination between 66 local and reference strains of Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease. Local strains were isolated from eight different geographical orchards and three cultivars in Tunisia. Cluster analysis and principal coordinate analysis of RAPD fingerprint data obtained with three primers (OPR-12, OPX-7 and OPX-14) separated the isolates into three groups. Group 1 contained isolates of the southeast of Tunisia and NCPPB 1479 from Serbia and PVBa 206 isolated in Italy. Group 2 comprised strains isolated from the north of the country whilst group 3 enclosed the majority of isolates obtained from five orchards located in the centre of the country and the strain CFBP 71. Results revealed that RAPDs failed to differentiate closely related strains of Pseudomonas savastanoi pv savastanoi, mainly those isolated in the same orchard although a clustering related to the geographical location was observed.


Session 2 /

PC122 S2

Kinetic biodegradation of 2,4-dichlorohydroxybenzene by soil microflora Noumeur, S. R., Daffri, A., Guenoune, S. and Bousseboua, H. Laboratoire de Génie Microbiologique et Applications, Faculté des Sciences de la nature et de la vie, Département des Sciences de la nature et de la vie, Université Mentouri/ Constantine, Algérie. E-mail : [email protected]

Abstract: Chlorophenols pollution becomes a serious environmental problem due to their persistence and toxicity, in particular the 2,4-dichlorohydroxybenzene which is widely used in pesticides production, wood preservatives and others indutrial applications. In soil, the 2,4dichlorohydroxybenzene results from the photolytic and biological decomposition of the 2,4D and also from the lessiving soil by wastewater previously polluted. A soil suspension and its various dilutions are used to inoculate a minimum growth medium supplemented with 2,4-dichlorohydroxybenzene as sole carbon and energy source. This culture is carried out in miniaturised fermentors, incubated at 30°C. Moreover, this system of culture makes it possible to measure qualitatively and quantitatively the possibly gases production. The concentration of 2, 4dichlorohydroxybenzene is measured spectrophotometrically. Sowing on solid agar medium plate is carried out for a first microbiological analysis of the microbial samples. Kinetic of microbial degradation of 2,4-dichlorohydroxybenzene by the total soil microflora reveals a lag phase of 8 hours. Beyond which we note a reduction in the concentration, relatively fast during the 72 hours, it is spread out until the 14th day. In the final analysis, the 2,4dichlorohydroxybenzene is degraded at the end of 56 days. More than 50% of the initial concentration is lost in the first 14 days interval without production of gas. This situation indicates that the substrate was not mineralized and could probably be transformed into by-products whose analysis is in hand. The research of the dominant bacterial groups implied in this phenomenon, gives as first result aerobic colonies of differents macroscopic aspects which remain to be identified. Key words: Biodegradation, 2,4-dichlorohydroxybenzene, soil microflora


Session 2 /

PC123 S2

Development of Biosensors for the Determination of Total Antioxidant Capacity in Olive Leaf Extract Çoban, S. and Bayraktar, O. Izmir Institute of Technology, Chemical Engineering, 35430 - Urla, Izmir-Turkey

Abstract: There are various methods used to determine the antioxidant capacity of biologically active components. Analytical methods used in the qualitative and quantitative determination of polyphenols in olive leaf involve techniques of chromatographic separation such as HPLC and GC-MS. However these techniques are expensive, reagent and time consuming. However, biosensors are attractive alternative techniques due to their unique characteristics. In this study, an amperometric laccase biosensor was developed for the detection of oleuropein, which contributes to the total antioxidant capacity in olive leaf. The biosensor was prepared by immobilization of laccase into the carbon paste. Different biosensors were prepared by changing the concentration of crosslinking agent and enzyme solution. So, effect of these parameters on biosensor performance was investigated. Extraction of phenolics from olive leaf is also an important part of this study. The extract was divided into two fractions, one is rich in oleuropein and the other is not. Therefore, biosensor performance could be investigated for fractions containing different type of phenolics. It is understood from this study that, oleuropein shows a good substrate behaviour for laccase enzyme. This study is very important because it shows how to detect oleuropein, the phenolic that determines the quality of olive leaf extract, in an easy and economic way.


Session 2 /

PC124 S2

Seq: tools for decision support 1

2

Sihem Tireche and Abdelaziz Tairi 1

mail : [email protected] 2

mail : [email protected] 1, 2

Laboratory Pétrochimique Synthesis:Ergonomics and Environmental protection. FHC University M’ hamed Bougara Boumerdès

Abstract: The risk related to the metal contamination is multiple: risk for human health, environmental, economic and, in the same way, reflected with a negative image of quotes «pollute ». This allows increasing multiple sources of metaliferous pollution in Isser Wadi. The study of the impact of industrial activity on the littoral of the wilaya of Boumerdès through the evaluation of the rejection of effluents of the complex (ORFEE) which rejects its waste water in the Wadi Isser (wadi exoreic). The complex has as a main activity the production and the marketing of domestic stainless steel products (Stainless) of various qualities. Last nine metal elements (Silver, Cadmium, Chromium, Copper, Iron, Manganese, Nickel, Lead, Zinc) are proportioned in Atomic absorption spectrometry per Flame (F-SAA). The results obtained were transposed on the system of evaluation of water quality (SEQwater), in order to evaluate physicochemical quality, its using aptitude and the natural functions of the aquatic environments. By identifying deteriorations which compromise biological balances or the uses. SEQ-Water authorizes a precise diagnosis of water quality and contributes to define the actions of corrections necessary to guarantee a ecological balance of the aquatic environment (durable development). The advantage of this tool is the ability of addressing with the qualified authorities and the industrialists to find necessary basic elements to their information, or to help them to make a decision to perform restoration policies . Key words: Surface treatment, effluents, Metal Pollution, Environmental Stress, SEQ-Water, durable Management.


Session 2 /

PC125 S2

Exploration of the molecular diversity of anaerobic municipal sludge digesters microflora 1,

1

1

1,2

1

1

S. Guermazi , E. Pelletier , R. Chouari , D. Rivière , D. Le Paslier , J. Weissenbach , 1

A. Sghir 1

CNRS-UMR 8030, Institut de Génomique, Génoscope et Université d’Evry Val d’Essonne, 2 rue Gaston Crémieux, 91057 Evry, France, 2

CIRSEE Suez Environment, Le Pecq, France

Abstract: The microbiota involved in anaerobic methanogenic sludge digestion is not well known and the process is still considered as a “black box”. In order to investigate the microbial diversity of micro-organisms implicated in this microbial process, we studied the diversity of sludge samples obtained from three anaerobic municipal digesters (Corbeil and Creil, France). A total of sixteen 16 rRNA gene libraries were constructed using archaeal and bacterial specific primers as also phylum-specific primers. The obtained sequences were subjected to phylogenetic and statistical analyses and were grouped into OTUs (Operational Taxonomic OTUs), based on 97% sequence similarity. The conducted analyses permitted the identification of four major bacterial phylogenetic groups variably distributed within the three digesters: Chloroflexi 24%, Bacteroidetes 20%, Proteobacteria 18% and Gram-positives 15%. Other minor groups were identified to be dominant within these digesters. For the archaeal domain, the same profile was obtained for the Evry and Corbeil digesters, with more than 70% of the obtained sequences affiliated to the new lineage Arc I. Within Creil digester, this lineage was absent, while the Methanosarcinales dominated (85% of the obtained sequences). Using the SONS program, the shared OTUs between the three digesters were identified. The linkage between sludge composition and the observed diversity will help us to understand microbial interactions within anaerobic populations and to optimise the anaerobic process.


Session 2 /

PC126 S2

Purification and characterization of new laccase produced by a thermophilic fungus Ben Younes, S., Mechichi, T. and Sayadi, S. Centre de Biotechnologie de Sfax, Laboratoire de Bioprocédés, Pôle d’Excellence Regional AUF, (PER-LBP) Route Sidi Mansour Km 6, B P « K » 3038 Sfax-Tunisia

Abstract: 37 thermophilic fungi were isolated from various Tunisian biotopes. Several ligninolytic activities such as extracellular oxydase, laccase, tyrosinase and peroxidase were tested. Preliminary investigation of laccase activity was done in ABTS solid medium. Further screening for laccase and other activities were achieved in agitated cultures. In this work, we will focus only the Laccase activity. 12 Laccase producing fungi were screened on solid medium containing ABTS. The production of laccase was confirmed in liquid culture medium by spectrophotometric measurement. Among the isolates, ST26 was selected as laccase hyperproducing micro-organism. This fungus has a better production in the presence of glucose and soya peptone as carbon and nitrogen sources respectively, and copper as inducter. The laccase enzyme was purified to electrophoretic homogeneity by ketone precipitation, size-exclusion chromatography and anion exchange chromatography with a final yield of 30 %. The laccase was homo-trimeric protein with a molecular mass of 28 kDa (SDS–PAGE) each monomer. The optimum pH of the enzyme varied from 5.0 for 2,6dimethoxyphenol and 2,2-azino-di(3-ethyl-benzthiazoline-6-sulfonate), to 6.0 for Hydroquinone and Guaiacol. Under standard assay conditions, Km values of the enzyme were 0.36 and 0.26 mmol l-1 towards DMP and ABTS, respectively. The laccase was inhibited by NaN3, SDS, DTT and p-coumarate but not by EDTA, NaF, L-Cys and NaBr. Laccase was not 2+

3+

2+

2+

stable in the presence of Fe . Also, the activity was inhibited by the ions Cr , Fe , Cu and 2+

Hg . The enzyme has an optimum temperature of 80°C and it was stable at pH 10.


Session 2 /

PC127 S2

Photosensitization study of photobactericidal effects of methylene blue mixed in wastewater and immobilised within the polymeric matrix 1-2

Sonia Sabbahi

1

1

1

*, Zoubeir Alouini , Meryam Jemli , Layla Ben Ayed and Abdellatif 2

Boudabbous 1

Laboratoire de parasitologie des eaux usées et des boues résiduaires, Institut National de Recherche en Génie Rural Eaux et Forêts. Rue Hédi Karray, B.P. 10, 2080, Ariana, Tunis, Tunisie Fax.21601717951; 2

Faculté des Sciences de Tunis, Département de BiologieCorresponding author : E-mail : [email protected]

Abstract: Agriculture in Tunisia faces acute problems of water quality and quantity caused by limited conventional water resources. One possibility to cope with low water resources is to purify wastewater for reuse. In this case, pathological microorganisms pose a potentially serious health risk to the population in those regions. To avoid health and ecological hazards caused by using effluents in crop irrigation, the treatment of domestic sewage may involve in its last stage a disinfection treatment, which inactivates pathogens. Disinfection of effluents can be achieved by a variety of methods, mostly using chlorine, hypochlorites, chlorine dioxide, ozone and UV light. Several physico-chemical treatments have been investigated as alternatives to chlorine. In particular, a photochemical procedure was used for disinfection of effluents with sunlight as the energy source; the oxygen dissolved in water as an oxidizing agent, a dye as an intermediary for the absorption and transfer of the light energy to the oxygen and/or organic matter molecules and the microorganisms as the oxidation target. The oxidative 1

.

-

species ( O2, OH, O2 , H2O2) which might be produced in this procedure irreversibly altered the vital cell constituents and resulted in lethal damage. Singlet oxygen by itself is a cytotoxic agent, at least against microorganisms. This has been shown by experiments where the dye did not contact the cell membrane or even penetrate into the cytoplasm. The inactivation of faecal coliforms using a combination of a photosensitizer [methylene blue (MB)] with sunlight was determined on a small scale. Comparative experiment was performed at three pH values: 5, 7 and 9 on faecal coliforms photoinactivation using MB. For MB, the increase of pH favours the high inactivation yield of bacteria reaching recommended microbiological quality guidelines for wastewater use in agriculture which are appropriate for crops likely to be eaten uncooked (≤ 3 log units of faecal coliforms per 100 ml). The faecal bacterial photosensitization increased with increasing time exposure to light (180 min). In parallel the sensitizer photobleaching was also followed under the same conditions. For MB, the pH 9 favours its degradation more than pH 5 and 7. The MB photobleaching at different pH values, in secondary wastewater, was studied spectrophotometrically. This demonstrated that a bathochromic shift of 10 nm and the absorption intensity diminished (hypochromic effect) took place after solar light exposure for 60 min. After 420 min of exposure to sunlight, we noted that the peak absorption shows total disappearance in the visible region in order to reappear in the UV region with a small intensity. So it can be suggested that the influence of sunlight time exposure on photodegradation is responsible to the formation of new products which can absorb in the UV region (325-400 nm). The increase of MB degradation at basic pH shows also that the .

-

+

formation of OH by reaction between OH and H , at basic pH, was at the origin of the increase of the photocatalytic efficiency. A wastewater sample (1 liter) was exposed to the visible irradiation for 60 min, the MB should be properly immobilised within the polymeric matrix in order to show the Type II photosensitization and the production of singlet oxygen on illumination rather than Type I electron transfer/ redox reactions and to avoid leaching of the compound into the environment. The faecal coliforms organisms were susceptible to the photodynamic action of the photosensitizer fixed to the support, using different MB concentrations. At acidic pH, MB immobilised or free in solution gave the similar photobactericial activity. On the other hand, at basic pH, MB free in solution gave the more good results on the photobactericidal effect than MB immobilized to the resin. Under these conditions, the residual concentrations of CF and SF are respectively 1.83±0.62 and 1.70±0.4 against 3.10±0.78 and 2.69±0.37, when the MB was immobilised to the support.


Session 2 /

PC128 S2

Effective scouring of cotton fabrics with a combination of pectinolytic, lipolytic and proteolytic enzymes Kalantzi, S., Mamma, D., Kalogeris, E. and Kekos, D.* Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., 15780, Athens, Greece *E-mail: [email protected]

Abstract: In contrast to harsh alkaline conditions conventionally used, bioscouring is an alternative enzymatic method to remove non cellulosic “impurities” from raw cotton prior to dyeing. This environmentally friendly treatment, in which lower quantity of water and energy is consumed, improves the wettability of cotton fabrics to water and dye liquor without affecting the cellulose backbone and thus avoiding fiber damage. In the present study a plain woven cotton fabric was scoured by a mixture of commercial enzyme preparations with pectinase, lipase and protease activities. A broad set of properties including weight loss, whiteness, hydrophilicity; crystallinity index (CI), degree of polymerization (DP) and color depth was applied to assess the effectiveness of bioscouring. In addition, the micromechanical properties of fabrics were estimated based on Kawabata Evaluation System measurements. Bio-scoured cotton fabrics exhibited significantly improved physicochemical as well as mechanical properties in comparison with untreated fabric. The duration of treatment was a key variable affecting the outcome of the process. Fabrics treated enzymatically for 2 hours displayed comparable characteristics and performed equally well to those subjected to the conventional alkaline procedure. The results indicated that a combination of pectinolytic, lipolytic and proteolytic enzymes could serve as an effective scouring agent.


Session 2 /

PC129 S2

Bioremediation: ecotechnology for the present century Susan Srinivas Rao Lecturer in enironmental sciences, koneru lakshmaiahcollege of engineering, vaddeswaram, andhra pradesh.E-mail: [email protected] Abstract: Bioremediation is one of nature's ways to purify the polluted environment and that degraded by the anthropogenic activities. Although the term 'bioremediation' may be recent, but the process is not new as its origin relates to the origin of life. When the first organism was stressed by certain compounds, it evolved the process to convert such compounds into less harmful forms by adopting certain detoxifying mechanisms in order to overcome the stress. The present day bioremediation technologies are based on the processes and potentials of almost all types of life forms, viz., plants, microorganisms and animals. Hence 'ecoremediation' is a term used to express the wider range of objectives and scope. To optimize the maximal benefit sustainability of such technologies, an effective policy directives supported by social perception is required and must necessarily incorporate the application of environmental impact assessments, intellectual property rights and cost-benefit analysis for commercial viability.


Session 2 /

PC130 S2

Biodegradation of azoïc dyes by micoorganisms isolated from textile wastewater 1

2

3

1

Walid Khemakhem , Adnane Moutaoukkil , Amina Bakhrouf , Emna Ammar and 2

Abdelaziz Soukri . 1

National Engineering School in Sfax, B.P. W, 3038 Sfax-Tunisia, E-mail : [email protected] 2

Faculty of Sciences Ain Choc, km 8, Road El Jadida, BP 5366 Maarif Casablanca, Morrocco 3

Faculty of Pharmacy, Avicenne street, 5000 Monastir, Tunisia

Abstract: The textile wastewater is a complex mixture of chemicals including high levels of suspended solids and heavy metals, with alkaline pH, discharged at relatively high temperatures. The organic pollutants are phenolic compounds (toluene, benzene and chlorobenzene) and naphthalene (substituted phenols). These have toxic effects. Physico-chemical treatment based on activated charcoal, ozonation, flocculation and reverse osmosis are used to decolorize the effluent. These methods are costly and biological treatment is more efficient to reduce coloration resulting from azoïc dyes. In this work, textile wastewater from Ksar Helal (Monastir, Tunisia) produced at 2800 3

m /day wee used. A yeast able to decolorize methyl red in 30 minutes at a rate of 96% was isolated and studied. Two other reactive dyes were tested and biodegraded. The isolate was -1

able to decolorize all of the three dyes with an enzymatic activity of 3.253 U mg total proteins. The enzyme decoloured a mono and di azoïc dyes by fission of azoïc band N=N, characteristic chomophore of the azoïc dyes. Optimal conditions of the enzymatic activity were determined (Temperature, pH and oxygenation). The SDS page revealed an area of decolouration in relation with the enzyme. Finally, the isolated yeast was used to purify neutralised textile wastewater and exhibited a decolouration and dye biodegradation, confirmed by analytical methods (physicochemical and spectroscopic: COD, UV-visible, IR, HPLC and GC/MS). Toxicity tests revealed a non toxic effluent after its treatment with the isolated yeast.


Session 2 /

PC131 S2

A tentative a double inoculation of Arachis hypogaea L by Glomus and Rhizobium in the areaof EL-KALA The Algerian North-East Touil Wided and Beddiar A Research laboratory in vegetable biology and environment - Faculty of Science - university Badji Mokhtar YEAR E-mail: [email protected]

Abstract: The interaction enters the mushroom mycorhizien Glomus sp , the bacterium Bradyrhizobium sp and the plant-host Arachis hypogaea L was studied in an experiment out of pots comprising four treatments of which a witness (plant-host, only), an inoculation with Glomus sp,another with Bradyrhizobium sp and the last receiving the two symbions. The observations were carried out on all the seedlings of each treatment. The principal parameters are: -parameters of growth: numbers average flowers by seedling, average height of the principal stem and weight of the fresh matter of the air part. -Parameters of output: numbers average and gauges pods. -Parameters of symbiosis: height and weight of the system racinaire, a number and fresh weight of the nodules. Key words: Arachis hypogaea L, Glomus, Bradyrhizobium,inoculation, endosymbiose.


Session 2 /

PC132 S2

Optimization of submerged culture conditions for xylanase production from Penicillium canescens Y. Bakri * and Y.Akeed Department of Molecular Biology and Biotechnology, AECS, P. O. Box 6091, Damascus, Syria *Corresponding Author; E-mail: ybakri@aec. org .sy

Abstract: The ability of the fungus Penicillium canescens to secrete xylanolytic activity in submerged culture was investigated. Xylanase production was enhanced by optimization of the type of carbon and nitrogen sources and their concentration. Among the various carbohydrates soluble studied, xylan was found to be the best inducer of xylanase (24.17 U/ml). The use of purified xylan as substrate is uneconomical; therefore, several natural agricultural residues were tested. The results show maximum activity was observed when barley straw 3% (W/V) was used as carbon source. (NH4)2SO4 (0.2%) was found to be best nitrogen source for optimum enzyme production (326 U/ml) after 5 days of incubation. Key words: Xylanase, Penicillium canescens, Submerged Culture.


Session 2 /

PC133 S2

Variability of the 16S-23S rRNA gene internal transcribed spacer of the Enterobacteriacae family Turki Y., Ouzari H., Jaoua L., Mehri I and Hassen A. Laboratoire Traitement et Recyclage, Centre des Sciences et Biotechnologie des Eaux., BP 95, 2050 Hammam Lif, Tunisie. E-mail : [email protected]

Abstract: In Prokaryotes, the rRNA genetic loci contain the genes for all three rRNA species, 16S, 23S, 5S genes which are separated by intergenic spacer regions (ITS). rRNA loci are present in 2 to 11 copies on the chromosomes of most bacterial species. Whereas a high degree of sequence homology existes for rRNA genes, the intergenic spacer regions show extensive sequence and length variations which can be used to charecterize bacteria at the genus, species, and subspecies levels. Modern typing methods are based on genotype characterization. A more promessing and simpler technique seems to be PCR ribotyping, which is based on the amplification of the spacer sequences between the 16S and 23S genes in the rRNA transcriptional units. In the present work we investigated the possibility of differentiating bacterial strains both at the biochimical tests, the serotype level and at the intraserovar level on the basis of the polymophism of the 16S-23S rRNA intergenic spacer region. For This, an universal primer targeting conserved sequences flanking the 3 ‘ end of the 16S and the 5 ‘ end of the 23S rRNA genes were used to amplify the 16S-23S rDNA internal transcribed spacer For over 200 strains of bacteria belonging to four genera and 12 species or serotypes, including Citrobacter, Enterobacter, proteus and Salmonella species. When the spacer amplification produits were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Key words: Enterobacteriacae, serotyping, ITS.


Session 2 /

PC134 S2

Relationship between the Coolia monotis (Meunier 1919) bloom and environmental factors in the North Lake of Tunis (Tunisia) 1, 2

Armi Zina

2

3

3

, Turki Souad , Ben Maiz Naceur & Trabelsi Elbahri .

1: Faculté des sciences de Bizerte (FSB). 2: Institut National des Sciences et Technologies de la Mer (INSTM). 3: Société de Promotion du Lac de Tunis (SPLT). Corresponding author: E-mail : [email protected] Adress: Institut National des Sciences et Technologies de la Mer, Port de pêche, 2060 La Goulette, Tunisie.

Abstract: During an annual cycle, between September 2005 and August 2006, the contribution to total cell phytoplankton density in the North Lake water was 36, 45 % and 61, 95 % of diatoms and dinoflagellates, respectively. We have detected many harmful species such as Pseudonitzschia spp, Dinophysis spp, Alexandium spp, Prorocentrum spp and Coolia monotis. Coolia monotis is a marine, planktonic, benthic, epiphytic and photosynthetic dinoflagellate with world-wide distribution. This species is known to produce toxins associated with Ciguatera Fish Poisoning (CFP) which are transferable to the humans through the food chain. In our area of study, Coolia monotis bloomed in the south part of the lake and presented its 5

. -1

highest concentration (5.10 Cells L ) in May 2006 (09/05/06) in station S4. The presence index and the relative abundance of this species during the period of study were respectively about 22, 8 % and 16, 2 %. The climatic factors were the principal stimulant of this florescence. A low tidal mixing energy with a relative high water temperature (22 °C) and a photoperiod about 12 hours/day helped for the stratification phenomena. -

Coolia monotis showed also a positive correlation with the highest concentration of NNO3 which was about 95 µM (r = 0, 21). Key words: Harmful phytoplankton, Coolia monotis, abiotic factors, North Lake of Tunis.


Session 2 /

PC135 S2

Functional and Safety Aspects of Enterococci Isolated From traditional Tunisian fermented meat 1

2

2

1

Zouhaier Ben Belgacem , Hikmate Abriouel , Antonio Gálvez and Mohamed Manai 1

Laboratoire de Biochimie et Biologie Moleculaire, Faculté des Sciences de Tunis, Campus Universitaire El2

Manar 2092, Tunis, Tunisie. Área de Microbiología. Fac. Ciencias Experimentales, Universidad de Jaén, Jaén, Spain.

Abstract: Enterococci compose the microbial association of a variety of fermented foods such as cheese, fermented meat and fermented vegetables. In an ecological study, 102 Gram-positive bacteria were isolated from artisanal Tunisian fermented meat and identified using Ribosomal DNA-based techniques including 16S–23S rRNA gene ISR polymorphism, PCR ISR-RFLP, RAPD, Genus/Species-specific PCR and, 16S rDNA sequencing. From these, 62 strains were identified as enterococci and showed two types of rrn operon, one with (M-ISR) and one Ala

without (S-ISR) tRNA in the ISR region. The functional properties of enterococci strains were investigated, as well as their safety aspects and probable role in food preservation. 22 of the strains produced antimicrobial activity against Listeria sp. and other food spoilage or pathogenic bacteria, and many of them tested positive upon PCR amplification of structural genes for enterocins A, B & P. Antibiotic resistance and others determinants of virulence were analyzed in all enterococci producing bacteriocins. All isolates were resistant to rifampicin, erythromycin, ciprofloxacin and levofloxacin. Vancomycin resistance was not detected. The incidence of virulence traits in enterococci strains was investigated with the objective of safety assurance of the particular fermented products. These results suggest that the enterococci isolated in this study may play a role in fermentation processes and confer interesting functional properties.


Session 2 /

PC136 S2

Introduction to Biotechnology Mohamed Farag E-mail : [email protected]

Abstract: Biotechnology is most briefly defined as the art of utilizing living organisms and their products for the production of food, drink, medicine or for other benefits to the human race, or other animal species. Technically speaking, humans have been making use of biotechnology since they discovered farming, with the planting of seeds to control plant growth and crop production. Animal breeding is also a form of biotechnology. More recently, cross-pollination of plants and cross-breeding of animals were macro-biological techniques in biotechnology, used to enhance product quality and/or meet specific requirements or standards. The discovery of microorganisms and the subsequent burst of knowledge related to the causes of infectious diseases, antibiotics and immunizations could probably be counted among man’s most significant, life-altering discoveries.


Session 2 /

PC137 S2

Characterization of thermophilic lactococci and enterococci from indigenous flora in fermented milk a,*

b

b

Farid Bensalah , Christine Delorme and Pierre Renault a

Faculté des Sciences, Département de Biologie, Laboratoire de Microbiologie Appliquée, Université Es-Sénia., Oran 31000, Algérie b

Génétique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas CEDEX, France

Abtract: Lactic acid bacteria (LAB) are widely used in food industry and their growth performance is important for the quality of the fermented product. By combining results from conventional isolation methods and molecular investigation of 16S rDNA and lactococcal/enterococcalspecific genes, we identify at strain level catalase negative gram positive thermoresistant cocci isolated from traditional ‘leben’, a 24-h fermented milk in arid area of west Algeria, in order to isolate new strains of potential interest in milk fermentation and assess their diversity within the wild microbial population. 40 strains phenotypically related to streptococci could be identified as belonging to the species Lactococcus lactis ssp. lactis, Enterococcus faecalis, Enterococcus faecium, and other Enterococcus species. No Streptococcus thermophilus strain was isolated. Ten different phenotype group were recognised, and the species content of these group were in some cases different from the expected features usually given in genus and species descriptions. In particular, 3 atypical lactococci, able to grow in 6.5% NaCl, at 42°C and showing a resistance to thermal stress were isolated. Selective SB medium was found to be a reliable technique, alternative to molecular techniques, allowing the discrimination of most enterococci from lactococci. New starter strains displying unusual properties for their species could be isolated from traditional ‘leben’ produced in isolated Algerian region. Study of more of this type should provide starter strains for innovation product. This study proposes a simple and reliable isolation method could be used at a first level to isolate number of such strains in different geographical area. Keywords : Streptococci, thermotolerant wild lactococci , enterococci , indigenous lactic acid bacteria , arid area , 16S rDNA.


Session 2 /

PC138 S2

Retention of metal micropolluants on activated carbon: application to waste treatment and environmental protection. Zeroual Sabrina, Hazourli Sabir and Guerfi Kamel. Laboratory water treatment and recycling of industrial waste, Department of Chemistry, University-Badji Mokhtar, Annaba BP 12, 23000. E-mail: [email protected]

Abstract : The concept of environment is new, it covers a wide heritage consisting of the entire physical environment, fauna and flora and everything that affects their lives. At the moment, or the problems of the environment and its protection attract the attention of researchers in the world, human societies are constantly exercise On the latter diverse form of pollution, which inevitably affect the quality of soil, air and water, in other words "the environment". Considering only the aquatic environment, water contamination by pollutants metal remains a concern for laboratory research; therefore, it is essential to take charge of this problem by developing means of decontamination that must be reliable, efficient and not expensive. In this context, we are interested in the method of adsorption which consists of fixing metal ions on the activated carbon. For their environmental application we tested the cadmium, the effect of physical-chemical parameters has been studied namely "pH, temperature, concentration initial…". The results show the carbon fabricated with a high temperature gives an efficiency of the order of 80%. This study aims to contribute to the protection of the environment for better health and the environment protected Key words: environment, water pollution, heavy metals, retention, activated carbon. References: [1]- J.O.M.BOKRIS, environment chemistry, plenum press, New York, 1977. [2]-R.WEINER, épuration des eaux résiduaires, édition Eyrollrs, 1975. [3]-A.HOUAS, I. BAKIR, M. KSIBI et E. ELALOUI, étude de l’élimination du bleu de méthylène dans l’eau par le charbon actif commercial CECA 40, J.Chim, Phys. 1999.


Session 2 /

PC139 S2

Lipid-lowering and antioxidative activities of phenolic extract and purified hydroxytyrosol recovered from olive mill wastewater in cholesterol-fed rats Ines Fki & Sami Sayadi Laboratoire des Bioprocédés, Pôle d’Excellence Régional AUF, Centre de Biotechnologie de Sfax, B.P. «K» 3038 Sfax, Tunisia

Abstract: In our previous studies, a phenolic-rich extract of olive mill wastewaters (OMW) was prepared under optimal conditions, using a continuous counter-current extraction unit and hydroxytyrosol was purified from the obtained OMW extract. The antioxidant activity of OMW extract and hydroxytyrosol was determined by a series of models in vitro. In this study, the hypocholesterolemic effects of hydroxytyrosol and OMW extract in rats fed a cholesterolrich diet were tested. Wistar rats fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks were used. Serum lipid levels, as well as malondialdehyde (MDA) and superoxide dismutase (SOD) and catalase (CAT) activities in liver were examined. Cholesterol-rich diet induced hypercholesterolemia was manifested in the elevation of serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C). Administration of a low-dose (2.5 mg/kg of body weight) of hydroxytyrosol and high-dose (10 mg/kg of body weight) of OMW extract significantly lowered the serum levels of TC and LDL-C while increasing the serum levels of high density lipoprotein cholesterol (HDL-C). Furthermore, the content of MDA in liver, heart, kidney and aorta, decreased significantly after oral administration of hydroxytyrosol and OMW extract compared with those of rats fed a cholesterol-rich diet. In addition, OMW phenolics increased CAT and SOD activities in liver. These results suggested that the hypocholesterolemic effect of hydroxytyrosol and OMW extract might be due to their abilities to lower serum TC and LDLC levels as well as slowing the lipid peroxidation process and enhancing antioxidant enzyme activity. Keywords: Antioxidant enzymes, Cholesterol-fed rat, Hydroxytyrosol, hypolipidaemic, Olive mill wastewaters extract, Serum lipid levels


Session 2 /

PC140 S2

Diversity of Tunisian saltern culturable aerobic bacteria and their performance in hypersaline wastewater treatment 1

2

3

4

Houda Baati , Neji Gharsallah , Ridha Amdouni , Abdelghani Sghir , Khaled 5

1

Medhioub and Emna Ammar 1


Faculty of Sciences in Sfax, P.O. 802, 3018 Sfax – Tunisia, 3

Tunisian General Company of Salterns (COTUSAL), Laboratory of Analyse, Road Gabes km 0.5 – 3018 Sfax, Tunisia, 4

University of Evry Val d'Essonne, CEA- Genoscope, France 5

Institute of Preparatory Engineering Studies; P.O. 805, 3018 Sfax –Tunisia, UR Etude et Gestion des environnements Urbains et Côtiers, LARSEN E-mail: [email protected]

Abstract : Microbial diversity was described through culturing, isolating and characterizing bacteria inhabiting solar saltern in Sfax (Tunisia). Forty bacterial isolates were subjected to phylogenetic analysis through 16S rRNA gene sequencing. The identification revealed two major groups, 36 strains of gamma-Proteobacteria (90%) and 4 strains of Firmicutes (10%). The gamma-Proteobacteria group consisted of several subgroups of the Halomonadaceae (57.5%), Vibrionaceae (15%), Altermonadaceae (10%), and Idiomarinaceae (7.5%). The genus Halomonas affiliated species (35%) was the most predominant among all of the isolates. Additionally, three new species: 183ZD08, 191ZA02 and 191ZA09 were found in the saltern, based on similarities of the 16S rRNA sequences to those of previously published species. The bacterial consortium isolated was used for preliminary treatment essay of an effluent generated by a factory processing marine products and characterized by high concentrations of organic compounds (COD of 4058 mg/l) and salt (80 g/l). The bacterial consortium was adapted to the substrate by a gradual increase of effluent concentration. The result showed the important role of this consortium in COD elimination. Therefore, the solar saltern of Sfax is an optimal environment for halophilic bacteria, where diverse viable bacterial communities can be used for hypersaline wastewater treatment.


Session 2 /

PC141 S2

Cinetique biodegradation of phenol by immobilized bacteria in calcium alginate Fethia Amrouche, Abdelkader Namane, Saliha Zeboudj and Amina Hellal* Ecole Nationale Polytechnique, Laboratoire des Sciences et Techniques de l’Environnement, BP. 182, Hassen Badi, 16200 Alger, Algérie Corresponding author: A. Hellal, * E-mail: hellal_ben @yahoo.fr

Abstract: Phenol is widely distributed as environmental pollutants due to its common presence in the effluents of many industrial processes, including oil refineries, ceramic plants, coal conversion processes and phenolic resin industries. Therefore, the removal of phenol from industrial aqueous effluents is of great practical significance for the environmental protection. By contrast with physical and chemical methods, biological methods of phenol removal are preferable in wastewater treatment process as the relatively low processing costs. Moreover, in spite of phenolic toxic properties, a number of microorganisms can utilize phenol under aerobic condition as sole source of carbon and energy. Several authors have isolated and characterized bacteria and fungi from industrial effluents for mineralizing phenol. However, little is known about the biodegradation of phenol by immobilized bacteria in calcium alginate gel. The main advantages in the use of immobilized cells in comparison with suspended ones include the retention in the reactor of microorganisms, protection of cells against toxic substances and cell recycle. In the present study, biodegradation of phenol using immobilized isolated bacteria in calcium alginate beads has been investigated. The rate of biodegradation by immobilized and freely suspended cells was compared. Compared with a free-cell culture, an immobilized-cell culture provided a better environment for the biodegradation of phenol due to the protective effect of gel matrix. Biodegradation was conducted with cell beads with −1

phenol concentrations ranging from 100 to 5000 mg. l . A stable removal efficiency was −1

achieved even at an inlet phenol concentration as high as 5000 mg l . On the other hand, immobilized cells can be recycled without losing their activity. Keys words: Phenol; biodegradation; isolated bacteria; immobilized cells.


Session 2 /

PC142 S2

Isolation of cellulase genes from Stachybotris microbispora: Three beta glucosidases from family 1 and 3 and one endoglucanase belonging to family 61 Salma Abdeljalil and Ali Gargouri Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, BP”K”3038, Sfax – Tunisie, E-mail: [email protected]

Abstract: Produced by several bacteria and fungi, cellulases constitute an enzymatic complex formed mainly by three classes of enzymes which include endoglucanases, cellobiohydrolases and beta-glucosidases. These enzymes are of great biotechnological interest. In fact, cellulases are used in food, brewery and wine, animal feed, textile and paper industries. Previously studies showed that the strain N1 from Stacchybotris microbispora, isolated in our laboratory, presents some interesting characteristics: secretion of cellulases which are able to act in a large spectrum of pH, from 5 to 9; production of several beta-glucosidases. We have constructed a genomic bank from the A19 mutant of N1. We isolated using PCR and NestedPCR, fragments of two different genes of beta-glucosidase belonging to family 1 and another from family 3. Thereafter, we carried out the screening of the genomic bank using those three probes. We isolated one hundred clones from the first round of hybridization followed by a second one with the same probes. Interestingly, the screening of the bank with the fragment of betaglucosidase (Family 3) led to the isolation of one clone containing an endoglucanase classified in family 61. Characterization of this gene will be presented.


Session 2 /

PC143 S2

Effect of nitrogen sources on pectinases production by the CT1 mutant of Penicilium occitanis Zamen Ben Romdhane, Noomen Hadj-Taieb and Ali Gargouri Laboratoire de « genetique Moleculaire des Eucaryotes » Centre de Biolechnologie de Sfax, BP. K. 3038, Sfax, Tunisia

Abstract: We are trying to improve the production of pectinases by the constitutive CT1 mutant of Penicilium occitanis using glucose in liquid media in combination with different nitrogen sources. The production of pectinases was not affected at low concentrations of glucose (0.51%) in the medium optimised in presence of NaNO3 and ammonium sulphate as nitrogen sources. At high concentrations of glucose (2% - 4%), the production of both endo-PC and exo-PC activities were strongly repressed and accompanied by a harmful effect on the mycelia growth. At the opposite, pectinolytic activities were enhanced when we used yeast extract and urea as nitrogen sources at high concentrations of glucose. On pectin, the growth and pectinolytic behaviour of our mutant, depending on the nitrogen sources, is not the same as on glucose. It was very interesting to note that the highest level of pectinases was reached on glucose and not on pectin. In addition, we give other evidences for the influence of, not only the quality/quantity of nitrogen and carbon sources but also of the pH of the medium.


Session 2 /

PC144 S2

Characterization of an L-arabinose isomerase from the Lactobacillus plantarum NC8 strain showing pronounced stability at acidic pH Wacim Bejar, Hichem Chouayekh, Moez Rhimi, Karim Jelleli, Malek Mseddi & Samir Bejar Laboratoire d’Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax BP “K” 3038 Sfax, Tunisie. E-mail: hichem.chouayekh @cbs.rnrt.tn, Tel.: +216 74 870451; fax: +216 74 870451;

Abstract: D-tagatose, a natural keto-hexose, is currently being introduced as low-calorie bulk sweetener. Its consumption does not promote tooth decay or elicit any increase in blood glucose level. The L-arabinose isomerases (L-AI) which catalyse in vivo the reversible isomerization of L-arabinose into L-ribulose can be employed in vitro for production of Dtagatose using D-galactose as substrate. The optimal pH of isomerisation by the hyperthermophilic L-AIs already characterized ranges from pH 7 to 8.5 while industrial application requires a slightly acidic pH range (~ 6) to reduce browning color and by-products formation. Due to their acid tolerance, the lactic acid bacteria could be interesting candidates to seek for L-AIs with high activity and stability at moderately low pH. In this study, the gene araA encoding the L-AI from Lactobacillus plantarum NC8 was cloned, sequenced and expressed in Escherichia coli. It encodes a polypeptide of 474 residues having 55% identities with L-AIs from Bacillus stearothermophilus US100 and Thermus sp. IM6501. The active form of the purified recombinant L-AI NC8 enzyme is a hexamer composed of six identical 55-kDa subunits (Chouayekh et al., 2007). The purified enzyme 2+

2+

was optimally active at 60 °C and pH 7.5. It required divalent cations such as Co and Mn for maximal activity and thermostability. The L-AI NC8 was exceptionally active and stable at acidic pH. Indeed, it exhibited 68% of its maximal activity at pH 5.5 and retained 89% of activity after 24 h incubation at pH 5. The apparent Km values of the enzyme for L-arabinose and D-galactose were 43.4 mM and 69.7 mM respectively; and its catalytic efficiency was -1

-1

approximately 10-fold higher for the physiological substrate L-arabinose (15.5 mM min ) -1

-1

than D-galactose (1.6 mM min ). The bioconversion yield of D-galactose to D-tagatose by the purified L-AI NC8 after 6 h at 60 °C at pH 7.5 was of 30%.


Session 2 /

PC145 S2

Organic pollution reduction of olive processing wastewater by moulds isolated from olive mill wastewater evaporation-ponds 1

2

1

Raja Jarboui , Néji Gharsallah and Emna Ammar 1


Faculty of Sciences in Sfax, P.O. 802, 3018 Sfax – Tunisia, UR Etude et Gestion des Environnements Urbains et Côtiers, LARSEN, E-mail: [email protected]

Abstract: Olive oil Mill Wastewater (OMW) raises a major environmental problem because of the annually large amount produced and the toxicity of the phenolic compounds present. The aim of this study is to investigate the moulds ability to degrade the OMW and to reduce their toxicity. Thirty moulds were isolated from the OMW evaporation ponds. First, moulds were tested to growing on diluted OMW based medium with a COD of 20 g/l. The removal of COD, phenols, glucose, proteins, and DO395nm and biomass were evaluated. Ten moulds were active and either decolorized and removed COD of OMW at rates superior to 50%. Aspergillus niger primary identified was selected and the optimized for OMW decolourizating and COD removing. Several carbon, nitrogen and salts substrates were also tested. Sucrose, (NH4)2SO4 and CaCl2 constituted the main substrates for this mould. The capacity of Aspergillus niger to reduce OMW pollution was tested on optimum medium supplemented with OMW at different dilutions (5%, 10%, 15%, 20%, 25% and 30%) characterized by COD of 6, 12, 18, 24, 30 and 36 g/l respectively. This mould showed a COD removal of 90, 82, 74, 65, 57 and 49 % and phenols removal to 96, 84, 79, 70, 60 and 55% respectively on the different OMW dilutions.


Session 2 /

PC146 S2

Optimization of Lipase-Catalyzed Synthesis of Acetylated Tyrosol by Response Surface Methodology Imen Aissa, Mouhamed Bouaziz, Hanen Ghamgui, Amel Kamoun, Nabil Miled, Sami Sayadi, and Youssef Gargouri National Engineering School of Sfax, Laboratory of Biochemistry and Enzymatic Engineering of Lipases, Roadof Soukra, 3038 Sfax-Tunisia, Centre of Biotechnology of Sfax , Laboratory of Bioprocesses, BP«K»3038, Sfax, Tunisia, National Engineering School of Sfax, Laboratory of Industrial Chemistry II, Road of Soukra, 3038 Sfax-Tunisia. E-mail: imenaissa @freesurf.fr

Abstract : Tyrosol is a phenol witch exhibit antioxidative activity, particularly abundant in fruits and olive oil. This phenol can prevent against several pathologies such as atherosclerosis and carcinogenesis. Tyrosol exhibit low liposolubility and low stability, so it is important to improve the stability of this compound via enzymatic esterification. A non-commercial immobilized lipase preparation from Staphylococcus xylosus was used to catalyse the alcoholysis reaction between the tyrosol and the ethyl acetate to synthetize the tyrosol acetate. Response surface methodology based on three-level, three-factor was employed to optimize the ethyl acetate to hexane volume ratio, the enzyme amount and the temperature. The molecule was characterized using spectrometric methods. This lipophilic analogue synthetized could be used as food antioxidant in oil-based formulae or cosmetic fields. Key works: Tyrosol, antioxidant, Lipase, Response surface methodology .


Session 2 /

PC147 S2

Proteolytic cleavage of ostrich and turkey pancreatic lipases: production of an active N-terminal domain Ben Bacha Abir, Gargouri Youssef, Mejdoub Hafedh and Miled Nabil Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Ecole Nationale d'Ingénieurs de Sfax, Tunisia.

Abstract : The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively). Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase. In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity. OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.


Session 2 /

PC148 S2

Use of reed in phytoremediation of wastewater Chaouch Radia INFP, Mardj Dib , Skikda Algérie

Abstract: Wastewater of a traction-engine factory was discharged without any prealable treatment. This water is burdened with many pollutants, organic and inorganic forms, in particularly heavy metals with special focus on the chromium. This element was present with concentration largely upper to the toxic level for fauna and flora. We have tested the possibility to reduce this toxicity by reed plantation along shore of brook where the waste water is dumped. This brook water is sometime used for irrigation. The first results showed on one side that reed plants can concentrate ( though the metabolism was disturbed ) great quantities of Cr without phytotoxic symptoms, and on the other side can cause drastic reduction of chromium water concentration. From these observations, we can suppose that reed plants are able to reduce low chromium pollution and constitute a solution to improve water quality. Key words : Water pollution, Heavy metals, Chromium, Phragmites communis


Session 2 /

PC149 S2

Storage stability of traditional butter oil produced fromSpontaneous fermentation cow’s milk Olfa Bali, Mohamed Ali Ayadia and Hamadi Attia Détartement de biologie, Unité analyses Alimentaires, Ecole Nationale d’Ingénieurs de Sfax, Route de Soukra, 3038 Sfax, Tunisia

Abstract: Fatty acid composition and thermal stability of traditional Tunisian butter oil (TTBO) were studied. Storage stability of samples was estimated by using the accelerated shelf-life testing method. Effect of heating on some quality characteristics of TTBO has been investigated and compared at different temperatures (60°C, 100°C and 130°C). Induction period of sample heated at 60°C was important compared to that at 100°C and 130°C. This result may indicate the sensitivity of TTBO to elevated temperature. Absorptivity at 232 and 270 nm, acidity and peroxide values increased rapidly after reaching the oxidation induction time. The temperature had a significant effect on the formation of oxidation products in TTBO. As a consequence, heat treatment produced alterations in the oxidative status of the butter oil that could affect the shelf-life. Key words: Traditional Tunisian butter oil; Fatty acid composition; Thermal stability


Session 2 /

PC150 S2

Effets de l’exposition aux radiations gamma sur quelques aspects de la biologie de salmonella. 1

2

2

1

3

3

Ben Miloud N. , El May A , Maatoug i , Barkallah I , Ruat S , Steinmetz G , 4

2

Marault m and Landoulsi A . Unité de Microbiologie et Biologie Moléculaire, Centre National des Sciences et Technologies Nucléaires, 1

Technopôle de Sidi Thabet , E-mail : [email protected] 2

Laboratoire de biochimie et Biologie Moléculaire, Faculté des Sciences de Bizerte Service de Biochimie post-génomique et Toxicologie Nucléaire, Commissariat à l’Énergie Atomique, site de 3

Marcoule Unité Caractérisation et Épidémiologie Bactérienne, Agence Française de Sécurité Sanitaire des Aliments 4

Maisons-Alfort

Abstract : Ce travail a eu pour objectif l’étude des effets des rayonnements ionisants de type gamma sur certains aspects de la biologie de Salmonella, peu de travaux ont réunis cette espèce et les radiations gamma. L’effet létal des radiations ionisantes a été associé chez d’autres espèces bactériennes, à un stress oxydant dû à la présence d’espèces réactives à l’oxygène et conduisant à des altérations des membranes cellulaires, des protéines et des acides nucléiques. Ainsi, nous avons procédé à une analyse de la viabilité de quatre sérovars de Salmonellasoumis à différentes doses d’irradiation allant de 0,5 à 2 KGy. Les résultats ont montré une diminution de la viabilité, doses dépendantes avec un comportement différentiel, statistiquement significatif, des différents sérovars vis-à-vis de l’irradiation. Afin de mettre en évidence d’éventuelles mutations radioinduites, au niveau du site de restriction des enzymes XbaI et BlnI couramment utilisées pour le typage de Salmonella, nous avons procédé à une analyse des profils de restriction des ADN des quatre sérovars, par électrophorèse en champ pulsé. Les résultats ont montré qu’aucune mutation n’est apparue au niveau des sites de restriction des enzymes utilisées, suite à une irradiation de 2KGy. L’étude de la sensibilité de Salmonella vis-à-vis des antibiotiques suite à une irradiation a permis de mettre en évidence une augmentation de cette sensibilité, visualisée par une augmentation statistiquement significative de la zone d’inhibition de certains antibiotiques. Ces augmentations sont doses dépendantes. Pour tenter d’expliquer le comportement différentiel des différents sérovars vis-à-vis des irradiations. Nous avons analysé par RT- PCR quantitative en temps réel, le niveau d’expression de l’ARNm des gènes de la catalase non-hémique KATN, de la protéine de choc thermique DNAK, de la sous-unité � de l’ARN polymérase ainsi que du 16S rRNA. Les résultats ont montré soit une répression ou plutôt une induction de certains gènes sous l’effet d’une irradiation de 2 KGy.


Session 2 /

PC151 S2

The fate of high concentrations of salt and ammonia on the detoxification of landfill leachates by selected white rot fungi Mariem Ellouze, Fathi Aloui and Sami Sayadi Laboratoire des Bioprocédés, Pôle d’Excellence Régionale AUF, (PER-LBP), Centre de Biotechnologie de Sfax, BP: « K », 3038, Sfax, Tunisie.E-mail: [email protected]

Abstract : Landfill leachates (LFL) are complex wastewaters with a variable composition depending on waste deposit characteristics and landfill age. LFL collected from Djebel Chekir (Tunisia) discharge area was found to be highly loaded with organic matter, ammonia, salts, heavy metals, phenols and hydrocarbons. It exhibited high toxicity to microorganisms and plants. Consequently, LFL represent a threat to the environment. This study attempts to develop a biological fungal process for the treatment and the detoxification of LFL using selected strains of Trametes trogii and Phanerochaete chrysosporium. Experimental results showed that COD removal efficiencies were 68% and 79% for P. chrysosporium and T. trogii, respectively when LFL underwent a twofold dilution. Also, a high reduction in the toxicity (%BI < 20%) was obtained. On the other hand, the study demonstrated that the presence of salts and ammonia at high concentrations was not the main cause of the fungal treatment limitation to 50% of LFL. In fact, the two strains tolerate important concentrations of salts and ammonia, exceeding those founded in raw LFL when cultivated on the synthetic media. This problem could be associated to the presence of toxic compounds such as phenolic and hydrocarbon compounds. Key words: Landfill leachates, White Rot Fungi, Detoxification, Tolerance.


Session 2 /

PC152 S2

Biofilms as an integrative method for the processing of wastewaters *

**

*

**

Popovici, S., Ichim, M., Pasarin, D. and

Ichim, L.,

*

National Institute for Research & Development in Chemistry & Petrochemistry-ICECHIM, **

Institute of Bioengineeing, Biotechnology and Environmental Protection-BIOING *

**

202 Splaiul Independentei, 060021 Bucharest, Romania, 10, Prof.Ion Bogdan streeet, 010539 Bucharest, Romania

Abstract: A compact, integrative bioremediation method for “grey” waters (household, hotel, restaurant etc. wastewaters) was developed, using genetically controlled microbial biofilms (Artrobacter, Ralstonia, Pseudomonas), fixed onto zeolites (clinoptilolite and mordenite). The estimated results after implementing the process are: the obtaining of purified water, which can be disposed in rivers or, by completion of the process, can be used as drinkable water. The biomass resulted from the re-conversion of the pollutant can be transformed into organic fertilizers. The technology proposed is based on bioprocesses, which finally could lead to: the design and development of some advanced environmentally-friendly procedures for wastewaters de-polluting and for the protection of natural ecosystems; the re-conversion of material energy from polluted wastewaters stereo-structures and the achievement of some sources bearing new values (organic fertilizer); the decrease of external environmental costs (by punctual de-pollution), a purpose in synergy with the European Union initiatives. The aerobe procedure zeolites-biofilms generates no pathogen microorganisms, in contact with the immersible substrate, and the zeolites action coupled with the biofilm allows a favorable spatial arrangement, which assures an intense bio-degradative enzymatic action. The use of biotechnologies based on biofilms fixed on zeolites should lead to the 3

decrease of: inputs and water polluting degree (up to maximum 125 mg O2/dm Chemical Oxygen Demand).


Session 2 /

PC153 S2

Biosorption of lead, copper and zinc by Penicillium sp.: Equilibrium and kinetic studies a

b

b

b

a*

L. Ezzouhri , E. Castro , M. Moya , F. Espinola and K. Lairini a Department of Biology, Faculty of Sciences and Techniques - Tangier, University Abdelmalek Essaadi. B.P. 416,Tangier, Morocco, E-mail address: [email protected] b Department of Chemical, Environmental and Materials Engineering, University of Jaén, Campus Las Lagunillas, 23071 Jaén, Spain

Abstract: A heavy metal resistant fungus Penicillium sp. was isolated from heavy metal contaminated sites in Tangier. The biosorption abilities of Penicillium sp. biomass were studied for lead, copper and zinc removal from aqueous solutions. Heat inactivated (killed) biomass was used in the determination of optimum conditions before investigating the performance of pretreated biomass. The influence of initial pH, contact time and initial metal ion concentration were evaluated on the biosorption studied. The maximum biosorption values capacities of Pb(II) Cu(II) and Zn(II) were found to be 23.5 mg/g, 13.03 mg/g and 5.5 mg/g at pH 5.5, 4.5 and 5, respectively. The multicomponent biosorption of Pb(II), Cu(II) and Zn(II) from binary and ternary mixtures was also described, compared to single metal ion situation in solution. The results showed that the Penicillium sp. is a potential engineering biosorbent for Pb(II), Cu(II) and Zn(II) removal from aqueous solutions.


Session 2 /

PC154 S2

Antibacterial Substances extracted from halophilic bacteria isolated from Dead Sea Abdul Jabbar N. Al-Shammari, Mazin K. Qato and Mohammad H. Semreen Faculty of Pharmacy, Al-Isra University, P.O.Box 33 Amman 11622 Jordan, E-mail: [email protected]

Abstract: Antibiotic like substances were extracted from culture of two halophilic strains isolated from black mud of Dead Sea in Jordan side. The substances inhibited the growth of gram positive and gram negative bacteria, representative by Staphylococcus aureus, Bacillus subtilis and E.coli. The antibacterial activities based upon the ability of the extracted substances to inhibit the growth of bacteria monitoring by Kriby-Baura method and Ihara method. No antibacterial activity was found when we used the saturated disc with supernatant of other halophilic isolates. Isolates Bm113 and PSM 142 supernatants shows clear zone of inhibition measured 19,22,and 16 mm on lawn of S.aureus,B.subtilis and E.coli respectively for strain Bm 113, and 21 24,and 17 for the same bacteria mention above for PSM 142. On second method which described by Ihara et al. (1997) the supernatant from two strains were added to the growth of indicator bacteria broth culture, after 18 hrs of incubation agar plates were examined for colonies growth. S.aureus were inhibited at 0.25-0.50 ug/ml, B.subtilis were inhibited by 0.75-1.0 ug/ml and E.coli 2.4-4.8 ug/ml. These preliminary results open the doors for new substance which never explore before, on other hands further investigation to identify the chemical nature and the mode of action need.


Session 2 /

PC155 S2

The biosynthesis of mycolyc acid in fermentation leading by temperature of corynebacterium glutamicum 1

2

2

2

2

A. Guermouche , M. Fick , E. Guedon , S. Delaunay and JL Goergen 1

2

Département de Biotechnologie, IGMO, Université Es-Sénia, Oran, Algérie. Laboratoire de Sciences de Génie Chimique – CNRS –2, avenue de la Forêt de Haye, BP 172, F-54505 Vandoeuvre-lès-Nancy, France.

Abstract: In mycobacteria, more particularly corynebacteria, the cellular wall fluidity modification depends on its composition in mycolic acids. The rate, structure, and composition modification of the mycolic acids double-layer, well known very impermeable, is a key factor to provide the glutamate excretion in the culture medium. The evolution of corynebacterium glutamicum 2262 lipidic contents was studied during the thermal technique of glutamate production. The alkaline extraction of the parietal lipids whose acids mycolic was carried out at identical temperature to that maintained in the bioreactor in discontinuous (33 and 39°C). After the thermal shock (at 39°C), whereas the production of glutamate started, the membrane contents changed significantly. In order to better characterize the mechanism of the glutamate excretion by Corynebacterium glutamicum 2262, we analyzed by chromatography the parietal lipids as well as cytoplasmic proteins of Corynebacterium glutamicum during the discontinuous glutamate producing process. Whereas the proteins 2D gel lets appear only one modification in the protein levels whithin thermal shock, the lipidic profile of the cell wall varies when they produce glutamate. Indeed three various additional spots appear in a chromatography analysis. The comparative study by chromatography, between the glutamate producing and noproducing cells let us observe a structural lipidic difference in the cell wall : in no producer variant (2262 NP) isolated in continuous culture with 39°C and in C. glutamicum cultivated with 33°C, we found the same lipidic profile. Keywords: mycolic acids – Temperature - glutamate excretion - membrane fluidity - thin layer chromatography.


Session 2 /

PC156 S2

Improvement of the antimicrobial activities of herbal infusion by fungal fermentation 1,2

Houda Battikh

1

, Emna Ammar, Kamel Chaieb and Amina Bakhrouf

Laboratoire de Contrôle et d’analyse des polluants chimiques et microbiologique, Faculté de Pharmacie, Rue 2

Avicenne, 5001 Monastir Unité de recherche Etude et Gestion des Environnements urbains et Côtiers ENIS, BP « W », 3038 Sfax

Abstract: Kombucha is a popular beverage among many traditional fermented foods across the world. Kombucha is also frequently called “tea fungus” in the literature, although there is actually no fungus involved in the fermentation but a symbiosis culture between bacteria and yeasts. Current strong and increasing interest and consumption of the product derives from its purported therapeutic benefits and particularly its antimicrobial potential proved. Kombucha is typically prepared by fermenting sweetened black tea with what is popularly known as a "tea fungus" at room temperature for 10 days. In this study we have fermented of six different Tunisian medicinal herbal extract by Kombucha consortium and tested its antimicrobial activity against a broad spectrum pathogenic bacteria (n=7) and a range of pathogenic fungi (n=7). The results were very promising and shown an improvement of the antibacterial and the antifungal potential of the herbal infusions by the Kombucha fermentation and demonstrated that the fermentation can be very useful in treatments of pathogenic microorganisms and particularly Candida strains. Key words: Kombucha, antibacterial and antifungal activities,Tunisian herbal extracts, fermentation, Candida


Session 2 /

PC157 S2

Effect Of Epigallocatechin gallate On The Antimicrobial ActivitiesOf β-lactams Against Different Types Of β-lactam Resistant Bacteria Ramadan Ahmed Al domany Department of Microbiology and Immunology, Faculty of Pharmacy Helwan University

Abstract: A total of 30 bacterial isolates including 10 Staphylococcus aureus, 10 Escherichia coli and 10 Pseudomonas aeruginosa were examined for their susceptibility to β-lactam antibiotics (ampicillin, amoxicillin, cephalexin, cefotaxime and imipenem) and epigallocatechin gallate to determine their MICs. Staphylococcus aureus isolates were also examined for their susceptibility to methicillin. Results indicated that the MICs of βlactams (ampicillin, amoxicillin, cephalexin and cefotaxime) against the tested S. aureus isolates ranged from 64 to 1024µg/ml while for imipenem ranged from 0.25 to 16 µg/ml. All the tested S. aureus isolates methicillin resistant and the MICs ranged from 64 to 256 µg/ml. The MICs of EGCg against all the tested S. aureus isolates were 100 µg/ml. The MICs of the tested β-lactams against the tested E. coli isolates were ranged from 32 to 512 µg/ml while for imipenem ranged from 0.5 to 32 µg/ml. The MICs of the tested β-lactams against the tested P.aeruginosa isolates were ranged from 64 to 1024 µg/ml while for imipenem ranged from 0.5 to 32 µg/ml. On the other hand, the MICs of EGCg against the tested E. coli and P.aeruginosa isolates were ≥800 µg/ml. All the tested bacterial isolates were examined for thier β-lactamase production using nitrocefin and the results indicated all the isolates were βlactamase producer. Interstingly, combination of one fourth the MIC of EGCg with β-lactams resulted in 3-7 times reduction in the MICs against the tested S. aureus isolates. On the other hand, only 1-3 times reduction in the MICs of βlactams were observed among the tested E. coli and P.aeruginosa isolates. Furthermore, combination of half the MIC of EGCg with β-lactams resulted in further reduction in the MICs with increasing in the antimicrobial activity against S. aureus isolates while the MICs of β-lactams were more or less affected for both E. coli and P.aeruginosa tested isolates. In conclusion, the combination of β-lactams with EGCg showed potent synergy with restoring the antimicrobial activities against β-lactamase producing S. aureus isolates in-vitro. The combinations of β-lactams and EGCg against β-lactamase-producing Gramnegative rods do indicate a limitation owing to the cellular location of β-lactamases. Therefore, the combinations may be worthy of further evaluation in vivo against MRSA infection.


Session 2 /

PC158 S2

Studies Leading to a Pilot Process for the Removal of Dibenzothiophene from Solutions Dr. Khalil Y. Mataqi Associate Research Scientist, Kuwait Institute for Scientific Research, Al-Shuwaikh, State of Kuwait

Abstract: Sulphur dioxide released into the atmosphere from the combustion of both crude oil and coal remains one of the most prominent and intractable issues of environmental concern, as it is a major contributor to the air pollution.The separation of dibenzothiophene (DBT) from hydroxybiphenyl (HBP) mixtures was attained by the adsorption of HBP with alumina and the elution of DBT with toluene. Alumina (5g) can adsorb HBP up to 40mM. The HBP was removed, in a controlled way, by elution with dichloromethane. DBT was extracted from DBT-toluene mixture with deionised water. A pilot system was designed after combining the above steps and characterized by: (1) no significant increase in the conversion of DBT to HBP when the air distribution (10%) was throughout the chemostat rather than as one central supply. (2) as the number of immobilised cells added to the system increases the conversion of DBT to HBP increases. To use all the DBT added to the system the DBT has been recycled. But when the recycled DBT compared with the freshly DBT (6.0mM) added to the system in the conversion to HBP the fresh DBT showed a constant conversion of HBP, whereas, the conversion to HBP was reduced when the recycled DBT was added. The limitations of the system were determined at different dilution rates (0.13-0.2 h-1) and the optimum dilution rate was found to be 0.13 h-1 with a 43.73% conversion rate of DBT to HBP. Furthermore, this system could account for the removal of DBT up to 21 mM.


Session 2 /

PC159 S2

Biochemical and molecular characterization of pathogenic vibrios in aquaculture Lajnef R (1, 3), El Bour M.(1), Gargouri R(2) and Hassen A (3) 1: Laboratoire de l’Unité Pathologique des Organismes Marins, INSTM Salambo. 2: Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax. 3: Laboratoire de Traitement et Recyclage des Eaux, Centre de Recherches et des Technologies des Eaux, Technopole Borj Cedria E-mail : [email protected]

Abstract: The aim of the present investigation was to clarify the identification of vibrio strains isolated from Tunisian aquaculture systems and to compare the identification results made by phenotypic and molecular tools. Ten vibrio strains isolated from an aquaculture system were characterized by phenotypic and molecular methods. According to their phenotypic properties, these strains had been identified as belonging to the genus Vibrio; whereas they diverged towards three genus: Enterococcus, Proteus and Vibrio by genotypic tools, so the use of 16S rRNA gene sequencing allowed differentiation among vibrio strains. The molecular methods, as 16S rRNA gene sequencing analysis and ribotyping are necessary for the separation of phenotypically close species to avoid miss identification. Key words: Vibrio; ribotyping, 16S rRNA, sequencing


Session 2 /

PC160 S2

Impact study and valorization of waste of metal furniture by the lca method Z. Kebbouche*, A.Tairi*, A. Cherifi* and S. Tireche *Laboratoire LSP : ergonomie et protection de l’environnement. Université de Boumerdès. Avenue de l’indépendance. 35000 Boumerdès E. Mail : zahia_kebb@ yahoo.fr N° tel : Kebbouche 072 57 37 56

Abstract: The search of a sustainable development leads some companies to deepen more and more an approach characterized by the integration of the ecological data since the start of industrial projects and, in particular, at the products design level. The most recognized tool at the moment, at an international level is the Life Cycle Assessment (LCA), which is subject to ISO 14040-14043 international normalisation. The LCA method allows a scientifique and objective evaluation of potential impacts of the product, considering the totality of its life cycle. The study carried out aims at quantifying the environmental impacts of an office, manufactured within “Algerian company of Metal Furniture CAMMO”, using the LCA method. On the whole, the companies of furniture are unaware of the various impacts of their products. More importantly, they are unaware of what is becoming of their products at the end of the lifetime. Among the most significant activities, we can cite: the surface treatments, painting, the machining and the transformation of metals. The principal impacts of these activities relate to the production of solid wastes, liquid effluents, or the atmosphere rejects. Other rejects, yet emitted out of the walls of the company, are also ascribable to the product, they are those generated by the suppliers of materials and components which take part in the composition of these products. After having defined the functional unit (ISO 14040), the study passes through an inventory of the emissions and raw materials, within the company and with the raw material suppliers (ISO 14041). To model the systems and to calculate the inventories of the life cycle of products, we have used the software Sima Pro 6.0. The Sima Pro is software of analysis of the life cycle of the products, developed by the Pré consultant in the Netherlands. The method of Eco-indicator 99 and CML, are used in order to characterize the impacts of the office. All the compartments are polluted: air, water and ground. Among the various stages of life that we have studied, the office has the most important impact at the time of the stage of extraction of the raw materials, the stage of manufacture and the stage of the end of lifetime which is unfortunately not dealt with. If we want to reduce these impacts, it is necessary to try to control those related to the phases of manufacture and to try to enhance the wastes of this product at the end of the lifetime. This impact study makes it possible to release the possibilities for environmental improvements of the product, which can touch several weak points of the product and especially can relate to all the range of the products manufactured within the company (all metal furniture).


Session 2 /

PC161 S2

The α-amylase gene from Bacillus subtilis US116 strain encoding an enzyme closely identical to that from Bacillus amyloliquefaciens but distinct in thermal stability Ezzedine Ben Messaoud, Sameh Ben Mabrouk, Sonia Jemli and Samir Bejar Centre de Biotechnologie de Sfax - Laboratoire d’Enzymes et de Métabolites des Procaryotes BP « K » 3038 Sfax, Tunisie. Tél. : 74 870 816, Fax : 74 870 818, E-mail : [email protected]

Abstract: The gene encoding for the α-amylase AMYUS116 was cloned and sequenced. The amino acid sequence of AMYUS116 exhibited an almost perfect homology with the aamylase BACAAM, excluding the residues N205 and N217 of AMYUS116 that were changed to H205 and I217 into BACAAM. Three mutant derivatives from AMYUS116 (N205H, N217I and N205H/N217I) were created by site directed mutagenesis and their physicochemical and kinetic properties were compared to those of the wild type enzymes. Therefore, the undertaken amylases mainly generated maltohexaose from starch and had radically the same kinetic parameters and optimum pH and temperature. They, however, were significantly distinct in thermal stability; AMYUS116 was more thermo-sensible since its half-life time at 80°C was 13 min while those of BACAAM and the double mutant were likewise 38 min. The single mutant amylases exhibited an identically intermediate thermal stability since their half-life times at 80°C were roughly 22 min. Of particular interest to the current search, the different thermal stability between AMYUS116 and BACAAM can lead to novel findings pertaining to protein stability, which can bring about new strategies for protein engineering. Basically, the comparative study of closely related amylases and the protein engineering of already existing ones are certainly important for they offer opportunities to understand the structure-function relationships of these biocatalysts.


Session 3

Session 3: Agricultural, Food and Marine Biotechnology -

Genetically modified crops Plant-microorganism interaction Biopesticides Marine biotechnology Biotechnological improvement of plants and food processes



Session 3



TC 1 S3

Rice Mutant Resources for Deciphering Developmental and Adaptive Processes Emmanuel Guiderdoni Plant Development and Genetic Improvement Unit (UMR DAP 1098), Cirad, Biological Systems Department, F-34398 Montpellier Cedex 5, France.

Abstract: Paralleling the international program of sequencing of the rice genome which culminated with the publication of the 390 Mbp chromosome sequence end of 2004, considerable efforts have been undertaken in this model cereal for generating insertion libraries, characterizing flanking sequences of inserts and gathering phenotype and sequence information in web-accessible databases. Several independent national initiatives in Korea, China, France and Taiwan launched in the late 1990s have led to the generation of more than 460,000 T-DNA insertion lines and the release of more than 113,000 Flanking Sequence Tags (FSTs) in public databases. It is presumed that the full characterization of all these lines with other concurrent initiatives using the maize Ac/Ds and En/Spm transposable elements and the tissue culture stimulated endogenous retrotransposon Tos17 will enable rice geneticists to find at least one insertion in any rice gene and several alleles in most of the genes. To contribute to the international effort of production of sequence-indexed insertion libraries in rice, we have generated a collection of 30,000 Nipponbare lines which are characterized by 25,000 and 15,000 flanking sequence tags of T-DNA and Tos17 inserts respectively. These lines are being field evaluated and seed increased in CIAT, Colombia and the phenotypes documented in the Oryza Tag Line database at http://urgi.versailles.inra.fr/OryzaTagLine/. This work was conducted in the frame of the national genomics initiative Génoplante (http://genoplante.com) and also supported by the National Sequencing Centre, Génoscope. In France, several research groups are now using these resources to decipher the molecular control of grain filling processes, response to biotic and abiotic stresses and of plant architecture determinants. Our group has focused its research on root development and plasticity in response to osmotic stress. Aside forward genetic screens which have yielded tagged mutants currently under analysis, a medium scale reverse genetics screen was conducted in the frame of projects supported by the Generation Challenge Program and the European Union. This screen included genes identified through expression profiling in osmotic- and salt stressed tissues (SSH microarrays) or orthologs of Arabidopsis developmental and stress-related genes, and also a long term, overall investigation of the role of the rice Leucine-rich repeat receptor like kinase (LRR-RLK) (320 genes), CLV3 like (30 genes) and WOX (15 genes) genes in development and biotic/abiotic stress response.



TC 2 S3

Basis of resistance to Bacillus thuringiensis toxins in Lepidoptera Juan Ferré, Salvador Herrero and Baltasar Escriche Department of Genetics, University of Valencia, Spain.

Abstract: The production of a parasporal crystal differentiates Bacillus thuringiensis (Bt) from other ubiquitous gram-positive and spore-forming bacteria, such as Bacillus cereus. This bacterium has been long characterized as an insect pathogen, and its toxic activity has been reported against several orders of insects, mainly Lepidoptera (butterflies and moths), Coleoptera (beetles) and Diptera (mosquitoes and blackflies). Bt produces a variety of insecticidal compounds which constitute the active ingredient of many bioinsecticides, but the most successful application of Bt is the development of insect-resistant plants, accomplished by transferring Bt genes to the plant genome. Since 1996, Bt crops expressing one or more Bt genes are planted commercially with great success. The main threat of this techonology is the evolution of resistance in insect populations. The first report on resistance to Bt appeared in 1985, when the general believe was that insect could never develop resistance against a “natural” insecticide. Since then on, the number of reports on insect resistance to Bt strains and toxins has grown steadily and so far 11 species of Lepidoptera, 2 of Coleoptera, and one of Diptera are known to have developed resistance to this bioinsecticide. Despite of this relatively long list, only one insect species, the diamondback moth, has evolved resistance in the field, although several times independently. More recently, the cabbage looper, Trichoplusia ni, has evolved resistance in Canadian greenhouses. It is important to determine the biochemical basis of resistance to help design the most appropriate management strategy, especially now that Bt-crops are being adopted globally. The study of the mechanism of resistance in many resistant strains has resulted in that the most common mechanism conferring high levels of narrow-spectrum resistance is an alteration in the binding site of Bt toxins. It is especially so in the cases of populations that have evolved resistance in the field or in greenhouses. However, a closer look at the reported binding site alterations indicates a heterogeneity in the type of Cry1A receptor modification.



TC 3 S3

Mechanisms mediating Ca2+ involvement in pathogen perception, programmed cell death and plant innate immunity Gerald Berkowitz Wei Ma, Rashid Ali, Andries Smigel, Robin Walker, Fouad Lemtiri-Chlieh, Rajeev Verma, Chris A. Gehring, and Gerald A. Berkowitz Agricultural Biotechnology Laboratory, University of Connecticut, Storrs, CT, USA

Abstract: A rise in plant cell cytosolic Ca2+ is an early step in pathogen perception and subsequent nitric oxide (NO)-mediated signaling cascades leading to plant innate immunity and H2O2 generation as part of a hypersensitive response (HR) leading to programmed cell death. Molecular events linking pathogen perception by plant cells to cytosolic Ca2+ rise are unknown. We identify steps upstream from inward Ca2+ current as involving pathogeninduced rise in cAMP and/or cGMP, and activation of a cyclic nucleotide gated channel (CNGC) leading to HR-associated NO and H2O2 generation. Pathogen associated molecular pattern (PAMP) molecules are highly conserved essential components of microbes that are recognized by plant cells initiating an innate immune response. The PAMP lipopolysaccharide (LPS) activates Ca2+ currents leading to NO and H2O2 generation, linking pathogen perception to the activation of CNGC channels. Plant cell cytosolic cAMP level is controlled by the integrated activities of adenylate cyclase (AC) (cAMP synthesis) and cAMP-phosphodiesterase (PDE) (cAMP breakdown). An AC inhibitor prevented LPS induced NO and H2O2 generation. In the absence of LPS, addition of either cAMP or a PDE inhibitor led to NO generation. Bioinformatic analysis identified leucine-rich-repeat receptors as candidate ACs and a putative guanylate cyclase (GC) domain in a known PAMP-receptor; AC and GC genes associated with this CNGC pathogen signaling have not yet been identified. In plants inoculated with a bacterial pathogen, block of cAMP synthesis prevented HR and impaired the rise in cytosolic Ca2+ upon pathogen perception. Our work suggests that pathogen perception during plant innate immunity may involve PAMP receptor mediation of cyclic nucleotide rise, initiating a signaling cascade involving downstream Ca2+, NO, and H2O2.



TC 4 S3

Functional characterization of genes involved in drought and salinity tolerance in plants Khaled Masmoudi, Faïçal Brini, Moez Hanin, Habib Khoudi Centre of Biotechnology of Sfax (CBS). Plant Molecular Genetics Unit. Route Sidi Mansour Km 6, B.P’K’ 3038 Sfax-Tunisia. Tel. +216-74 440 816 ext.1092; Fax: +216-74 440 818; email: [email protected]

Abstract: Drought and salinity are major constraints on crop production and food security, and adversely affect entire countries over several years and result in serious social, economic, and environmental cost. Wheat production in the Mediterranean region is limited mainly by the availability of water resources. Water-deficit stress caused by drought and soil salinization adversely affects plant growth and crop productivity. Adaptation of plants to salt stress (i.e. resumption of growth after exposure to high soil salinity) requires cellular ion homeostasis involving net intracellular Na+ and Cl- uptake and subsequent vacuolar compartmentalization without toxic ion accumulation in the cytosol. Sequestration of Na+ ions into the vacuole through the action of a vacuolar membrane Na+/H+ antiporter and H+-Pyrophosphatase pump is one mechanism that confers salt tolerance to these organisms. Accumulation of organic solutes due to dehydration is another mechanism by which plants physiologically adapts to plant water deficit. Dehydrins are involved in the adaptation to water and osmotic stress. The full-length cDNAs of wheat Na+/H+ antiporter, TNHX1, V-H+-pyrophosphatase, TVP1, and dehydrin, DHN-5 were cloned, sequenced and functional characterized. Transgenic Arabidopsis plants overexpressing the wheat candidate genes are much more resistant to high concentrations of NaCl and to water deprivation than the wild-type strains. These transgenic plants grow well in the presence of 200 mM NaCl and also under water deprivation regime, showed a faster recovery from mannitol treatment, while wild type plants exhibit chlorosis and inhibition of growth. The leaf water potential was less negative for wild type than for transgenic plants. This could be due to an enhanced osmotic adjustment in the transgenic plants. Moreover, these transgenic plants accumulate more Na+ and K+ in their leaf tissue than the wild type plants. The toxic effect of Na+ accumulation in the cytosol is reduced by its sequestration into the vacuole. Water loss rate under drought or salt stress was higher in wild type than transgenic plants. Increased vacuolar solute accumulation and water retention could confer the phenotype of salt and drought tolerance of the transgenic plants (Brini et al., 2005; 2007a, b, c). Overexpression of the isolated genes from wheat in Arabidopsis thaliana plants is worthwhile to elucidate the contribution of these proteins in the tolerance mechanism to salt and drought. A similar strategy could be one way to develop transgenic staple crops (wheat) with improved tolerance to these important abiotic stresses. Transgenic wheat plants overexpressing the candidate genes mentioned above are being produced. Genetically engineered drought- and salt-tolerant plants could provide an avenue to the reclamation of farmlands lost to agriculture because of salinity and a lack of rainfall.



TC 5 S3

Extraction of “green labelled” pectin from plant by-products Agata Zykwinska, Maud Panouillé, Jean-François Thibault and Estelle Bonnin INRA, UR 1268 Biopolymères, Interactions, Assemblages, F-44000 Nantes, France

Abstract : Plant cell walls are constituted of proteins and polysaccharides, among which cellulose, hemicelluloses and pectins. Pectin is a family of complex and heterogeneous polysaccharides that can be viewed as multiblock co-biopolymers. Several blocks are associated one with another, among which: homogalacturonan (HG), xylogalacturonan (XGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII), arabinan, and (arabino)galactan., which can be extracted from plant cell walls and used in food industry as additives due to their gelling, stabilising and thickening properties. At the industrial scale, pectins are extracted in acidic conditions most often from apple pomace and citrus peels. However, acidic extraction generates large amounts of effluents that required to be treated. Moreover, the consumer demand for “green” products stimulates the search for alternative means of extraction. Therefore, enzymes could represent an alternative and environmentally friendly way to extract pectins. Following this idea, the use of proteases and cellulases preparations was investigated in order to extract “green labelled” pectins from plant by-products. Different enzymatic preparations were characterized by their activities towards proteins, cellulose and pectins. These preparations were then tested regarding pectin extraction from different plant by-products (chicory roots, citrus peel…). The yield of enzymatic extraction of pectins was lower (12%) compared to acidic one (28%). However, the enzyme-extracted high-methoxy (HM) pectins presented better gelling properties in the presence of sucrose at low pH (3) than the acid-extracted pectins. These HM pectins can be demethylated using Pectin Methyl Esterases (PMEs). Demethylation could be done after protease and cellulase extraction (2-step treatment) or PME can directly be added to the protease and cellulase mixture (1-step treatment). In both cases, low-methoxy (LM) pectins present good gelling properties in the presence of calcium. It is therefore possible to obtain “green labelled” HM and LM pectins with real functional properties. Acknowledgment: We gratefully acknowledge funding from the Commission of the European Community, Program Priority Food Quality and Safety, FOOD-CT-2005-006922, “Reducing Food Processing Waste”.



TC 6 S3

Genetic transformation of forest trees Franche, C. Equipe Rhizogénèse Symbiotique, UMR DIA-PC, IRD (Institut de Recherche pour le Développement), 911 avenue Agropolis, BP 5045, 34394 Montpellier Cedex 5, France.

Abstract: In contrast to other agronomic and horticultural species, trees possess several physiological characteristics such as long life cycles, wood production, phases of dormancy combined with storage processes, and a long distance between the shoot and the root. Biotechnology thus plays a major role in attempts to improve tree characteristics, and since the first successful transformation of a tree species in 1987, transgenic trees have become essential tools for forest tree biotechnology and tree physiology. Our laboratory has developed genetic transformation procedures based on Agrobacterium for the actinorhizal forest tree Casuarina glauca. This tropical species is nodulated by the nitrogen-fixing actinomycete Frankia and also forms symbiotic association with both ecto- and endomycorrhizal fungi. In arid and semi arid regions with poor forest ressources, Casuarina plays a major role in sustaining soil fertility and crop production, in controlling efficiently wind and water erosion, and in providing fire wood. One major goal of our laboratory is to study the plant genes that are expressed specifically or at enhanced levels in actinorhizal nodules. In this context, transgenic trees are a major tool for the study of the regulatory mechanisms that control the expression of the actinorhizal symbiotic genes, for dissecting the roles of specific proteins in nodule development, and for comparing regulatory mechanisms of legume and actinorhizal symbiotic genes. The understanding of the different types of nitrogen-fixing nodules will help to develop future strategies to modify lateral root development on non-symbiotic plants to enable some to associate with nitrogen-fixing bacteria.



TC 7 S3

BaSysBio, a systems biology approach for Bacillus Subtilis modeling S. Pérès and F. Molina SysDiag FRE3009 CNRS BIO-RAD Complex system modelling and engineering for diagnosisCap delta/Parc euromédecine 1682 rue de la Valsière CS 61003, 34184 MONTPELLIER Cedex 4 00 33 (0)4 67 16 66 03 Fax: 00 33 (0)4 67 16 66 01, [email protected]

Abstract: BaSysBio European Integrated Project (15 partners) aims to achieve major breakthroughs in the understanding of the regulation of gene transcription in bacteria at a global scale. The highly dynamic gene regulation is mediated by transcription factors (TF) that trigger or repress the expression of their target genes. Transcription control is embedded into a hierarchical flow of information from genes to phenotype in which many regulatory steps can occur. BaSysBio adopts a systems biology approach in which quantitative experimental data will be generated for each step of the information flow, and will fuel computational modelling. High-throughput technologies (living cell arrays, tiling DNA microarrays, multidimensional liquid chromatography proteomics and quantitative metabolomics) will be developed in conjunction with new computational modelling concepts to facilitate the understanding of biological complexity. Models will simulate the cellular transcriptional responses to environmental changes and their impact on metabolism and proteome dynamics. The iterative process of simulations and model-driven targeted experiments will generate novel hypotheses about the mechanistic nature of dynamic cellular responses, unravel emerging systems properties, and ultimately provide an efficient roadmap to tackle novel, pathogenic organisms. Our contribution to BaSysBio project relates to multi-scale functional network modelling dealing with complex biological data. We use the bioΨformalism (1) based on elementary action to provide dynamics models and simulation relevant with biological knowledge. Such modelling approach is combined with elementary mode network analysis. (1) Maziere P., Granier C. and Molina F. A biological processes description scheme based on elementary bricks of action. J. Mol. Biol. (2004) 339, 77–88 This work is supported by BaSysBio EU FP6 project



TC 8 S3

Biotechnology, morphogenetic plasticity of floral tissues and sex determinism in dioecious plant species Noureddine Drira Faculté des Sciences de Sfax, Laboratoire des Biotechnologies Végétales Appliquées à l’Amélioration des Cultures

Abstract: In some plants such as Date palm, the sexes are separated leading to male and female plants. This sex separation is mediated by sexual chromosomes. The investigation of in vitro development of flower initials in these plants allows the identification of the different morphogenetic behaviours susceptible to be induced by specific factors of the medium (growth factors/ hormones, trophic factors and gamma ray irradiation). The study of flower initials can favour several opportunities in changing the architectural model of the plant and can reveal the reel degree of flower ramification that was previously contracted to a maximum during evolution. On the other hand, the flowers developed on plants regenerated in vitro from leaf tissues are usually subjected to abnormalities and variations. However, the floral material displayed a high stability when it is used as source of in vitro multiplication. Finally, the hermaphrodite state imposed experimentally to flowers and female plants allows a better understanding of sex genesis.



TC 9 S3

Plant Biotechnology in Egypt: Current Status and Future Scenarios Mahmoud M. Saker Prof. & Head of Plant Molecular Genetics Group (PMGG) & Coordinator of Nobel Project, National Research Center (NRC), El-Behouth St., Dokki, Cairo, Egypt, P.O. Box: 12622, Tel: + (202) 33371362 ext. 2408/2285, + (202) 33357897 (direct), Fax: + (202) 33370931, Mobile phone: + (2) 010 1771691, [email protected]

Abstract: Mankind has used plants as main source of food, raw materials for industrial applications and energy for thousands of years. Also, from the earliest stages of civilization, plant extracts have been used to obtain chemicals, materials and drugs to cure diseases. The natural high degree of biodiversity existed among plant genetic resources in early stages of history, provided mankind with sustainable food, clothes and medicines throughout the successive eras. As result of extensive breeding programs, extensive applications of agrochemicals (green revolution), reclamation of new lands and urbanization, we are suffering nowadays from biodiversity crises and our food crops became susceptible to pests and sensitive to abiotic stresses. Moreover, global warming, desertification, shortage of water resources and un-renewable energy and continued increment in population of developing countries are new emerging problems facing mankind nowadays. Fortunately, there is still one room to increase food crop production and to grow food crops in new lands, which were unsuitable for cultivation before through biotechnology. To fight hunger and malnutrition, drought and desertification, shortage of non-renewable energy and epidemic killer diseases, new non traditional food and fruit crops, with improved nutritional quality and with low water demand should be developed and considered as staple food crops, to replace the existing water consuming crops. This review highlights the current status and future scenarios of plant biotechnology activities going on at National Research Center (NRC) of Egypt. These activities can be divided into three main eras as follows: 1- 1st era (mid eighty's-mid ninety's) has been focused on establishment of human resources (HR) and lab facilities, rapid mass micropropagation, in vitro production of secondary metabolites and production of biocontrol agents and biofertilizers. 2- 2nd era (1995-2005) has been focused on establishment of regeneration and transformation systems of some economically important crops, establishment of bioreactor-based production of bioactive substances, DNA fingerprinting, cryopreservation and molecular markers. 3- 3rd era (2005-2015) is again focusing on HR and changing brain drain into brain gain (Nobel project), production of transgenic plants, cloning of plant genes, effective and better utilization of native germplasm, molecular farming and energy crops.


Oral Communications

Session 3


Session 3

OC1 S3

Global quantitative analysis of protein expression and phosphorylation status in the liver Medaka exposed to microcystin-LR Karim Mezhoud1, Danièle Praseuth2, Simone Puiseux-Dao1, Jean-Christophe Fran¸cois2, Cécile Bernard1, Marc Edery1_ 1 USM505: ´ Ecosystèmes et interactions toxiques. 12, rue Buffon, F-75231, Muséum national d’Histoire naturelle, Paris cedex 05, France. 2 CNRS, UMR5153, Paris, F-75231 France; Inserm, U565, Paris, F-75231France; Muséum national d’Histoire naturelle, USM0503, Paris, F-75231,France

Abstract: Microcystins (MCs) are hepatotoxins with a potent inhibitor activity of protein phosphatases PP1 and PP2A. These non-ribosomal peptides are getting more and more attention due to their acute toxicity and potent tumor-promoting activity. These toxins are produced by freshwater cyanobacteria. We report a toxicological study conducted on aquatic animal models such as the medaka fish. To date, the detailed mechanisms underlying the toxicity of microcystins are unknown. MC-leucine-arginine (MC-LR) is the most toxic and the most commonly encountered variant of MCs in aquatic environment. It has been used for toxicological investigations on the liver of intoxicated medaka. We performed differential proteome analyses of MC-LR-treated and untreated medaka fish in order to investigate the mechanisms of establishment of early responses to the toxin. The identification of proteins involved in these early responses might constitute candidates as biomarkers of MC-LR exposure. Cytosolic proteins from livers of exposed or non-exposed medaka were resolved by 2D electrophoresis and detected using stains specific for phosphoproteins and for whole proteinacoeous content. Overall, fifteen spots were found to vary significantly on the proteomic 2D maps or on the phosphoproteomic 2D maps. Of these 15 proteins, only two could not be identified by mass spectrometry. Among the other identified proteins, phenylalanine hydroxylase and keratin 18 (type I) showed variations in phoshoryl content in agreement with inhibition of PP2A activity after exposure of the fish to MC-LR. The other identified proteins exhibited variations in their expression level. The identified proteins appear to be involved in cytoskeleton assembly, cell signalling, oxidative stress and apoptosis. The functional implications of responses to MC-LR exposure of these proteins are discussed. The methodology described in this report should be widely generalizable to a number of tissues and organisms, thus helping in the search for biomarkers of MC-LR contamination. Keywords : Toxicology - Microcystin-LR - Medaka - Proteomics - Phosphorylation - Mass spectrometry.


Session 3

OC2 S3

Comparative study of the response of growth, nitrogen fixation and carbohydrate metabolism in Medicago cilaris lines cultivated under salt stress Ben Salah I.1, Albacete A.2, Martínez Andújar C.2, Zribi F.1, Pérez-Alfocea F.2& Abdelly C.1* 1 2

Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, CBBC, BP 901, 2050 Hammam-Lif Tunisie ; Department of Plant Nutrition, CEBAS-CSIC, Campus de Espinardo, 30100 Mucia Spain

Abstract: In this study, two lines of Medicago ciliaris (TNC1.8 and TNC11.9) were used. After germination and inoculation with bacterial suspension, seedlings were grown in pots filled with vermiculite under semi-controlled conditions in a glasshouse. The saline treatment (100mM of NaCl) was applied after the appearance of nodules and plants were harvested at the end of the vegetative period (58 days after sowing). Under conditions of salt stress, the two lines showed a decrease of total biomass production, but TNC 1.8 was less affected by salt than TNC 11.9. Chlorophyll content was not changed in TNC 1.8 by contrast with TNC 11.9. Except leaves of TNC 1.8, all organs biomass were affected in the two lines, but TNC 1.8 exhibited the higher potentialities of biomass production of these organs. Nitrogen fixation, estimated by the measurement of acetylene reduction activity, was also decreased in the two lines, and it was more sensitive to salt than growth parameters. TNC 1.8 exhibited constantly the higher values of nitrogen fixation. Reduced nitrogen contents of all organs were not affected in the tolerant line by contrast with the sensitive one. Under stress conditions, sucrose transport towards young leaves, root tips and nodules of the sensitive line was affected. The same trend was observed in the tolerant line except for young leaves, where the sucrose allocation was not affected. Sucrolytic activities (sucrose synthase, alkaline, vacuolar and parietal invertases) were not affected in young leaves of the tolerant line by contrast with the sensitive line, in which these enzymes activities were reduced. Salt affected roots sucrolytic activities of the two lines with the same intensity. This constraint induced also perturbations in nodule sucrolytic activities in the two lines: It inhibited sucrose synthase but this inhibition was more accentuated in TNC 11.9; alkaline/neutral activity was not altered in TNC 1.8 whereas it decreased more than half in TNC 11.9. Therefore, we can say that in absence of nitrogen source, salt stress reduced Medicago ciliaris growth not only by its depressive effect on nitrogen fixation but also by its depressive effect on the transport and the use of sucrose by growing tissues. The tolerance of TNC1.8 to salt could be attributed to i) the performance of its photosynthetic organs thanks to the maintain of an adequate supply in sucrose and a better use of these substrates and also ii) a better use of these photoassimilats by nitrogen-fixing organs.


Session 3

OC3 S3

Over expression of an alien ZnFAl gene from A. littoralis in tobacco enhances salt and drought stress tolerance Rania Ben Saad, Nabil Zouari, Walid Ben Ramdhan, Jalel Azaza, Afif Hassairi Abstract: Stress perception and signal transduction leading to tolerance, involve a complex interplay of different gene products. Proteins with the A20/AN1 zinc-finger domains are present in all eukaryotes and are well characterized in animals, but little known about their function in plants. We have cloned a gene “ZnFAl” coding for an A20/AN1 zinc-finger protein from Aeluropus littoralis (a halophyte grass). The study of its expression profile in A. littoralis showed that this gene is induced by salt, drought, ABA, cold, Heat, and SA. In order to validate its role in conferring abiotic stress tolerance, we have overexpressed this gene in tobacco plants under the 35S promoter. The F2 generation plants of three transgenic lines are tolerant to salt and drought stress not only in seed-germination but also in vegetative stage. In fact, these plants were able to continue their life cycle and produced viable seeds; however the wild type plants died under the used stress conditions. Thus, ZnFAl gene seems to be an important candidate gene to improve drought and salt tolerance in plants. We have also isolated its promoter by chromosome walking. The in silico analysis of this promoter had shown the presence of some cis-acting elements involved in stress-responsive gene expression. This promoter seems to be tissue specific since the GUS activity was detected only in vascular tissues in stress treatments. Key words: A. littoralis, ZnFAl, abiotic stress, transgenic tobacco


Session 3

OC4 S3

Improvement of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and Manduca sexta by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes Tounsi1, S., Ebn Aoun1, A., Blight2, M. and Jaoua1, S. 1

Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box “K”, 3038. Sfax, Tunisia Centre de Génétique Moléculaire, Bât. 26, CNRS, 91198 Gif-sur-Yvette, France E. mail: [email protected]

2

Abstract: The use of bacteria endowed with entomocidal properties for biological control of insect pests has risen. For such purposes, heterologous expression in P. temperata strain K122, of two B. thuringiensis cry genes encoding Cry1Aa (1) and Cry1Ia delta-endotoxins (2) active on Lepidoptera was studied (3). Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1 x 106 cells ml-1. Recombinant P. temperata strains expressing the cry1Aa and/or cry1Ia genes of Bacillus thuringiensis have been constructed. The two cry genes, encoding delta-endotoxins, were placed under the control of the lac promoter and IPTG dependent expression in P. temperata was demonstrated. The expression of the cry genes in K122 resulted in a clear improvement of oral toxicity. This improvement was of 6.2, 6.6 and 14.6 fold for the strains K122(pBCcry1Aa), K122(pBScry1Ia), K122(pBCcry1Aa + pBScry1Ia) respectively. On the other hand, when tested against M. sexta, after 48 hours post-ingestion of the same number of cells of respectively K122(pBS), K122(pBCcry1Ia) and K122(pBScry1Aa), the weight of M. sexta surviving larvae treated with K122 was 2 to 3 fold higher than those of larvae treated with the two other recombinant strains. This work demonstrates that the heterologous expression of B. thuringiensis cry genes in P. temperata can be used to improve and broaden its host range for insect control. References:

1- Tounsi Slim, J’mal Anis, Zouari Nabil and Jaoua Samir (1999) Biotechnol. Lett. 21, 771-775. 2- Tounsi Slim, Zouari Nabil and Jaoua samir (2003) J. Appl. Microbiol. 94, 1-6 3- Tounsi Slim, Ebn Aoun Ammar, Blight Mark, Rebaî Ahmed and Jaoua Samir (2006) J. Invert. Pathol. 91: 131135.


Session 3

OC5 S3

Application of Proteolysis, Histopathological and immunohistopathological Analyses to Study the Mode of Action of Bacillus thuringiensis δ-endotoxins on Prays oleae and E. kuehniella larvae Rouis, S., Chakroun, M., Saadaoui, I. and Jaoua, S. Centre of Biotechnology of Sfax, Laboratory of Biopesticides. P. O. Box « K » 3038 Sfax-Tunisia

Abstract: Prays oleae is one of the most important olive tree pathogenic insect in the Mediterranean basin and particularly in Tunisia. The mode of action of Bacillus thuringiensis kurstaki Cry insecticidal toxins in Prays oleae midgut was investigated for the first time. The importance of Bacillus thuringiensis δ-endotoxins proteolysis in determining their potency against Prays oleae was confirmed in vitro. By immunohistochemistry, the in vitro and in vivo binding of these toxins to Prays oleae larvae midgut was studied and evidenced a midgut columnar cell vacuolization, microvilli damage, epithelium cell content passing into the larvae midgut. The in vitro study of the interaction of Prays oleae midgut proteins with biotinylated Cry toxins allowed the prediction of four suitable receptor proteins in Prays oleae (Rouis et al., 2007a). On the other hand, a comparative study of different steps in the mode of action of the individual Bacillus thuringiensis kurstaki BNS3 Cry1Aa and Cry1Ac δ-endotoxins on E. kuehniella larvae was performed in order to investigate the origin of the difference in the response of this larvae to each of the latter. Proteolytic activation was shown to be one of the main steps impaired in E. kuehniella tolerance to Cry1Aa. The absence of two proteinase activities as well as an altered activity level observed in the case of Cry1Aa would be the consequence of proteinase-mediated tolerance of E. kuehniella to this toxin. In situ binding and histopathological effect analyses allowed concluding that the binding of the toxin to BBMV receptors is the key step in E. kuehniella tolerance to Cry1Aa toxin (Rouis et al., 2007b). References: a- Rouis S, Chakroun M, Jaoua S.: Comparative Study of Bacillus thuringiensis Cry1Aa and Cry1Ac deltaEndotoxin Activation, Inactivation and In Situ Histopathological Effect in Ephestia kuehniella (Lepidoptera: Pyralidae). Mol Biotechnol. 2007; 27: b- Rouis S, Chakroun M, Saadaoui I, Jaoua S.: Proteolysis, histopathological effects, and immunohistopathological localization of delta-endotoxins of Bacillus thuringiensis subsp. kurstaki in the midgut of lepidopteran olive tree pathogenic insect Prays oleae. Mol. Biotechnol. 2007; 35:141-8.


Session 3

OC6 S3

Optimal Growth Conditions for the Production of β-Carotene and Glycerol from a Halophilic Microalga Dunaliella sp. Isolated from the Dead Sea, Jordan. Nader Fareid Abusara and Abdul-Karim Jaber Sallal Department of Applied Biology, Jordan University of Science and Technology, P. O. Box: 3030, Irbid, Jordan

Abstract : Microalga Dunaliella sp. was isolated and identified from scattered ponds of the southern part of the eastern shores at the Dead Sea. The most suitable media for the isolated Dunaliella sp. was found to be M1 (modified BG-11) which gave the highest growth and β-carotene production. Dunaliella cells were grown optimally at 20˚C while most cells died at 40 and 50˚C. The effects of different physical (temperature and light intensity) and chemical (different nitrogenous and sulfate compounds) factors were tested. The best salinity for Dunaliella growth was 2.5 % NaCl, while the maximum β-carotene to Chlorophyll a ratio was found high salinities: DSw-M1 (1:1), 10% NaCl and DSw-M1 (3:1). Glycerol production was found to increase linearly as NaCl concentration increases. The highest glycerol to Chlorophyll a ratio was obtained at cells grown in DSw-M1 (3:1), DSw-M1 (1:1) and 20% NaCl concentration. By using NaNO3 at 40 mg N l-1 concentration as a nitrogen source and MgSO4 at 25 mg l-1 concentration as a sulfate source the maximum growth and β-carotene production was obtained in comparison with other nitrogenous and sulfate concentrations. In response to different light intensities the maximum growth was obtained at 61 μmol s-1 m-2, and the maximum β-carotene production was at 200 μmol s-1 m-2, while the maximum β-carotene to Chlorophyll a ratio was recorded in cells grown at 1000 μmol s-1 m-2. Glycerol production was only affected by salt concentration factor.


Session 3

OC7 S3

Efficient, Simple and Rapid Transformation of two Turkish Cowpea (Vigna unguiculata L.) Cultivars by Agrobacterium tumefaciens Aasim, M., Khawar, K.M. and Özcan, S. Department of Field Crops, Faculty of Agriculture, University of Ankara, 06110 Dıskapi, Ankara, Turkey

Abstract : Cowpea (Vigna unguiculata L.) is a summer annual, short duration legume crop welladapted to the drier regions, where other food legumes fail to perform well. It is primarily used as vegetable in the form of green leaves, green pods, fresh shelled green peas and shelled dried peas. It is also used as fodder, cover crop and green manure. The study presents efficient shoot regeneration and genetic transformation of two Turkish cowpea cultivars using immature cotyledon explants by culturing them on MS medium containing different concentrations of BAP with and without NAA. Regeneration and shoot elongation behavior varied depending upon the concentration of NAA and genotype. MS medium containing 0.5 mg/l BAP in cv. Akkiz and 0.75 mg/l BAP in cv. Karagoz without NAA was more potent for shoot regeneration from immature cotyledon explants. Moreover, presence of NAA had promotory effect in cv. Akkiz and inhibitory effect in cv. Karagoz on shoot length. This protocol was used for genetic transformation of the two cultivars by Agrobacterium tumefaciens LBA 4404 pRGGBAR to obtain herbicide resistant plants. Large number of transgenic plants were produced within a short period of two months and set seeds. Transgenic plants growing in greenhouse were confirmed by GUS and PCR analysis.


Session 3

OC8 S3

In vitro and in vivo virus free potato seed production using Elisa Reader Maia Kukhaleishvili, Kakha Nadiradze, Eka Bulauri; Nino Murvanidze; Tamar Chipashvili and Nana Phirosmanashvili Abstract: The potato harvest losses caused by viruses, phytopatogenic fungi and pests are quite significant. By all accounts, late blight and bare patch are the most common pathogenic microbes in Georgia. Late blight is caused by fungus Phytophthora infestans. Its occurrence is not always consistent and depends on climate (intensiveness of precipitation). Late blight morbidity rates are rather high ranging from 15% to 35%. After two storage rounds, they rise even further up. Bare patch is caused by fungus Rhisoctonia. It is a quite widespread disease. The long cold spring is good for its massive propagation, with morbidity rates reaching 20-30%. During the progress of bare patch, the plants turn yellow and leaves are rolling up. Contamination of potato plantlets is a major reason of weakness or downfall of potato plants, leading to reduction of harvest. In addition to the pathogenic microbes, the potato harvest is affected by pests such as Colorado beetle, nematodes etc. In Georgia, a variety of chemicals is used against pests and phytopathogenic fungi: zenkor, ridomil, prestige, tattoo, calipso etc. Our farmers use a new generation chemical produced by Bayer cropscience. They apply this chemical to the plants 6-7 times. The extent of waste is considerable, while yields cannot be mended because the farmers do not take the climatic conditions into account. In Georgia, production of virus less potato seed s is very problematic. Volume of elite seed potato production is limited. However, potato consumption per capita is 67 kg annually. Therefore, estimated demand on seed potatoes is 12 000 ton. Local seed potato farmers are producing only 600-700 tons. For this reason, on the market, there is a huge demand on imported seed potatoes (main suppliers are Russia, Armenia and EU). Seed potato import causes hard currency outflow from the country, and dependence on seed potato exporters. On the other hand, in Georgia there is all recourses for own seed potato production, especially in Kvemo Kartli (Dmanisi and Tsalka districts) and Samtskhe-Javakheti (Akhaltsikhe, Akhalkalaki, Ninotsminda districts) regions, which are located above 1 500 m elevation and are traditional potato farming regions. using of ELISA READER for analyzing and identification viruses we will strenghten our research activities in purposes producting virus free potato seed The strategic objective of the project will be to minimize damage wrecked by phytophthora, bare patch and Colorado beetle to potato harvest. We have studied the chemicals currently in use in Georgia. Once their optimal concentrations are identified Farmers will receive relevant recommendations on how to use the chemicals properly. We believe that systemic approach should be employed for the potato protection starting from the soil preparation and planting material and ending with the post-harvest handling. This approach would be comprised of agrotechnic, physiological, physical and chemical activities. Thus, it is necessary to change application scheme of the chemicals. Recommendations that we could elaborate would, together with implementation of other agrotechnical measures, significantly help farmers minimize potato harvest losses caused by phytophthora and other pests. 1. Identify optimal concentrations of currently available commercial chemicals against late blight and bare patch at different stages of plant growth. 2. Identify time periods of chemical spraying with due consideration of climatic conditions. 3. Identify concentration and operation time of the chemicals via their application at different stages of Colorado beetle propagation. 4. Identify optimal alternation scheme of the chemicals applied against Colorado beetle to prevent development of resistance in the latter. Testing of the currently available chemicals against phytopathogenic fungi and pests, identification of right concentrations, identification of application time periods and norms considering climate conditions. Elaboration of the new system of application. Elaboration of recommendations. Collaboration with farmers.


Session 3

OC9 S3

Cloning and Expression of Human Papillomavirus type 16 Capsid Protein L1 in Arabidopsis thaliana Sonia M’hirsi El Adab1, Aymen Ezzine1, Iness Ben Khedija1, Lotfi Chouchane2 and M. Najib Marzouki1 1: Bioengineering Unit 99UR 09-26, INSAT BP 676, 1080 Tunis-cedex. 2: Laboratory of Molecular Immuno-Oncology, Faculty of Medicine of Monastir. [email protected]

Abstract: Plants are currently used as cost-effective and safe heterologous expression system for the production of experimental vaccine. Here, we describe the expression of the HPV16 L1 protein in Arabidopsis thaliana. The HPV16 major capsid gene L1 coding sequence was extracted by digestion from the plasmid pMOS-L1 HPV16 containing the template sequence, and cloned into the binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector were cloned under the control of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase nptII gene, which allowed the selection of transformed plants using kanamycin. The Arabidopsis plants were transformed by floral dip method, using Agrobacterium tumefaciens GV3101, which harboured the plant expression plasmid. The generated transgenic Arabidopsis plants were selected on a kanamycin added MS medium and the integration and the stability in three successive generations of the L1 gene in the plant genome were confirmed by PCR amplification. The evidence for the mRNA expression was acquired by RT-PCR analysis. In order to identify farther integration of transgene into the genome of Arabidopsis thaliana, Southern blot assay was carried out and showed that the HPV16 L1 gene was integrated stably into the genome of the transformed Arabidopsis plants. Western blot analysis showed that transformed Arabidopsis plants express the HPV16 L1 protein1. Thus, HPV16 L1 protein expressed in transgenic Arabidopsis plants can be potentially used as an edible vaccine. 1

: Expression of Human Papillomavirus type 16 major capsid protein L1 in transgenic Arabidopsis thaliana Sonia M’hirsi El Adab, Aymen Ezzine, Iness Ben Khedija, Lotfi Chouchane and M. Najib Marzouki (2007)

Plant Molecular Biology Reporter 25, p 133-144. Sonia M’hirsi El Adab and Aymen Ezzine contributed equally to this work.


Session 3

OC10 S3

The induced state of resistance in inflorescence of Vitis vinifera L. by a Plant growth promoting bacteria, Burkholderia phytofirmans strain PsJN against Botrytis cinerea Pers. Ait Barka E.1, S. Compant1, S. Paquis1, F. Baillieul1, J. Nowak2, and C. Clément1 1

Laboratoire de Stress, Défenses et Reproduction des Plantes, Unité de Recherche Vignes et Vins de Champagne, UPRES EA 2069, UFR Sciences, Université de Reims Champagne-Ardenne, 51687 Reims Cédex 2, France; 2 Department of Horticulture, Virginia Polytechnic Institute and State University, 0327-301 Saunders Hall, Blacksburg, VA 24060, USA

Abstract : Interaction between plants and beneficial bacteria can have a profound effect on plant growth and development, health status, yield, and soil quality. These microorganisms can presensitise plant cell metabolism, so upon exposure to stress, these pre-sensitised or “primed” plants are able to respond more quickly and more efficiently than non primed plants and thus better withstand the challenge. Burkholderia sp. strain PsJN, is an effective plant growth-promoting bacterium isolated as a contaminant from Glomus vesiculiferum-infected onion roots. The bacterium promotes growth of potato, vegetables, and grapevine via reduction of the inhibitory hormone ethylene by high level of ACC deaminase secreted by PsJN. Strain PsJN also showed biocontrol activities against gray mould since it can act as an effective protector against in vitro and in vivo growth of Botrytis cinerea. Furthermore, previous work showed that in clonal multiplication of grapevine via nodal explants taken from stock plants pre-inoculated with PsJN, the bacteria are transmitted through successive subcultures of plantlets, without a need for re-inoculation. The bacterium has been detected in root and stem in vitro cultures grapevine roots under growth chamber conditions. Incidence of colonization by plant growth-promoting bacteria Burkholderia phytofirmans strain PsJN on resistance of Vitis vinifera L. cv. ’Chardonnay’ was also studied in vivo using fruiting cuttings harboring preflowers buds. Results of this experiment demonstrated, the ability of plant-growth promoting bacterium isolated from onion roots, B. phytofirmans strain PsJN, to induce grapevine defense gene expressions at the root level and also systemically in preflowers buds, after soil inoculation,. Several genes encoding PR proteins were significantly induced after bacterization with PsJN bacteria in comparison to control treatment at both root and inflorescence levels. This enhanced capacity leads to an induced state of resistance in flowers to face injuries caused by Botrytis cinerea. Our results demonstrate that the disease can thus be diminished by 60% after soil inoculation with the beneficial bacteria, Burkholderia phytofirmans strain PsJN. This work describes for the first time a systemic induction of gene expression in inflorescence tissues which leads to an enhanced capacity of bacterized plants for protecting flowers against phytopathogens.


Session 3

OC11 S3

Identification And Molecular Characterization Of Durum Wheat Map Kinase Phosphatase 1 (Tmkp1). Zaidi I1, Hanin Moez2, Masmoudi Khaled3. Centre of Biotechnology of Sfax, Laboratory of plant molecular Genetics Road of Sidi Mansour Km 6, P. O. Box « K » 3038 Sfax-Tunisia. [email protected]

Abstract: Plant cell response to salt and drought stresses is complex and depends on many signal transduction pathways, among which the MAPK (mitogen activated protein kinase) pathway. The activation of the MAPKs is not a simple switch and both duration and magnitude of activation is crucial in determining the outcome of the cellular reaction. Thus it seems likely that dephosphorylation of the MAP kinases is vital for their control. This is achieved by the removal of phosphate groups by protein phosphatases such as MAPK phosphatases (MKP). In Arabidopsis thaliana, a MKP (AtMKP1) was shown to be a negative regulator of the MAPK pathway involved in plant salt stress response. In this work, we isolated wheat cDNA highly homologous to AtMKP1 (TMKP1). The TMKP1 cDNA has an ORF of 2259 bp and encodes a protein of 752 a.a. Sequence analysis shows that TMKP1 protein is highly homologous to the rice MAPK phosphatase OsMKP (83% homology) and other plant MKPs including maize and Arabidopsis. Gene expression analysis of TMKP1 was performed by RT-PCR on two Tunisian wheat varieties, either tolerant (Oum Rabiaa) or sensitive (Mahmoudi) to salt and drought stresses. Our data show a differential regulation of TMKP1 by salt and drought stress since it is repressed in Oum Rabiaa and induced in Mahmoudi. Since the abscissic acid (ABA) is one of the main phytohormones mediating stress response, its involvement under salt stress treatment was investigated in Mahmoudi. Our results indicate that the salt induction of TMKP1 is ABA dependent since the use of naproxen (an inhibitor of the ABA biosynthesis pathaway) together with NaCl, abolishes this induction. In order to understand the cellular function of this protein, we studied the possible interactions with two wheat MAP kinases (TMAPK 3 and TMAPK6) in vivo and in vitro. Our data show specific interactions between TMKP1- TMPK3 and TMKP1TMKP6. These interactions require at least the N-terminal part of TMKP1, including the first 132 amino acids.


Session 3

OC12 S3

Rapid and Highly efficient micropropagation of Two Turkish woads Isatis constricta Davis and I. aucheri Boiss. under in vitro conditions Khawar K.M1., Ozel C.A., Ulug A., Sur I., Kizilates E., Ozlu G., Hadimogulları B., Cam D., Arslan O. 1

Department of Field Crops, Faculty of Agriculture, University of Ankara, Ankara, Turkey, Department of Biology Eduation, Faculty of Education, Gazi University, Teknikokullar, Ankara, Turkey(*Email, [email protected], Telephone No. 00903125961540, Fax No. 0090312318266)

Abstract: Many woad species grow in Turkey. The study reports in vitro multiplication of two woad species Isatis constricta Davis and I. aucheri, both of which are perennial plants and are also identified as potent inhibitor of prostaglandin and leukotriene. with anti inflamaotory potential. Isatis species have glucobrassicin. which has potential to treat cancer. The plant has strong root system and aid to prevent soil erosion by providing good soil cover. Furthermore; especially at rosette stage. Because of endemic nature of the plant it has not been exploited extensively. In vitro techniques such as micropropagation has proved useful tool for propagation of number of plants. There has been no report of in vitro propagation of these plants. This has also affected Agrobacterium mediated genetic transformation studies. The main objective of the study was to develop a methodology for the rapid improvement and propagation of this plant through tissue culture using leaf and petiole explants. The study reports effects of Cytokinins and auxins on multiple shoot regeneration in these species using leaf and petiole explants. A comparison of results between two cultivars showed clear effect of plant growth regulators on shoot regeneration potential of explants and genotypes. In general terms plant regeneration capacity of I. constricta was better compared to I. aucheri. The regenerated shoots of I constricta could be rooted in MS medium containing low concentrations of auxins. However, regenerated shoots of I. aucheri could only be rooted after pulse treatment with auxins. All of the rooted plants were acclimatized in the greenhouse and looked after carefully to obtain flowers and seeds.


Session 3

OC13 S3

The effect of crude aqueous Juniperus excelsa leaf and fruit extracts on some human pathogenic bacteria Lina Al-Amir, Prof. Khalil ALMaarri* and Yasmeen Junaid * College of Agriculture, Damascus University, and General Commission of Biotechnology, Plant Biotechnology Laboratory, Damascus, Syria. Email : [email protected]

Abstract: Crude aqueous extracts of J.excelsa fruits and leafs were prepared from samples collected during winter of the year 2006 and spring, summer and autumn of 2007 along differing geographical regions of Syrian mountains. Studying the effect of these extracts indicated they inhibited the growth of some pathogenic bacterial species. Bacteria were isolated from clinical specimens collected from patients. The bacteria were characterized according to routine lab diagnostic measures and the API E20 and API Staph kits were used to confirm the species' identification. Identified bacteria were further tested for their susceptibility towards nine antibiotics. Escherichia coli exerted high resistance towards all crude extracts of leafs and fruits of J.excelsa though the different E.coli strains varied in their resistance towards the antibiotics tested. Results showed a variation in the minimal inhibitory concentrations (MIC's) of the crude extracts of leafs towards Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. The concentrations of crude extracts tested ranged from 1000mg /ml to 50μg /ml for both leafs and fruit. The crude aqueous extract of leafs was obtained from young and old aged leafs during different seasons and they showed a variation in their MIC values toward S.aureus and S.epidermidis for in old aged leafs the values ranged between 5-17.5 mg /ml and in young aged leafs between 3-5 mg /ml. As for P.aeruginosa the MIC value in young aged leaf extracts was 35 mg /ml and in old aged leafs it was 50 mg /ml for summer samples while winter samples showed no effect neither did the fruit extracts up to the highest concentration used (1000 mg /ml ). It could be concluded that the crude aqueous extracts of J.excelsa leafs had an inhibitory (bactericidal) effect on some Gram negative bacteria but not on E.coli, and that MIC values of the crude extracts differ according to bacterial strains, plant age and seasonal changes.


Session 3

OC14 S3

Evidence of the involvement of the C-terminal region of Bacillus thuringiensis Cry1Ac in delta-endotoxin crystallization Dammak Karray M, Tounsi S and Jaoua S* Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box: K, 3038. Sfax, Tunisia E. mail: [email protected]

Abstract: Bacillus thuringiensis is a gram-positive soil bacterium that forms parasporal crystals during sporulation that are mainly composed of one or several insecticidal proteins highly specific for different insect larvae and nematodes. The Cry1 proteins typically form bipyramidal crystals, are ~130 kDa in MW, and several closely related proteins may be present in a single crystal. The mechanism of formation of the crystalline inclusions is not well understood. The ability of Cry1A delta-endotoxins to form crystals has been attributed to the presence of the Cterminal part. In order to provide evidence for the involvement of the C-terminal region of Bacillus thuringiensis Cry1Ac delta-endotoxin in its crystallization, we have generated deletion (Cry1AcΔEcoRI) and mutations (Cry1Ac*) in the N-terminal region of Cry1Ac and studied the expression of the corresponding altered delta-endotoxins in both Cry- and Cry+ B. thuringiensis strains. When produced in an acrystalliferous strain, both of Cry1Ac* and Cry1AcΔEcoRI could not form crystals. But, when they were expressed in the crystalliferous strain, BNS3, they cocrystallize with the endogenous delta-endotoxins generating bipyramidal crystals with modified shapes. These results revealed that the C-terminal part of Cry1Ac was really involved in crystal formation. Such findings let us trying to promote the crystallization of a naturally secreted Cry1Ia protein. Therefore, we have fused the Cry1Ac C-terminal part with the Cry1Ia N-terminal one. We demonstrated that the corresponding chimeric protein named Cry1Iac could co-crystallize with the other BNS3 Cry1A expressed delta-endotoxins.


Session 3

OC15 S3

Cloning, sequencing and expression of cry1A genes of a new Bacillus thuringiensis strain highly toxic to Lepidopteran larvea Saadaoui, I., Rouis, S. and Jaoua, S.* Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box: K, 3038. Sfax, Tunisia E. mail: [email protected]

Abstract: Much attention has been given to Bacillus thuringiensis, an aerobic, spore-forming, gram-positive bacterium that has been developed as a microbial insecticide. Different varieties of this bacterium produce a crystal protein that is toxic to specific groups of insects. B. thuringiensis strain BLB1 is one of the Bacillus thuringiensis collection of the Laboratory of Biopesticides (Jaoua et al. 1996). The transmission electron microscopy of the crystals produced by the BLB1 strain showed a small cubic crystal and a big bipyramidal one. Bioassay of BLB1 crystal proteins resulted in an LC50 of 70.32 ng of toxin per mg of flour against third instar E. kuehniella with confidence limits of (32-108 ng/mg), smaller 2 folds than LC50 of the reference strain HD1. These results demonstrate the higher toxicity of BLB1 and prove its big importance in the biological control. PCR amplification of cry genes showed that BLB1 has cry1E in addition to the HD1 cry genes content (cry1Aa; cry1Ab; cry1Ac; cry2Aa and cry1Ia). Taking into account the crucial role of cry1A genes on the toxicity against lepidopteran larvae (Tounsi et al., 2006), we investigated these genes by cloning, sequencing, expression and toxicity study experiments. Moreover, comparative study of the δ-endotoxin/spore ratio for BLB1 and HD1 strains, allowing the estimation of the production level per spore, suggested that BLB1 production is the very high. References: JAOUA S, ZOUARI N, TOUNSI S and ELLOUZ R (1996) FEMS Microbiol. Lett. 145, 349-354 TOUNSI S, DAMMAK M, ZOUARI N, REBAÎ A and JAOUA S (2006) Biological Control 37: 243246.


Session 3

OC16 S3

Biotechnological Approaches for Improvement of Garlic (Allium sativum L.) Haque, M.S., Parvin, L., Momtaj, M.H., Begum, S.N. and Karim1, M.A. Department of Biotechnology and Department of Crop Botany, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh 1 Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh 2202, Bangladesh E-mail: [email protected]

Abstract: Garlic is considered genetically closed and genetic improvement through traditional breeding is blocked due to sexual sterility. Induction of somaclonal variation and development of gene transfer techniques seem to be ideal for garlic improvement. In the first part of this study, garlic plants were regenerated and the in vitro clones were subjected to RAPD to detect possible somaclonal variations among them. Regenerants obtained directly from root explants showed a certain extent of somaclonal variations which were cultivar dependent. Local clones exhibited higher variation (3.7 %) compared to the exotic one (1.5 %). Certain changes in DNA level were found in both genotypes suggesting that there exists a mutation sensitive part in the garlic genome that could be exploited to induce somaclonal variation at large number to select desirable trait. To establish a suitable and reproducible protocol of Agrobacterium-mediated gene transfer, a protocol was first developed for regeneration via callus formation and then, GUS (βglucuronidase) gene expression in garlic callus was observed using histochemical GUS assay. Highest callus formation both in root tips (82%) and basal discs (88%) was found on 2.0 mg/L 2,4-D. Callus induction was higher (72.70%) in exotic garlic. Basal disc of local variety showed 63.33% shoot regeneration, whereas root tips of exotic variety showed 92.58% shoot regeneration. In the GUS assay, conspicuous GUS positive (blue color) regions were detected on the entire surface of the calli. Exotic variety showed the better response than local variety to GUS assay. The percentage of GUS positive calli increased with the increase of co-cultivation period, at a constant pre-culture period. The highest percentage (100%) of GUS positive explants was found in co-cultivation period of three days. Further study is underway to develop a reliable protocol of gene transfer in garlic.


Session 3

OC17 S3

In vitro antioxidant studies on phenolic and alkaloid extracts of two Fumaria Algeria species. Fadila Maiza Benabdesselam 1*, Khalida Bogouffa 1, Khodir Madani 1, Mohamed Chibane 1 1

Laboratory 3BS, Faculty of Nature and life Sciences, Bejaia University, Bejaia, 06000. Algeria. *Fadila [email protected]

Abstract: Fumaria capreolata(L.) and Fumaria bastardii(L.) (family Fumariaceae) have been used in the indigenous system for the treatment of various diseases . Our study aimed first to identify by RPHPC the phenolic and alkaloid contents of the two species and second to examine their antioxidant activity using 2,2 diphenyl 1 pierylhydrazyl (DPPH) radical scavenging, reducing power and lipid peroxidation methods. Key words: Fumaria capreolata L.. , Fumaria bastardi L.., isoquinoline alkaloids, phenolic compounds, HPLC. Antioxidantactivity 1.Introduction The genus Fumaria belongs to the family Fumariaceae, is widely distributed in Northern Africa. Phytochemical studies of have showed the occurrence of isoquinoline alkaloids in various species of Fumaria and in others genera belonging to the Papaveraceae family (Preininger 1986). Many isoquinoline alkaloids have exhibited interesting pharmacological activities (Cui et al 2006). The present study aims to identify by RPHPLC the alkaloid and phenolic contents of Fumaria capreolata and Fumaria bastardii ,and to investigate their in vitro antioxidant potential . 2- Materials and Methods 2.1.Plant material :Aerial parts of Fumaria capreolata and Fumaria bastardii were used for the study. 2.2. Extraction of alkaloids :Extraction of isoquinoline alkaloids was carried on according to the method of Sousek (Sousek et al 1996) 2.4- Extraction of phenolic compounds :Extraction of phenolic compounds for HPLC analysis and antioxidant studies was carried on using the method of shahid and his collaborators (in Shirwakar et al. 2006) 2.5. Identification of active substances: Alkaloids and phenolic acids were identified by RP HPLC 2.6. Antioxidant activities : the antioxidant activities of the extracts were made using 2,2 diphenyl 1 picrylhydrazyl (DPPH) radical scavenging, reducing power and lipid peroxidation methods. 3. Results : Protopine,Stylopine,Fumaritine,Fumariline and adlumuceine were identified in the two species. F.bastardii shows presence of Corytubérine, and Parfumine while F.capreolata contains Palmitine ,Cryptonine and Fumaricine. Both plants extracts exhibited strong total antioxidant activity however extract of F.bastardii was more potent then F.capreolata one. 4.Conclusion: The more efficiency of F.Bastardii can be related to it’s richness on active substances . The traditional use in Folk medicine of the genus Fumaria can be related to it’s isoquinoline alkaloids and phenolic compounds. 5.References Cui W.H., Iwasa K.,Tokuda H., Kashihara A., Mitani Y., Hasegawa T., Nishiyama Y., Moriyasu M., Nishino H., Hanaoka M., Mukai C. and Takeda K.(2006). Phytochemistry, 67, (1): 70-79. Preininger V. (1986). In:Alkaloids, 29, 1-98,Brossi A ed. Academic press London. Soušek J., Guédon D.,Adam T., Bochorăkovă H., Tăborskă E., Vălka I and Simănek V. (1999) Phytochemical Analysis, 10 :6 -11 Shirwakar A., Shirwakar A.R., Rajendran K. and Punitha I.R.S.(2006). Pharm. Bull., 29(9):1906-1910.


Session 3

OC18 S3

Characterization of a novel Vip3-type toxin of Bacillus thuringiensis strain BUPM95 Mesrati-Abdelkefi L., Tounsi S., Kamoun F. and Jaoua S. Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P.O. Box “K”, 3038. Sfax, Tunisia E. mail: [email protected]

Abstract: Bacillus thuringiensis, a naturally occurring gram-positive bacterium, can produce several proteins that have insecticidal properties affecting a selective range of insect orders. A vip3-type gene named vip3LB has been isolated from B. thuringiensis strain BUPM95. It was carried by the large plasmid containing the cry1Ia gene of B. thuringiensis (1). Its nucleotide sequence predicted a protein of 789 amino acids residues with a calculated molecular mass of 88.5 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that vip3LB is a new vip3-type gene (1), presenting several differences with other vip-type genes. The corresponding secreted vegetative insecticidal protein was active against lepidopteran insects. By primer extension analysis, we have identified the transcription start point of the vip3LB gene (2). Upstream from this tsp, was found a nucleotide sequence partially homologous to the consensus sequence for the EσE holoenzyme of B. subtilis. The transcriptional activity from the promoter was detected at the vegetative stage of growth starting at mid-log phase as well as during the middle stage of sporulation (2). The vip3LB gene was cloned into pET-14(b) vector and overexpressed in E. coli. The expressed Vip3LB protein found in the cytoplasmic fraction was purified and used to produce anti-Vip3LB antibodies. Using the gut juice of Prays oleae, purified Vip3LB bound to 65 kDa protein whereas Cry1Ac bound to 210 kDa midgut receptor. These results support the use of Vip3LB as a biological control pest especially when a resistance to Cry toxins was detected. References: 1- Mesrati-Abdelkefi lobna, Tounsi Slim and Jaoua Samir (2005) FEMS Microbiol. Lett. 244, 353-358. 2- Mesrati-Abdelkefi lobna, Tounsi Slim, Fakher kamoun and Jaoua Samir (2005) FEMS Microbiol. Lett. 247, 101104.


Session 3

OC19 S3

Seasonal variation in antimicrobial activity of crude extracts of the green alga Ulva rigida (Ulvaceae) Trigui, M., Besbes, A. and Jaoua, S. Laboratory of Biopesticides, Centre of Biotechnology of Sfax, P.O. Box: K, 3038. Sfax, Tunisia. E.mail: [email protected]

Abstract: In recent years, algae extracts have attracted a great deal of scientific interest due to their potential as a source of natural biologically active compounds. Efforts have been made to explore the potential of some algae for the treatment of infectious diseases in order to substitute standard pharmaceutical remedies. The present work aimed to investigate the antimicrobial activity of the crude extract of Ulva rigida and the influence of the growth season and geographical location on its biological activity. Crude extracts of Ulva rigida were tested for their antimicrobial activity against Gram-positive bacteria (5 species), Gram-negative bacteria (7 species) and fungi (2 species). The agar well diffusion test, was used to determine the sensitivity of the tested samples, whereas the well microdilution was used to determine the minimal inhibition concentrations (MIC) and the minimal bactericidal concentration (MBC) of the active sample. The assays revealed different susceptibilities of the bacterial phyla under investigation to Ulva extracts. For example, Gram (+) Bacillaceae were generally more sensitive than Gram (-). Ulva extracts showed a high activity against phytopatogenic bacteria as Agrobacterium and Erwinia bacterium species. Ethanolic extract from Ulva rigida inhibited the growth of Candida albicans whereas none of these extracts disrupted the growth of Aspergillus niger. In order to study the variation of the antimicrobial activity with growth season, hydroalcolic extracts from the algae collected monthly (April 2006 through Juin 2007) were tested against two Gram (+) and a Gram (-) bacteria. The antimicrobial activity of the crude extract from Ulva rigida collected in February had the highest antimicrobial activity. This activity was also affected by the geographical location. The overall results provide promising baseline information for the potential use of the crude extracts from Ulva rigida in the treatment of bacterial and fungal infections.


Session 3

OC20 S3

Effect of inherited sterility and Bacillus thuringiensis on mortality and reproduction of Phthorimaea opercullela Zeller (Lepidoptera: Gelechiidae) Hayat Makee*, Mohammed Tlas, Samer Amer And Jamal Abdulla * Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria. P.O. Box 6091, Damascus, Syria.

Abstract: The effect of a commercial formulation of Bacillus thuringiensis (Dipel® 2X) upon F1 progeny of irradiated and unirradiated Phthorimaea operculella male parents was investigated. The F1 progeny of irradiated parents was more susceptible to B. thuringiensis than that of unirradiated parents. A combination of irradiation and B. thuringiensis led to higher mortality in F1 progeny of P. operculella. The LC50 was 0.406 g/100ml for F1 progeny of unirradiated parents, but 0.199 g/100ml for those of irradiated parents. There was a great reduction in the fecundity, fertility and pupal weight of F1 progeny of irradiated parents compared to those unirradiated parents. Such reduction was increased by applying higher concentration of B. thuringiensis. Our study proved that a combination between inherited sterility technique and B. thuringiensis could give a good controlling result against P. operculella.


Session 3

OC21 S3

Genetic diversity of rhizobial isolates obtained from root nodules of Arachis hypogaea in northwestern Morocco. M. Rabie El-Akhal1,2, Ana Rincón2, Francisco Arenal2,3, M. Mercedes Lucas2, Nouredin El Mourabit4, José J. Pueyo2 , Said Barrijal1. 1

Equipe de Recherche Valorisation Biotechnologique des Microorganismes (ERVBM), Département des Sciences de la Vie, Faculté des Sciences et Techniques, Tanger, Morocco 2 Department of Plant Physiology and Ecology, Instituto de Recursos Naturales, Centro de Ciencias Medioambientales, CSIC, Serrano 115-bis, E-28006 Madrid, Spain 3 Current address: PharmaMar SAU. Microbiology Dept. R&D. Avda. de los Reyes, 1. Pl. La Mina Norte 28770, Colmenar Viejo, Madrid, Spain. 4 Institut National de Recherche Agronomique, Tanger, Morocco. Corresponding author: [email protected]

Abstract: The molecular characterisation of rhizobial strains isolated from root nodules of Arachis hypogaea growing in north-western Morocco was performed. Bacterial isolates were characterised using several molecular techniques (16S-ARDRA, fingerprinting and sequencing) and phylogenies were inferred from their 16S gene sequences. Based on the ARDRA analysis, isolates were classified into two major groups: slow-growers and fast-growers, and a high genetic variability among isolates was observed by fingerprinting. Phylogenetically, most isolates grouped with the genus Bradyrhizobium and Rhizobium. This work provides, for the first time, a study about the diversity of rhizobia able to nodulate peanut in Morocco. This characterization provides a basis for the selection of peanutnodulating rhizobia which may have applications in formulating appropriate inocula for improving peanut crop yield on Moroccan soils. Keywords: Arachis hypogaea, peanut, phylogeny, rhizobia.


Session 3

OC22 S3

Overcome of Photorhabdus limitations of production and overproduction of corresponding bioinsecticides by fermentation Wafa Jallouli, Nabil Zouari And Samir Jaoua Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P. O. Box « K » 3038 Sfax-Tunisia, [email protected]

Abstract: Photorhabdus temperata is a bioluminescent bacterium that lives in mutualistic association with entomopathogenic nematodes of the genus Heterorhabditis. Previous studies have demonstrated that the bacterium is characterized by VBNC state, polymorphism Vsm and phase variation. Such cell states reduce this potency as bioinsecticide. In order to overcome such limitations in P. temperata cells, quality was improved by adequation of a new medium ensuring stability of cells through Vsm and phase variation and reduction of VBNC state. Produced cells retain oral toxicity against the insect larvae Ephestia kuehniella. Overproduction of Photorhabdus viable cells by fermentation and optimization of economical production media were developed. Inoculum quality played a key role in fermentation process. Furthermore, providing dissolved oxygen at 40% oxygen saturation in the medium and the pH of 7, increased biomass production of 20%. In order to reduce the cost of P. temperata bio insecticide process production, a new medium composed of soya bean and diluted sea water was optimised. Into 7,5 l fermenter, doubled biomass production and cultivability were obtained compared to that in the optimized model medium. Interestingly, this new formulated medium was shown to improve oral toxicity against E. kuehniella. This should contribute to a significant reduction of the cost of P. temperata bio insecticide production. The correction of oxidative stress by addition of catalase or sodium pyruvate upon the inocultation of bacteria on agar plates promotes the recovery of nonculturable cells. High level of toxicity was observed with the whole culture after physical lysis. water instead of sea. A higher (70%) or lower aeration level (15%) led to cell lyses from 62 h incubation time.


Session 3

OC23 S3

Development of a new generation of Bacillus thuringiensis bioinsecticides based on asporogenic and oligosporogenic mutants Ben Khedher, S., Zouari, N. and Jaoua, S. Centre of Biotechnology of Sfax, Laboratory of Biopesticides, P. O. Box « K » 3038, Sfax-Tunisia E.mail address: [email protected]

Abstract: The bioinsecticides, particularly those based on the bacterium Bacillus thuringiensis, having been drawing increasing attention are considered as a good alternative to cure the undesirable effects of chemical insecticides. In order to increase the stability and the toxicity of the crystals of B. thuringiensis, we were developed a new generation of bioinsecticides issued from sporeless mutants of B. thuringiensis. 40 asporogenic and oligosporogenic mutant strains were isolated after treatment of a wild strain of B. thuringiensis BNS3 (Jaoua et al., 1996) by nitrous acid. The screening of mutants on the basis of the production of delta-endotoxins in glucose and in starch based media (Zouari and Jaoua, 1997; Ghribi et al, 2007) showed an improvement in their delta-endotoxins production. Adaptation of sporeless strains to heat shock and sodium chloride addition were investigated and their involvement in bioinsecticide production improvement was studied. The delta-endotoxin production was significantly improved by sodium chloride addition (7 g l-1) with an overproduction of about 24 % but it was slightly improved by heat treatment at 42°C during 10 min. However, combination of the two stressors was often accompanied by a decrease in delta-endotoxin production. On the other hand, an economical medium containing only soya bean in 4 fold diluted sea water was formulated to produce similar delta-endotoxins titer to that in the optimised medium which is of a great importance from the practical point of view. References : - Jaoua, S., Zouari, N., Tounsi, S. and Ellouz, R. 1996. Study of the delta-endotoxins produced by three recently isolated strains of Bacillus thuringiensis. FEMS Microbiol. Letters, 145: 349-354. - Zouari, N. and Jaoua, S. 1997. Purification and immunological characterization of particular delta-endotoxins from three recently isolated strains of Bacillus thuringiensis. Biotechnology Letters, 19: 825-829. - Ghribi, D., Zouari, N., Trigui, W. and Jaoua, S. 2007. Use of sea water as salts source in starch and soya bean based media, for the production of Bacillus thuringiensis bioinsecticides. Proces Biochemistry, 42 (3): 374-378.


Session 3

OC24 S3

Improved transformation of tobacco and wheat plants via modification of homologous recombination frequency Boyko, A., Greer, S. and Kovalchuk, I. University of Lethbridge, Department of Biological Sciences, Lethbridge, Alberta, T1K 3M4, Canada

Abstract: Our lab aims to improve plant transformation by improving tissue regeneration efficiency and increasing frequency of transgene integration. We attempted to improve transformation of dicots and monocots. As a model for dicots, we used Nicotiana tabacum (tobacco), and for monocots Triticum aestivum (wheat). In case of tobacco we used Agrobacterium-mediated transformation of leaf disks, whereas in case of wheat direct somatic embryogenesis technique after micropartical bombardment. We have attempted nearly fifty different combinations of micro-, macro-elements and hormones and found several that substantially improve transformation rates. Simple modifications in the growth media (MS for tobacco and DSEM for wheat) such as the increase in the level of ammonium and/or potassium nitrate resulted in 2-3fold increase in the number of regenerated calli (toacco) or production of primary embryos (wheat). Similarly, this lead to a 2-5-fold increase in the number of regenerated transgenic tobacco and wheat plants. This approach allowed to generate one transgenic tobacco plant per single 2x2 cm leaf disk. We also found that avoiding selection, such as antibiotic or herbicide resistance, results in dramatic increase in the number of regenerated calli/embryos. When regenerated plants were checked for the transgene expression, it was found that 8% of all tobacco plants were transgenic. This report suggests that improvement of transformation efficiency can be achieved by simple modification of the amount or ratio of basic chemicals in the regeneration media.


Session 3

OC25 S3

A High-Throughput Multiplex Method adapted for GMO Detection M. Chaouachi (1, 2, 3), M. Romaniuk(2), A. Bartegi (3), V. Laval(2) , D. Brunel(1) and Y. Bertheau(4) (1) Centre National de Génotypage (INRA-CNG) Plateforme SNP, 02 Rue Gaston Crémieux 91057. Evry cedex. France. [email protected] (2) INRA, Laboratoire de Méthodologies de la Détection des OGM, Unité PMDV, Route de Saint Cyr, RD10, 78026 Versailles cedex. [email protected] (3) Laboratoire de Biochimie & Interaction Moléculaires O2/UR/09-01 Institut Supérieur de Biotechnologies. Monastir, Tunisie. [email protected] (4) INRA, Route de Saint Cyr, RD10, 78026 Versailles cedex. [email protected]

Abstract: According to European and third countries regulations, any product with an accidental or technically unavoidable presence above a threshold of authorized GMO per ingredient shall be labeled. To comply with these regulations, the European Union and third countries supports several research programs, such as Co-Extra, aiming at the development and the validation of reliable, standardized, specific, qualitative and quantitative GMO detection methods. Moreover, the recent repeated observation of unauthorized GMOs on the market pointed out the need of detecting unknown GMOs in a cost- and time effective way. For this, a recent technology called SNPlexTM with a high capacity of multiplexing (till 48 targets in one tube) was adapted and optimized to GMO detection. This paper describes a new SNPLexTM assay to detect both authorized and unauthorized GMOs. This SNPlexTM assay is based on the combined, and preferably simultaneous, detection of several short DNA sequences of taxa, GMO constructions and controls such as donor organisms. Performance criteria such as the specificity and the sensitivity were assessed. The absolute and the relative limits of detection ranged from 10 to 40 ng and from 0.5 to 2% respectively. It is the first time a multiplex analysis allowing detection of 48 markers in a tube is described.


Session 3

OC26 S3

Influence of differentiation state, salt stress and Methyl jasmonate on in vitro production of cucurbitacins from tissue cultures of Ecballium elaterium and Cucumis prophetarum Sakr1,2,M.M; Souad El Gengaihi3; kamel,A3 and Mai M. Farid2 1-Dept. of Plant Biotechnology, National Research Center, Dokki, Cairo, Egypt 2-Plant Molecular Genetic Group, Nobel Project, National Research Centre, Dokki, Cairo, Egypt 3- Dept. of Medicinal and Aromatic Plants, Pharmaceutical Sciences Division, National Research Center, Dokki, Cairo, Egypt

Abstract:

Ecballium elaterium and Cucumis prophetarum are rare wild cucurbits known with their tetracyclic triterpenoids which posses antitumer , antiviral and anti-inflammatory activities. Studies on in vitro culture of the Egyptian landraces indicated that the culture medium supplemented with 0.1-0.5 mg/l NAA in combination with BA at 2 mg/l is the best medium for callus proliferation from hypocotyl and cotyledon explants of aseptically grown seedlings. MS medium supplemented with 2 mg/l BA, 60 mg/l adenine sulfate and 170 mg/l KH2PO4 is the best medium for shoot proliferation after a phase of callus formation. In case of Ecballium elaterium, optimum cucurbitacin content, represent 144% of the content of the intact plant (three week old seedling), recorded in callus culture. Meanwhile, the total cucurbitacin content (3.583%) of Cucumis prophetarum callus culture represents 190% and 84.7% of intact plant at three weeks and flowering stages respectively. Cucurbitacins content of suspension cultures were elicited by application of different concentrations of NaCl (salt stress) and Methyl jasmonate (elicitor). The obtained results indicated that salt stress can be applied successfully to elicit cucurbitacins accumulation in suspension cultures of Cucumis prophetarum. The same strategy is not recommended for Ecballium elaterium suspension cultures. Methyl jasmonate elicit the biosynthesis and accumulation of cucurbitacins in suspension cultures of Cucumis prophetarum. The highest yield of cucurbitacins (0.1898 %) was reached after three days of culturing in liquid medium contained 200 µM MeJA. This level of cucurbitacin is about 11 fold more than that of Ecballium elaterium and Cucumis prophetarum which can be successfully used for the production cucurbitacin in vitro.


Session 3

OC27 S3

Cloning of full length cDNA for Vitis vinifera SIP/raffinose synthase related to salt stress response Daldoul, S1,2., Höfer, M2.,Chenenaoui, S1,2., Abdelwahed, G1 and Mliki, A1. 1

C.B.B.C/ Laboratoire de Physiologie Moléculaire de la Vigne ;B.P.901, 2050 Hammam-Lif, Tunisia. RLP-Agroscience GmbH/Alplanta-Institute for Plant research ; Breitenweg 71,67435 Neustadt and der Weinstraße, Germany.

2

Abstract: In order to characterise the complement of salt-responsive genes in a tolerant Tunisian grapevine, we have constructed subtractive cDNA libraries from the variety Razegui. So far, a cDNA fragment corresponding to a mRNA of a seed imbibition protein(SIP)/raffinose synthase was obtained by differential screening. This gene displays high expression levels under moderate salt stress conditions in Razegui when compared to the sensitive variety (Syrah). The corresponding transcript has been detected in leaves and roots. Sequence alignment reveals 88% homology (and 79% identity) with alpha galactosidase from Cucumis melo and high similarity to seed imbibition proteins from other plant species which contain a raffinose synthase catalytic domain. Raffinose synthase catalyses the biosynthesis of raffinose from galactinol and sucrose. Raffinose family oligosaccharides are supposed to function as osmoprotectants in abiotic stress tolerance in plants. Full length cDNAs of VvSIP/raffinose synthase from Razegui were cloned. They contain a 2325 bp open reading frame encoding for a polypeptide of 774 amino acids. The present data suggest that the VvSIP1/raffinose synthase could be involved in the defense mechanism against abiotic stress in Razegui. Therefore, the functional characterization of this gene would be of great interest to confirm this result.


Session 3

OC28 S3

Alteration of ABA mediated resistance against alternaria blight in Brassica through osmotin gene transformation Gohartaj1, Dinesh Pandey1, Anil Kumar1 K.C.Bansal2 and G.K.Garg1 G.B. Pant University of Agriculture and Technology, Pantnagar1, NBPGR, IARI New Dehli2

Abstract: Alternaria blight is a recalcitrant disease and several components play important in pathogenesis. It was reported earlier that etiological agent of the disease is destrxun B is elaborated by the fungus Alternaria brassicae along with abscisic acid and thought that ABA might be perturbing the course of disease onset after the fungal attack.The alternaria toxin mediated cell death might be further aggravated by the ABA. Osmotin, PR-5 Protein is known to be induced in several abiotic stresses when introgressed in tobacco and potato also extended resistance against fungal infection through perturbation of signal transduction pathways. In view of the above attempt was made to transfer osmotin in to Brassica and study the effect of osmotin over-expression on the perturbation of signal transduction proteins involved in programmed cell death. Interestingly transformed calli resisted the ABA effect. ABA treated shoots showed the loss of chlorophyll and degeneracy of shoots. Further, ABA mediates the cell death pathway by induction of p53 and caspase like proteins. However over -expression of osmotin alters the signaling pathway which suppresses the p53 and caspase like protein.


Session 3

OC29 S3

Selection of potato mutants tolerant to salinity using in vitro and DNA marker techniques Al-Safadi B*. Arabi MIE, MirAli N, Al-Daoude A, Nabulsi I Department of Biotechnology and Molecular Biology, AECS PO Box 6091, Damascus, Syria. [email protected]

Abstract: In vitro cultured explants from potato (Solanum tuberosum) cvs. Draga, Diamant, and Spunta were irradiated with gamma ray doses of 25, 30, and 35 Gy. Growing plantlets were subsequently propagated to obtain enough explants for in vitro selection of plants tolerant to salinity. Around 1300 MV4 plantlets from the three cultivars were subjected to selection pressure. MV4 explants were cultured on an MS medium supplemented with NaCl in varying concentrations ranging from 50 to 200 mM. Surviving plantlets were propagated and recultured to insure their tolerance to salinity. Salt tolerant plantlets were acclimatized and transferred to pots and grown under greenhouse conditions. Mutant and control plants were later subjected to a second selection pressure by irrigating them with water containing NaCl in concentrations ranging from 50 to 250 mM. Cultivar Spunta produced the highest number of tolerant plants. Four plants of Spunta appeared to be tolerant to salinity whereas only one plant from Diamant was tolerant and no plants from cultivar Draga were tolerant. In an attempt to determine DNA loci involved in the salinity tolerance, 8, 6 and 4 mutants of the three potato cultivars, Draga, Spunta and Diamant found to be salt tolerant in the previous study were used for analyzes. In this study, two molecular marker techniques, RAPD and ISSR were employed. More than one hundred PCR fragments that were absent in the control (Draga, Spunta or Diamant) and present in the relevant mutations were obtained, gel extracted, cloned in pBluescriptII SK+ and sequenced using plasmid specific primers. However, database search using Blast X did not result in any significant match between the acquired sequences and loci believed to be involved in salt tolerance. Further work using different marker technique such as cDNA-AFLP will be employed to define these loci.


Session 3

OC30 S3

Overexpression of the Vegetative Insecticidal Protein of Bacillus thuringiensis during vegetative growth and sporulation phases Sellami, S., Jamoussi, K., Mesrati Abdelkefi, L., and Jaoua, S*. Centre of biotechnology of Sfax, Laboratory of Biopesticides, P. O. Box « K » 3038 Sfax-Tunisia *Email: [email protected]

Abstract: The Gram positive bacterium Bacillus thuringiensis produces during sporulation, crystalline proteins encoded by cry genes. The expression of cry1Ac gene is under the control of sporulation-linked dual promoters, BtI and BtII which are responsible for the accumulation of highly level of Cry protein in mother cell. Beside delta endotoxins, several strains of Bacillus thuringiensis produce during vegetative growth phase, a second generation of insecticidal proteins active against lepidopteran larvae (Mesrati et al., 2005A, 2005B); Vegetative Insecticidal Protein (VIPs). In the present work, the time course for the expression of Vip3LB protein of the wild strain BUPM95 was analysed by western blot. It was demonstrated that VIP3LB can be detected in concentrated supernatants, reaching its maximum level at the middle of the logarithmic phase and remaining at high levels during and after sporulation, indicating the stability of VIP3LB. The vip3LB was cloned and expressed under the control of the two strong sporulationdependent BtI and BtII promoters of the cry1Ac into the shuttle vector pHTblue, the new plasmid was called pHT-spo-vip3LB. We have demonstrated that the synthesis of VIP3LB expressed from BUPM95 (pHT-spo-vip3LB) was improved and remained stable compared with the wild strain BUPM95 (pHTBlue), owing to the addition of BtI and BtII promoters. References: Mesrati, A.L., Tounsi, S. and Jaoua, S. (2005) (A) Characterization of a novel vip3-type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid. FEMS Microbiol. Lett. 244, 353-358. Mesrati, A.L., Tounsi, S., Kamoun, F. and Jaoua, S. (2005) (B) Identification of a promoter for the vegetative insecticidal protein-encoding gene vip3LB from Bacillus thuringiensis. FEMS Microbiol. Lett. 247, 101-104.


Session 3

OC31 S3

Production of azadirachtin through in vitro culture of neem (Azadirachta indica A. Juss) Muhammad Rafiq* and Muhammad Umar Dahot Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro, Pakistan E-mail: [email protected]

Abstract: Neem originated azadirachtin is widely used as a natural biopesticide due to its non-toxic and eco-friendly nature. Currently azadirachtin is extracted from seeds of wild and cultivated plants because their chemical synthesis is either extremely difficult or economically infeasible and its availability is dependent upon a reliable supply of quality seeds. The biotechnological production of valuable secondary metabolites through plant cell or organ cultures is an attractive alternative for the extraction of whole plant material. Plant cell culture can provide a potential production of azadirachtin alternative to traditional cultivation methods or chemical synthesis routes. In order to establish neem callus and suspension cultures for azadirachtin production, different concentrations and combinations of plant growth regulators (2, 4-D, NAA and BAP) were studied using immature flowers, leaves, nodular stem sections, immature and seeds as explants. The highest callus development was observed when immature flowers were inoculated on MS basal medium with addition of 1.0mg/L 2,4-D, 1.0mg/L BAP, 0.2mg/L NAA and 10% sucrose solidified with 0.4% phytagel incubated at 25±2° C. The highest neem cell suspensions were obtained by using immature flower derived callus on MS medium with addition of 1.0mg/L 2,4-D, 0.2mg/L BAP and 3% sucrose in 50ml containing 250ml flasks, placing at shaking incubator at 25±2° C in 24h dark photoperiod. Different carbon sources (sucrose, glucose, etc.) and nitrogen sources (NH4NO3, HNO3, Urea, etc.) were used to observe the increased production of azadirachtin. The biomass fresh and dry weights as well as azadirachtin contents of sample cell cultures were determined.

Abbreviations: 2, 4-D: 2, 4-Dichlorophenoxy acetic acid; NAA Nepthalene acetic acid; BAP: 6benzyl aminopurine; MS Murashige and Skooge (1962) medium


Session 3

OC32 S3

Microbial Technology Of Bacillus Thuringiensis Var. Kurstaki (Berliner) Formulation Against Defoliator Spodoptera Litura (Fabricius) In Groundnut (Arachis Hypogea). Palaniappan. S and Satheesh. J School of Biotechnology, SRM University Chennai, Tamil nadu – 603203, India. Email: [email protected]

Abstract: Crop losses world wide due to insect pests is estimated about 15%. Among pests, the tobacco caterpillar Spodoptera litura is to be one of the deadliest pest in all crops. .The residual affects of the postcodes and induced resistance in insects has promoted the use of microorganism as an alternative. The natural pathogenic microorganisms which control the pests is Bacillus thuringiensis. Among the several crops in south India, the most commercial crop species like groundnut (Arachis Hypogea) and castor plants was mostly infected with Spodoptera litura larvae. The bacterial formulation fits very well to the groundnut ecosystem as the required micro climatic factors such as humidity to cause epizootics on the larvae are very much available and there is, therefore, much scope for use of Bacillus thuringiensis formulation in groundnut. In the light of the above, the present investigation was undertaken to develop a bentonite Bacillus thuringiensis formulation to be used against the groundnut defoliator Spodoptera litura. This paper aims to study about the efficacy of Bacillus thuringiensis against Spodoptera litura in groundnut species under laboratory and pot culture conditions. This paper also contains formulation of bentonite based bacterial pesticide, mode of action of Bacillus thuringiensis in insect pest and the bionomics of Spodoptera litura in both natural and artificial diet. Key words: Bacillus thuringiensis, Spodoptera litura, Arachis Hypogea, Bentonite.


Session 3

OC33 S3

Molecular investigation of brittle leaf disease affected date palm (phoenix Dactylifera L) leaves. Saidi Mohammed Najib1, Gargouri-Bouzid Radhia1 and Noureddine Drira2 l

Laboratoire des Biotechnologies Végétales Appliquées à l'Amélioration des Cultures, Ecole Nationale d'Ingénieurs de Sfax, BP «W» Route Soukra Km 4, 3038 Sfax, Tunisia. 2 Faculté des Sciences de Sfax, Route de Soukra km 4 - B.P N° 802, 3018 Sfax, Tunisia E-mail address: [email protected]

Abstract: Despite its great ecological and socio-economical importance is desert oases; Date palm (phoenix dactylifera L.) is being decimated, especially in Tunisia, by a new disease called brittle leaf disease (BLD). The causal agent of this disease is still unknown. However many hypotheses have been elaborated to identify the causal agent of this disease. Indeed the presence of viruses, bacteria (phytoplasmas), and fungi was inverstigated. Moreover, mineral element deficiency in soil, and leaves was also reported. The aim of this work is to identify up-regulated and down-regulated EST from BLD affected date palm, after optimization of RNA isolation from leaves. Samples from different disease stages were subjected to this analysis. A differential Display experiment and subtractive hybridization were carried out. Isolated ESTs that seem to be differentially expressed in the diseased tissues are sequenced. They showed important homology with some abiotic and biotic induced genes from many plant species.


Session 3

OC34 S3

Protective Whey Proteins From Camel (Camelus Dromedarius) Milk Halima El-Hatmia, Jean-Michel Girardetb, Jean-Luc Gaillardb, Touhami Khorchania and Hamadi Attiac a

Institut des Régions Arides, Laboratoire d’Elevage et de Faune Sauvage, 4119 Medenine, TUNISIE Laboratoire des BioSciences de l’Aliment, U.S.C. INRA 885, Université Henri Poincaré – Nancy 1, B.P. 239, 54506 Vandœuvre-lès-Nancy CEDEX, FRANCE C Ecole Nationale d’Ingénieurs de Sfax, Unité d’Analyses Alimentaires, B.P. W, 3038 Sfax, TUNISIE b

Abstract: The quantitative distribution of individual proteins greatly differs between species, and the distribution in camel milk is unique and particular. Whey proteins from camel milk were examined on their relative distribution, as compared to whey proteins of cow milk, and with special interest in proteins with possible antimicrobial activity. Presence and identify of whey proteins (WPs) were monitored using SDS-PAGE technique. Camel milk contains a variety of protective proteins that contribute to bacterial growth inhibition. Some of these proteins were not found in cow milk, or only in minor amounts, such as the novel peptidoglycan recognition protein (PGRP), the camel whey basic protein (CWBP), the whey acidic protein (WAP), or lactophorin (PP3). Due do the lower protein content, and a larger contribution of whey proteins, camel milk could be an interesting alternative in infant milk formula. Absence of β-lactoglobulin, which may result in intolerance of an infant towards cow milk, a low amount of lactoperoxydase, which seems to be down-regulated in human milk early in lactation, a high amount of α-lactalbumin, which has a high nutritional value. Lactoferrin, which is also found at a high level in human milk, similarly to human milk, could be an advantage in utilisation of camel milk in dairy products for infants and people with allergy against cow milk product. In addition to the low amount of flore of camel milk, as well as the high content of immunoglobulins (IgG1, IgG2, IgG3) may also explain why there are reports of medicinal properties, and why some nomads use camel milk as a remedy against diarrhoea. It was concluded that camel and cow milk differ strongly in the composition of whey protein fraction. The different composition of the camel milk whey, as compared to milk of cows, was suggested to be a result of genetic and environmental factors. Camel’s milk is one of the key foods available in arid and sub-arid regions where it covers a substantial part of the quantitative and qualitative nutritional needs.


Session 3

OC35 S3

Primary cultures of heart cells from the clam Ruditapes decussatus as an alternative method for ecotoxicological studies H. Hanana1, M. Droguet1, H. Talarmin1, J P. Pennec1, B. Boussaid2 and G. Dorange1 1

EA « Facteurs nerveux et Structuration tissulaire» - Institut de Synergie des Sciences et de la Santé, CHU Morvan 6 avenue Foch 29609 Brest Cedex, France 2 Institut National des Sciences et Technologies de la Mer, centre INSTM La Goulette, Port de pêche 2060 La Goulette, Tunisie E-mail : [email protected]

Abstract: Cells were isolated from the heart of the clam Ruditapes decussatus and routinely cultured. Fibroblastic adherent cells were identified as cardiomyocytes due to i) their spontaneous beating ii) their positive reaction with antibodies against sarcomeric tropomyosin, sarcomeric αactinin, myosin and troponin iii) their electrophysiological properties analysed by patch-clamp. These cultured cells were used as an experimental in vitro model to evaluate the effect of a dinoflagellate toxin, the okadaic acid (OA), on cell viability, cell signalling and electrophysiological properties. Our results showed that even at levels as high as 1000 nM after 24h of treatment, OA produced no cytotoxicity as demonstrated by MTT assay. OA increased the level of inward calcium currents as measured by patch clamp. Moreover, after an incubation of 2h, OA induced at 500 nM and 1000 nM a significant phosphorylation of the stress activated P38 Mitogen Activated Protein Kinase (MAPKs), as evaluated by ELISA. The increase of calcium influx followed by the activation of P38 explained that OA was not toxic for cells in culture. Thus, it is possible to conclude that clam heart cells in culture could be used as an alternative method to study the effects of marine pollutants.


Poster Communications

Session 3


Session 3 /

PC1 S3

Determination of genetic stability of micropropagated Apricot cv Ajami and Torinel rootstock using Random Amplified Polymorphic DNA (RAPD) markers AL-Qudah A.*, Shibli R.** and K. ALMaarri* * College of Agriculture, Damascus University, and General Commission of Biotechnology, Plant Biotechnology Laboratory, Damascus, Syria. ** College of Agriculture, Jordan University of Science and Technology, Irbid, Jordan.

Abstract : An efficient micropropagation procedure for Ajami cultivar and Torinel rootstock of Apricot, cultivated in Syria, was developed by using shoot tip cultures. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of micropropagated plants of Apricot compared to mother plants. 20 RAPD primers were used to amplify DNA from in vivo and in vitro plant material to assess the genetic fidelity.4 primers were useful to detect the polymorphism among studied material. The result showed 100% similarity between mother plant and vitoplants in Apricot cv "Ajami" and Torinel rootstock. All RAPD profiles from micropropagated plants were monomorphic and similar to those of field grown control plants. No variation was detected within the micropropagated plants.


Session 3 /

PC2 S3

Extraction de pentoses de co-produits de plantes saccharifères par traitement thermique couplé à une hydrolyse acide ou enzymatique H. Boussarsar, B. Rogé & M. Mathlouthi Labotatoire de Chimie Physique Industrielle, UMR FARE, Université de Reims Champagne-Ardenne, BP 1039 – 51687 Reimd Cedex2, France, E-mail : [email protected]

Abstract : Les matériaux lignocellulosiques issus de co-produits de plantes saccharifères constituent une ressource renouvelable dont la valorisation s’intègre bien dans un projet agroindustriel d’exploitation de la plante entière. Un tel projet doit s’attacher à minimiser les rejets et protéger l’environnement. Ainsi, lorsqu’on envisage d’extraire du xylose à partir de coproduits riches en xylanes, on préfèrera partir d’une matière première déjà gonflée d’eau et chaude comme c’est le cas pour la bagasse à la sortie des moulins de sucrerie de canne plutôt que de matériau sec qu’il faut humidifier. Parmi les traitements appliqués pour solubiliser et hydrolyser les xylanes de la fraction hémicellulosique, différents traitements thermiques, du chauffage modéré à l’explosion à la vapeur sont décrits dans la littérature. De même des traitements chimiques comme l’hydrolyse acide, l’hydrolyse alcaline ou la combinaison de solvant organique et d’acide inorganique ont été utilisés. D’autres techniques comme l’explosion à l’ammoniaque (AFEX) ou au CO2 sont également connus. Nous avons abordé le choix du traitement de valorisation du matériau lignocellulosique avec le souci d’extraire le moins d’impuretés possible. En effet, l’augmentation de la coloration et de la concentration en monomères dans le sirop final de cristallisation du xylose implique une purification comprenant chromatographie séparative et adsorption de colorants. Dans ce travail, nous présentons les résultats des traitements hydrothermiques associés à une hydrolyse acide ou enzymatique de la bagasse. L’hydrolyse acide permet d’obtenir un rendement d’extraction de xylose élevé (80% environ) mais une coloration 5 fois plus élevée que le traitement hydrothermique seul et une pureté inférieure à 70%. L’hydrolyse enzymatique donne quant à elle, un plus faible rendement d’extraction avec cependant moins de coloration et d’impuretés gênantes pour la cristallisation du xylose. Pour obtenir le xylose par hydrolyse enzymatique couplée à un prétraitement hydrothermique, il faut un couple de catalyseurs enzymatiques : une endo-xylanase pour solubiliser les hémicelluloses et une exo-xylosidase pour dégrader les oligo-xylanes en xylose. Les conditions d’utilisation d’une endo-xylanase thermorésistante sont décrites et les perspectives d’application de ce travail présentées.


Session 3 /

PC3 S3

Population genetic structure of moroccan wild medfly (ceratitis capitata): polymorphism within and between populations. A.A. Alaoui*, A. Imoulan*, A.W. Tadlaoui*, Z. El Alaoui talibi*. A. El Meziane* * * Laboratoire de Biotechnologie de la Valorisation et la Protection des Agro-Ressources, Faculté des Sciences et Techniques-Guéliz, Marrakech. E-mail: [email protected]

Abstract : The Medfly, Ceratitis capitata is a devastating pest and extremely harmful to a large range of fruit trees, it is ranked first among economically important fruit fly species in the world. For example, The United States is eager to see medfly populations reduced in other countries because if they become established here, it could cost as much as $1.5 billion dollars a year due to export sanctions, lost markets, treatment costs and crop losses. In our country, where the argan forest; an endemic host plant in Morroco; is known as a reservoir to the medfly, that is medfly control is more complicated. Population genetic structure study of medfly is a key for an integral control strategy. In the purpose to evaluate the genetic diversity of the Medfly population, DNA of 120 individual, sampled from six subpopulations in three argan forest, has been extracted and analyzed using 5 primers. Using 70 polymorphic loci developed from RAPD markers. The matrix ‘presence / absence’ of bands was obtained from agarose gels. This allows comparing the degree of differentiation and polymorphism within and between populations studied. UPGMA dendrograms was drawed according to the genetic distance measured between each subpopulation pair in addition to Bactrocera olea; a very closely related species that was used as an out-group. The correlation between geographic and genetic distance is verified. In general total variability is explained by very high polymorphisms within subpopulation. Selection, flux migratory and other evolutionary constraints have been proposed to discuss and to interpret the results. Keys words : Genetic diversity, RAPD, Ceratitis capitata, Population genetic structure.


Session 3 /

PC4 S3

Valorization of tuna waste as substitution of fish meal in diets for Nile tilapia, Oreochromis niloticus (L, 1758) reared in geothermal waters of Southern Tunisian. 1

2

2

Saber Abdelkader Saidї , Mohamed Salah Azaza , Mohamed Majdeddine Kraїm , Jos van 4

1

Pelt and Abdelfattah El Feki 1. Faculty of Science of Sfax; 2.National institute of Marine Sciences and Technologies. 3. University Hospital Gasthuisberg, Leuven, Belgium.

Abstract : In order to develop low cost nutritionally balanced diets for Nile Tilapia (Oreochromis niloticus), attempts have been made to replace conventional feed ingredients with less expensive, by-product unconventional protein sources, from industrial residues. The present work was conducted over 45 days-period to evaluate waste of tuna transformation (WTT) as fish meal (FM) substitute in diet for Nile Tilapia fingerlings. The WTT was included in the diets at various levels of 0%, 10%, 20% and 30% (diets A0, A1 A2 and A3, respectively). Four isonitrogenous and isocaloric diets were prepared and fed fish initially weighing 2.16 g with three replications for each treatment. There were no significant differences (P>0.05) in growth performance among fish fed with diets A0, A1 and A2. However, fish fed with diet A3 had significantly lower growth (P10 cells l ), and October-2006 (>10 cells l ). The dominant accompanying species to such blooms are always small flagellates (over 4

-1

10 cells L ) which characterize oligotrophic systems. This group was mainly represented by Prasinophyceae (Pyramimonas sp., Micromonas sp. and Tetraselmis sp.), Cryptophyceae (Plagioselmis sp. and Hillea sp.) and Prymnesiophyceae (Chrysochrochromulina sp.). These species seem to be the best-adapted to the environmental conditions defined by the Pseudonitzschia spp. blooms in Bizerte lagoon. Key words: Pseudo-nitzschia blooms, phytoplankton assemblage, Bizerte lagoon.


Session 3 /

PC92 S3

Determination of the distribution area and assessment of the extention surfaces of brown algae Padina pavonica in the north of Tunisia Ksouri, J., Elferjani, H. and Mensi, F. National Institute of Sea Sciences and Technologies (INSTM), 29 rue Général Khéréddine 2015 Le Kram – Tunisie, E-mail : [email protected]

Abstract : The discovery in 1992 of interesting pharmacological properties for Padina pavonica has propelled this brown algae to the rank of valuable. The present work, done in the setting of a convention between the INSTM and the Society “Marine Algae of Tunisia” in July-August 2006, deals with the determination of the distribution area and the extension surfaces of this species in Cape Zébib (North of Tunisia). The adopted technique is based on the observation by diving along 13 transects sweeping the whole zone of study. At the level of this site, the species is observed in an inshore strip along 1600 m between the coast and the isobathe 10 m, on a total surface of 132 ha; it presents itself as the populations having different rates of recovery (TR). Putting this in mind, the surface covered by Padina pavonica represents only 33,7% of its distribution area, equivalent to 44,5 ha. The dense populations (TR: 60-80%) and those not highly populated (TR: 40-60%), observed respectively between 0 - 1,5 m and 1,5 - 4 m, are majority and constitute 72,4% of the efficient surface.


Session 3 /

PC93 S3

Expression study of LOX 3 gene during the phytopathogenesis of Alternaria brassicae Sarwar Ali, Javed Ahmad, S. Marla, Anil Kumar, Murray Grant1 and Gohar Taj Department of Molecular Biology & Genetic Engineering, G.B.Pant university of Ag & Tech., Pantnagar (U.S.Nagar) 263145, Uttarakhand, India, University of Exeter, Exeter, U.K. E-mail : [email protected]

Abstract : Rapeseed mustard is one of the important oil seed crop of India . Major caused of low productivity of Brassica is Alternaria blight caused by Alternaria brassicae. Plant shows defense reactions at the time of infection. Main defense component is phytoharmones like jasmonic acid, salicylic acid Ethylene etc. Their synergic, antagonistic and cross talk provide defense to the plant against pathogen. JA pathway is responsible for giving resistance to plant at the time of pathogenesis. In our study we have given emphasis to JA signaling. We have concentrated on bio-synthatic pathway of JA from linolenic & linoleic acid. These acids are primary precursor molecule for synthesis of JA. LOX enzyme catalyzes the hydroperoxidation of polyunsaturated fatty acid containing a cis, cis1,4pentadiene conjugated double bond system. LOX derived fatty acid, hydroperoxide are precursor molecules of JA which may play regulatory role. In order to investigate role of LOX 3 gene from Arabidopsis thaliana the amplicon was transformed into DH5α . The Brasica juncea var.RLM198 was grown in glass house conditions and plant were infected by inoculating Alternaria brassicae spores. The total RNA was isolated from inoculated as well as control plants at different time intervals. In subsequent study isolated RNA were used for northern hybridization with a prob (LOX3 amplicon).However no positive signals were seen after hybridization that indicate, LOX3 gene does not express at this stage or might be expressed at later stage of infections.


Session 3 /

PC94 S3

Tocopherols and phytosterols compositions of Coriandrum sativum seeds 1

2

1

2

1

Jazia Sriti , Thierry Talou , Wissem Aidi Wannes , Muriel Cerny and Brahim Marzouk 1

Aromatic and Medicinal Plant Unit, Biotechnologic Center in Technopark Borj-Cédria, Hammam-Lif, Tunisia 2

Agro-industrial chemistry laboratory UMR 1010 INRA/INP, ENSIACET University. 118 street of Narbonne, Toulouse, France.

Abstract : Coriander (Coriandrum sativum L.) is a plant belonging to the Umbelliferae family and is native to the Mediterranean area. The objective of this work is to characterize the Tunisian coriander through its phytosterols and tocopherols compositions. Fifty µl of internal standard (dihydrocholesterol) were added to 140 mg of oil. Then, 3 ml of KOH 1M in ethanol were added and mixed to each sample and a heating at 75°C was maintained during 30 min. After cooling at the ambient temperature, 1 ml of distilled water and 6 ml of isohexane were added to the mixtures, followed by a decantation and the recovery of hexane phase was analyzed by gas chromatography (GC). Sterols analysis by gas chromatography requires their silylation by the addition of 1ml Nmethyl-N-trimethylethylsilyl-heptafluorobutyramide (MSHFBA) mixed with 50µl 1-methyl imidazol. Sterols were analyzed by gas chromatography (GC) using a Perkin Elmer, apparatus equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. Total sterol (TS) amount was estimated to be 36.53mg/g oil. Stigmasterol, β-sitosterol, Δ5, 24-stigmastadienol and Δ7-stigmasterol were found to be the sterol markers. Tocopherols were analysed by high-performance liquid chromatography on a silica column using as elution system isooctane/ isopropanol (99.5:0.5v/v). The γ-tocotrienol was predominant with 238.40 µg/g oil equivalent to 72.8% of total tocopherols. Keys words: Coriandrum sativum L., seed, fatty acids, sterols, stigmasterol, tocopherols


Session 3 /

PC95 S3

Weight Loss in Rats upon Consumption of High-altitude Algae from Peruvian Andes Nunez, J., and Mendoza, A. Faculty of Medical Sciences. Universidad Nacional de Ancash, Huaraz. Av. Centenario 200, P.O. Box 70, Huaraz-Peru. E-mail: [email protected]

Abstract : The green-blue algae cushuro (Nostoc Sphaericum vaucher) grow spontaneously in lakes and rivers at high altitude and it is eaten for native dwellers from Peruvian sierra. The study was to evaluate the effects of its ingestion, in rats eaten at libitum; and to learn about the composition of its fatty acid contents. Results: a) Chromatographic analysis of fatty acids: saturated: 14:0, 15:0, 16:0 y 18:0; monounsaturated: 16:1 (cis9), 18:1 (cis9) y 18:1 (cis11); polyunsaturated: 16:2 (cis9,cis12), 18:2 (cis9,cis12) y 18:3 (cis9,cis12,cis15). The plentiful fatty acids are 15:0, 16:0, 16:1 and 18:3. The fatty acids 15:0 and 18:1 (cis11) are uncommon in biology and not important in human nutrition. b) Animal feeding studies: weanling rats were feed with a normal standard diet (13.8 % protein and 38 % sugar) with addition 5 % of dry cushuro weight, during two months. They had a significantly weight loss (p = 0.000006), a significantly excreta production (p = 0.000266) and a moderate increase of water drinking (p = 0.032697) than rats with a standard diet only. The ingestion of solid food was similar in two groups (p = 0.19999). These data suggest that the alga cushuro seem to contain antinutrients in the enough amount which may influence weight loss in rats and it influence negatively in the nutrient absorption of a normal diet.


Session 3 /

PC96 S3

Nutritional status of leaf proteins extracted from some terrestrial weeds. Shanker, J. and Singh, D Division of Biochemistry K.A. Postgraduate College C.S.J.M. University Allahabad India

Abstract : Biochemical studies were made to determine nutritional status of some terrestrial weeds (leaves). The leaf protein concentrate (L.P.C.) fractionated from Kans (Saccharum spontaneous Linn.), Motha, (Cyperus rotiundus Linn.) and Mandusi (Phalaris minor Retz) taken for biochemical composition. It was observed that the crude protein content was much more higher in the L.P.C. of all the three weeds than those observed in the leaves. Similarly the essential amino acid contents were found in adequate quantity except methionine in the L.P.C. of these terrestrial weeds. Similar observations were also recorded for the dry matter, crude fiber, crude fat and ash content in the leaves and L.P.C. of these weeds. The results indicate the these terrestrial weeds may be utilized as a promising source for the extraction of leaf protein concentrate, and thus its utilization as a non conventional protein rich food, feed and in medico pharmaceutical field. It can be an ideal supplementing +

+

food for the people whose condition necessitates drastic restrictions on the intake of Na & K , and for the people with gastrointestinal problems. L.P.C. can be added to nonalcoholic beverages to increase their nutritional value and to supplement some cereals.


Session 3 /

PC97 S3

The economic role, agricultural and environmental Myriapoda in Algeria; future prospects Kahina houd1 & Ouided Daas-Maamcha2 1Département agronomy - University Center of El Tarf - El Tarf. 2Laboratoire of Animal Biology Applied - Department of Biology, Faculty of Sciences - Badji Mokhtar University - Annaba. E-mail: [email protected]

Abstract : The Myriapoda constitutes a very important group of soil fauna; the impact of their activities is fundamental to the structure, fertility and soil composition. The order of Chilopoda represents a group of Myriapoda, located at atop the food chain are excellent natural agents of biological control of pests of crops, bio indicators of pollution because they accumulate trace metal elements in their exoskeleton. Specimens of this group of invertebrates represent therefore, terrain models to environmental interest. To find the biological diversity of this group, little known in Algeria as potential agents of biological control, the dynamics of this population and the environmental conditions, soil and climate that control the distribution of this group, we undertook A study of the biodiversity of these Chilopoda in various habitats, and this, during the annual period from April 2006 and March 2007. The study physico-chemical soil showed significant differences in terms of particle size and pH. The observation of characters morpho-anatomical specimens revealed harvested according to the classification criteria, the presence of 11espèces attached to six (6) family: Scolopendridés, Lithobiidés, Scutigéridés, Geophilidés, Iulidés, Polydesmidés. The analysis of the temporal distribution of Chilopoda shows a very marked seasonal variation, it is characterized by a gradual decline in the biomass in the summer and this is likely related not only to seasonal variations in climate, but also the conduct of reproductive cycles of each species. Key words: Chilopoda, Soil, Environment, Agriculture, Development


Session 3 /

PC98 S3

Phytochemical Analysis of Polyphenols Extract of Lavandula stoechas and its Interaction with Bovine Serum Albumin Protein Moussi, K., Madani, K. and Chibane, M. Abderrahmane Mira University of Bejaia, Faculty of Natural Sciences and Life, Department of Nutritional Sciences, Laboratory of Biomathematics, Biochemistry, Biophysics and Scientometrics. Bejaia 06000, Algeria. Tel / Fax: 034214762, E-mail: [email protected]

Abstract : Lavandula stoechas is an aromatic species of the area of Adekar (Bejaia). It belongs to the family of Lamiaceae and to the Lavandula genus. Its traditional use led us to study this plant within the framework of the valorization of its natural substances. For that, we carried out the extraction of polyphenols of the leafs and the flowers of this shrub, in particular a phytochemical analysis, an interactional study of the methanolic extract with the Bovine Serum Albumin protein and antibacterial study. These analyses showed that the extract is characterized by a rate of 7.25 % of polyphenols with non-polar majority (56.05 %), 1.46 % of flavonoïdes, absence of tannin and by the existence of flavones glycosides identified by revelation from the paper chromatography and colorimetric methods. The interactional nature of the extract with the Bovine Serum Albumin protein is influenced by the concentration of extract, the ionic forces and time. These effects and non-polar nature of the extract of plant were confirmed by simulation of the interaction of the flavones glycosides with a similar human Serum Albumin protein, showing that the interaction occurs mainly by hydrophobic bonds. Finally, two bacterial strains are sensitive to the extract


Session 3 /

PC99 S3 +

+

Cloning and functional characterization of a plasma membrane Na /H antiporter gene from durum wheat variety (TdSOS1). 2

1

3

2

1

K. Feki1, F.J. Quintero , M. Ayadi , B. Chalhoub , J.M. Pardo and K. Masmoudi . 1

Laboratory of Plant Molecular Genetics, Centre of Biotechnology of Sfax, Tunisia. Instituto de Recursos Naturales y Agrobiologia de Seville, Spain. 3 Research in vegetable genomics Unit, Evry, France Postal address: Centre of Biotechnology of Sfax, Road of Sidi Mansour Km 6, P. O. Box « K » 3038 SfaxTunisia, E-mail: [email protected] 2

Abstract : Abiotic stress, including drought, high salinity or cold, affect severely agricultural productivity, causing around 70% of yield loss. Among abiotic stresses, salinity is the major constraint of crop productivity because it can not only decrease crop yield, but also limit expansion of agriculture onto previously uncultivated land. Following a salt stress, the cell reacts by activating or repressing several genes and signal transduction pathways. Among them, there is the Salt Overly Sensitive (SOS) where the target protein is the plasma membrane +

Na+/H+ antiporter (SOS1). The protein SOS1 is highly specific for Na transport and therefore +

+

cannot transport other monovalent cations, such as K or Li . To further investigate the importance of the SOS1 protein in salt stress, we have isolated the full length cDNA SOS1 (3.5Kb) from durum wheat variety (TdSOS1). The ORF of TdSOS1 is cloned and predicted to encode a protein of 127 kDa. Hydrophobicity plot analysis showed that the N-terminal portion of TdSOS1 is highly hydrophobic and it has 12 predicted transmembrane domains in the C-terminal part. Sequence comparison of TdSOS1 showed high homology with rice SOS1 (OsSOS1). The in vitro phosphorylation experiment showed that the hyperactive kinase SOS2T/D∆308 phosphorylate TdSOS1 in two essential residues (Ser1136-Ser1138). In order to study the functional analysis of the endogenous promoter of TdSOS1, we isolated the promoter sequence, using a genomic BAC library of bread wheat (CV. Chinese spring). Two BAC clones (1020I and 454A20) containing the SOS1 gene were identified. The promoter region was isolated by inverse-PCR from the BAC clones. We studied the expression pattern of TdSOS1 in leaves, sheaths and roots of two durum wheat varieties; Oum Rabiaa (tolerant) and Mahmoudi (sensitive). We found variability in TdSOS1 expression level in different organs in the two wheat varieties.


Session 3 /

PC100 S3

Antifertility and antifeedant activities of gibberellic acid against Locusta migratoria migratoria (Orthoptera; Acrididae) Abdellaoui K., Ben Halima-Kamel M., and Ben Hamouda M. H UR 04 AGR04: Invertébrés, micro-organismes et malherbes nuisibles: méthodes de lutte alternative, Institut Supérieur Agronomique de Chott-Mariem 4042- Sousse- Tunisie, E-mail : [email protected]

Abstract : Migratory locusts represent a major group of insect pests in African. Experiments were conducted to assess the effect of gibberellic acid (GA3), plant growth regulator, on the reproductive activity and the feeding behaviour of Locusta migratoria migratoria (Orthoptera; Acrididae) with 125, 625 3125, 4125, 5125 and 6125 ppm. The effects of GA3 on the reproductive potential of L. migratoria were investigated to exposing the freshly emerged (0-1 day old) male and female adults to these concentrations. Fecundity and per cent fertility were recorded on the basis of the total number of eggs oviposited and hatched. To study the food consumption and utilisation in L. migratoria nymphs, we used the five gravimetric index defined by Waldbauer (1968) and Montgomery (1983). The assessment for reproductive potential was made on the basis of reduction in fecundity and fertility of laid eggs and measured as shortening of the longevity. The maximum reduction in fecundity and percent fertility was observed with 4125 and 6125 ppm. Therefore, GA3 was responsible to slowing down the sexual maturity, to prolong the punter rhythm, to decrease the weight of the insects and the ovaries and to delay the development of the ovaries of L. migratoria. Indeed, the result showed that GA3 affected the morphometric parameters of ovaries of L. migratoria. In the same way, feeding deterrence was observed against GA3. Nymphs treated ate less food and subsequently their approximate digestibility (AD) was lower than the control. The efficiency of conversion of digested (ECD) was low, leading to a loss of weight among nymphs. Overall analysis of the data points to dual function of this compound; the antifertility and antifeedant actions. Key words: L. migratoria, gibberellic acid, antifertility, antifeedants.


Session 3 /

PC101 S3

Combined action of salinity and exogenic glutamic acid on the endogenous proline in the Atriplex halimus L. Dahli K., Belkhodja M. Département de Biologie, Centre universitaire Mustafa Stambouli de Mascara, Algérie E-mail: [email protected]

Abstract: The objective of this work is the study of the effect of salinity composed of NaCl+CaCl2 at concentrations 300 and 600 meq.l-1 of nutritive solution enriched or not with the glutamic acid at 25, 50 and 100 mM on the endogenous proline in the young plants of Atriplex halimus L. It is well-known that the glutamic acid is a precursor in the synthesis of proline acting as osmorégulateur under the under the abiotic constraints. The first experimental protocol concludes that the accumulation of the proline appears in the leaves according to their position in the plant. Indeed, the leaves at the top of the plant are much more proline-rich than the middle or the bottom. Also, the results show that the amino acid accumulates more in the leaves than in the stems and the roots of the plants stressed or not. This accumulation of the amino acid changing when the concentration of NaCl+CaCl in the medium increases. Enrichment in proline appears much more when 2

glutamic acid is brought to the salt solution. For this purpose, the results show that when the glutamic acid is brought to 50 and 100 mM in -1

the salt solution at 300 meq.l -1

of NaCl+CaCl , the proline accumulates in the leaves. 2

However, under the 600 meq.l treatment of NaCl+CaCl the contribution of the glutamic acid 2,

to 100 mM caused a slowdown in the accumulation of the amino acid. Key words: Salinity, Proline, Glutamic acid, Atriplex halimus.


Session 3 /

PC102 S3

Analysis of morphological parameters in pistachio nuts (Pistacia vera L.) khadija Farès, Ferdaous Guasmi , Leila Touil, Tebra Triki and Ali Ferchichi Abstract : The pistachio tree (Pistacia vera L.) is one of the fruit-bearing species known in the past of the world. In Tunisia, this culture is practised since antiquity. The main world producers of pistachio nuts are Iran, USA, turkey and Syria. Commercial exploitation of pistachio commenced in the 1930s in Iran, which still remains the largest producer, providing 56.10% of the world’s production. The second largest pistachio producer is USA, where kerman is the most commonly grown cultivar. It covers over 90% of the total country production of pistachios. In both Iran and USA, pistachio plantations are irrigated whereas in turkey there is no irrigation yet in place for this crop. Within the Pistacia genus, Pistacia genus, Pistacia vera (also called “Green Gold Tree”) is the only edible and worldwide marketable species. Since the 18 Century and during the Ottoman Empire, pistachio was the most appreciated crop by the local rules (“Beys”).Pistachio nuts are used as dessert and in the confectionery industry to prepare cakes and sweets. The nuts contain about 60% of fatty acids (oleic and linoleic acid) and 22% of proteins it’s also rich in vitamin A, B1, B2, B3,B5,B6, B9, B12 . Their energy value is twice as important as that of sugar or butter. Pistacia vera is also used in Tunisia as a rootstock. Wood and leaves are used as agricultural implements and fodder. The trees can be used for erosion and desertification control or as ornamental plants. For all these different uses, Pistacia vera can be considered a truly valuable multipurpose species. In Tunisia, the identification and the characterization of the varieties of pistachio tree was the research object many. The work completed in the South areas of the country with Chenchou, Sfax revealed the presence of several cultivars with different characteristics morphological, agronomic and ecological. The identification and the characterization of some pistachio cultivars in South of Tunisia revealed a remarkable diversity between them, thanks to morphological markers (length sheets, leaf area, length of fruit, forms of final leaflet, their length and by chemical markers (content of vitamins). For some cultivars, the length of fruits is between 16.3 cm and 20.4 cm, the length of the final leaflet is 5.88 cm to 8.42 cm its oscillates from elliptic 2

2

to round and the average leaf area varies between 17.13 cm to 34.05 cm in all varieties. Key words: Pistachio nuts (Pistacia vera L.), morphological, diversity


Session 3 /

PC103 S3

Comparative phenology and growth of perennial grasses Cenchrus ciliaris L. and Stipa lagascae established under soil water deficit a*

a

b

c

a

Saloua Jaballah , Aly Gribaa , Chadya Guenaoui , Khadija Kraiem and Ali Ferchichi a

Laboratoire d’Aridoculture et des cultures Oasiennes, Institut des Régions Arides : Médenine (IRA), 4119, Tunisie b

Laboratoire d’Ecologie pastorale, Institut des Régions Arides : Médenine (IRA), 4119, Tunisie c

Institut National d’Agronomie de Tunisie (INAT - Tunis), *E-mail : [email protected]

Abstract : The perennial herbaceous plants represent one of the most interesting resources but their persistence depends on their ability to cope with acute drought. The survival of plants in extreme conditions must therefore be studied to understand what strategies and mechanisms are involved in the perennial grasses from arid area. The aim of the present study was to compare the adaptative responses of both species Cenchrus ciliaris L. and Dactylis glomerata (cultivar Kasbah). We analyse the impact of water deficit on phenology and growth through some morphological traits. The results show that water deficit affects the biological cycle and there is a difference between species. In addition, the morphological traits are significantly affected (P