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5th International Conference on Chemical Technology 10. – 12. 4. 2017 Mikulov, Czech Republic

www.icct.cz PROCEEDINGS

of the 5th International Conference on Chemical Technology

www.icct.cz

Proceedings of the 5th International Conference on Chemical Technology 1st edition, October 2017

EDITORS Martin Veselý University of Chemistry and Technology, Prague Technická 5 166 28 Praha 6 Zdeněk Hrdlička University of Chemistry and Technology, Prague Technická 5 166 28 Praha 6 Jiří Hanika Institute of Chemical Process Fundamentals of the CAS, Prague Rozvojová 1/135 165 02 Praha 6-Suchdol Jaromír Lubojacký Jistebník 242 742 82 Jistebník, Czech Republic All communications submitted were duly reviewed by the scientific committee.

PUBLISHED AND DISTRIBUTED BY Czech Society of Industrial Chemistry Novotného Lávka 5 116 68 Prague, Czech Republic in cooperation with Czech Chemical Society Novotného Lávka 5 116 68 Prague, Czech Republic

PRINTED BY Ocean Design, Prague, Czech Republic

ISBN: 978-80-86238-65-4 (Print) ISBN: 978-80-86238-62-3 (Online) ISSN 2336-811X (Print) ISSN 2336-8128 (Online) Copyright © 2017 Czech Society of Industrial Chemistry

SCIENTIFIC COMMITTEE Assoc. Prof. Ing. Jaromír Lederer, Ph.D. | UniCRE Litvínov (President) Ing. Jaromír Lubojacký, MBA | Ixa Consulting, Jistebník (Vice-President) Prof. Ing. Jiří Hanika, DrSc., dr. h. c. | ICPF of the CAS, Prague (Vice-President) Prof. Ing. Martin Bajus, DrSc. | STU Bratislava Prof. Ing. František Babinec, Ph.D. | RISCO Brno Prof. Ing. Zdeněk Bělohlav, Ph.D. | UCT Prague Prof. Dr. Ing. Karel Bouzek | UCT Prague Assoc. Prof. Ing. Karel Ciahotný, Ph.D. | UCT Prague Prof. Ing. Jiří Drahoš, DrSc., dr.h.c. | CAS Prague Prof. Dr. Fabio Fava | Department of Civil, Chemical, Environmental and Materials Engineering of the University of Bologna Assoc. Prof. Ing. Tomáš Herink, Ph.D. | Unipetrol RPA Litvínov Prof. Ing. Milan Hronec, DrSc. | STU Bratislava Prof. Ing. Ľudovít Jelemenský, DrSc. | STU Bratislava Prof. Ing. Tomáš Jirout, Ph.D. | CTU in Prague Prof. Ing. Jan John, Ph.D. | FJFI, CTU in Prague Prof. Dr. Augustin F. Martinez | Universidad Politécnica de Valencia Prof. Dr. Dmitry Yu. Murzin | Åbo Akademi University, Turku, Finland Prof. Ing. Petr Kalenda, Ph.D. | FCHT University of Pardubice Prof. Elena Korotkova | Tomsk Polytechnic University Assoc. Prof. Ing. Milan Králik, Ph.D. | VUChT Bratislava Prof. RNDr. Bohumil Kratochvíl, Ph.D., DSc. | UCT Prague Prof. Ing. Petr Mikulášek, Ph.D. | FCHT, University of Pardubice Ing. Jozef Mikulec, Ph.D. | VÚRUP Bratislava Prof. Ing. Josef Pašek, DrSc. | UCT Prague Assoc. Prof. Dr. Ing. Petra Patáková | UCT Prague Ing. Zbyněk Průša | Agrofert, Deza Valašské Meziříčí Prof. Ing. Milan Pospíšil, Ph.D. | UCT Prague Ing. Miloslav Slezák, Ph.D. | FCHT, University of Pardubice Ing. Ivan Souček, Ph.D., MBA | Association of Chemical Industry in Czech Republic Prof. Ing. Ján Šajbidor, DrSc. | FCHPT, STU Bratislava Prof. RNDr. Jitka Ulrichová, Ph.D. | UP Olomouc Ing. Radomír Věk | Lovochemie Lovosice Prof. Ing. Kamil Wichterle, DrSc. dr.h.c. | VŠB TU Ostrava Jacek Olszacki, Ph.D. | PKN ORLEN, Poland

PROGRAM COMMITTEE Ing. Jaromír Lubojacký, MBA | Ixa Consulting, Jistebník (President) Assoc. Prof. Ing. Jaromír Lederer, Ph.D. | UniCRE Litvínov (Vice-President) Prof. Ing. Jiří Hanika, DrSc., dr. h. c. | ICPF of the CAS, Prague (Vice-President) Prof. Ing. Zdeněk Bělohlav, Ph.D. | UCT Prague Assoc. Prof. Ing. Karel Ciahotný, Ph.D. | UCT Prague Prof. Ing. Petr Kalenda, Ph.D. | FCHT, University of Pardubice Prof. Dr. Ing. Karel Bouzek | UCT Prague Assoc. Prof. Dr. Ing. Petra Patáková | UCT Prague Prof. RNDr Bohumil Kratochvíl, Ph.D., DSc. | UCT Prague Ing. Miloslav Slezák, Ph.D. | FCHT, University of Pardubice Prof. Ing. František Babinec, Ph.D. | RISCO Brno Prof. Dr. Ing. Dalibor Vojtěch | UCT Prague Assoc. Prof. Petra Lovecká, Ph.D. | UCT Prague Prof. Ing. Dušan Baran, Ph.D. | UCT Prague

CONTENTS BIOTECHNOLOGY AND BIOREFINARY USE OF NOVEL WASTE PRODUCTS IN THE MICROBIAL PRODUCTION OF LACTIC ACID Drahokoupil M., Paulová L., Patáková P.

11

STILBENOID DETERMINATION IN MUST, WINE AND WINERY WASTE SAMPLES Gharwalova L., Koukalova M., Cejkova A., Kolouchova I., Masak M.

16

KINETICS OF KERATIN ACIDIC HYDROLYSIS IN BATCH AUTOCLAVE Hanika J., Kastanek P., Rouskova M., Sabata S., Solcova O.

21

TERTIARY STRUCTURE PREDICTION OF POTENTIAL EFFLUX-PUMP PROTEIN IN CLOSTRIDIUM BEIJERINCKII NRRL B-598 Koscova P., Sedlar K., Kupkova K., Kolek J., Patakova P., Provaznik I.

26

INFLUENCE OF PLATINUM AND PALLADIUM NANOPARTICLES ON THE GROWTH OF BACTERIUM PSEUDOMONAS AERUGINOSA Koukalová M., Čejková A.

31

EFFECT OF AMPHOTERICIN B AND CHITOSAN ON CANDIDA SPP. BIOFILM FORMATION Kvasničková E., Paldrychová M., Lokočová K., Masák J.

36

ANTIBIOTIC RESISTANCE OF PSEUDOMONAS AERUGINOSA Lokočová K., Masák J.

42

EFFECT OF BIOLOGICALLY ACTIVE SUBSTANCES ON CANDIDA BIOFILM Lokočová K., Paldrychová M., Masák J.

47

AN ENERGY INDEPENDENT SOLAR DRYING SYSTEM FOR FOOD PRODUCTS DRYING Noori A.W., Royen M. J., Haydary J.

51

EFFECT OF NON-THERMAL PLASMA ON PSEUDOMONAS AERUGINOSA QUORUM SENSING SYSTEM Paldrychová M., Scholtz V., Kvasničková E., Masák J.

57

ANTI-BIOFILM EFFECT OF VITIS VINIFERA EXTRACT ON THE CANDIDA GENUS Paldrychová M., Kolouchová I., Masák J.

62

BIODEGRADATION OF CHICKEN FEATHER BY POLYHYDROXYALKANOATES ACCUMULATING BACTERIA Pernicová I., Šuráňová Z., Innemanova P., Obruča S.

67

CENTRIFUGAL PUMP CHARACTERISTICS COMPUTATION AND RELIABILITY EVALUATION AT VARIABLE SPEED DRIVEN Qazizada M. E., Pivarčiová E., Bialy W.

72

FATTY ACIDS PROFILE ANALYSIS IN FRESH SUSPENSION OF JAPONOCHYTRIUM SP. Rouskova M., Maleterova Y., Hurkova K., Kastanek P., Hanika J., Solcova O.

78

EXTRACTION OF ANTIOXIDANTS FROM FRESH WINE GRAPES MARC Rouskova M., Jiru M., Krmela A., Hanika J., Solcova O.

83

ANALYSIS OF CLOSTRIDIUM BEIJERINCKII NRRL B-598 CODING REGIONS USING RNA-SEQ DATA OF A CLOSELY RELATED STRAIN Sedlar K., Branska B., Kupkova K., Koscova P., Kolek J., Vasylkivska M., Patakova P., Provaznik I.

87

3

SUPERCRITICAL FLUID EXTRACTION: A GREEN METHOD FOR THE ISOLATION OF VALUABLE SUBSTANCES FROM THE VARIOUS BIOLOGICAL MATRICES Topiar M., Sajfrtova M., Solcova O., Hradecky J., Hurkova K., Hajslova J.

92

DIFFICULTIES IN ISOLATING TOTAL RNA FROM SPORULATING SOLVENTOGENIC CLOSTRIDIA Vasylkivska M., Patáková P.

97

ECONOMY OF CHEMICAL INDUSTRY UTILIZING A MULTIDIMENSIONAL DISCRIMINANT ANALYSIS IN CREATING NEW FINANCIAL ANALYSIS PREDICTION MODELS FOR INDUSTRIAL ENTERPRISES Baran D., Pastýr A.

103

APPLICATION OF OF LCA METHOD IN THE RUBBER INDUSTRY Baran D.

109

ECONOMICAL ASPECTS OF POPULATION AGING Botek M.

114

CONSUMER CHEMICALS BUYING PROCESS AND WAYS TO IMPROVE IT Branska L., Pecinova, Z., Patak, M., Horecká, K.

118

THE LEVEL AND DEVELOPMENT OF VALUE PRODUCTIVITY IN SELECTED ENTERPRISES OF CHEMISTRY INDUSTRY COMING FROM THE CRISIS 2011-2015 Klečka J.

124

APPLICATION OF PROJECT MANAGEMENT METHODS USABLE IN THE IMPLEMENTATION PHASE FROM THE PERSPECTIVE OF SELECTED CHEMICAL COMPANIES Kostalova J., Tetrevova L.

130

IMPACT OF HARVEST PERIOD ON NATURAL RUBBER QUALITY Kutnohorská O., Živec J.

136

CZECH PUBLIC ATTITUDES TO THE CHEMICAL INDUSTRY Lostakova H., Jelinkova M., Ostransky F.

142

VERIFICATION OF CAUSAL CONSISTENCY IN FACTORIAL DESIGN A CASE STUDY FROM AMMUNITION PERFORMANCE OPTIMIZATION Jadrná M., Macak T.

148

USE OF ECO-DESIGN PRINCIPLES IN CHEMICAL PRODUCT INNOVATIONS Munzarová S., Vávra J., Bednaříková M.

155

THE DEMAND FORECASTING PROCESS IN CZECH CHEMICAL COMPANIES Patak M., Branska L., Pecinova Z., Kvochova I.

161

COST MODELS IN ENVIRONMENTAL MANAGEMENT Strachotová D, Dyntar J.

167

CSR ACTIVITIES OF CHEMICAL COMPANY – A CASE STUDY FROM PLASTIC INDUSTRY Vavra J., Bednarikova M., Munzarova S., Prochazkova K.

179

THE RANGE OF SERVICES IN THE B2B MARKET WITH PRODUCTS OF THE CHEMICAL INDUSTRY Vlckova V., Lošťáková H.

185

NEW MANAGEMENT WAREHOUSE SYSTEM DESIGN Zemanová P., Botek M., Strachotová D

191

4

INORGANIC TECHNOLOGY PERFLUORINATED SULFONATED ACIDS AS A POLYMER ELECTROLYTE AND ELECTRODE COMPARTMENTS IN PEM WATER ELECTROLYSIS Bystron T., Mališ J., Brožová L., Zhigunov A., Bouzek K.

198

HIGH SURFACE CARBON ELECTRODES FOR CAPACITIVE DEIONISATION Giurg A., Denk K., Paidar M.

204

SYNTHESIS AND PROPERTIES OF SUPERABSORBENT COPOLYMER OF ACRYLIC ACID, ACRYLAMIDE AND KONJAC GLUCOMANNAN POLYSACCHARIDE Hermann P., Svoboda L., Bělina P.

210

THE EFFECT OF MINERALIZERS ON PIGMENTARY PROPERTIES OF CASSITERITE PIGMENTS DOPED BY MANGANESE IONS Karolová L., Dohnalová Ž.

215

WO3 PHOTOANODES PREPARED BY SPRAY PYROLYSIS FOR PHOTO-ELECTROCHEMICAL WATER SPLITTING Zlámal M., Krýsa J.

220

PHOTOELECTROCHEMICAL AND BLOCKING PROPERTIES OF TITANIA LAYERS PREPARED BY SPRAY PYROLYSIS Krýsová H., Zlámal M., Krýsa J., Kavan L.

225

JET MILL VERSUS PLANETARY MILL – COMPARISON OF PROPERTIES OF MILLED ZnFe2O4 PIGMENT Luxová J., Podzemná V., Šulcová P.

230

SYNTHESIS AND POSSIBILITIES OF UTILIZATION OF ORTHOFERRITES WITH PRASEODYMIUM Luxová J., Šulcová P.

236

MECHANICAL AND STRUCTURAL PROPERTIES OF METAKAOLIN-BASED GEOPOLYMER MORTARS IN EXTERNAL ENVIRONMENT Matulová L., Steinerová-Vondráčková M.

241

THE MIXED OXIDE IN Bi2O3-ZnO-CeO2 SYSTEM PREPARED BY SOLID STATE REACTION Těšitelová K., Šulcová P.

246

THE VARIOUS METHODS OF PREPARATION OF THE PEROVSKITE COMPOUND SrCeO3 BY LIQUID METHODS Těšitelová K., Burkovičová A., Hablovičová B., Dohnalová Ž., Šulcová P.

250

MATERIAL ENGINEERING TESTING BIOACTIVITY OF CHEMICALLY MODIFIED POLYMERS Benkocká M., Kolářová K., Matoušek J., Kolská Z.

256

PREPARATION OF SPHEROIDIZED AND NANO-STRUCTURAL SPINELS BY THE SPPS METHOD Brozek V., Lukac F., Medricky J., Musalek R., Maslani A., Mastny L., Brodil R.

261

MATERIAL DESIGN PROBLEMS OF PLASMA-CHEMICAL REACTORS FOR DISPOSAL PERFLUORINATED COMPOUNDS Brožek V., Březina V., Mastný L., Kubatík T.F., Živný O.

267

LINEAR GRATINGS IN CHALCOGENIDE GLASS THIN FILMS PREPARED VIA HOT EMBOSSING Buzek J., Schroeter S., Palka K., Vlcek M.

273

5

MIXED ALKALI EFFECT IN LITHIUM-SODIUM PHOSPHATE GLASSES MODIFIED WITH TUNGSTEN OXIDE Kalenda P., Koudelka L., Mošner P., Nikolić J., Moguš-Milanković A.

278

NANOSTRUCTURED ITO LAYERS FOR QCM GAS SENSORS Seidl J., Jirešová J., Hofmann J., Holánek P.

284

STUDY ON THE THERMOELECTRIC PROPERTIES OF POLYCRYSTALLINE SnSe DOPED WITH As Šraitrová K., Kucek V., Ruleová P., Plecháček T., Drašar Č.

287

SYNTHESIS OF MAGNETIC MICROPARTICLES FOR ISOLATION OF DNA FROM PCR PRODUCTS Zolal A., Štika M., Syslová K.

292

OIL, GAS, COAL, FUEL, BIOFUELS STUDY OF CATALYTIC HYDROTREATMENT OF PYROLYSIS BIO-OIL Auersvald M., Straka P., Shumeiko B., Staš M.

299

S-VALUE: A PRACTICAL APPROACH TO THE PROCEDURE FOR QUANTIFYING THE INTRINSIC STABILITY OF ASPHALTENES IN RESIDUAL FRACTIONS Černý R., Hamerníková J., Jíša P., Hidalgo J.M., Vráblík A.

304

MATHEMATICAL MODEL OF FISCHER-TROPSCH SYNTHESIS Filip L., Zámostný P., Rauch R.

310

CATALYTIC HYDROTREATING OF LIGHT CYCLE OIL AND ITS MIXTURE WITH ATMOSPHERIC GAS OIL Hidalgo J.M., Vajglová Z., Vráblík A., Černý R., Lederer J.

316

STUDIES ON THE COPPER OXIDE OXIDATION CAPACITIES UNDER VARIOUS CONDITIONS Hudský T., Ciahotný K., Berka J.

322

AGEING OF BITUMINOUS BINDERS IN MIXTURE WITH DIFFERENT TYPES OF MASTIC Matoušek L., Jíša P., Černý R.

327

INFLUENCE OF REACTION CONDITIONS AND ADDITION OF WASTE COOKING OIL TO FEEDSTOCK FOR BITUMEN SEMI-BLOWING ON PROPERTIES OF PRODUCTS Jíša P., Černý R., Matoušek L.

332

THEORETICAL PRINCIPLES OF PYROLYSIS OF LIGNOCELLULOSIC BIOMASS Shumeiko B., Auersvald M., Staš M., Kubička D.

337

ABLATIVE FAST PYROLYSIS – PROCESS FOR VALORIZATION OF RESIDUAL BIOMASS Schulzke T., Conrad S.

344

REVIEW OF PYROLYSIS BIO-OIL APPLICATIONS Staš M., Auersvald M., Shumeiko B., Kubička D.

350

THE EFFECT OF PRESSURE ON HYDROTREATING OF RAPESEED OIL Váchová V., Straka P., Blažek J., Kubička D., Šimáček P.

355

MODELLING OF FLUIDIZED BED HYDRODYNAMICS FOR A CA-LOOPING SYSTEM Vodička M., Hrdlička J., Opatřil J., Skopec P.

361

EVALUATION OF MARINE FUELS STABILITY BY ULTRAVIOLET RADIATION Vráblík A., Hidalgo J. M., Černý R.

367

6

PETROCHEMICALS AND ORGANIC TECHNOLOGY I SUBSTITUTION OF TRICHLOROETHYLENE AS EXTRACTION SOLVENT IN CRUDE CAPROLACTAM REFINING Fojtášková J., Koumar J., Leštinský P., Maršolek P., Maxa M., Obalová L.

374

REGENERATION OF IL SOLVENTS USED FOR TERT-BUTYL ALCOHOL–WATER MIXTURE SEPARATION Graczová E., Šulgan B., Steltenpohl P.

379

SYNTHESIS OF NEW FULGIDES AND THEIR SPECTRAL CHARACTERISTICS Kišac M., Machalický O.

385

REACTION CONDITIONS EVALUATION OF HETEROGENEOUS CATALYSED ALDOL CONDENSATION OF FURFURAL WITH ACETONE Kocík J., Horáček J., Velvarská R., Kadlec D., Kolena J., Lederer J.

389

HYDROGENATION OF C6 DIENIC COMPOUNDS USING RUTHENIUM CATALYSTS Kotova M., Karlíčková A., Vyskočilová E., Červený L.

395

LIQUID PHASE DEHYDROGENATION OF ISOPROPANOL Muntágová Ľ., Wenchich Š., Králik M., Végh Zs., Kučera M., Bíro P.

401

CATALYTIC AND THERMAL CLEAVAGE OF β-PINENE OXIDE AND MYRTENOL Patera J., Paterová I., Hladíková P., Wolfová M.

406

PREPARATION AND UTILIZATION OF Pd PD MODIFIED BIFUNCTIONAL CATALYSTS BASED ON LAYERED DOUBLE HYDROXIDES FOR 4-TERT-BUTYL-α-METHYLHYDROCINNAMIC ALDEHYDE PREPARATION Paterová I., Makovcová L., Červený L., Kovanda F.

411

PHENYLACETYLENE AS AN ACID CATALYST FOR ORGANIC REACTIONS Sekerová L., Frýbová M., Vyskočilová E., Červený L.

416

ETHANOL SEPARATION BY SOLVENT EXTRACTION USING [TDTHP][NTf2] IONIC LIQUID: ALTERNATIVES OF EXTRACTION SOLVENT REGENERATION Steltenpohl P., Graczová E.

421

Mg-Al-Zn MIXED OXIDES AS EFFECTIVE CATALYSTS IN ALDOL CONDENSATIONS Vrbková E., Varlamov A., Tišler Z., Vyskočilová E., Červený L.

427

PHARMACEUTICAL TECHNOLOGY EFFECT OF ALCOHOL ON TRAMADOL HYDROCHLORIDE RELEASE FROM CONTROLLED RELEASE FORMULATIONS CONTAINING CO-PROCESSED DRY BINDERS Myslíková K., Komersová A., Lochař V., Mužíková J.

433

TECHNOLOGY OF 4TH GENERATION PLATINUM CYTOSTATICS WITH ADAMANTYL LIGANDS Svoboda J., Mikoška M., Syslová K.

439

EFFECT OF HYDROPHILIC POLYMERS ON DISSOLUTION PROFILES OF BINARY MIXTURES Školáková T., Slámová M., Patera J., Zámostný P.

445

EFFECT OF DILUENT PARTICLE SIZE ON FLOW PROPERTIES AND HOMOGENITY OF BLENDS FOR DIRECT TABLET COMPRESSION Zámostný P., Majerová D., Bartáková M.

450

7

POLYMERS, COMPOSITES VANADYL COMPLEX WITH TETRADENTATE MACROCYCLIC LIGAND AS A DRIER FOR SOLVENT-BORNE ALKYD PAINTS Charamzová I., Honzíček J., Vinklárek J.

457

PREPARATION OF NANOSTRUCTURED SURFACES USING UV RADIATION AND THEIR ANALYSIS Knapová T., Neubertová V., Kolská Z.

462

REMOVAL OF CAESIUM FROM A SOLUTION OF BORIC ACID BY USING ZEOLITE Kůs P., Foubíková A., Skala M., Koloušek D., Kotowski J.

466

CHLORINE DIOXIDE BLEACHING OF SODA RAPESEED PULP Potůček F., Říhová M.

470

DISPLACEMENT WASHING OF KRAFT SPRUCE PULP COOKED TO LOW KAPPA NUMBER Potůček F., Rahman M. M.

476

ECO FRIENDLY POLYMER SYSTEMS BASED ON POLYVINYL ACETATE AND SACCHARIDES Puková K., Machotová J., Mikulášek P., Rückerová A.

482

ANIONIC POLYMERIZATION AND PHYSICAL PROPERTIES OF THE POLYMERS MADE FROM TRANS-ΒFARNESENE Trnka T., Pleska A., Yoo T., Henning S.K.

488

SECURE MANAGEMENT PROCESSES, ACCIDENTS PREVENTION INHERENTLY SAFER DESIGN OF A NOVEL INDUSTRIAL SCALE REACTOR FOR ALKYLPYRIDINE DERIVATIVES PRODUCTION Janošovský J., Kačmárová A., Danko M., Labovský J., Jelemenský Ľ.

492

MULTILEVEL DATA ANALYSIS IN COMPUTER AIDED HAZARD IDENTIFICATION Janošovský J., Danko M., Labovský J., Jelemenský Ľ.

497

CERIUM DIOXIDE NANOPARTICLES – ECOLOGICAL RISK ASSESSMENT Kobetičová K., Krejsová J.

503

RISK ANALYSIS BASED ON THE CRITICALITY CLASSES AND THEIR DETERMINATION USING ACCELERATING RATE CALORIMETRY Mašín J., Ferjenčík M., Šelešovský J.

506

WASTE PROCESSING, AIR AND WATER PROTECTION, TECHNOLOGIES FOR THE DECONTAMINATION OF SOILS DIRECT NO DECOMPOSITION OVER K-PROMOTED Co-Mn-Al MIXED OXIDES Bílková T., Pacultová K., Obalová L.

513

UTILIZATION SORBENT ON LIGNOCELLULOSE MATERIALS BASE FOR REMOVES HALOGENATED DYES INCREASING PARAMETER AOX FROM MODEL EFFLUENT WATER Filipi M.

517

SORPTION OF SELECTED HEAVY METALS FROM OXALIC ACID SOLUTION USING DIFFERENT TYPES OF SORBENTS Chlupáčová M., Kůs P., Parschová H.

521

8

APPLICATION OF ION EXCHANGE RESINS TO RECOVERY OF METALS FROM DECONTAMINATION SOLUTIONS Chlupáčová M., Kůs P., Parschová H.

526

MULTISPECIES AQUATIC MICROCOSM AS A TOOL FOR CHEMICAL ASSESSMENT Kobetičová K., Krejsová J.

531

CESIUM DOPED Co3O4 SPINEL FOR N2O DECOMPOSITION Michalik S., Pacultová K., Obalová L.

536

CHLORINATED AROMATICS – OCCURRENCE, USAGE AND METHODS OF DEGRADATION Pérko J, Weidlich T.

540

SAFE DISPOSAL OF METALLIC MERCURY FROM PHASED-OUT MERCURY CELLS Raschman R., Zápotocký L., Říhová M.

545

SIMULANTS OF CHEMICAL WARFARE AGENTS FOR TESTS OF THERMAL DESORPTION TECHNOLOGY Šváb M., Zápotocký L

550

REMOVAL OF ACID DYES FROM AQUEOUS EFFLUENTS BY ACTION OF IONIC LIQUIDS Weidlich T., Václavíková J., Šimek M.

557

PETROCHEMICALS AND ORGANIC TECHNOLOGY II CONTRIBUTION METHOD FOR KINETIC PARAMETERS ESTIMATION APPLIED TO THE STEAM CRACKING MODELING Karaba A., Zámostný P.

562

9

BIOTECHNOLOGY AND BIOREFINARY

10


USE OF NOVEL WASTE PRODUCTS IN THE MICROBIAL PRODUCTION OF LACTIC ACID Drahokoupil M., Paulová L., Patáková P. Department of Biotechnology, University of Chemistry and Technology, Prague, Czech Republic [email protected]

Abstract The objective of our project was to design and optimize the process of microbial production of lactic acid using an alternative, inexpensive substrate with the goal to achieve high yield and concentration of lactic acid produced by the strain Lactobacillus casei 198. Firstly, the chicken feather was investigated as a low-cost source of nitrogen. Feather was hydrolysed with sodium hydroxide at a concentration of 2, 5, 10, 15 and 20 % by weight and used as a replacement of all nitrogen sources in MRS medium. Taking the growth of biomass and the final concentration of lactic acid as the main criterions, 20% hydrolysate of chicken feather was chosen for the next experiments, where it was used not only as a source of nitrogen but also as an neutralizing agent. Using this hydrolysate caused minimal dilution of the total volume of the culture medium in the bioreactor. The final concentration of lactic acid in the medium was 48 g l-1.

Introduction Lactic acid is a chemical compound with a very wide range of use. It is naturally produced by the lactic fermentation of sugars (e.g. in milk, cheese, sauerkraut, etc.) and has been used for a long time, for example, in bakery, brewing, tanning, preparation of lemonades, non-alcoholic beverages or dyeing and finishing of textiles. In the cosmetics industry, it is used for its moisturizing effects on the skin, in the pharmacy as an easily absorbable bioactive substance carrier. It is also used in ointments, mainly because of its antiseptic properties. New utilization of lactic acid includes its use as a precursor for the synthesis of propylene glycol and acrylic polymers or also in production of environmentally friendly solvents. Last but not least, the lactic acid polymer is a suitable material for replacing traditional plastics which main precursor is petroleum1. In addition to their origin from renewable sources, other major advantages of lactic acid polymers include, for example, their relatively easy degradability. The most widespread microorganisms capable of producing lactic acid are lactic acid bacteria. These bacteria belong to an artificially formed group of microorganisms that was created on the basis of metabolic similarity, then the final product of metabolism is lactic acid at least 50 %2. Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus, Enterococcus and Bifidobacterium are included in this group. Common features of these families are facultatively anaerobic to anaerobic lifestyle, Gram-positive cell wall structure and, in most cases, inability to form a spore3. Lactic acid bacteria are ubiquitous except in extreme conditions. The main place of their occurrence is the gastrointestinal tract of animals and humans, their oral cavity and the urovaginal tract. A microorganism of the genus Lactobacillus, the strain Lactobacillus casei 198, was selected for our project. This representative was selected from a large number of strains studied during the previous research in the Laboratory of Microbial Processes at the Department of Biotechnology UCTP4. In case of this producer, the highest yield of total lactic acid or its L- isomer, which is generally more valuable than D-isomer, was found. The cost of the substrate for lactic acid production is one of the most important economic aspects of the whole process. Previously used food raw materials, such as molasses (sugar-based waste, maize starch or whey) (dairy products), are now receding because of high prices or competition with food production and therefore there are being sought cheaper substrates for lactic acid production. These are usually waste products from agriculture. In the past, for example, it has been shown that a good substrate for fermentation production of lactic acid could be soy or wheat straw hydrolysate or other lignocellulosic substrates 5. Suitable materials used for the production of lactic acid should meet the following criteria: low cost, high availability, low contaminants content, high rate of lactic acid production, high yields of the product, and the possibility of fermentation without complicated and expensive substrate treatments6.

Simulation and experiment Alkaline hydrolysis of chicken feather Chicken feather was hydrolysed with sodium hydroxide at a concentration of 2, 5, 10, 15 and 20 % by weight and used as a neutralizing agent. Feathers, 2 g were placed into 250 mL Erlenmeyer flasks with 100 mL of 2, 5, 10, 15 or 20 % by weight NaOH and incubated for 24 h at 70 °C and 130 rpm. The hydrolysis time was


11

dependent on the concentration of the hydroxide used (with increasing hydroxide concentration, consumption of time was declining) 7. Screening fermentation flasks For our experiment, screening tests were performed in flasks at first. The inoculum was prepared from 100 ml sterile MRS medium and 1 ml of cryopreserved Lb. casei 198. The flask was then placed in a thermostat heated to 37 °C for about 18 hours. The inoculum always constituted 10% of the total volume of the culture medium. 45 ml of complete sterile MRS medium was prepared, which was seeded with 5 ml of inoculum. Cultivation was carried out statically in a thermostat at 37 °C. After 24 hours, the pH of the culture medium was adjusted to pH 6.5 with the hydrolysate containing a given NaOH concentration and the necessary amount of glucose was added. Subsequently, samples were taken for HPLC and optical density of cell suspension, and the flasks were again placed to the thermostat for next 24 hours. After 48 hours, samples were taken again for the respective analyses and the whole experiment was terminated. Fermentation in bioreactor From previous results, a neutralizing solution for the production of lactic acid was chosen to be a feather hydrolysate prepared with 20% NaOH. 450 ml of complete MRS medium was prepared, which was inoculated with 50 ml of inoculum. Cultivations were carried out in two stirred LabFors laboratory reactors at 37 °C, mixing 200 rpm, without bubbling CO2. In the first reactor the pH was regulated with 20 % by weight NaOH solution and in second bioreactor the feather hydrolysate was used for keeping constant pH. After 17 hours of culturing, 20 g of 500 g l-1 sterile glucose solution was added to each reactor, to replenish carbon source. After 21 hours, the carbon source in the form of a sterile glucose solution was fed into the reactors by means of peristaltic pumps. The dilution rate was calculated to be 27 ml of glucose per hour. During the culture, samples were taken for HPLC and measuring optical density. After 36 hours of culture, the glucose stock solution was depleted and the entire experiment was terminated.

Discussion and result analysis Results for fermentation flasks Table I Results obtained in fermentation flasks

Sample

Feather

Glucose

Glucose

Lactic acid

Lactic acid

NaOH

Volume of

concentration

concentration

concentration

concentration

[%]

hydrolysate

after 24h

after 48h

after 24h

after 48h

[ml]

[g l-1]

[g l-1]

[g l-1]

[g l-1]

2

15

14.18

0

14.28

33.17

5

8

15.72

0

18.53

34.01

10

4.5

17.37

0.24

20.88

40.94

15

3.5

16.35

0.34

20.18

40.24

20

2

16.04

0.45

19.37

47.96

hydrolysate Feather hydrolysate Feather hydrolysate Feather hydrolysate Feather hydrolysate

Feather hydrolysates prepared using 20 % by weight NaOH solution seem to be the best choice for pH neutralization, since the highest concentration of lactic acid (47.96 g l-1) was achieved, and there was minimal dilution of the culture medium, see Table I. In further experiments, only these feather hydrolysates prepared using 20 % by weight NaOH were used.

12


Results for fermentation in bioreactor Table II Results obtained with 20% NaOH as a neutralizing agent 20 % by weight NaOH Time

Glucose concentration

Lactic acid concentration

[hours]

[g l-1]

[g l-1]

0

20.03

3.25

3

16.36

7.20

16

0

22.57

Table III Results obtained with chicken feather hydrolysate as a neutralizing agent Chicken feather hydrolysate Time Glucose concentration Lactic acid concentration [hours] [g l-1] [g l-1] 0 19.67 2.71 3 12.88 5.29 16 0 23.01 Table IV Summarizing results of yield and productivity of lactic acid by fermentation in bioreactors. 20 % by weight NaOH Chicken feather hydrolysate YKM/GLC in batch fermentation, g g¸-1 0.96 1.03 YKM/GLC in fed-batch fermentation, g g-1 0.64 1.05 Productivity of fermentation, g l-1 h-1 1.69 2.20 Table II and III summarize the concentrations of lactic acid and remaining glucose for batch culture. A continuous feed of carbon source in the form of a concentrated glucose solution was introduced at 16 hour. Since the glucose and neutralizing agent contribute to volumetric changes in the reactor, the total mass balance has to be performed to evaluate lactic acid production. From the total mass balance it was calculated that the total yield of lactic acid using neutralization with sodium hydroxide was 29.5 g. Using the feather hydrolysate, the total yield of lactic acid was 40.7 g. Total results summarizing the yield and productivity of lactic acid during different types of bioreactor cultivation are shown in Table IV. The results show that feather hydrolysate is a suitable neutralizing agent for the production of lactic acid. The overall course of cultivation is displayed in Figure 1 - 3.


13

NaOH

20

Feather

Concentration, g l-1

Batch fermentation

Fad-batch fermentation

15

10

5

0

0

5

10

15

20

Time, hours Figure 1. Concentration profile of the consumption of glucose during fermentation in bioreactor.

40

NaOH

35

Feather

30

Batch fermentation

Fad-batch fermentation

Mass, g

25

20 15 10 5 0

0

5

10

15

20

25

Time, hours Figure 2. Concentration profile of the lactic acid production during fermentation in bioreactor. In Figures 1 and 2, we can see the course of glucose consumption and lactic acid production during batch culture and then during the feeding with concentrated glucose solution. From the data obtained, it is obvious that the course of lactic acid production using both neutralizing agents was very similar, but at the end of the cultivation, the production of lactic acid using the hydrolysate of the feathers was higher than that with the NaOH solution alone. This phenomenon could be caused by depletion of nitrogen source in case of neutralization with NaOH while using alkaline feather hydrolysate prevented this depletion.

14


18

16

Concetration of glucose, g l-1

45

pH Glucose

40

Feather hydrolysate

35

14

30

12

25

10 20

8

15

6

10

4

5

2 0

ph Value of hydrolysate, ml

20

0

5

10

15

20

0

Time, hours Figure 3. Comparison of glucose consumption, pH change, and addition of feather hydrolysate as a neutralizing agent during fermentation in a bioreactor. Thanks to our online measurement of neutralizing agents and online pH regulation, it is easy to estimate the consumption or depletion of carbon source in the form of glucose as shown in Figure 3. Thanks to this knowledge, carbon can be replenished to the culture medium in time to prevent substrate depletion, and this may result in better production of lactic acid.

Conclusion From the data obtained in screening experiments in the flasks, it can be concluded that the optimal concentration of NaOH for preparation of feather hydrolysate and neutralization is 20 % by weight. Using this hydrolysate as a neutralizing agent in the production of lactic acid in the bioreactor, the obtained data suggest that feather hydrolysate appears to be a better neutralizing agent for the production of lactic acid compared to simple use of NaOH. With online measurement of consumption of neutralizing agent and on-line pH regulation, it was possible to predict the exhaustion of the carbon source in time to prevent substrate depletion during fermentation.

Acknowledgement This work was preformed thanks to financial support of the project BIORAF No. TE01020080 of the Technological Agency of the Czech Republic, project LTACH-17006 of the Inter-Action Inter-Excellence program of Ministry of Education, Youth and Sport of the Czech Republic and to Financial support from specific university research (MSMT No 20-SVV/2017).

References 1. 2. 3. 4. 5. 6. 7.

Martinez F. A. C., Balciunas E. M., Salgado J. M., Gonzalez J. M. D., Converti A., Oliveira R. P. S.: Trends Food Sci. Tech. 30, 70 (2013). Robinson R. K.: Encyclopedia of Food Microbiology. Academic Press, New York 2000. Vos P., Garrity G., Jones D., Krieg N. R., Ludwig W., Rainey F. A., Schleifer K. H., Whitman W.: Bergey´s Manual® of Systematic Bacteriology. Springer-Verlag, New York 2009. Chmelík J.: Production of lactic acid as a precursor for manufacture of bioplastics. UCTP, Prague 2016. Wang J., Wang Q., Xu Z., Zhang W., Xiang J.: J Microbiol. Biotechn. 25, 26 (2015). Wee Y., Kim J., Ryu H.: Food Technol. Biotech. 44, 163 (2006). Stiborova H., Branska B., Vesela T., Lovecka P., Stranska M., Hajslova J., Jiru M., Patakova P., Demnerova K.: J. Chem. Technol. Biotechnol. 96, 1629 (2016).


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STILBENOID DETERMINATION IN MUST, WINE AND WINERY WASTE SAMPLES Gharwalova L., Koukalova M., Cejkova A., Kolouchova I., Masak M. Department of Biotechnology, UCT, Technicka Street 5, 16628 Prague [email protected]

Abstract Resveratrol, 3, 5, 4‘- trihydroxystilbene, is a natural polyphenolic compound that exhibits a wide range of biological activities, especially antimicrobial and antioxidant. So far, the most important natural source of resveratrol for humans is wine from blue grapes. Significant amounts of resveratrol and its glycoside polydatin are also present in the wine waste, which is formed during the production of wine (stems, marc, lees), and during green works in the vineyard (green shoots, leaves, canes). Antioxidants contained in this waste could be further used in pharmaceutical or food industry. The aim of this study was to determine the content of stilbenoids in wine and wine waste from blue and white Vitis vinifera varieties. Stilbenoids were extracted with 40% ethanol. Stilbenoids in the samples were determined using HPLC with a DAD detector. The extracts with high concentration of antioxidants could be further tested on selected opportunistically pathogenic microorganisms in suspension and biofilm populations.

Introduction Grapevine is one of the most commonly grown fruit crops in the world. The grape is traditionally promoted as healthy food based on the high content of polyphenolic antioxidants present mostly on the skin and seeds of the berry. Most of its worldwide production is used in winemaking. Waste and by- products of wine making are produced during the growing of grapes as well as winemaking. Winemaking produces waste in two significant periods: harvest and postharvest. The amount of solid waste generated during harvest is greatly higher than during post- harvest1. The wine industry produces yearly millions of tons of waste that might represent and ecological and economical issue with a direct impact on the local environment. Since 2009, the European Union strongly encourages winemakers to manage their waste more sustainably in the norm EN 491/2009. Their reuse in agriculture, as well as for the production of novel products, is becoming the subject of numerous studies. The waste products of winemaking could represent a cheap source for the extraction of natural antioxidants, providing the opportunity of increasing the value of such waste materials. These could be further reused in food, cosmetic and pharmaceutical industry2. Their further application requires an evaluation of the polyphenolic composition. Phenolic compounds are the most widespread plant secondary metabolites. They are natural antioxidants, commonly found in wine, must and by- products of winemaking industry3, 4. The largest group of phenolic compounds is formed by flavonoids. These include flavanols, flavan-3-ols or anthocyanins. Stilbenoids, another important group of phenolic compounds, are secondary metabolites that are produced by plants in response to stressful conditions (mechanical damage, UV) or in response to fungal infections. A major source of these compounds is Vitis vinifera. The most significant stilbenes are considered to be resveratrol and its glycosidic form, polydatin5. Resveratrol (3, 4’, 5- trihydroxystilbene) exists in two isomeric forms: cis- and trans-, whereas the transform prevails in plants. Resveratrol is sold as a dietary supplement thanks to its biological and antioxidant properties6. The most significant sources of resveratrol for humans are wine and must. The present work aimed to evaluate the stilbenoid content of various vine waste material (stems, shoots, canes, lees) as well as wine and must from two Czech vineyards. These findings could help to determine whether Czech must and young wine could present a sufficient source of resveratrol for the consumers and also whether the waste from winemaking could be further utilized as an inexpensive source of stilbenoids.

Experiment Chemicals Ethanol 96% (v/v) p.a. (Penta, Czech Republic) Ethanol 96% (v/v) denatured, Acetonitrile for HPLC super gradient (VWR Chemicals, USA), trans-resveratrol, ≥ 99% GC (Sigma Aldrich, Germany), trans-polydatin ≥ 95% HPLC (Sigma-Aldrich, Germany) pinosylvin ≥ 97% HPLC (Sigma-Aldrich, Germany) pterostilbene (Cayman Chemical Company, USA). Sample preparation

16


Winery waste, must and wine from processing of white and blue varieties of Vitis vinifera were analysed. These samples were obtained from either Saint Claire Vineyard in Troja or Vineyard Grebovka, both situated in Prague, Czech Republic. Samples of stems and green shoots were grinded to the size of 1-3 mm; samples of canes were first dried to constant mass and then grinded; lees were centrifuged before the extraction. Green shoots were obtained in August 2016; stems, must and young wine in September/October 2016, canes and wine in January/February 2017 (harvest year of the wine samples was 2016). A solid- liquid extraction was applied to obtain phenols. The ratio was determined based on the nature of the matrix. The optimized conditions for phenolic extraction were 40% (v/v) ethanol for 24 hours at room temperature and 96% (v/v) ethanol in the case of lees. The determination of dry matter of stems, canes and shoots was carried out at 105°C to constant mass. The cane and stem samples were prepared by drying in a laboratory oven (40 °C, to constant weight) and subsequent milling. A Muller- Thurgau cane sample (Grebovka) was subjected to three types of extract preparations: drying and subsequent milling to the size of 1-3 mm; (22°C); milling to the size of 1-3 mm without previous drying; cutting to the size of 0.5-1.0 cm by garden clippers. HPLC HPLC separation and quantification was realized with an 1100 series HPLC system with a DAD detector (Agilent, USA) and a reversed- phase 125x 4 mm Watrex, Nucleosil 120-C18 column. Samples of the harvest years 2015 and 2016 were analysed after filtration through cellulose acetate membrane filters (0.45 μm, Sartorius Stedim Biotech, Germany). The calibration curve was prepared from pure standards (concentrations between 1-10 mg l– 1 ) of trans- resveratrol, trans- polydatin, pterostilbene and pinosylvin, which were dissolved in 40 % (v/v) ethanol. These were kept in darkness at the temperature of 4°C. Concentrations of trans- resveratrol, trans- polydatin, pterostilbene and pinosylvin were determined by reversephase HPLC using a gradient of acetonitrile and demineralized water. The proportion of the acetonitrile in the mobile phase was increased during the analysis time from 10% up to 95 % 7, see Tab. I. Table I The conditions and the composition of the mobile phase gradient, A- acetonitrile, B- demineralized water Time (min) A B 0 10 90 25°C 5 20 80 Sample injection: 20 μl 35 50 50 Flow rate: 1 ml min–1 40 95 5 Stop time: 46 min 45 95 5 Post time: 5 min The compounds were identified using a photodiode array detector (DAD), a spectrophotometric detector type with adjustable wavelength in the UV/VIS region. The wavelength of 306 nm was evaluated as the optimum wavelength for detection of resveratrol and its analogues, as it is in near the absorption maxima of these compounds8.

Discussion and results analysis The quantitative analysis of Vitis vinifera samples provided by Czech vineyards, Vineyard Grebovka and Saint Claire Vineyard in Troja, showed that the most significant sources of stilbenoids from the tested waste samples were canes from winter pruning (Fig. 1). These are one-year old shoots that ensure the flow of assimilates from the places of their synthesis (leaves) to their places of use (berries). The transport of assimilates together with stilbenoids occurs both in the phloem and xylem, where the accumulation of these substances was already proved9. The resveratrol content in green shoots ranged from 0 to 2.38 mg kg–1 of dry matter (DM), while the polydatin content in shoots was higher (9.41-36.36 mg kg–1 DM). The resveratrol and polydatin content in stems from vineyard Troja were in the range of 4.90-7.04 mg kg–1 DM and 1.78-2.73 mg kg–1 DM, respectively (Tab. II). Similar results for stilbenoid content in green shoots were obtained by Lachman et al. 10. The stilbenoid content depends on the extraction method used. Farhadi et al. 11 reported that trans-resveratrol content in cane samples ranged from 0.9 to 2.6 mg kg–1 DM, using methanolic HCl colution as an extracting agent. Also a study by Soural et al.12 proved that the amount of extracted stilbenoids depends on the choice of the extraction procedure.


17

Figure 1. Stilbenoid determination in waste from Vitis vinifera Pinot Gris (T- vineyard Troja, G- vineyard Grebovka) Table II Stilbenoid content in waste samples (shoots, stems, lees) Resveratrol Polydatin Sample type Variety Vineyard (mg kg–1 DM) (mg kg–1 DM) Shoots Muller- Thurgau Grebovka 0.00 19.11 Shoots Muller- Thurgau Troja 0.00 23.38 Shoots Pinot Gris Grebovka 2.38 24.23 Shoots Pinot Gris Troja 1.75 28.96 Shoots Pinot Noir Grebovka 2.22 30.25 Shoots Pinot Noir Troja 1.88 36.36 Stems Muller- Thurgau Troja 2.73 5.46 Stems Pinot Gris Troja 1.78 4.90 Stems Pinot Noir Troja 2.70 7.04 Lees Pinot Gris Grebovka 2.48 5.27 The stilbenoid content was higher in cane extracts when the canes where dried before being milled. In MullerThurgau extracts, the concentration of trans- resveratrol was 62.8 mg kg–1 DM if the canes were cut by garden clippers; 95.4 mg kg–1 DM if the canes were only milled without previous drying; 132.3 mg kg–1 of dry matter if the canes were dried before milling. Since the concentrations of trans- polydatin in the dried and milled canes were much lower (4.92 mg kg–1 DM) than in canes which were cut or just milled (9.74 and 9.03 mg kg–1 DM, respectively), it seems possible that the drying process caused the glucose molecule to be cleaved off. Similarly, a higher trans- resveratrol content in leaves was obtained after drying in an oven by Lachman et al. 10. Trans- resveratrol and trans-polydatin were detected in all the examined samples of canes; analytes pinosylvine and pterostilbene were not detected in any of the samples. By comparing the results for cane samples of each of the vineyard, it is possible to track the relation between stilbenoids in the specific varieties (Muller- Thurgau, Pinot Gris) and the microclimate of the vineyard (Fig. 2). However, these relations are not significant enough to affect the reuse of the waste as a source of phenolic compounds. The stilbenoid content in Vitis vinifera depends on the humidity of the soil, UV light or fungal infections13, 14. Several studies have proved that the phenolic content in the plant and wine depends on the variety15, 16 and growing location10. The stilbene contents could be possibly further increased in postharvest time exposure to UV light, ozone and other abiotic stresses. The highest trans-resveratrol content was in Gewürztraminer canes from Troja (153.79 mg kg–1 DM). Polydatin was the more prevalent stilbenoid in canes from blue varieties (except for Pinot Noir from Grebovka vineyard), while resveratrol was more prevalent in white varieties (except for Muller- Thurgau from Saint Claire Vineyard in Troja). These numbers suggest that canes are an inexpensive source of antioxidant and antimicrobial substances.

18


Furthermore, their reuse is easier than that of other tested waste samples due to their easy storability given by its high percentage of dry matter (56-81%).

Figure 2. Stilbenoid determination in cane samples from winter pruning (T- vineyard Troja, G- vineyard Grebovka) The results for stilbenoid determination in must and wine proved that the concentrations of resveratrol and its glycoside rise with higher ethanol content (Fig. 3). As expected, the stilbenoid content was higher in red wines than in white and rose wines. During white the production of white wine, the skins and seeds of the grapes are removed before fermentation. Red wine, on the other hand, undergoes a maceration period during which the polyphenols are partially extracted. The elevated levels of resveratrol during the ethanol fermentation are in accordance with its physico-chemical properties, since it is more soluble in an alcoholic solution17. The amounts of stilbenoids in must from the blue variety Pinot Noir were comparable to the stilbenoid content in the white wine Pinot Gris and are therefore suitable as a natural non- alcoholic source of polyphenolic antioxidants.

Figure 3. Stilbenoid determination in wine and must samples (Pinot Noir and Pinot Gris) from vineyard Grebovka


19

Conclusion Waste generated in the winemaking process, especially canes from winter pruning, could be further used as a valuable source of stilbenes. A higher trans- resveratrol content can be achieved by drying canes in an oven. The drying process would be also useful in the preservation process. Must from blue Vitis vinifera varieties could be a significant non- alcoholic source of stilbenoids in the human diet.

Acknowledgement Financial support from specific university research (MSMT No 20-SVV/2017).

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

17.

Oliveira M., Queda C.,Duarte E.: Water Sci Technol, 60, 1217 (2009). Oliveira M.,Duarte E.: Front Environ Sci En, 10, 168 (2016). Arvanitoyannis I.S., Ladas D.,Mavromatis A.: Int J Food Sci Technol, 41, 475 (2006). Santamaría B., Salazar G., Beltrán S.,Cabezas J.L.: Desalination, 148, 103 (2002). Waterhouse A.L.: Ann NY Acad Sci, 957, 21 (2002). Smidrkal J., Filip V., Melzoch K., Hanzlikova I., Buckiova D.,Krisa B.: Chem Listy, 95, 602 (2001). Cho H.S., Lee J.H., Ryu S.Y., Joo S.W., Cho M.H.,Lee J.: J Agric Food Chem, 61, 7120 (2013). Kolouchová-Hanzlı ́ková I., Melzoch K., Filip V.r.,Šmidrkal J.: Food Chem, 87, 151 (2004). Bruno G.,Sparapano L.: Physiol Mol Plant Path, 69, 195 (2006). Lachman J., Kotíková Z., Hejtmánková A., Pivec V., Pšeničnaja O., Šulc M., Střalková R.,Dědina M.: Hort Sci, 43, 25 (2016). Farhadi K., Esmaeilzadeh F., Hatami M., Forough M.,Molaie R.: Food Chem, 199, 847 (2016). Soural I., Vrchotová N., Tříska J., Balík J., Horník Š., Cuřínová P.,Sýkora J.: Molecules, 20, 6093 (2015). Melzoch K., Hanzlíková I., Filip V., Buckiová D.,Šmidrkal J.: Agric conspec sci, 66, 53 (2001). Adrian M., Jeandet P., Douillet-Breuil A.C., Tesson L.,Bessis R.: J Agric Food Chem, 48, 6103 (2000). Cantos E., Espín J.C.,Tomás-Barberán F.A.: J Agric Food Chem, 50, 5691 (2002). Ortega T., De La Hera E., Carretero M.E., Gómez-Serranillos P., Naval M.V., Villar A.M., Prodanov M., Vacas V., Arroyo T., Hernández T.,Estrella I.: Eur Food Res Technol, 227, 1641 (2008). Pezet R.,Cuenat P.: Am J Enol Viticult, 47, 287 (1996).

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11. 12. 13. 14. 15. 16.

KINETICS OF KERATIN ACIDIC HYDROLYSIS IN BATCH AUTOCLAVE Hanika J. 1, Kastanek P.2, Rouskova M.1, Sabata S.1, Solcova O.1 Institute of Chemical Process Fundamentals of the CAS, Rozvojová 135, 165 02 Prague 6, Czech Republic Ecofuel Labs., Ocelarská 9, 190 00 Prague 9, Czech Republic [email protected]

1 2

Abstract The contribution brings novel data on the non-traditional process presented during previous ICCT 2015 conference1. According to the patent application2 the hydrolysis of waste chicken feathers was performed in a reaction mixture containing water and a soluble acid catalyst with the pKa value lower than 4, in a stirred batch reactor for 0.5 to 10 hours, in the temperature range from 90 to 130°C and at the pertinent vapour pressure of reaction mixture. Kinetics of preparing a mixture of proteins and amino acids by hydrolysis of a waste material containing chicken feathers was investigated in this study.

Introduction Hydrolytic splitting of peptide bond in a keratin protein structure in waste feathers results in a mixture of low molecular weight peptides and amino acids. This aqueous solution can be easily mixed with compost or added as a component in a plant’s dressing. Thus, it enables recycling of biogenic elements in an agricultural process. The hydrolysate has a promising use for plant’s protective sprays against stress, caused by e.g. increased intensity of sun exposure, lack of moisture, etc. Proteins hydrolysis is accelerated in an acid or alkaline environment and by elevated temperature. Use of inorganic or mineral acids is disadvantageous policy, because resulting hydrolysate prior its application requires a neutral adjustment of pH value. Hydrolysis can be processed in the presence of organic acids and/or using carbon dioxide dissolving in water and creating the necessary slight acidic environment by their dissociation. Its concentration in water can be increased using a higher pressure in the reactor. Simultaneous proteins and fat hydrolysis of chicken feathers were carried out at increased temperature and in the presence of carbon dioxide (partial pressure 1 - 2 MPa) in this study. Bench scale tests were performed using a mixed autoclave (volume 2.5 lt., typical reaction time 5 hr). This procedure was described in the patent application2. Knowledge of kinetic aspects of the feather hydrolysis and process parameters operation window are inevitable for a process scale up.

Experiment Low molecular proteins and amino-acids formation by keratin hydrolysis was investigated in this study. Experiments were carried out using fried chicken feathers from Rabbit Co. Trhový Štěpánov. This waste material (typically 300 ml, weight 75 g) was put together with 1 litter tap water into pressure autoclaves, volume 2.5 and 2.0 litters, respectively. In some tests dried and cut feather was used, too. In series of experiments the effect of hydrolysis time, temperature (or corresponding reaction mixture vapour pressure) was investigated. Starting the kinetic tests an inert gas (preferentially carbon dioxide) was introduced to the reactor in order to minimize undesired transformation of batch components by oxidation. Reaction time between 3 to 7 hours was applied (including heating up and cooling down of the reactor, i.e. 2 hours). Reaction temperature was maintained at 130 oC which corresponded to pressure 16.9 bars. The starting temperature for keratin and feathers fat hydrolysis was supposed to be 110 oC. At the end of the test after a reactor cooling down the reaction product was separated by filtration to liquid hydrolysate (pH of the hydrolysate was 6.4 in all cases) and solid waste which was dried to investigate a mass balance equilibrium of hydrolysis. The mass balance of the individual tests was nearly identical. The mass deficit was undoubtedly caused by handling losses as emptying the autoclave, separation of reaction products, filtration and drying of the solid waste. Liquid filtrate was analysed using HPLC/MS and GC/MS methods. The total content of soluble peptides of low molecular weight and amino acids distribution were determined in the hydrolysate for all samples. Several samples were used for screening of feather fat hydrolysis products like free


21

fatty acids, mono-, di- and tri-glycerides of different fatty acids as well. The effect of reaction time on hydrolysate colour was tested using IR-FT method. In some experiments the dried waste was used for the 2 nd step hydrolysis.

Kinetics of feathers hydrolysis Time function of proteins concentration in hydrolysate of frozen dry feathers is illustrated in Figure 1. The first experiment corresponding to reaction time zero was aimed to find a reaction conversion during the first step of operation. In this starting test the reaction mixture was only heated up to temperature 110 oC and then cooled down to ambient temperature. The subsequent tests were done at the same temperature for different reaction time. The results showed that the reaction time of 3 h was sufficient to achieve the equilibrium protein’s concentration in the hydrolysate. The equilibrium is probably limited by their solubility in reaction mixture, containing many other components – amino-acids, free fat acids and fatty acid glycerol’s.

Figure 1. Proteins concentration versus reaction time of hydrolysis at 130 oC and 1.69 MPa Formation of the soluble proteins and amino acids in the hydrolysate proceeds just in begin of the keratin hydrolysis, as well. The time dependence of the total content of amino acids is shown in Figure 2. It is interesting that during hydrolysis of dry feathers occurs a moderate decrease in summa of amino acids content. This fact can be associated with a parallel thermal decomposition some of them.

Figure 2. Summa of amino-acids concentration versus time of hydrolysis at 130 oC and 1.69 MPa

22


On the other hand, concentration of aspartic acids in the hydrolysate slightly increases to its limited value, see Figure 3.

Figure 3. Aspartic acid concentration in hydrolysate versus reaction time at 130 oC and 1.69 MPa

Distribution of the individual amino acids in hydrolysates prepared during various reaction times is shown in Figure 4. In contrast to the increase in content of the aspartic acid hydrolysate it was possible to identify more dominant folder in the first phase hydrolysis – bioconversion glutamic acid and arginine. It's unclear why a large number of these two amino acids is created in autoclave by simply heating at temperature 110 oC only with subsequent cooling (i.e. short time of hydrolysis at low temperature).

Figure 4. Representation of amino acids in the hydrolysate, depending on reaction time (0 to 4 h)

According to the representation of the low molecular weight proteins in the hydrolysate the optimum reaction time is equal approx. 3 h, while the content of amino acid reaches the equilibrium values already in the first stage of the hydrolysis. After 1 h hydrolysis their summa concentration in the hydrolysate in the course of decomposition reactions gradually decreases.


23

It cannot be excluded that the achievement of a steady-state representation of the amino acids in the hydrolysate can be related to the limited solubility of the individual amino acids. The reason consists in a complex hydrolysate composition. The higher reaction conversion of keratin is reached with increasing reaction time. The intensity of a yellow discoloration of the hydrolysate was observed in this case. Figure 5 brings measured UV-VIS-FT spectra of hydrolysates (reaction time from 7 h -top to 3 h -bottom) after their filtration. This simple analytical method can be recommended as a reaction product quality test using absorbance value at light wave 350 nm as an example. Further, it should be stressed that very good absorbance of hydrolysates predetermine their application as anti-stress agent for plants controlling sun light intensity.

Reaction time from 3 to 7 h

Figure 5. Effect of reaction time on absorbance spectrum of hydrolyzates (UV-VIS/FT)

Products of feather fat hydrolysis As was mentioned above, parallel reaction to proteins transformation is feather fat hydrolysis which takes place in the reaction system as well. The following Figure 6 shows the relative representation of free fatty acids, monoacylglycerols, triglyceroles of fatty acids and the total content of the following components. Two different autoclaves volume 2.0 and 2.5 litres were used for these experiments. A very good reproducibility of the experiments was stated. It's worth noting a fact that the reaction product did not contain any diacylglycerols and only a tiny amount of monoacylglycerols. The presence of free fatty acids (FFA) as a good surfactant has a positive effect on hydrolysate application in agriculture as spraying agent of plants. A surface active agent supports a very good wettability of plant’s sheets. This effect, together with the sun light intensity prevention (see above), is appreciate very much as well.

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Figure 6. comparison of the concentrations of lipids in hydrolysates of hydrolysates from autoclaves and 2.0 2.5 L capacity

Conclusion Recycling economy is a strong imperative of the current mankind epoch and a focus on a bio-element’s recycling should stay in the first line. It was shown that the waste chicken feathers from agriculture and food industry represent very good raw material not only for intensification of this segment of economy but also for synthesis of chemical specialties, representing by amino-acids and low molecular proteins. The next steps in research and development of the chicken feather hydrolysis should be focussed on the process scale up and final products formulations as well.

Acknowledgement Financial support from the Technology Agency of the Czech Republic under the Competence Centre BIORAF (project No. TE01020080) and Strategy AV21, Foods for the future is very much acknowledged. Team of Department of Food Analysis and Nutrition, University of Chemistry and Technology Prague is greatly acknowledged for sample analysis.

References 1. Hanika J., Šolcová O., Kaštánek P.: Pressure hydrolysis of protein in waste of chicken cartilage and feathers in the presence of carbon dioxide, Proc. 3rd Internat. Conf. Chem. Technol. ICCT, Mikulov, April 13-15, 2015, pp. 435-438. 2. Hanika J., Šolcová O., Hajšlová J., Zachariášová M., Jírů M., Kaštánek P., Bízková Z., Hrstka Z., Fulín T.: A method of preparing a mixture of proteins and amino acids with a predominant content of aspartic acid, CZ Pat. 306431 (2016), PV-2015629.


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TERTIARY STRUCTURE PREDICTION OF POTENTIAL EFFLUX-PUMP PROTEIN IN CLOSTRIDIUM BEIJERINCKII NRRL B-598 Koscova P.1, Sedlar K.1, Kupkova K.1, Kolek J.2, Patakova P.2, Provaznik I.1 Department of Biomedical Engineering, Brno University of Technology, Brno, Czech Republic Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic [email protected]

1 2

Abstract The fields of computational biochemistry and structural biology are growing rapidly. With various computational tools the whole workflow for in silico modeling of three-dimensional protein structures can be compiled. Here, we present a method for tertiary structure prediction for arcB1, a putative efflux pump gene, recently identified in the genomic sequence of Clostridium beijerinckii NRRL B-598. The strain C. beijerinckii NRRL B-598, formerly misidentified as C. pasteurianum, is an oxygen tolerant heterofermentative anaerobe with the ability of acetone-butanol fermentation, with butanol as the main product. Unfortunately, the mechanism of butanol efflux remains unknown. The translated AcrB1 protein sequence had a high similarity to the already described efflux-pump of Escherichia coli. To study these sequences in a structural context, we constructed a homology model for C. beijerinckii NRRL B-598 and compared it with the corresponding experimentally determined structure from E. coli. The predicted protein model helps us to understand its function and confirm that acrB1 is a gene for nonspecific efflux pump — a potential pump capable of butanol efflux.

Introduction In order to determine the function of a protein, we should undoubtedly consider its tertiary structure — the atom arrangement along with the covalent bonds and non-covalent interactions stabilizing the molecule structure1. The three-dimensional shape of the protein definitely corresponds to its amino acids chains. However, two proteins with different primary structures can have the same function, and the tertiary structure is significantly more evolutionarily conserved than the primary one2. Many protein structures have been already determined by different experimental methods: X-ray crystallography, NMR spectroscopy or electron microscopy3. Nowadays, there are more than 125,000 available structures in the PDB database (Protein Data Bank)4 and the number is still increasing. However, predicting the shape of a protein experimentally is very difficult, expensive, and time-consuming. If an experimentally determined structure is not available or the proteins are not amenable to these techniques, computational methods can provide us with a useful model5. Unfortunately, the task of predicting protein tertiary structure is not simple. There are several methods for predicting the tertiary and quaternary structure of a protein from its primary sequence: homology (comparative) modeling, threading, ab initio methods, protein-protein docking, etc6. These methods can be separated into two categories: (i) template-based methods (comparative modeling, threading), which depend on the available protein structures, and (ii) template-free methods, also called ab initio or de novo methods, which are useful for prediction of protein structures without any template7. There is a variety of computer programs and web servers for predicting the structure of proteins8. For prediction of the tertiary structure of arcB1, we used a putative efflux pump gene of bacterial strain Clostridium beijerinckii NRRL B-598, formerly misidentified as C. pasteurianum9 — an oxygen tolerant heterofermentative anaerobe. C. beijerinckii NRRL B-598 is a non-type, non-pathogenic, spore-forming, mesophilic, rod-shaped bacterium and its complete genome is available in the National Center for Biotechnology Information (NCBI) GenBank database10 (https://www.ncbi.nlm.nih.gov/genbank/) under accession number CP011966.2. Pilot studies of C. beijerinckii NRRL B-598 imply its great biotechnological potential for acetone-butanol fermentation, with production of butanol as the main product11. In previous studies, the acrB1 gene of C. beijerinckii NRRL B-598 has been identified to be a potential nonspecific efflux pump — a pump capable of butanol efflux. Such kinds of pumps could be utilized to increase butanol production while lowering butanol toxicity for cells. The translated AcrB1 protein sequence had shown a high similarity to the already described efflux-pump protein of Escherichia coli. Therefore, a molecular model of the potential protein responsible for butanol efflux has been designed through homology modeling12 with an objective to confirm its role.

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Materials and methods Homology modeling The translated protein sequence of multidrug transporter AcrB1 from C. beijerinckii NRRL B-598 (GenBank: ALB45939.1) was obtained from NCBI Protein database (https://www.ncbi.nlm.nih.gov/protein) in FASTA format. The protein sequence has been translated from annotated coding region in the NCBI GenBank database (CDS Region in Nucleotide: CP011966.2, 3346823-3349948). In the study presented in this paper, we used Molecular Operating Environment (MOE)13 software, version 2015.10, available from the Chemical Computing Group (CCG; www.chemcomp.com) for the homology modeling. This research tool contains multiple powerful applications for protein structure modeling purposes. The whole modeling process consists of a homology search, building a homology model, and final evaluation of the obtained homology model. In the first step we searched for the template structure using the MOE PDB Search application and a generalized version of the FASTA methodology14. Next, we refined the alignment between sequence and the template using the MOE Align application, and then built a homology model using the MOE Homology application. Finally, we checked the geometry of the model using the MOE Protein Geometry application and compared the Ramachandran diagram15, serving as a simple but important indicator of the three-dimensional structure quality. Comparison to protein from E. coli After we determined the tertiary structure of multidrug transporter AcrB1 from C. beijerinckii NRRL B-598, we compared the homology model with the experimentally obtained structure by X-ray crystallography of the protein from E. coli, which showed the closest identity to our protein. We used the MOE Superpose application for this purpose and identified conserved residues in both structures.

Results and discussion Homology search Homology PDB strategy including HMMER calculations (evaluation against the Hidden Markov Model for the potentially matching family) resulted in five reported hits indicating protein families that were suitable templates for homology modeling (Table I). Table I Hit protein families reported by MOE PDB Search application. E values stands for expectation value, EHMMER values for HMMER E values. Lower values correspond to better scores. PDB Code 3NE5 4U8V 4U8V 4R86 3AQP

Description Cation efflux system protein CusA Multidrug efflux pump subunit AcrB Multidrug efflux pump subunit AcrB RND family aminoglycoside/multidrug efflux pump Probable SECDF protein-export membrane protein

E 8.3 x 10-61 8.0 x 10-45 8.0 x 10-45 2.1 x 10-9 3.0 x 10-1

EHMMER 1.8 x 10-122 2.9 x 10-30 1.3 x 10-025 9.6 x 10-014 5.2 x 10-005

As a template structure we selected the top homologous protein family with the highest similarity score to the target sequence – the family is represented by cation efflux system protein CusA from E. coli. In total, the family included six protein chains (PDB 3NE5.A, PDB 4K0J.A, UniProt C1AA57, UniProt A0A068NLB8, UniProt E4TBG0, UniProt Q0F3G6). Building a homology model We compared the six found protein chains using the sequence identity matrix, which shows a similar identity in all the chains to the target sequence, as can be seen in Table II. For further analysis we selected 3NE5 as a template structure (Cation efflux system protein CusA) with known protein structures available in the PDB database.


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Table II The sequence identity matrix of all protein chains from the top homologous protein family to the target sequence. The percentage sequence identity shows the similarity of all compared chains.

In total ten homology models were calculated in the Amber10:EHT forcefield with a Reaction Field (R-Field) treatment of solvation electrostatics. A calculated refined final model with its specifications is shown below (Figure 1). Model specifications Contact Energy Packing Score GB/VI U Esol Eele Evdw Ebond Atom Clashes BB Bond Outliers BB Angle Outliers BB Torsion Outliers Rotamer Outliers

-2009.3264 2.4194 -76859.2188 16718.7402 -16343.5664 -41796.9766 1567.35144 56947.3477 261 342 845 753 28

Figure 1. The final homology model of AcrB1 from C. beijerinckii NRRL B-598 after its refinement with its specifications. Evaluating the Homology Model We have investigated the geometric quality of the refined model using a Ramachandran diagram, with results shown in Figure 2. In this picture, a model structure is compared to the protein structure in E. coli. Although the calculations are not ideal, most of the core residues are in the permitted zone of plots. Obviously, both diagrams show similar patterns.

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A)

B)

Figure 2. Phi-Psi plot. (A) Homology model. (B) Efflux complex from E. coli (3NE5). Comparing the Homology Model with the X-ray Structure After evaluation of the homology model by a Ramachandran diagram, we also performed the comparison of the predicted homology model with the experimentally (X-ray) determined structure of AcrB from E. coli (3NE5). The result is visualized in Figure 3 and shows that the homology model is very close to the experimentally resolved structure at most of the residues.

Figure 3. Comparison of the homology model (grey) with the X-ray Structure of AcrB from E. coli (3NE5) (black).

Conclusion The study of protein size, shape, topology, composition, and important bonding connections is necessary to understand a protein function. A novel gene arcB1 was identified in the genomic sequence of Clostridium beijerinckii NRRL B-598. In order to understand its function and therefore its potential use in biorefinery, we constructed a model of the tertiary structure for the final protein. The protein shows a high similarity to the efflux-pump gene of Escherichia coli. We used this fact for construction of a comparative model for C. beijerinckii NRRL B-598 to analyze the sequences in the structural context. For this pilot modeling study, we


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used protein-modeling tools available from the MOE software. After the tertiary structure was determined, we compared the predicted protein structure to the experimentally determined E. coli structure of AcrB protein obtained from PDB (3NE5). This analysis helped us to identify conserved residues in both structures and the predictions showed comparability to E. coli efflux pump proteins. We confirmed the similarity of AcrB1 protein to C. beijerinckii NRRL B-598 and E. coli based on predicted structural features from the amino acid sequences by comparing their Ramachandran plots. In order to evaluate the quality of our model, we will perform additional evaluations with different programs. The tertiary structure model is a rich source of information that enables us to build up a picture of the probable conformational properties and our findings and experiments will be very useful for our future research.

Acknowledgement This work has been supported by grant project GACR 17-00551S.

References 1. 2. 3. 4. 5. 6.

12. 13. 14. 15.

Bettelheim F. A.: Introduction to organic and biochemistry (2011). Illergård K., Ardell D. H., Elofsson A.: Proteins Struct. Funct. Bioinforma. 77, 499 (2009). Berman H. M.: Nucleic Acids Res. 28, 235 (2000). RCSB PDB. Yearly Growth of Total Structures. Xu Y., Xu D., Liang J.: Computational Methods for Protein Structure Prediction and Modeling 2 (2006). Oliva B., Cai W., Planas-Iglesias J., Bonet J., Manuel A., Feliu E., Gursoy A.: Structural Bioinformatics of Proteins (2012). Fiser A.: Computational Biology, 673, 73 (2010). Eswar N.: Nucleic Acids Res. 31, 3375 (2003). Sedlar K., Kolek J., Provaznik I., Patakova P.: J. Biotechnol. 244, 1-3(2017). Clark K., Karsch-Mizrachi I., Lipman D. J., Ostell J., Sayers E. W.: GenBank. Nucleic Acids Res. 44, D67–D72 (2016). Lipovsky J., Patakova P., Paulova L., Pokorny T., Rychtera M., Melzoch K.: Fuel Process. Technol. 144, 139– 144 (2016). Bourne P. E., Weissig H.: Structural bioinformatics. (2003). Molecular Operating Environment (MOE), 2015.10. Pearson W. R.: Methods in Enzymology. 266, 227–258 (1996). Ramachandran G. N., Ramakrishnan C., Sasisekharan V.: J. Mol. Biol., 7, 95 (1963).

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7. 8. 9. 10. 11.

INFLUENCE OF PLATINUM AND PALLADIUM NANOPARTICLES ON THE GROWTH OF BACTERIUM PSEUDOMONAS AERUGINOSA Koukalová M., Čejková A. University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6 – Dejvice [email protected]

Abstract Currently, there is an important and widely discussed problem of the resistance of certain microorganisms to conventional antibiotics. This problem is caused not only by defensive mechanisms, but is supported by the fact that most of the bacterial strains have the ability to colonize surfaces and create structured communities known as biofilms. Microorganisms in biofilm have different phenotype and can be up to thousand times more resistant compared to their planktonic counterparts. Due to the high resistance of biofilm to antibiotics, new approaches in inhibition of biofilm growth are needed. Metal nanoparticles represent one of possible alternatives in developing new effective antimicrobial agent. In recent years, metal nanoparticles have great attention due to their specific properties, given by their small size. As a result, nanoparticles can easily penetrate the cell membrane into the cytosol, where they can interact with macromolecules, which may be lethal to the cell. From available studies, it is clear that nanoparticles are materials that combine the effect of many antimicrobial agents and therefore the potential for resistance to their effects is unlikely. In this study, the effect of platinum and palladium nanoparticles was studied against the biofilm growth of bacteria Pseudomonas aeruginosa, one of the most common pathogens that cause many medicinal complications.

Introduction Almost every microorganism prefers to colonize surfaces and grow in a biofilm form of growth. Cells in the biofilms have different phenotype, compared with their planktonic counterparts, they produce extracellular polymeric substances (EPS) that form surrounding matrix. Matrix is one of the reasons why biofilms are up to thousand times more resistant to external influences, like antibiotics or host immune system. Thus, biofilms can provide a source of systemic chronic infections1, 2. Nanoparticles (NPs) are classified as particles with one dimension within the 1–100 nm size range. Silver nanoparticles (AgNPs) are of pharmaceutical interest because of their potential to replace synthetic antimicrobial drugs. While they have been found to have antimicrobial properties, understanding the broader implications of the effect of new NPs, such as characterization of how novel NPs interact with microorganisms is required3. There are many reports available on the use of noble metals like silver, gold, and platinum etc. and their analogs for medicinal uses. In Ayurveda (Indian medical system), the extremely fine powder of metals (e.g. silver, gold, copper, mercury, iron or zinc) with enhanced therapeutic activity known as ‘‘bhasma’’ have been commonly used for the treatment of various disorders. It is generally used to fight against asthma, anemia, cough, chronic fever, urinary and uterine disorder, bronchitis, pneumonia, bleeding etc. Moreover, these bhasmas also have anti-aging property, reduces dark circles around eyes and fatigue 4. Apart from these applications, heavy metals like silver, gold and platinum showed potential uses in the field of medicine due to their antimicrobial activities and hence used in many antimicrobial formulations 5. Biomedical applications of platinum nanoparticles (PtNPs) include cancer diagnostics, anti-cancer agents (induce DNA damage in cancer cells), experimental treatment of Parkinson's disease and osteoporosis, use in HIV and tuberculosis treatment, specific nitrogen oxides or cholesterol biosensors5, 6, 7, 8. The most important properties of platinum nanoparticles predisposing to these applications are their catalytic activity and stability, surface plasmon resonance capability, and the ability to form complexes with nucleic acids and other biopolymers. However, toxicity for humans and microorganisms is not yet sufficiently documented, and the same type of NPs often show contrasting effects2, 5. Available studies indicate its dependence on nanoparticle concentration and stabilization agent, and an increasing tendency with decreasing size, probably related with increased ability to penetrate the cell and possible organelles5. Palladium is one of the most used transition metals. It is grayish white in colour and has the greatest reactivity in the group of precious metals. Palladium is used in the production of industrial catalysts as a sensor for detecting different analytes (e.g. hydrogen peroxide in milk) or decomposing harmful substances in the environment. While the use of palladium is quite extensive, its advantages in using nanoforms are still hidden. Therefore, it is necessary to determine toxicity of palladium nanoparticles (PdNPs) and also to test their potential for antimicrobial application3.


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However, the detailed antibacterial mechanisms of NPs have not been thoroughly explained, and the same types of NPs often present contrasting effects2. Here, we examine the inhibitory effects of PdNPs and PtNPs toward biofilm of two strains of bacteria Pseudomonas aeruginosa (DBM 3081 and DBM 3777), that is one of the top three causes of opportunistic and nosocomial human infections.

Materials and methods Microbial strains and cultivation conditions Two strains of gram-negative bacteria Pseudomonas aeruginosa DBM 3081 and DBM 3777 were obtained from the Collection of Yeasts and Industrial Microorganisms (DBM) of University of Chemistry and Technology, Prague. Stock cultures were stored at -70 °C and pre-cultured, for 24 hours aerobically at 37 ˚C or 30 °C before each experiment, in Luria Broth (LB) medium. The experiments were also carried out in this growth medium. Nanoparticles Platinum and palladium nanoparticles were obtained from the Department of Solid State Engineering, UCT Prague. Both types of nanoparticles have broad size of 2 nm and were stabilized in the glycerol. Nanoparticles were stored at room temperature. Inhibitory effect of NPs to bacteria The influence of NPs on biofilm formation was carried out in commercially available pre-sterilized, polystyrene, flat-bottomed, 96-well microtiter plates (TPP AG, Switzerland). To each well of the plate, 100 μL of standard cell suspensions in Luria-Bertani (LB) medium (OD600 nm = 0.6) and appropriate volume of nanoparticles to reach the concentration were added. In addition, LB medium was added so the total volume was 300 µL. Nanoparticles free controls were included. Plates were incubated for 24 hours at 37 °C and 30 °C respectively. Total biomass evaluation Each well of the plate was washed twice with phosphate-buffered saline (PBS) and 200 µL of crystal violet solution (0.1 %) was added for 20 minutes. After that time, each well was washed again with PBS and 200 µL of ethanol (96 %) was added for 10 minutes. Using the microplate reader Infinite M200 PRO (Tecan Group Ltd., Switzerland) absorbance of 100 µL of that solution at 580 nm was measured. Experiments were performed with eight replicate wells for each concentration. Cell viability Each well of the plate was washed twice with PBS and 50 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution in PBS (1 mg mL-1) with 60 µL of glucose solution in PBS (57.4 mg mL-1) were added and incubated at appropriate temperature (37 °C and 30 °C respectively) in the dark until crystals of formazan appeared. After MTT incubation, 100 µL of washing solution was added and plate was shaking for 30 minutes to dissolve crystals of blue formazan. Using the microplate reader Infinite M200 PRO (Tecan Group Ltd., Switzerland) absorbance of 100 µL of that solution at 570 nm was measured. Experiments were performed with eight replicate wells for each concentration. This method is used to determine minimum biofilm inhibitory concentration (MBIC80), where the cell viability is reduced in more than 80 %, compared to the control. The effect of nanoparticles was compared to negative controls without any nanoparticles. The control in all experiments was assigned as 100% to allow direct comparison of nanoparticles influence.

Results We examined the inhibitory effect of several concentrations of NPs to the biofilm growth of two strains of P. aeruginosa. With the lowest concentrations of NPs used, we observed higher or same rate of growth compared with the control. It can be seen from the figures that for both types of nanoparticles, with the increasing concentration the inhibitory effect increases against both P. aeruginosa strains. For PdNPs, MBIC80 was determined at 50 mg L-1 for P. aeruginosa DBM 3081 (Figure 1) and for P. aeruginosa DBM 3777 at 70 mg L1 (Figure 3). While using PtNPs, MBIC80 for P. aeruginosa DBM 3081 was determined at the concentration of 86 mg L-1 (Figure 2) and for P. aeruginosa DBM 3777 at 200 mg L-1 (Figure 4). Comparing the obtained results, PdNPs have higher inhibitory effect while using lower concentrations than PtNPs. It is also apparent that P. aeruginosa DBM 3081 is more sensitive to the effect of nanoparticles than P. aeruginosa DBM 3777. Results on Figure 3 show, that lower PdNPs concentrations resulted in more than twice higher total biomass compared to

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the control. This is probably due to the reaction of cells to stress conditions caused by PdNPs, while cells produce an increased amount of EPS to protect themselves. This trend is also noticeable with PtNPs (Figure 4).

Figure 1. Influence of PdNPs on the growth of P. aeruginosa DBM 3081; Total biomass,

Metabolic activity

Figure 2. Influence of PtNPs on the growth of P. aeruginosa DBM 3081; Total biomass,

Metabolic activity


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Figure 3. Influence of PdNPs on the growth of P. aeruginosa DBM 3777; Total biomass,

Metabolic activity

Figure 4. Influence of PtNPs on the growth of P. aeruginosa DBM 3777; Total biomass,

Metabolic activity

Conclusions Investigating the interactions of metal NPs with microorganisms is an important step in potential biological applications. Our major result is that PdNPs are more efficient than PtNPs against both strains of P. aeruginosa. Obtained results also showed differences in inhibition even within two strains of the same microorganism. However, further studies are required considering to the complex effect and mechanisms of nanoparticles. Though there are many mechanisms attributed to the antimicrobial activity shown by nanoparticles, it is not fully understood and cannot be generalized as the nanoparticles are found to act on different organisms in different ways. Therefore, detailed research and comparative study of strain-specific variability is required to determine the bactericidal efficiency of metal nanoparticles.

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Acknowledgement This work was supported by the Czech Science Foundation (GACR) project 17-15936S.

References 1. 2. 3. 4. 5. 6. 7. 8.

Silva S., Pires P., Monteiro D. R., Negri M., Gorup L. F., Camargo E. R., Barbosa D. B., Oliveira R., Williams D. W., Henriques M.: Medical mycology, 51, 178 (2013). Wang L., Hu C., Shao L.: International Journal of Nanomedicine, 12, 1227 (2017) Adams C. P., Walker K. A., Obare S. O., Docherty K. M.: PLoS ONE, 9, 85981 (2014) Pal D., Sahu C. K., Haldar A.: Journal of Advanced Pharmaceutical Technology & Research, 5, 4 (2014) Rai M., Ingle A. P., Birla S., Yadav A., Santos C. A. D.: Critical Reviews in Microbiology, 42, 696 (2015) Capeness M. J., Edmundson M. C., Horsfall L. E.: New Biotechnology, 32, 727 (2015) Rick J., Tsai M. C., Hwang B.: Nanomaterials, 6, 5 (2016) Yamada M., Foote M., Prow T. W.: WIREs Nanomed Nanobiotechnol, 7, 428 (2015)


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EFFECT OF AMPHOTERICIN B AND CHITOSAN ON CANDIDA SPP. BIOFILM FORMATION Kvasničková E., Paldrychová M., Lokočová K., Masák J. University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Czech Republic [email protected]

Abstract Nowadays, antibiotics are often ineffective in biofilm-associated infections treatment. It leads to the use of drugs in higher doses and emergence of related side effects symptoms. This is the reason why researchers are trying to find new alternative substances which are able to overcome this problem. One of the possibly suitable compounds is polysaccharide chitosan, which has significant antimicrobial and also anti-biofilm properties. This study is focused on the investigation of amphotericin B and polysaccharide chitosan anti-biofilm effect on Candida albicans DBM 2164, Candida parapsilosis DBM 2165 and Candida krusei DBM 2163. We investigated metabolic activity of biofilms using MTT assay and total biofilm biomass using crystal violet staining. It was proved that chitosan has significant effect on inhibition of metabolic activity of Candida spp. biofilm cells.

Introduction The chronic biofilm-associated infections are still often appearing1 and the high resistance of biofilms to typically used antibiotic makes the medical care more complicated and often ineffective 2-3. The most common isolated representatives of yeasts from a variety of human infections are Candida albicans, Candida parapsilosis, Candida krusei, Candida tropicalis and Candida glabrata4. C. albicans is the most described opportunistic pathogenic yeast, which may occur in the oral cavity, genital tract or bloodstream and causes fatal candidiasis in immunodeficient patiens5-6. Indeed, Candida parapsilosis, Candida krusei, Candida tropicalis and Candida glabrata have increasing tendency to cause various infections5. C. parapsilosis causes many types of diseases include endocarditis, meningitis, fungemia, vulvovaginitis and urinary tract infections7. Nowadays, there exist strains of the C. parapsilosis which are sensitive to antifungal agents (e.g. amphotericin B), but others are already resistant8. C. krusei is considered to be less pathogenic than other Candida spp. but this species is generally more hydrophobic, which plays an important role in the colonization of patients’ tissues. Therefore, C. krusei causes human infections such as endophthalmitis, fungemia and endocarditis9. The high frequency in use of antibiotics is the reason for still often emerging resistance and thus inefficiency of traditionally used drugs8. In contrast, natural substances with anti-biofilm properties, such as flavonoids, polyphenols, stilbenes and polysaccharides are one of the possibilities that can be solution in the treatment of these infections10. For example polysaccharide chitosan, a hydrophilic biopolymer which is present in the outer shell of crustaceans has a broad antifungal activity11-12 and it has even an activity against growth and biofilm formation of filamentous fungi13-14. The purpose of this study is to investigate anti-biofilm activity of natural polysaccharide chitosan in vitro in comparison with amphotericin B as new possibility in the treatment of Candida spp. infections.

Materials and Methods Microbial strains The representatives of Candida spp., Candida albicans DBM 2164, Candida parapsilosis DBM 2165 and Candida krusei DBM 2163 were kindly provided by Collection of Microorganisms of Institute of Biochemistry and Microbiology, UCT Prague. Cultivation conditions The inoculum was pre-cultured aerobically at 30 ˚C, 100 rpm (from stock cultures stored at -70 °C) before each experiment (C. albicans in YPD medium, C. parapsilosis and C. krusei in Malt Extract medium), harvested after 24 h (C. albicans) or 48 h (C. parapsilosis and C. krusei), then centrifuged at 9000 g for 10 minutes and resuspended in appropriate growth medium. Antifungal agents Amphotericin B and chitosan were purchased from Sigma-Aldrich. Amphotericin B was dissolved in appropriate growth medium (final concentration 1500 mg l-1). Chitosan (final concentration 1000 mg l-1) was dissolved in

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appropriate growth medium with content of acetic acid (the final concentration was maximally 2 %). Each substance was stored, maximally for one week, at 4 °C until used. Biofilm formation Biomass concentration was set on OD600nm=0,600±0,020. Each well of 96-wells polystyrene microplate was inoculated by 200 μl of this suspension. Biofilm formation was held at 30 °C, stirred 150 rpm. After 24 h of cultivation the samples were washed three times with saline and antifungal agents were added in sterile growth medium. After next 24 h samples were washed three times with saline and the biofilm eradication by amphotericin B or chitosan was determined using MTT assay or crystal violet staining. Anti-biofilm activity The effect of amphotericin B or chitosan on eradication of selected Candida spp. biofilms was determined by MTT assay and crystal violet staining. Metabolic activity of biofilm was determined by addition of 50 μl MTT (in concentration 1 g/l) and 60 µl glucose (in concentration 57.4 g/l) to the each well for 1-4 h. Then 100 µl of solution (40 % DMF in 2 % acetic acid and 16 % SDS) was added to each well. This solution washed out the crystals of formazan (formed by MTT conversion by metabolic active cells) from the biofilm cells. The absorbance was measured at 570 nm using a microtiter plate spectrophotometric reader (Tecan, Switzerland). The total biofilm biomass was determined by staining the biofilm with 100 μl 0.1 % crystal violet, incubated for 20 minutes at room temperature and washed by addition of 200 μl of 96 % ethanol for 10 minutes. The absorbance was measured at 580 nm.

Results and Discussion In Fig. 1-6 is depicted the effect of amphotericin B or natural polysaccharide chitosan on biofilm eradication of all tested yeast strains. These strains are sensitive to the action of both selected substances. Chitosan has comparable activity to the typically used amphotericin B. We proved that chitosan has anti-biofilm activity which corresponds with literature11, 15. It has primarily the ability to inactivate biofilm cells. The 80 % of biofilm cells of C. albicans are inactivated by 50 mg l-1 of amphotericin B (Fig. 1) or 250 mg l-1 of chitosan (Fig. 2) and C. krusei is inactivated by 100 mg l-1 of both substances (each used alone) (Fig. 5,6).

Metabolic activity of biofilm, 570 nm Total biofilm biomass, 580 nm

1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Amphotericin B, mg l-1 Figure 1. The effect of different concentrations of amphotericin B on C. albicans DBM 2164 biofilm eradication; cultivation 24 h, eradication 24 h, 30 °C, 150 rpm, YPD medium; ( ) MTT – metabolic activity of biofilm, ( ) KV – total biofilm biomass


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1,0 Metabolic activity of biofilm, 570 nm Total biofilm biomass, 580 nm

0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Chitosan, mg l-1

Figure 2. The effect of different concentrations of chitosan on C. albicans DBM 2164 biofilm eradication; cultivation 24 h, eradication 24 h, 30 °C, 150 rpm, YPD medium; ( ) MTT – metabolic activity of biofilm, ( ) KV – total biofilm biomass


Chitosan decreases the metabolic activity of biofilm cells by 80 % in concentration 200 mg l-1 in the case of C. parapsilosis biofilm (Fig. 4), while amphotericin B in concentration 1400 mg l-1 (Fig. 3). On the other hand, amphotericin B is able to more decrease total biofilm biomass of C. parapsilosis than chitosan (total biofilm biomass was less than one half of control samples in the higher tested concentration of amphotericin B). 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0

Amphotericin B, mg l-1 Figure 3. The effect of different concentrations of amphotericin B on C. parapsilosis DBM 2165 biofilm eradication; cultivation 24 h, eradication 24 h, 30 °C, 150 rpm, Malt extract medium; ( ) MTT – metabolic activity of biofilm, ( ) KV – total biofilm biomass

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1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0

Chitosan, mg l-1


Figure 4. The effect of different concentrations of chitosan on C. parapsilosis DBM 2165 biofilm eradication; cultivation 24 h, eradication 24 h, 30 °C, 150 rpm, Malt extract medium; ( ) MTT – metabolic activity of biofilm, ( ) KV – total biofilm biomass 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0

Amphotericin B, mg l-1 Figure 5. The effect of different concentrations of amphotericin B on C. krusei DBM 2163 biofilm eradication; cultivation 24 h, eradication 24 h, 30 °C, 150 rpm, Malt extract medium; ( ) MTT – metabolic activity of biofilm, ( ) KV – total biofilm biomass The visualization of C. parapsilosis biofilm affected by the presence of amphotericin B (1400 mg l-1) or chitosan (200 mg l-1) in the growth medium is demonstrated in Fig. 7. The images of biofilm correspond with data obtained by other methods (MTT assay and crystal violet staining). Amphotericin B (1400 mg l-1) influences the total biofilm biomass more than chitosan (200 mg l-1). Chitosan has significant effect on biofilm structure, but the amount of biomass is the same as in control sample. On the other hand there is still the fact that amphotericin B was used in much higher concentration than chitosan.


39


1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0

Chitosan, mg l-1 Figure 6. The effect of different concentrations of chitosan on C. krusei DBM 2163 biofilm eradication; cultivation 24 h, eradication 24 h, 30 °C, 150 rpm, Malt extract medium; ( ) MTT – metabolic activity of biofilm, ( ) KV – total biofilm biomass

Figure 7. Visualization of amphotericin B or chitosan effect on inhibition of C. parapsilosis DBM 2165 biofilm formation; (A) control sample (without any substances), (B) 1400 mg l-1 of amphotericin B, (C) 200 mg l-1 of chitosan; cultivation 24 h, 30 °C, 150 rpm, Malt extract medium, scale up 200 µm

Conclusion We confirmed anti-biofilm activity of chitosan against Candida spp., especially its ability to inactivate biofilm cells. But it is not possible to conclude the uniform activity against each strain of this genus. Due to amphotericin B anti-biofilm activity in high concentrations we consider these results to be important. The use of chitosan in mutual combination with amphotericin B will be the next step of our research. This process may bring us desired results.

Acknowledgment Financial support from specific university research (MSMT No 20-SVV/2017) and Czech Science Foundation (GACR) project 17-15936S.

References 1. Jefferson K. K.: FEMS Microbiol. Lett., 236 (2), 163-173 (2004). 2. Thebault P., Lequeux I., Jouenne T.: J. Wound Tech., 21 (6), 20-23 (2013). 3. Watnick P., Kolter R.: J. Bacteriol., 182 (10), 2675-2679 (2000). 4. Donlan R. M.: Clin. Infect. Dis., 33 (8), 1387-1392 (2001). 5. Kuhn D. M., Chandra J., Mukherjee, P. K., Ghannoum, M. A.: Infect. Immun., 70 (2), 878-888 (2002).

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6. Chandra J., Kuhn, D., Mukherjee P., Hoyer, L., McCormick T. M. A. Ghannoum.: J. Bacteriol., 183, 5385-23 (2001). 7. Trofa D., Gácser A., Nosanchuk J. D.: Clin. Microbiol. Rev., 21 (4), 606-625 (2008). 8. van Asbeck E. C., Clemon, K. V., Stevens D. A.: Crit. Rev. Microbiol., 35 (4), 283-309 (2009). 9. Samaranayake Y. H., Samaranayake L. P.: J. Med. Microbiol., 41 (5), 295-310 (1994). 10. Řezanka T., Masák J., Čejková A.: Stud. Nat. Prod. Chem., 38, 269-303 (2012). 11. Martinez L. R., Mihu M. R., Tar M., Cordero R. J. B., Han G., Friedman A. J., Friedman J. M., Nosanchuk J. D.: J. Infect. Dis., 201 (9), 1436-1440 (2010). 12. Silva-Dias A., Palmeira-de-Oliveira A., Miranda I. M., Branco J., Cobrado L., Monteiro-Soares M., Queiroz J. A., Pina-Vaz C., Rodrigues A. G.: Med. Microbiol. Immunol., 203 (1), 25-33 (2014). 13. Kvasničková E., Paulíček V., Paldrychová M., Ježdík R., Maťátková O., Masák J.: World J. Microbiol. Biotechnol., 32 (11), 187 (2016). 14. Cota‐Arriola O., Cortez‐Rocha M. O., Rosas‐Burgos E. C., Burgos‐Hernández A., López‐Franco Y. L.: Plascencia‐Jatomea M.: Polym. Int., 60 (6), 937-944 (2011). 15. Kvasnickova E., Matatkova O., Cejkova A., Masak J.: J. Microbiol. Methods, 118, 106-112 (2015).


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ANTIBIOTIC RESISTANCE OF PSEUDOMONAS AERUGINOSA Lokočová K.1, Masák J.1 University of Chemistry and Technology Prague (Department of Biotechnology), Technická 5, 166 28, Prague 6, Czech Republic

1

[email protected]

Abstract A biofilm can be defined as a community of microorganisms enveloped in protective matrix of EPS (expolymeric substances) adhering to various surfaces. The cells in the biofilm are significantly more resistant to environmental influences than suspension cells and they also show resistance to antibiotics and disinfectants. Microbial biofilms cause chronic human diseases (e.g. pneumonia in cystic fibrosis patients, endocarditis, middle ear infections and implant-/catheter-associated infections) and the treatment is very difficult. Therefore, great effort is made to find different antibiotics or natural substances, which may be used in the treatment of biofilm infections. This study is aimed at monitoring the ability of polymyxin B in comparison with first-line antibiotics (gentamicin and ceftazidime) to disrupt biofilm formation of opportunistically pathogenic gram-negative bacteria Pseudomonas aeruginosa ATCC 10145 and ATCC 15442. The biofilm was observed in 96-well microtiter plates using MTT tetrazolium reduction assay to determine the metabolic activity of the cells in biofilm and crystal violet (CV) staining method to quantify the amount of biofilm biomass. The highest antibiofilm effect of polymyxin Bwas confirmed in the case of gram-negative bacteria P. aeruginosa ATCC 10145.

Introduction Pseudomonas aeruginosa is an opportunistic human pathogen that causes respiratory infections, urinary tract infections and chronic infections in immunocompromised patients. P. aeruginosa biofilm is highly regulated by biological and physicochemical signalling. Complex bacterial communities produce exopolymers (e.g. extracellular polysaccharide, proteins and extracellular DNA) that protect bacteria from the host immune response. This gram-negative bacterium has various mechanisms of resistance to antibiotics1. The aminoglycoside antibiotic gentamicin has many desirable properties for the treatment of gram-negative bacillary infections. Aminoglycosides disrupt bacterial protein synthesis through binding to prokaryotic ribosomes.Resistance of Pseudomonas spp. consists in decrease in drug uptake and accumulation2. The third generation cephalosporins includes e.g. cefotaxime and ceftazidime3. The antibiotic ceftazidime crosses the bacterial wall, interferes withthe synthesis of cell wall and causes cell lysis of the pathogens4. One of the mechanism of resistance to ceftazidime is alteration of the cell-wall permeability of gram-negative bacteria5. Polymyxin B is currently used as clinical drug. The polymyxin antibiotics are progressively considered as the final option for therapy of infection that are caused by resistant bacteria. However, the concerns on their neurotoxicity and nephrotoxicity still persist6. The goal of our experimental work was monitoring the ability of polymyxin B in comparison with first-line antibiotics (gentamicin and ceftazidime) to disrupt biofilm formation of opportunistically pathogenic gramnegative bacteria Pseudomonas aeruginosa ATCC 10145 and ATCC 15442.

Materials and Methods Bacteria strains The representative of gram-negative bacteria, Pseudomonas aeruginosa (strains ATCC10145 and ATCC 15442), were obtained from the American Type Culture Collection. Cultivation conditions Stock cultures were stored at -70 °C in 50% glycerol solution. Strains of P. aeruginosa were preculturedaerobically at 37 °C in Luria-Bertani (LB) liquid medium (stirred at 150 rpm for 24 h). Then the cultures were centrifuged at 9000 g for 10 minutes and re-suspended in LB medium. Antibiotics The antibiotics polymyxin B, ceftazidime and gentamicin were dissolved in LB medium to a final concentration (polymyxin B – 5, 7.5, 10, 12.5, 15 mg/L; ceftazidime – 1, 25, 20, 100, 150 mg/L; gentamicin – 5, 10, 15, 20, 25 mg/ L).

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Biofilm cultivation in 96-well microtiter plates The cultivation was carried out in 96-well polystyrene microtiter plates. To each well 210 µl of standard cell suspension of P. aeruginosa (OD600nm = 0.6) and 70 µl of the antibiotic solution were added. Microtiter plates were incubated for 24 h at 37 °C and stirred at 150 rpm. Then non-adherent cells were removed by washing with saline solution (three times). After 24 h we observed anti-adhesive properties of antibiotics against biofilms adhered to polystyrene microtiter plates using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay to determine the metabolic activity of the cells in biofilm and crystal violet (CV) staining method to quantify the amount of biofilm biomass. Optical density (OD) was measured at 600 nm to quantify the cells present in the surroundings of P. aeruginosa biofilm. All experiments were measured in eight replicates.

Results and Discussion The results of experiments are shown in Figures 1-6. We observed the effect of polymyxin B in quite low concentrations (10 mg L-1) for both tested strains of P. aeruginosa (Figure 1; Figure 2). The metabolic activity of the cells was determined by MTT assay (spectrophotometric measurement at 570 nm) and inhibition over 80 % was observed. The other tested antibiotics had lower activity than polymyxin B. The antibiotic ceftazidime at concentration of 25 mg L-1 inhibited the metabolic activity of adhered cells over 80 % only in P. aeruginosa ATCC 10145 (Figure 3). The second strain (ATCC 15442) was found less susceptible to ceftazidime (Figure 4). Its metabolic activity was decreased by 50 %. It was found that gentamicin has significant anti-adhesive activity against both strains, P. aeruginosa ATCC 15442 (Figure 6) and ATCC 10145 (Figure 5). The highest inhibition of the metabolic activity by gentamicin was determined in the concentration 15 mg L-1 for P. aeruginosa ATCC 15442 (Figure 6). The amount of suspension cells present in the surroundings of P. aeruginosa biofilm (measurement of OD at 600 nm) and total biofilm biomass stained by crystal violet (spectrophotometric measurement at 580 nm) were most affected by polymyxine B. CV

Total biofilm biomass inhibition, % Metabolic activity of biofilm inhibition, % Amount of suspenzion cells inhibition, %

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60

40

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Figure 1. Effect of polymyxin B on adhesion of P. aeruginosa ATCC 10145; expressed as the inhibition of total biofilm biomass (CV), metabolic activity of biofilm (MTT) and the suspension cells (OD)


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CV


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MTT

OD (600 nm)

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60

40

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10 12.5 15 Concentration, mg L-1 Figure 2. Effect of polymyxin B on adhesion of P. aeruginosa ATCC 15442; expressed as the inhibition of total biofilm biomass (CV), metabolic activity of biofilm (MTT) and the suspension cells (OD)

CV


100

MTT

OD (600 nm)

80

60

40

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50 100 150 Concentration, mg L-1 Fig. 3 Effect of ceftazidime on adhesion of P. aeruginosa ATCC 10145; expressed as the inhibition of total biofilm biomass (CV), metabolic activity of biofilm (MTT) and the suspension cells (OD)

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1

25


CV


100

MTT

OD (600 nm)

80

60

40

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0

1

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50 100 150 Concentration, mg L-1 Fig. 4 Effect of ceftazidime on adhesion of P. aeruginosa ATCC 15442; expressed as the inhibition of total biofilm biomass (CV), metabolic activity of biofilm (MTT) and the suspension cells (OD)

CV


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60

40

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Fig. 5 Effect of gentamicin on adhesion of P. aeruginosa ATCC 10145; expressed as the inhibition of total biofilm biomass (CV), metabolicactivity of biofilm (MTT) and the suspension cells (OD)


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CV

MTT

OD (600 nm)


100

80

60

40

20

0

5

10

15 20 25 Concentration, mg/L Fig.6 Effect of gentamicin on adhesion of P. aeruginosa ATCC 15442; expressed as the inhibition of total biofilm biomass (CV), metabolic activity of biofilm (MTT) and the suspension cells (OD)

Conclusion We carried out experiments focused on the anti-adhesive properties of the antibiotics against P. aeruginosa biofilms. It was shown that polymyxin B is more efficient than ceftazidime and gentamicin. The first-line antibiotic ceftazidimehadno significant effect on P. aeruginosaATCC 10145 adhesion. Compared to the first-line antibiotics, polymyxin B may be more effective. Modern medicine needs to find ways of treating biofilm infections and has prompted a gradual move away from traditional antibiotic treatments and toward nonantibiotic therapies.


References 1. 2. 3. 4. 5. 6.

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Ochoa S. A., Cruz-Cordova A., Rodea G. E., Cazares-Dominguez V., Escalona G., Arellano-Galindo J., Hernandez-Castro R., Reyes-Lopez A., Xicohtencatl-Cortes J.: Microbial Res., 172, 68 (2015). Mingeot-Leclercq M. P., Glupczynski Y., Tulkens P. M.:Antimicrob Agents Chemother, 43, 727 (1999). Pfeifer Y., Cullik A., Witte W.:Int. J. Med. Microbiol., 300, 371 (2010). Kalman D., Barriere S. L.: Tex Heart Inst J., 17 (3), 203 (1990). Martens M. G.: Obstet Gynecol Clin North Am, 16, 291 (1989). Zavascki A. P., Goldani L. Z., Li J., Nation R. L.: J Antimicrob Chemother, 60, 1206 (2007).


EFFECT OF BIOLOGICALLY ACTIVE SUBSTANCES ON CANDIDA BIOFILM Lokočová K.1, Paldrychová M.1, Masák J.1 University of Chemistry and Technology Prague (Department of Biotechnology), Technická 5, 166 28, Prague 6, Czech Republic

1

[email protected]

Abstract The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that consist of polysaccharides, proteins, lipids and nucleic acids.EPS mediate microbial adhesion to various surfaces and also provide the mechanical stability of biofilms. Biofilm cells often show resistance to commonly used antibiotics. The most common isolated yeasts species from nosocomial infections are for example Candida parapsilosis, Candida krusei, Candida albicans and Candida glabrata. In order to find suitable substances that are able to affect these resistant structures, a number of natural substances have been tested. This work is focused on testing a biologically active substance with anti-biofilm effect. For this purpose the natural substance chitosan was selected to study its potential to disrupt the biofilm formation and stability of the opportunistic yeasts Candida parapsilosis DBM 2165, Candida krusei DBM 2163 and Candida albicans DBM 2164. The cultures were cultivated in polystyrene microtiter plates. The metabolic activity of the cells in biofilm was determined using MTT assay. Crystal violet staining was used to quantify the amount of biofilm biomass. The highest effect of chitosan (at the concentration of 5 mg L-1) on the metabolic activity of cells in biofilm was confirmed in the case of C. parapsilosis.

Introduction The most commonly isolated yeast from medical devices is Candida albicans, but other Candida species such as Candida parapsilosis and Candida krusei are also frequently identified. These opportunistic pathogens are known to adhere to various materials for example catheters and cause many infections. Candida biofilms are highly resistant to antifungal drugs1. There is a growing interest in the use of natural substances for therapeutic purposes due to their biological activities2. Chitosan is natural polysaccharide biopolymer derived by deacetylation from chitin (major component of the crustacean exoskeletons e.g. crabs, shrimps and crawfish). This compound has excited a lot of interest due to biomedical applications. Chitosan is used as an antimicrobial agent against many bacteria, yeasts and filamentous fungi4. Further studies brought positive information about the anti-biofilm activity of chitosan, by reducing the metabolic activity of preformed Candida biofilms up to 90 %5. This work was aimed at monitoring the ability of chitosan to disrupt biofilm formation of opportunistically pathogenic yeasts Candida parapsilosis DBM 2165, Candida krusei DBM 2163 and Candida albicans DBM 2164, which are able to cause human infections.

Materials and Methods Yeasts strains The representative strains of yeasts, Candida parapsilosis DBM 2165, Candida krusei DBM 2163 and Candida albicans DBM 2164, were obtained from Collection of Microorganisms of Institute of Biochemistry and Microbiology UCT Prague. Cultivation conditions Stock cultures were stored at -70 °C in 50 % glycerol solution. All strains of yeasts were grown in Yeast Extract – Peptone – Dextrose (YPD) medium for 24 h at 30 ˚C, stirred at 150 rpm. Biological active substances The biological active substance chitosan was dissolved in YPD medium to a final concentration. The required amount of chitosan was dissolved in 99% acetic acid to occupy a maximum of 2 % of the total volume of the final solution. Biofilm cultivation in 96-well microtiter plates The standard cell suspension of Candida strains (OD600nm = 0.6) was cultivated in 96-well polystyrene microtiter plates (210 µl per well) with 70 µl of the antibiotic solution for 24 h at 30 °C and stirred in an orbital shaker (150 rpm). The metabolic activity of the cells in biofilm was determined using MTT assay (measurement of


47

absorbance at 570 nm). Crystal violet staining was used to quantify the amount of biofilm biomass (measurement of absorbance at 580 nm). MTT assay The suspension cells formed in the surroundings of Candida biofilm were transferred and measured at 600 nm and the wells of polystyrene microtiter plate were washed three times with a saline solution. The MTT and glucose solutions were added to biofilm on the bottom of wells (the final concentrations of MTT was 0.45 mg mL-1, glucose 31.3 g L-1) and incubated for 1 to 4 hours (dependent on the microorganism) on a microtiter plate shaker at 150 rpm. After the incubation a solubilization solution was added (100 µl) to each well and incubated for 30 minutes at 230 rpm to dissolve the formazan crystals. The absorbance of coloured solution (100 µl) was measured at 570 nm (eight replicate wells). Crystal violet staining The total biofilm biomass was quantified using crystal violet staining method. The suspension was removed from wells by washing three times with the saline solution. The crystal violet solution (200 µl) was added to each well. The wells were washed again three times with saline after incubation for 20 minutes at room temperature. Then 200 µl volume of 96 % ethanol was added and after 10 minutes the absorbance of coloured solution (100 µl) was measured at 580 nm (eight replicate wells).

Results and Discussion The effect of chitosan on Candida biofilm was observed. The results of experiments expressed as the inhibition of metabolic activity are shown in Figures 1-3. In the Figure 1 is depicted the effect of chitosan on C. albicans, in Figure 2 on C. parapsilosis and in Figure 3 on C. krusei.

Metabolic activity of biofilm inhibition, %

100 80 60 40 20 0

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50

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150 200 250 Concentration, mg L-1

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Figure 1. Effect of chitosan on adhesion of C. albicans DBM 2164; expressed as the inhibition of metabolic activity of biofilm (MTT) It was proved that studied yeast strains are sensitive to the chitosan. We determined the inhibition of the metabolic activity by chitosan in all studied strains. The highest effect on reduction of the metabolic activity of biofilm was confirmed in C. parapsilosis DBM 2165. The metabolic activity was decreased by 80 % in concentration 5 mg L-1, which was determined by MTT assay (Figure 2). On contrary, the total biofilm biomass (stained by crystal violet) showed inductive character (data not shown). Chitosan had similar effect on the inhibition metabolic activity and biofilm formation of C. krusei. In this case, chitosan at concentration of 20 mg L-1 inhibited the metabolic activity of the cells by 80 % (Figure 3).

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The third representative, C.albicans, was found less susceptible to chitosan. We confirmed the inhibitory effect on the metabolic activity at concentration of 250 mg L-1. The variable effect was observed in the amount of total biofilm biomass (data not shown). The natural substance chitosan affected the inhibition metabolic activity of all three tested representatives of Candida. 100 80 60 40 20 0

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20 30 Concentration, mg L-1

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Figure 2. Effect of chitosan on adhesion of C. parapsilosis DBM 2165; expressed as the inhibition of metabolic activity of biofilm (MTT)

100 80 60 40 20 0

0.1

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Figure 3. Effect of chitosan on adhesion of C. krusei DBM 2163; expressed as the inhibition of metabolic activity of biofilm (MTT)

Conclusion In our study, the determination of the anti-biofilm activity of chitosan was investigated. The natural compound chitosan decreased the metabolic activity of Candida species biofilms. The highest effect of chitosan on the


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metabolic activity was proved in the case of C. parapsilosis biofilm. Our results show that chitosan potentially can be developed as a therapeutic agent for the treatment of Candida species infections.


References 1. 2. 3. 4. 5.

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Kumamoto C. A.: Curr Opin Microbiol,5 (6), 608 (2002). Koh Ch., Sam Ch., Yin W., Tan L. Y., Krishnan T., Chong Y. M., Chan K.: Sencors, 13 (5), 6217 (2013). No H. K., Park N. Y., Lee S. H., Meyers S. P.: Int J Food Microbiol, 74 (1-2), 65 (2002). Kong M., Chen XG., Xing K., Park H. J.: Int J Food Microbiol, 144 (1), 51 (2010). Silva-Dias A., Palmeira-de-Oliviera A., Miranda I. M., Branco J., Cobrado L., Monteiro-Soares M., Queiroz J. A., Pina-Vaz C., Rodrigues A. G.: Immunol Med Microbiol, 1, 25 (2014).


AN ENERGY INDEPENDENT SOLAR DRYING SYSTEM FOR FOOD PRODUCTS DRYING Noori A.W.1,2, Royen M. J.1,2, Haydary J.1 Institute of Chemical and Environmental Engineering, Slovak University of Technology in Bratislava, Radlinského 9, 81237 Bratislava, Slovakia, Tel: +421905794876, email:[email protected] 2 Faculty of Chemical Technology, Kabul Polytechnic University, Kart-e Mamoorin, Kabul, Afghanistan 1

Abstract In this work, an energy independent solar drying system for the study of food products drying at higher altitudes and specific climate conditions is introduced. As a model material, sliced tomato was selected because of its short shelf live, high humidity and its potential to be a high value dried product. To achieve complete protection of fruit and vegetable against sunlight, birds, insects, rain and dust during the drying process, indirect solar dryer system was used. The solar dryer system design includes a rectangle section (100x60x40 cm) chamber and a flat solar collector (150x60x10 cm) with the surface area of 0.9 m2. Air flow was induced by a fan installed at the inlet of the collector and powered by a photovoltaic solar panel and a battery system. The temperature and humidity of air were monitored at the collector inlet, collector outlet and the drying chamber outlet. The key element of the collector is a 10.5m long rectangle section aluminum pipe (5.5x3.5 cm) coated with an absorption layer. The dryer capacity is around 3 kg of wet material (sliced tomato) per a batch. The average air temperature increase in the collector was 30 oC during the winter season. The air relative humidity decreased from 21% to 15% after passing through the collector. The moisture of a tomato slice decreased from the initial value of 92% down to 22% during the experiment time (30 h). Quality of tomatoes dried using the designed solar dryer differs significantly from those dried by other common methods, like an open sun drying system, in color as well as in texture. The equilibrium moisture content of the product was reached after 30 h in the time period of 18-22 of December 2016 when the outside maximum temperature was 17.6 °C. The tomato weight decreased from 333g to 33.15g; the weight loss being approximately 90%. The heated air temperature and humidity at the dryer inlet and outlet were influenced by the change of the ambient temperature and humidity during the day. Variation of the drying rate with the change of the ambient temperature and humidity was observed.

Introduction Afghanistan is an upland and landlocked country with dry continental climate, low air pressure and low air humidity. Economy of the country is based on agribusiness1. The agriculture sector is the second biggest sector after the services in Afghanistan. The major Afghan agriculture products are cereals, fruits, nuts, vegetables and medicinal plants1. The major export agriculture product is dried fruits (27%), medicinal plants (10%) and fresh fruits (7%)4. Herbs, dry vegetables, fresh and dry fruits are the most famous products of Afghanistan in the Asian markets. Most of the agriculture products require some kind of preservation to enhance their shelf life since the production usually exceeds the market demand during the harvest season. Drying is the most used and most suitable method for agriculture products preservation7. Drying of agricultural products is an energy-intensive operation. High prices and shortages of fossil fuels increase the emphasis on the use of solar energy as an alternative energy source, especially in developing countries 3. Globally, the solar energy usage capacity increases at the rate of 10 – 60% annually for many technologies5. A solar dryer can be an alternative to the hot air and open sun drying methods, especially in locations with good sunshine during the harvest season8. Hot air solar drying systems may be classified as direct, indirect and mixed modes7. Solar drying systems need a shorter time for the drying process completion than the open sun light systems8. Afghanistan is one of the countries with a huge potential for using solar energy as it has more than 300 sunny days per year, with the average annual solar direct normal radiation higher than 5 [kWh/m2/day]. Fig. 1, adapted from5, shows the country’s map of normal solar radiation. Nowadays, the solar energy in Afghanistan is used for cooking, lighting, water heating and drying process 2. However, traditional open sun drying methods without control of quality and hygiene conditions are applied. The principal objective of this work was the design an energy independent solar drying system for food products drying in the indirect mode to achieve complete protection of fruit and vegetable against sunlight, birds, insects, rain and dust during the drying process producing standard dried products for worldwide market. As a model material, sliced tomato was selected, because of its short shelf live, high humidity and its potential


51

to be a high value dried product. Tomato (lycopersicum esculentum) is an herbaceous plant which is part of the family Solanaceous, like potato6.

Figure 1. Map of sunlight intensity for all areas of Afghanistan.

Solar Dryer There are different types of dryers with different structure and different work principles for agriculture products (fruits and vegetables) drying. All dryers are formed of two basic parts: collector and drying chamber. In the collector, the sun radiation heats the air to the desired temperature while in the drying chamber, the product is dried by heated air which is passing through the product beds1.

Figure 2. Characteristics of solar dryer collector and chamber.

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A scheme of the designed solar dryer system is shown in Fig. 2 and it is pictured in Fig. 3. The system includes a rectangle section (100x60x40 cm) chamber and a flat solar collector (150x60x10 cm) with the surface area of 0.9 m2. Air flow was induced by a fan installed at the inlet of the collector and powered by a photovoltaic solar panel and a battery system. The temperature and humidity of air were monitored at the collector inlet, collector outlet and the drying chamber outlet. The key element of the collector is a 10.5m long rectangle section aluminum pipe (5.5x3.5 cm) coated with an absorption layer. The dryer capacity is around 3 kg of wet material (sliced tomato) per a batch. The average ambient pressure during the measurement days was 80-82 kPa.

Figure 3. Solar dryer: a) complete device photo; b) chamber back and front side photo; c) device inner side; d) exhaust fan and sensor location at the outlet of the chamber; e) sensor location at the inlet of the chamber.

Results and discussion This experiment was done in the time period of 18-22 of December 2016, in the winter season, when the outside maximum temperature was 17.6 °C. The equilibrium moisture content of the product was reached after 30 hours of active drying. Fig. 4, representing the mass loss versus time, shows the 90 % mass loss of the sliced tomato; the tomato weight decreased from 333 g to 33.15 g.

Figure 4. Mass loss of product vs. drying time


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The device worked just for 5 hours each day, on the mass loss curve each gap stands for the 19 hours off-time per day. The decrease of the tomato relative moisture is shown in Fig. 5. The moisture of tomato slices decreased from the initial value of 92% down to 22% during 30 h of active drying.

Figure 5. Decrease of wet material relative moisture during the drying time. Air humidity changed during the process; while the ambient relative humidity was 21%, the air relative humidity decreased to 15% after passing the collector, and it increased to 40 - 70% at the chamber outlet. As results from Fig. 06, the change of temperature during the day had influenced also the ambient air humidity. It is most visible for the air leaving the drying chamber.

Figure 6. Humidity changes in three different locations in the device during the drying time Drying rate is another characteristic of the drying process shown in Fig. 7. The drying rate changed during the day; its maximum value corresponded with the maximum temperature achieved at noon.

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Figure 7. Dependence of the drying rate on time The collector efficiency is presented in Fig. 8. The maximum increase of the air temperature after passing the collector was from 17°C up to 47°C. Also the temperature changes during the day are visible in this figure. The maximum temperature difference (maximum collector efficiency) corresponds with that at noon, when the sun radiation angle is more perpendicular to the collector surface.

Figure 8. Collector efficiency shown by air temperature increase after passing the collector

Conclusions

Under the climatic conditions of Afghanistan (Kabul) at the period of the experiment (end of December 2016, winter, ambient day temperature: 10-17 oC, air relative humidity: 15-20%, air pressure: 80-82 kPa), five days were sufficient for sliced tomato drying in an energy independent indirect solar dryer. The average air temperature increase after passing through the collector was 30 oC. The air relative humidity decreased by around 7% after passing through the collector and increased by around 30% after passing through the drying chamber. Approximately 90% of the product weight were lost during the drying process. Lower relative humidity and lower ambient pressure of upland geographical locations enable effective indirect solar drying also during the winter time.

Acknowledgement

This work was supported by the project SAMRS/2016/AFG/01/01 of the Slovak Agency for International Development Cooperation.


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References 1. 2.

3. 4. 5. 6. 7. 8. 9.

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Adolfo G. F. P.: Ener. Proc., 57, 2984 – 2993 (2014). Ministry of Agriculture Irrigation and Livestock, Prject report, (DCI-FOOD/2008/020-138/228-825; GCP/AFG/063/EC), Kabul, Afghanistan (2012). Manaa S., Younsi M., Moummi N.: Ener. Proc. 36, 511 – 514 (2013). Rostami R., Khoshnawa S. M., Lamit H., Streimikiene D., Mardni a.: Renew .Sust. Energ .Rev., 76, 14401464 (2017). Shukla A. K., Sudhakar K., Baredar P.: Reso-Effi. Tech., in Print, (2017) Srisittipokakun N., Kirdsiri K., Kaewkhao J.: Proc. Eng., 32, 839 – 846 (2012) Tiris C., Tiris M. Dincer I.: Appl. Rher. Eng. , 16(2), 183-187 (1996) Varun., Sunil., Sharma A., Sharma N.: Proc. Eng., 38, 3260 – 3269 (2012) World bank.: Rep. No. AUS9779, Kabul, Afghanistan (2014)


EFFECT OF NON-THERMAL PLASMA ON PSEUDOMONAS AERUGINOSA QUORUM SENSING SYSTEM Paldrychová M.1, V. Scholtz2, E. Kvasničková1, J. Masák1 1 2

UCT Prague (Department of Biotechnology), Technická 5, 166 28, Prague 6 UCT Prague (Department of Physics and Measurements), Technická 5, 166 28, Prague 6

Abstract One of the most important nosocomial pathogens is Pseudomonas aeruginosa, gram-negative bacterium often resistant to antimicrobials commonly used in medical practice. Pseudomonas aeruginosa thus presents a challenge to develop treatments targeted e.g. on some of the important virulence factors, instead of the cell viability. P. aeruginosa regulates the expression of virulence factors, such as biofilm formation or exotoxin pyocyanin production via quorum sensing (QS) mediated by diffusible N-acyl-homoserine lactone (AHL) signals. A number of quorum sensing inhibitors (QSI), mostly derived from nature, which are structurally related to the signal molecules or accelerate the degradation of these molecules, have been described. The aim of our study was to evaluate the ability of non-thermal plasma (NTP) to interfere with the QS system of P. aeruginosa. Agrobacterium tumefaciens NTL4 (pZLR4) was used for determination of the AHLs levels after NTP treatment. This biosensor, which doesn´t produce its own AHLs, contains an inserted plasmid that is responsible for the expression of β-galactosidase in presence of exogenous AHLs. This enzyme cleaves added X-Gal to a blue product which can be measured spectrophotometrically. By the above mentioned method, we proved that NTP significantly affected the P. aeruginosa QS system (levels of AHLs were after NTP application lower compared to control).

Introduction Pseudomonas aeruginosa is gram-negative bacterium, an opportunistic pathogen and one of the main causes of the nosocomial infections. It poses a risk especially for the patients suffering from cystic fibrosis, patients with third degree of burns, and also for the patients who have various types of chirurgical implants in their body (infections of urinary tract after urinary catheter implantation are quite common). Intrinsic resistance of P. aeruginosa to antimicrobials is caused by its lowly permeable outer membrane, expression of membrane efflux pumps and production of many degradative enzymes. These facts presents a challenge to development of treatments targeting some of the important virulence factors which are produced during the infection process, instead of cell viability1. Biofilm formation provides bacterial cells protection against various stresses including antibiotics. Due to the enzymes production (such as elastase, protease), bacteria can destroy host tissue or suppress the host immune response (alkaline protease). Delayed inflammatory response to bacterial invasion can also be caused by the production of exotoxin pyocyanin. A key role in the regulation of expression of all of these virulence factors plays quorum sensing (QS). In Pseudomonas aeruginosa at least three QS systems, which are highly organized and interconnected, have been described2. Las and rhl quorum sensing systems are mediated by N-acyl-homoserine lactone (AHL) signals3. The third, quinolone-based quorum QS was described in 1999 by Pesci and his coworkers4. The fourth QS system was described relatively recently as IQS. It should be mentioned that in clinical isolates, IQS can replace the functions of las QS2. P. aeruginosa QS system can be affected by many substances, isolated from natural sources, e.g. by ajoene, sulfur compounds contained in garlic5, salicylic acid6 or monoterpenes isolated from Citrus reticulate7. N-acylhomoserine lactone (AHL)-dependent QS systems of P. aeruginosa can also be inhibited by non-thermal plasma (NTP) treatment8. Plasma is defined as neutral, ionized gas composed of particles (photons, electrons, positive and negative ions, free radicals and excited or non-excited molecules) which are in permanent interaction9. Numerous research groups already published promising results of experiments in which NTP was used for microbial inactivation10 (for example application in dental care11). Ziuzana et. al. described the potential of NTP to reduce the virulence of P. aeruginosa by deactivating the QS controlled virulence factors pyocyanin and elastase B12. The aim of our study was to evaluate the ability of NTP generated by cometary corona with a metallic grid to interfere with the (AHL)-dependent QS system of P. aeruginosa (strains DBM 3777, ATCC 15442, DBM 3181 and ATCC 27853) and subsequent biofilm formation. We applied Agrobacterium tumefaciens NTL4 (pZLR4) biosensor to detect AHL signals in bacterial spent culture supernatants, after the treatment. We proved that NTP significantly affected P. aeruginosa (AHL)-dependent QS system. Levels of AHLs were lower after the NTP application compared to control in all four strains.


57

Material and methods Bacteria strains and culture condition Strains of Pseudomonas aeruginosa DBM 3777 and DBM 3181 were kindly provided by Department of Biochemistry and Microbiology UCT Prague. Strains of P. aeruginosa ATCC 15442, ATCC 27853 and biosensor strain Agrobacterium tumefaciens (Rhizobium radiobacter) NTL4 (pZLR4) ATCC BAA – 2240 were obtained from the American Type Culture Collection. Stock cultures were stored at -70 °C in 50% glycerol solution. All P. aeruginosa strains were grown in Luria-Bertani (LB) liquid medium at 37 °C. Biosensor strain was grown in AB minimal medium (100 µl of 20% w/v sterile glucose solution per 100 ml and 5 mg l-1 of gentamicin were added to the medium before use) at 30 °C. Biofilm cultivation in polystyrene microtiter plates Aliquots of 280 µl of standard bacterial suspension (OD600 nm = 0.60 ± 0.02) in LB medium were cultivated in polystyrene 96-well microtiter plate for 24 h at 37 °C, in an orbital shaker (150 rpm). To detect N-acyl-homoserine lactone (AHL) signals during the cultivation, samples (spent bacterial culture supernatants) were collected in regular intervals. Biofilm cultivation on carries made from titanium alloy (Ti6-Al4-V) and non-thermal plasma treatment For the P. aeruginosa biofilm cultivation (24 h, 37 °C, LB medium), carriers made from titanium alloy (Ti6-Al4-V) were used. Non-adherent cells were removed by washing three times with saline. Biofilm-growing cells on carriers were exposed to NTP treatment generated by cometary corona with a metallic grid (Fig. 1) for 15 and 30 minutes, and then cultivated in fresh LB media for 24 h. To quantify the cells formed in the surroundings of P. aeruginosa biofilm, measurement of optical density (OD) at 600 nm was used. Spent bacterial culture supernatants were collected and stored at -20 °C for N-acyl-homoserine lactone molecules detection by biosensor. Anti-biofilm effect of NTP was evaluated by crystal violet (CV) staining.

Fig. 1.

The arrangement of the device for non-thermal plasma generation (according to Scholtz et al. 2013)

Biosensor assay for the AHL levels determination To detect signal molecules from spent bacterial culture supernatants (after the NTP treatment) Agrobacterium tumefaciens NTL4 (pZLR4) biosensor was applied. A. tumefaciens NTL4 (pZLR4) does not produce its own AHLs, but the reporter gene is induced when exogenous signal molecules are present. Induction of reporter gene leads to production of β-galactosidase, which can be measured spectrophotometrically (660 nm) by X-Gal usage13. The procedure has been modified according to Singh and Greenstein 14. Aliquots of 50 µl of standard biosensor suspension (OD400 nm = 0.50 ± 0.02) in AB medium containing glucose and gentamicin were cultivated in presence of P. aeruginosa supernatants in polystyrene 96-well microtiter plate for 16 -18 h at 30 °C, in an orbital shaker (150 rpm). After cultivation, 50 µl of lysis buffer (4.351 g l-1 MgCl2.6H2O, 200 mg l-1 cetyltrimethylammonium bromide and 800 mg l-1 NaN3 dissolved in phosphate-buffered saline, pH 7.4) was added in to each well. After shaking for 90 minutes, 5 mg of X-Gal dissolved in 1 ml of DMSO was mixed well with 4 ml of lysis buffer, and 50 µl of this solution was added in to the each well. Absorbance of the blue product was measured at 660 nm, after one hour from the start of reaction.

58


Crystal violet staining The total biofilm biomass formed on carriers made from titanium alloy (Ti6-Al4-V) was quantified using crystal violet staining. Non-adherent cells were removed from carriers by washing three times with saline. To all carriers, 2 ml of 0.1% crystal violet filtered solution were added. After 20 minutes of incubation at room temperature, the carriers were washed again three times with saline to remove unbound dye. Addition of 2 ml of 96 % ethanol was used to elute the bound dye. After 10 minutes, a 100 µl volume of colored solution was transferred to the microtiter plate to measure absorbance (580 nm).

Results and Discussion We have investigated the production of QS signals in P. aeruginosa DBM 3777. A significant increase in AHL production was observed between 4 and 24 hour of P. aeruginosa DBM 3777 cultivation (Fig. 2) in microtiter plate. It is expressed as the level of blue product (spectrophotometric measurement at 660 nm) in Fig. 2. After 48 h of biofilm cultivation, stationary phase was reached, and the level of AHLs remained constant, as well as the amount of suspension cells (measurement of optical density at 600 nm) and total biofilm biomass stained by crystal violet (spectrophotometric measurement at 580 nm).

Fig. 2.

Change in the AHLs levels, amount of suspension cells (OD) and total biofilm biomass (CV) during biofilm cultivation of P. aeruginosa DBM 3777

(AHL)-dependent QS systems of P. aeruginosa (DBM 3777, ATCC 15442) were affected by non-thermal plasma treatment. We proved that NTP has anti-virulence activity as well as in Ziuzina, et al. 12 study, because corresponding decrease in AHL production by P. aeruginosa (DBM 3777, ATCC 15442) and total biofilm biomass were observed after exposure of NTP for 30 minutes (Fig. 3). Inhibition of β-galactosidase activity was stronger in P. aeruginosa ATCC 15442 (by at least 23 %), as well as the inhibition of the biofilm formation (by 61 %), which was determined by crystal violet staining (Fig. 3). We also observed decrease of AHLs levels in clinical isolates P. aeruginosa DBM 3181 and P. aeruginosa ATCC 27853 (Fig. 4). The production of AHL decreased by more than half in P. aeruginosa DBM 3181 due to NTP treatment (30 min). However, we did not confirm the inhibitory effect on suspension cells formed in the surroundings of P. aeruginosa biofilm and on the total biofilm biomass. Values after NTP exposure obtained by optical density measurement (600 nm) and values obtained by spectrophotometric measurement after crystal violet staining (580 nm) does not differ much from the control. After applicationof the NTP (15 min), the absorbance values (580 nm) indicated a slight increase in biofilm formation in both clinical isolates compared to control (Tab. I). Especially in P. aeruginosa clinical isolates may the function of las or rhl QS system be taken over by another QS system. IQS quorum sensing system was discovered relatively recently, its quorum sensing signal molecule was described as 2-(2-hydroxyphenyl)thiazole-4-carbaldehyde. Due to this signal, the expression of virulence factors (in this case biofilm formation) is maintained even when other QS molecules production is suppressed 15, 2.


59

Fig. 3.

Change in the AHLs levels in P. aeruginosa (DBM 3777, ATCC 15442), expressed as the inhibition of βgalactosidase activity (AHL) and total biofilm biomass formed on carriers made from titanium alloy (Ti6Al4-V) inhibition (CV) after NTP treatment (30 min).

Fig. 4.

Change in the AHLs levels in P. aeruginosa DBM 3181 and P. aeruginosa ATCC 27853 (clinical isolates), expressed as β-galactosidase activity (660 nm), after the NTP treatment (15 and 30 min), compared to control

Table I The influence of the NTP treatment (15 and 30 min) on P. aeruginosa (DBM 3181 and ATCC 27853) growth, expressed as the amount of suspension cells (OD) and total biofilm biomass (CV) compared to control control

P. aeruginosa DBM 3181 P. aeruginosa ATCC 27853

60

15 min

30 min

OD (600 nm)

CV (580 nm)

OD (600 nm)

CV (580 nm)

OD (600 nm)

CV (580 nm)

1.85 ± 0.04

0.35 ± 0.06

1.83 ± 0.09

0.42 ± 0.12

1.74 ± 0.10

0.37 ± 0.12

1.64 ± 0.39

0.23 ± 0.05

1.67 ± 0.07

0.36 ± 0.01

1.81 ± 0.02

0.26 ± 0.16


Conclusion A considerable amount of data has been gathered to demonstrate the role of quorum sensing in the onset of P. aeruginosa virulence. (AHL)-dependent QS systems are responsible for biofilm formation and associated infections development. We proved that NTP significantly affected QS system of all four P. aeruginosa strains (DBM 3777, ATCC 15442, DBM 3181 and DBM 27853) used in this study. However, NTP did not affect subsequent biofilm formation of clinical isolates DBM 3181 and ATCC 27853. This effect may be due to activity of another signal molecule that can take over the function of AHL signals, which is quite common in P. aeruginosa. In the future study, we will use the NTP treatment with antibiotics, as combination therapy appears to be a useful tool.

Acknowledgment Financial support from specific university research (MSMT No 20-SVV/2017)

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Gellatly S. L., Hancock R. E.: Pathog. Dis., 67, 159 (2013) Lee J., Zhang L.: Protein & cell, 6, 26 (2015) Pearson J. P., Pesci E. C., Iglewski B. H.: Journal of bacteriology, 179, 5756 (1997) Pesci E. C., Milbank J. B., Pearson J. P., McKnight S., Kende A. S., Greenberg E. P., Iglewski B. H.: Proc. Natl. Acad. Sci., 96, 11229 (1999) Jakobsen T. H., van Gennip M., Phipps R. K., Shanmugham M. S., Christensen L. D., Alhede M., Skindersoe M. E., Rasmussen T. B., Friedrich K., Uthe F.: Antimicrob. Agents Chemother., 56, 2314 (2012) Bandara M. B., Zhu H., Sankaridurg P. R., Willcox M. D.: Invest. Ophthalmol. Visual Sci., 47, 4453 (2006) Luciardi M. C., Blázquez M. A., Cartagena E., Bardón A., Arena M. E.: LWT-Food Sci. Technol., 68, 373 (2016) Flynn P. B., Busetti A., Wielogorska E., Chevallier O. P., Elliott C. T., Laverty G., Gorman S. P., Graham W. G., Gilmore B. F.: Scientific reports, 6 (2016) Alkawareek M. Y., Gorman S. P., Graham W. G., Gilmore B. F.: Int. J. Antimicrob. Agents, 43, 154 (2014) Scholtz V., Kvasničková E., Julák J.: Acta Phys. Pol., A, 124 (2013) Fridman G., Friedman G., Gutsol A., Shekhter A. B., Vasilets V. N., Fridman A.: Plasma Processes Polym., 5, 503 (2008) Ziuzina D., Boehm D., Patil S., Cullen P., Bourke P.: PloS one, 10 (2015) Steindler L., Venturi V.: FEMS Microbiol. Lett., 266, 1 (2007) Singh M. P., Greenstein M.: Journal of microbiological methods, 65, 32 (2006) Lee J., Wu J., Deng Y., Wang J., Wang C., Wang J., Chang C., Dong Y., Williams P., Zhang L.-H.: Nat. Chem. Biol., 9, 339 (2013)


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ANTI-BIOFILM EFFECT OF VITIS VINIFERA EXTRACT ON THE CANDIDA GENUS Paldrychová M.1, Kolouchová I., Masák J.1 1

UCT Prague (Department of Biotechnology), Technická 5, 166 28, Prague 6

Abstract Fungal infections are often caused by the yeasts from the Candida genus. Representatives of the Candida genus grow predominantly as surface-attached stable communities known as biofilms. These cells are highly resistant to antibiotics, which were initially developed to inhibit the growth of planktonic cells. Nowadays a considerable part of research is devoted to finding tools which specifically influence the biofilm-growing cells, in particular the application of natural substances. In this study, we investigated the influence of crude extract from the blue grapes of Vitis vinifera enriched by resveratrol from Polygonum cuspidatum root on the planktonic and biofilmgrowing cells of Candida genus. We used microcultivation device to determine minimum inhibitory concentrations (MIC80) and we applied MTT assay to estimate the cell viability. To quantify and visualize the biofilm formed after 24 hours of the cultivation in the presence of the extract the Cellavista device and crystal violet staining were used. The lowest crude extract concentration preventing cell adhesion and subsequent biofilm formation determined for C. albicans was 1111 mg l-1. This concentration corresponds to the content of resveratrol 100 mg l.

Introduction In contrast to the extensive studies focusing on bacterial biofilms, very limited number of research groups study yeast biofilms. Biofilm-growing cells of Candida albicans are an integral part of the oral and gastrointestinal human microflora or urogenital tract1. Unfortunately C. albicans has a whole arsenal of virulence factors that cause persistent infection in susceptible individuals. These virulence factors include the production of proteases, adhesins and also the ability of ”phenotype switching”1, 2. The phenotype switching is controlled by the QS system in C. albicans and it is mediated by farnesol and tyrosol. Farnesol represses filamentation and biofilm formation, on the other hand tyrosol stimulates the formation of germ tubes and the development of hyphae. Hyphae formation is important for C. albicans onset of virulence3. Susceptible individuals may also be attacked by Candida parapsilosis, this yeast is also associated with nosocomial infections, especially with infections related to vascular devices4. Candida krusei is also an opportunistic pathogen, often resistant to fluconazole and has decreased susceptibility to amphotericin B. This yeast is associated with the highest mortality rate, in comparison with infections caused by other Candida species5. Nowadays, the activity of polyphenols against a broad range of microorganisms, e.g. Candida genus is intensely studied as a possible alternative to conventional antibiotic therapy. Antimicrobial activity of many natural substances, as well as polyphenols are known to increase significantly when applied as mixtures, rather than individual compounds. Flavonoids (one of the polyphenolic groups) increase the bioavailability of coadministered substances6. In Xu, et al. 7 study antimicrobial activities of pure catechin and quercetin, they were not so effective as the usage of crude phenolic extracts from Vitis rotundifolia against food borne pathogens. In current study, we applied crude extract from the blue grapes of Vitis vinifera enriched by resveratrol from Polygonum cuspidatum root on the planktonic and biofilm-growing cells of Candida albicans, Candida parapsilosis and Candida krusei. We compared the effects of this extract, in which resveratrol content is 9 %, with the effects of pure resveratrol and fluconazole.

Material and methods Bacteria strains and culture condition Strains of Candida albicans DBM 2164 and Candida parapsilosis DBM 2165 were kindly provided by Department of Biochemistry and Microbiology UCT Prague. Strain of Candida krusei CCM 8271 was obtained from the Czech Collection of Microorganisms (Faculty of Science, Masaryk University, Brno). Stock cultures were stored at -70 °C in 50% glycerol solution. All Candida strains from the DBM collection were grown in YPD medium at 30 °C. All Candida strains from the CCM were grown in YPD medium at 37 °C. Biologically active agents Extract from the blue grapes of Vitis vinifera enriched by resveratrol from Polygonum cuspidatum root was kindly provided by Interpharma Praha, Czech Republic. It has a proved amount of resveratrol (9 %) and contains other

62


biologically active substances (resveratrol glycosides, procyanidins, catechin, quercetin and quercetin glucoside). Stock solutions were prepared by dissolving in DMSO. Pure resveratrol was purchased from Sigma-Aldrich and dissolved in DMSO. The content of DMSO was lower than 1% in all assays, to not interfere with the growth of yeast. To allow comparison, the numerical value of concentration of Vitis vinifera extract represents the concentration of resveratrol in all experiments. Fluconazole was purchased from Sigma-Aldrich and was dissolved in sterile YPD medium before use. Minimum inhibitory concentration (MIC80) determination The influence of Vitis vinifera extract (0 – 2222 mg l-1) on yeast growth was investigated by micro-cultivation in microtiter plate in Bioscreen C device (Oy Growth Curves Ab Ltd., Finland). A 30 µl volume of standard cell suspension of yeast (OD600nm = 0.10 ± 0.02) was added into each well. Controls without Vitis vinifera extract were included. Each experiment was performed in five replicates. According to the definition by Andrews (2001) minimum inhibitory concentrations (MIC80) were determined, as the lowest concentration that causes at least 80% decrease in growth after overnight cultivation8. Biofilm cultivation in polystyrene microtiter plates Aliquots of 280 µl of standard yeast suspension (OD600nm = 0.60 ± 0.02) in YPD medium were cultivated in the presence of Vitis vinifera extract, resveratrol and fluconazole in polystyrene 96-well microtiter plate (TPP AG, Switzerland), for 24 h at 30/37 °C, in an orbital shaker (150 rpm). Concentration ranges were 0 – 2222 mg l-1 for Vitis vinifera extract, 0 – 200 mg l-1 for resveratrol and 0 – 100 mg l-1 for fluconazole. Controls without agent were also included. Each experiment was performed in eight replicates. MTT assay The metabolic activity of biofilm-growing cells was determined by MTT assay according to Riss, et al. 9. Nonadherent cells were removed from wells by washing three times with saline. To the each well 50 μl of MTT (1 mg/ml) and 60 µl glucose (57.4 mg/ml) were added. After 3 hours, 100 µl of solubilization solution was added to each well to dissolve formazan crystals (formed by biofilm-growing cells, which are metabolically active, from MTT). Then 100 µl volume of this solution was transferred to the microtiter plate (Gama, Czech Republic) and the absorbance (570 nm) was measured using a microtiter plate reader (Tecan, Switzerland). Crystal violet staining The total biofilm biomass formed in polystyrene microtiter plate was quantified using crystal violet staining. Nonadherent cells were removed from wells by washing three times with saline. To the each well, 200 µl of 0.1% crystal violet filtered solution were added. After 20 minutes of incubation at room temperature, the wells were washed again three times with saline, the saline was then replaced by 200 µL of 96% ethanol and after 10 minutes a 100 µl volume of colored solution was transferred to the microtiter plate (Gama, Czech Republic) to measure absorbance (580 nm) by microtiter plate reader (Tecan MTP, Switzerland). Light microscopy - Cellavista device From eight parallels one representative sample was selected and the area populated by biofilm was visualized by Cellavista device, as previously described Kvasnickova, et al. 10. Cellavista device (Roche, Switzerland) is a fully automated inverted microscope, which allows analysis of the 96-well microtiter plates.

Results and Discussion Candida albicans DBM 2164 We have investigated the Candida albicans DBM 2164 biofilm formation. After two hours, the yeast cells adhere to the surface of the carrier, micro-colonies appear after four hours of biofilm cultivation. Growing community of yeast are being created after eight hours of cultivation (Fig. 1A). The middle phase of biofilm formation (12 h) is characterized by extracellular polymeric substances (EPS) production. We investigated the biofilm formation in the presence of the Vitis vinifera extract (resveratrol content 0 - 200 mg l-1) after 24 hours of cultivation by Cellavista device. The lowest applied concentration (resveratrol content 50 mg l-1) significantly affected the C. albicans DBM 2164 adhesion (Fig. 1B) and the metabolic activity of adherent cells (Fig. 2A). The lowest crude extract concentration preventing cell adhesion and subsequent biofilm formation determined for C. albicans by MTT assay, was 1111 mg l-1 (data not shown). This concentration corresponds to the content of resveratrol 100 mg l-1. After the application of pure resveratrol (50 mg l-1), such significant decline in metabolic activity of the adherent cells (as after the crude extract application) was not observed (Fig. 2B). This effect can be caused


63

by additional substances (e.g. trihydroxyoctadecanoic acid) which are presented in the Vitis vinifera extract. Nigam, et al. 11 showed that 3-OH-oxylipins, can affect quorum sensing system of Candida albicans. A: 0.25 h

Fig. 1.

2h

4h

8h

B: control

B

0.4 0.3 0.2 0.1 0 control

50

resveratrol in Vitis vinifera extract (mg l-1)

metabolic activity of biofilm (570 nm)

metabolic activity of biofilm (570 nm)

200 mg l-1

Candida albicans DBM 2164 biofilm formation visualized by Cellavista device (A), the influence of Vitis vinifera extract (concentration of resveratrol 50 and 200 mg l-1) on C. albicans DBM 2164 biofilm, biofilm cultivation in the presence of biologically active substances (BAL) 24 h, 30 °C (B), scale bar 100 µm A

Fig 2.

50 mg l-1

0.4 0.3 0.2 0.1 0 control

50

pure resveratrol (mg l-1)

The influence of Vitis vinifera extract - concentration of resveratrol 50 mg l-1 (A) and the influence of pure resveratrol on C. albicans DBM 2164 metabolic activity of adherent cells determined by MTT assay (B), biofilm cultivation in the presence of biologically active substances (BAL) 24 h, 30 °C

Candida parapsilosis DBM 2165 In initial screening we compared the activity of Vitis vinifera extract (content of resveratrol 0 – 150 mg l-1) with fluconazole (0 – 100 mg l-1) commonly used antifungal agent, against suspension cells of C. parapsilosis DBM 2165. The minimum inhibitory concentration (MIC 80) of resveratrol was not found in this concentration range (Tab. I). Lee et al. (2005)12 determined this value for Candida albicans at 250 mg l-1. The fluconazole MIC80 value (25 mg l-1) coincides with the MIC range (0,5 – 65 mg l-1) reported in Swinne et al. study 13. It is generally known that biofilm-growing cells are more resistant to treatment with commonly used antibiotics (fluconazole) than their suspension counterparts. Our results also confirmed this assumption. After the application of fluconazole (50 mg l-1), metabolic activity of biofilm-growing cells was inhibited by only 50 %, and after the application of Vitis vinifera extract (resveratrol content 50 mg l-1) by 66 % (Fig. 3). Table I Minimum inhibitory concentration (MIC80) of biologically active agents for suspension cells of Candida parapsilosis DBM 2165 Biologically active agents Vitis vinifera extract (resveratrol concentration) Fluconazole

64

MIC80 (mg l-1) >150 25


metabolic activity of biofilm inhibition (%)

resveratrol

fluconazole

100 80 60 40 20 0 5O

75

100

biologically active agents (mg Fig. 3

l-1)

The influence of Vitis vinifera extract (resveratrol content 50 – 100 mg l-1), and fluconazole (50 – 100 mg l-1) on the metabolic activity of the Candida parapsilosis DBM 2165 biofilm, determined by MTT assay, biofilm cultivation in the presence of biologically active substances (BAL) 24 h, 30 °C, values are expressed as inhibition rate of metabolic activity compared to control

Candida krusei CCM 8271 We have shown (by crystal violet staining) that additional susbstances presented in Vitis vinifera extract may improve the anti-biofilm effect of resveratrol. Biofilm formation of C. krusei CCM 8271 was more significantly inhibited by the effect of Vitis vinifera extract than pure resveratrol (Fig. 4). Procyanidins, compounds which are presented in Vitis vinifera extract might be the possible reason for increased activity of Vitis vinifera extract. In the Riihinen study (2014)14 procyanidins applied in mixture inhibited biofilm formation of S. mutans, while the corresponding pure substances showed reduced activity.

total biofilm biomass inhibition (%)

resveratrol in Vitis vinifera extract

pure resveratrol

100 80 60 40 20 0 50

75

100

biologically acitive agents (mg Fig. 4

150

200

l-1)

The influence of Vitis vinifera extract (resveratrol content 50 – 200 mg l-1), and pure resveratrol (50 – 200 mg l-1) on the total biofilm biomass of Canida krusei CCM 8271, determined by crystal violet staining, biofilm cultivation in the presence of biologically active substances (BAL) 24 h, 37 °C, values are expressed as total biofilm biomass inhibition compared to control


65

Conclusion The extract from Vitis vinifera grapes enriched by resveratrol from Polygonum cuspidatum root reduced the area populated by biofilm-growing cells from the Candida genus effectively. Significant inhibitory effect on the total biofilm biomass as well as on the metabolic activity of Candida cells were proven. We noticed the more pronounced inhibitory effect of Vitis vinifera extract on biofilm-growing cells than for pure resveratrol. This effect can be caused by additional substances (e.g. trihydroxy-octadecanoic acid or procyanidins) presented in the Vitis vinifera extract. In C. parapsilosis DBM 2165 we observed higher efficacy of Vitis vinifera extract, compared to fluconazole (for biofilm-growing cells). The lowest Vitis vinifera extract concentration preventing cell adhesion and subsequent biofilm formation was determined for C. albicans DBM 2164 and corresponds to the content of resveratrol 100 mg l-1. In the future study, the effect of trihydroxy-octadecanoic acid on Candida albicans quorum sensing system will be evaluated as well as the effect of different Vitis vinifera extracts on this biofilm.

Acknowledgment Financial support from specific university research (MSMT No 20-SVV/2017)

References

6. 7. 8. 9. 10. 11. 12. 13. 14.

Calderone R. A., Fonzi W. A.: Trends Microbiol., 9, 327 (2001) Kruppa M.: Mycoses, 52, 1 (2009) De Sordi L., Mühlschlegel F. A.: FEMS Yeast Res., 9, 990 (2009) Garzoni C., Nobre V., Garbino J.: Eur. J. Clin. Microbiol. Infect. Dis., 26, 915 (2007) Cantón E., Pemán J., Valentín A., Bosch M., Espinel-Ingroff A., Gobernado M.: Diagn. Microbiol. Infect. Dis. 62, 177 (2008) Seleem D., Pardi V., Murata R. M.: Arch. Oral Biol., 76, 76 (2017) Xu C., Yagiz Y., Hsu W.-Y., Simonne A., Lu J., Marshall M. R.: J. Agric. Food Chem., 62, 6640 (2014) Andrews J. M.: J. Antimicrob. Chemother., 48, 5 (2001) Riss T. L., Moravec R. A., Niles A. L., Benink H. A., Worzella T. J., Minor L.: Cell viability assays 2015 Kvasnickova E., Matatkova O., Cejkova A., Masak J.: J. Microbiol. Methods, 118, 106 (2015) Nigam S., Ciccoli R., Ivanov I., Sczepanski M., Deva R.: Curr. Microbiol., 62, 55 (2011) Lee S., Lee H., Min H., Park E., Lee K., Ahn Y., Cho Y., Pyee J.: Fitoterapia 76, 258 (2005) Swinne D., Watelle M., Nolard N.: Rev. Iberoam. Micol. 22, 24 (2005) Riihinen K. R., Ou Z. M., Gödecke T., Lankin D. C., Pauli G. F., Wu C. D.: Fitoterapia, 97, 78 (2014)

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1. 2. 3. 4. 5.

BIODEGRADATION OF CHICKEN FEATHER BY POLYHYDROXYALKANOATES ACCUMULATING BACTERIA Pernicová I.1,2, Šuráňová Z.2, Innemanova P.3,4, Obruča S.1,2 Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic Institute of Food Science and Biotechnology, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic 3 Dekonta, a.s., Dřetovice 109, 273 42 Stehelčeves, Czech Republic 4 Institute of Environmental Studies, Faculty of Science Charles University in Prague, Benátská 2, 128 01, Prague 2, Czech Republic [email protected] 1 2

Abstract Millions of tons of feather, important waste product of poultry-processing industry, are disposed of annually without any further benefit. Therefore, the main aim of this work is to study biodegradation of chicken feather by selected Pseudomonas strains revealing keratinase activity and which are also capable of polyhydroxyalkanoates accumulation. Non-treated chicken feather was used as the sole carbon substrate for cultivation of bacteria Psudomonas putida KT2440 and two strains isolated from petroleum polluted areas which were identified as Pseudomonas fulva and Pseudomonas gessardii. Enzymatic activity of proteinase and keratinase was determined in cultivation media. Pseuodomonas putida demonstrated the highest degradation enzymatic activity. Further, bacterial culture grown on waste feather can be used as inoculum for the production of PHA using waste frying oil as a substrate and and octanoic acid as a precursor of MCL-PHA. In this condition, Pseudomonas gessardii creates 6 and 8 carbon PHA.

Introduction Polyhydroxyalkanoates (PHA) are biodegradable and biocompatible “green” polymers, which are synthesized by a wide variety of bacteria. Generally, PHAs are friendly to environment and, moreover, they possess suitable chemical physical properties to be considered as an alternative the commonly used plastics produced from petrochemical resources. PHA polymer can be used as bottles, containers or other packaging, but also in medicine (surgical sutures), in the pharmaceutical industry as a biodegradable scaffolds for drugs or hormones targeted delivery. In this context, their biodegradability is their main advantage. PHAs can be divided according to the number of carbon atoms in the monomer. First group is called short chain length (SCL) and monomer consists of 3 until 5 carbon. Monomer unit with 6 – 14 carbon is called medium chain length (MCL) 1. Bacteria accumulate PHAs as intracellular granules when external carbon source is present in excess and other nutrients such as nitrogen or phosphorus are limiting. The granules of PHA are further used by bacteria as a source of carbon and energy when extracellular carbon sources are exhausted 2. Important waste by-product of poultry-processing industry is millions of tons of feather. Classical methods of elimination of feather, such as incineration or landfilling, are non-ecological and even they are banned in some countries. Feathers are rich in keratin which contains cysteine, arginine, threonine and hydrophobic amino acids, keratin has high nutrient potential. Feather keratin could be used in animal feeds, fertilizers or feed supplements. Individual amino acids could be obtained, for example, by degradation of keratin by keratinaseproducing bacteria, keratinase are responsible for degradation of keratin 3, 4. In this work we aimed at potential biodegradation of waste chicken feather by Pseudomonas putida KT2440 and two strains isolated from petroleum polluted areas Pseudomonas fulva and Pseudomonas gessardii. Further, bacterial biomass grown of waste feather was subsequently used for MCL-PHA production.

Materials and method Non-treated chicken feather was used as the solo carbon substrate during cultivation of bacteria in mineral medium. Cultivation was carried out for 7 days. Total loss of feather, biomass and pH was determined in cultivation media. Supernatant of cultivation medium was used for determination of enzymatic activity. Bacterial culture growth in mineral medium with chicken feathers was used as inoculum for PHA accumulation. Enzymatic activities of proteinase and keratinase were determined spectrophotometrically in cultivation media with feather as the sole substrate. Bacterial culture with feather was used as inoculum for the accumulation of PHA. Cultivation for the accumulation of PHA was performed in mineral medium with 20 g∙l-1 oil and 2g∙l-1 octanoic acid. Biomass yields were determined gravimetrically and PHA content in bacterial biomass was measured by GC-FID.


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Table I Composition of MCL-PHA production medium Composition of production medium NH4SO4 Na2HPO4 ∙ 12 H2O KH2PO4 MgSO4 ∙ 7 H2O MES (solution of trace) Vegetable oil After 24 hours Octanoic acid

g∙l-1 1 or 0 11.1 1.05 0.2 1 ml∙l-1 20 2

Results Cultivation of the bacterial strains on the waste chicken feather as the sole carbon source (20 g∙l-1) was carried for 7 days. During the cultivation, biomass growth, pH change, total loss of feather and enzymatic activity were measured. It was confirmed that all the three bacteria are able to degrade and utilize waste feather. Generally, the course of feather degradation did not correlate with bacterial biomass. Even though the bacterial biomass decreased at the end of the week, the total loss of feather continued during whole experiment. Among bacterial strains tested, Pseudomonas putida demonstrated the highest degradation ability

Figure 1. Total loss of feather - PP – Pseudomonas putida, D2 – Pseudomonas gessardii, D3 – Pseudomonas fulva Enzymatic degradation of feather is accompanied by peptide and amino acid release, biodegradation reduces the volume and weight of the feather and also changes its appearance. After the cultivation, the feathers changed into slurry mass with small pieces of non-degraded feather. When degraded by Pseudomonas putida, feather was almost completely spread.

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Figure 2. Feather after biodegradation, PP – Pseudomonas putida, D2 – Pseudomonas gessardii, D3 – Pseudomonas fulva Degradation of feather is causes of proteinase, enzymes that degrade proteins, in particular by keratinase which are responsible for degradation of keratin – the main component of feathers. Therefore, the enzyme activities of proteinases and keratinases were measured in the supernatant of the culture medium. The time course of enzyme activities for the tested bacterial strains is different. The highest proteinase activity was observed in Pseudomonas putida and reached its maximum on the 5th day of cultivation. On the contrary, the lowest activity was detected in Pseudomonas fulva which correspond to its slow degradation of feather. The structure of keratin is highly resistant to proteolytic cleavage and requires high keretinase activity. The highest keratinase activity was again observed in Pseudomonas putida which is associated with its highest loss of feathers. The keratinase activity of all bacteria declined since the 5th day of cultivation; this may be due to inhibition of bacterial strains as a consequence of an increase of pH. Proteinase and keratinase present in culture supernatant can be considered as interesting side product of microbial biodegradation of waste chicken feather.

Figure 3. Enzymatic activity - PP – Pseudomonas putida, D2 – Pseudomonas gessardii, D3 – Pseudomonas fulva Furthermore, our intent was to link feather degradation with PHA production. The production of PHA directly in the degradation medium is not possible due to the high nitrogen content. That’s why the biomass was grown in the degradation of feather as inoculum to produce PHA. PHA was produced using 20 g∙l-1 of waste frying oil as a carbon substrate and 2 g∙l-1 octanoic acid was added to the culture medium to induce production MCLPHA. Moreover, production was also supported by the complete nitrogen limitation in the production media (ammonium sulfate was completely omitted). Each day of production, the percentage of PHA in biomass was determined by gas chromatography. P. fulva (D3) accumulated MCL-PHA consisting of 6C and 8C monomers in medium without (NH4)2SO4 but its biomass was higher in medium with inorganic nitrogen source. Oppositely, Pseudomonas putida had higher biomass in media in which nitrogen source was completely omitted and also in this case the bacterial strains MCL-PHA content in bacterial biomass reached 53 wt. % (8C) in medium without inorganic nitrogen source.


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Figure 4. Biomass – accumulation of MCL-PHA, PP – Pseudomonas putida, PP_without (NH4)2SO4 Pseudomonas putida in cultivation medium without (NH4)2SO4, D2 – Pseudomonas gessardii, D2_without (NH4)2SO4 - Pseudomonas gessardii in cultivation medium without (NH4)2SO4, D3 – Pseudomonas fulva, D3_without (NH4)2SO4 - Pseudomonas fulva in cultivation medium without (NH4)2SO4

Figure 5. Total PHA content in biomass, PP – Pseudomonas putida, PP_without (NH4)2SO4 - Pseudomonas putida in cultivation medium without (NH4)2SO4, D2 – Pseudomonas gessardii, D2_without (NH4)2SO4 - Pseudomonas gessardii in cultivation medium without (NH4)2SO4, D3 – Pseudomonas fulva, D3_without (NH4)2SO4 Pseudomonas fulva in cultivation medium without (NH4)2SO4

Conclusion All the tested bacterial strains: Psudomonas putida, Pseudomonas fulva and Pseudomonas gessardii were able to degrade feather and produce keratinase as an interesting side product which can be simply isolated from the supernatant. Pseudomonas putida revealed the highest degradation ability (loss of feather reached almost

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28%) and had also the highest enzymatic activity. Bacterial biomass stemming from feather biodegradation can be subsequently used for MCL-PHA production using waste frying oil as a substrate. Using this strategy and P. putida, very high MCL-PHA (8C) content in bacterial biomass (53 %) can be reached.

Acknowledgement This work was supported by the project Materials Research Centre at FCH BUT—Sustainability and Development no.LO1211 and national COST project LD15031 of the Ministry of Education, Youth and Sports of the Czech Republic and by the project GA15-20645S of the Czech Science Foundation (GACR).

References 1. 2. 3.

Krueger et al., Electronic Journal of Biotechnology, 2012, vol. 15, No. 3. Obruca et al., Folia Microbiologica, 2010 vol. 55(1), 17-22 p. Godheja et al., Journal of Bioremediation & Biodegradation, vol. 5. 2014

4.

Stiborova et al., Journal of Chemical Technology and Biotechnology, Vol. 91, 1629–1637 p.


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CENTRIFUGAL PUMP CHARACTERISTICS COMPUTATION AND RELIABILITY EVALUATION AT VARIABLE SPEED DRIVEN Qazizada M. E.1, Pivarčiová E.1, Bialy W.2 Technical University in Zvolen, Faculty of Environmental and Manufacturing Technology, Department of Machinery Control and Automation Technology, Masarykova 24, 960 53 Zvolen, Slovakia 2 Silesian University of Technology, Faculty of Organization and Management, Institute of Production Engineering, 41-800 Zabrze, ul. Roosevelta, Poland [email protected] 1

Abstract In this paper, reliability of a centrifugal pump at different rotational speeds and flow rates are discussed. This article concentrates, methods related to the reliability analysis of pumping system operation by a frequency converter computer controlled located in interface box. Centrifugal pumps are often controlled by adjusting their rotational speed, which affects the resulting flow rate and output pressure of the pumped fluid. Initially, the determination of energy efficiency– and reliability–based limits for the recommendable operating region of a variable speed–driven centrifugal pump is discussed. The main focus is on the free axis type centrifugal pumps, but the studied methods can also be feasible with axial flow pump, gear pump and peripheral flow pump, if allowed by their characteristics. In addition the head and flow of fluid transported at different frequencies of rotation, to produce a map of reliability characteristic curves to verify the similarity rules for pumps recommendable operating region of a variable speed driven (VSD) discussed.

Introduction From a reliability point of view centrifugal pumps has received considerable attention in recent years. Centrifugal pumps are one of the most important components in any apparatus which have to deal with fluids as essential part of its industry. The reliability and maintainability of centrifugal pump systems have in the overall device availability plays a very important role of a suitable maintenance strategy (Sentyakov et al, 2013). The comprehensive evaluation of pumps reliability not only requires a detailed evaluation of the history and operation of the pumps, but also an evaluation of the machinery support and operation functions procedure (Bloch, 1998). The main objective of research is the effect of rotational speed on the pump energy consumption and reliability. The results show that the recommendable operating region of a VSD centrifugal pump can be determined with energy efficiency and reliability-based criteria, and the limits of this operating region are strongly dependent on the rotational speed of the pump. This article is focusing on the free axis type centrifugal pumps, but the studied methods can also be feasible with axial flow pump, gear pump and peripheral flow pump, if allowed by their characteristics (Ahonen, 2011).

Theoretical overview and applied formulas A brief overview on centrifugal pump characteristics such as net head, power, efficiency, net position suction head, and Laws of Similarity are discussed: Net head H is the height of the fluid column in the open pipe after the pump (Blišťan 2011). The device characteristic indicates the delivery head, which is necessary for delivering the fluid against the existing resistances in the piping for any flow rates (Benra, 2013).

𝐻𝐻 =

𝑃𝑃2 −𝑃𝑃1 𝜌𝜌𝜌𝜌

(1)

Where P1 and P2 shows the pressure drop of the pump system, ρ is fluid density, g is the acceleration due gravity. Or specific work єw done by the pump is:

𝜀𝜀𝑤𝑤 = 𝐻𝐻𝐻𝐻 =

𝑃𝑃2 −𝑃𝑃1 𝜌𝜌

(2)

The power imparted into a fluid will increase the energy of the fluid per unit volume. Thus the power relationship is between the conversion of the mechanical energy of the pump mechanism and the fluid elements within the pump (Larralde, 2010). The power curves show the energy transfer rate as a function of flow. Distinction is made between three kinds of power (Chalghoum, 2016): • Supplied power from external electricity source to the motor and controller.

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•

Shaft power transferred from the motor to the shaft.

• Hydraulic power transferred from the impeller to the fluid The power given to the fluid Pw is denominated hydraulic power or output power and calculated:

𝑃𝑃𝑤𝑤 = 𝜌𝜌𝜌𝜌𝜌𝜌𝜌𝜌

(3)

Where H is the head, Q is flow rate of liquid, ρ is fluid density, g is the acceleration due gravity. The mechanical power Pf given to the pump by the activator motor is denominated control power or input power and can be calculated as:

𝑃𝑃𝑓𝑓 = 𝜔𝜔 𝑇𝑇 = 2π nT 60

Pf =

2𝜋𝜋 60

(4)

𝑛𝑛𝑛𝑛

(5)

The ω is the angular axis speed in [rad sec ], n frequency of rotation in [r m ] (rpm, revolutions per minute (abbreviated rpm, RPM, [rev min-1], or [r min-1]) is a measure of the frequency of rotation, and T the torque in the axis in [Nm], to obtain the power in [W] (Haidary, 2013). Pump efficiency η is defined as the ratio of the power imparted on the fluid by the pump in relation to the power supplied to drive the pump (Blišťan, 2011).

𝜂𝜂 =

𝑃𝑃𝑤𝑤 𝑃𝑃𝑓𝑓

=

–1

𝜌𝜌𝜌𝜌𝜌𝜌𝜌𝜌

-1

(6)

2𝜋𝜋 𝑛𝑛𝑛𝑛 60

Where Pw is hydraulic power. Pf is mechanical power, ρ is fluid density, g is the acceleration due gravity. If there are no losses, Pw = Pf, and the efficiency is 100 %, but it is not the case in practice. NPSH has two parts: NPSH required (NPSHR) and NPSH available (NPSHA). (NPSHR) is a function of the pump and it is defined as (Qazizada et al 2016): 𝑃𝑃1 𝜌𝜌𝜌𝜌

𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑅𝑅 = (

+

𝑤𝑤12 2𝑔𝑔

−

𝑃𝑃∗ ) 𝜌𝜌𝜌𝜌

(7)

Where P1 and w1 are the pressure and velocity at the inlet of the pump and P* is the transported liquid’s steam pressure (Sentyakov et al, 2013). Velocity is calculated as: Q

(8)

𝜋𝜋 4

(9)

w=A

Where A, [m ] is the circular surface area of the impeller which is calculated as: 2

𝐴𝐴 = 𝐷𝐷 2

Where D [m] is impeller diameter (Wang, 2016). The pump operation can also be quantified by calculating its specific energy consumption Es [Wh m3], which is especially useful to determine the energy efficiency of different pump flow control methods (Europump, 2004 & Hovstadius, 2005). Typically, Es equals the consumed electric energy per flow volume, which can be determined by:

Es =

Pin Q

(10)

𝑛𝑛 𝑛𝑛𝑛𝑛

(11)

Where Pin is the electric power consumed by the pumping system. The similarity laws express the mathematical relationship between the several variables Involved In pump performance. They apply to all types of centrifugal and axial flow pumps and are reasonably accurate as long as there is some vane tip overlap (Bloch & Allan, 2010).

𝑄𝑄 = ( ) 𝑄𝑄𝑛𝑛 n 2 nn 𝑛𝑛 3

H = ( ) Hn 𝑃𝑃 = ( ) 𝑃𝑃𝑛𝑛 𝑛𝑛𝑛𝑛

𝑛𝑛 2 𝑛𝑛𝑛𝑛

𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑅𝑅 = ( ) 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑅𝑅𝑅𝑅

(12) (13) (14)

Where n is the present rotational speed of the pump and the subscript n denotes the operational value at the nominal speed nn of the pump (Karassik, 1998). The similarity rules are calculated using equations (11, 12, 13, and 14) and using experimental data are shown in (Table I) that can be used to estimate the effect of fluid change, efficiency, speed or size of any dynamic turbo machine (pump or turbine) from a geometrically similar family. The units of flow rate Q [l min-1], head H [m], mechanical power Pf given to the pump [m] and net position suction head required NPSHR [m] are defined. Table I Affinity rules of centrifugal pump


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Table I Affinity rules of centrifugal pump n 2 3 4 5 6

Qn [l min-1] 16,91 15,34 13,65 11,67 10,672

Hn [m] 7,744 6,607 5,833 4,894 4,137

Pfn [W] 288,6 239,45 179,88 150,9 115,97

NPSHRn [m] 1,00 0,856 0,714 0,567 0,502

Values for specific and suction specific speed are often calculated using inconsistent units, and even the applied equations may vary.

nq =

nss =

n√Q H0.75

(15)

n√Q

(16)

NPSHR 0.75

Where the unit of rotational speed is [r m ], the flow rate unit is [m s ], and the head values are in meters (Gülich, 2008). -1

3

–1

Practical part Calculation of centrifugal pump specification values according to mentioned equations: At the begging of measuring experimental data we found tav, for obtaining of average temperature, we measured water temperature t1 = 21 [°C] at the beginning and t2 = 23 [°C] at the end of the experiment, then tav = 22 [°C]. According to average temperature acceleration of gravity and density of water are chosen from physical properties’ table of water, 9,81 [m s–1] 997,9 [kg m–3] respectively. Each measured values calculated only three steps for the number of rotation speed (2600 – 3000 [r m-1]). In 3000 [r m-1] each measured value of Q in (Table II) calculated the net head, output power, input power, and pumping efficiency applying equations (1, 2, 3 and 5). NPSHR is a function of the pump and it calculated via equation (7). For calculating of velocity w [m s–1] and for circular surface area A we used equations (8), (9) respectively. Calculating specific energy consumption Es, equation (10) which is especially useful to determine the energy efficiency .Ratio of (required and available) net suction position of head, specific speed nq and suction specific speed nss is calculated by applying equations (15), (16), All above measured values for 3000 [r m-1] is shown in (Table II) the same process of measurement is done for different rotational set of frequency converter, for (2800 and 2600 [r m-1]) until ten steps revolutions per minute respectively (Edibon, 2014). Table II Calculated data of experimental values N o 1 2 3 4 5 6 7 8 9 10

Q [l min–1] 18,12 17,7 16,38 15,34 13,61 12,16 10,94 8,71 6,77 4,55

N o 1 2 3 4 5 6

Q [l min–1] 16,53 16,06 14,29 13,68 11,96 11,3

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H [m]

Pw [W]

Pf [W]

η%

8,88 8,99 9,09 9,40 9,29 9,29 9,29 9,50 9,80 9,70

26,2 25,9 24,2 23,5 20,6 18,4 16,5 13,5 10,8 7,20

355 311,0 301,5 292,1 282,7 273,3 263,8 254,4 241,9 226,1

7,4 8,3 8,0 8,0 7,3 6,7 6,2 5,3 4,4 3,1

H [m] 7,6 7,9 8,1 8,1 8,4 8,4

Pw [W] 20,6 20,8 19,0 18,2 16,5 15,6

Pf [W]

η%

299 281 284, 284, 269, 272,

6,9 7,4 6,6 6,4 6,1 5,7

n = 3000 [r m-1] NPSHR W [m s–1] [m] 0,427 1,072 0,417 1,031 0,386 0,908 0,361 0,818 0,320 0,682 0,286 0,580 0,257 0,503 0,205 0,383 0,159 0,301 0,107 0,233 -1 n = 2800 [r m ] NPSHR W [m s–1] [m] 0,389 0,922 0,378 0,880 0,336 0,733 0,322 0,687 0,281 0,567 0,266 0,525

Es [W h m–3] 44,1 35,0 38,0 39,4 47,4 55,5 63,9 91,7 132 260

NSPHA/ NPSHR 9,37 9,74 11,0 12,2 14,7 17,3 19,9 26,1 33,2 43,0

Es [W h m–3] 43,6 39,3 49,5 54,0 61,2 70,1

NSPHA/ NPSHR 10,8 11,4 13,6 14,6 17,7 19,1

nq

nss

10,1 9,92 9,46 8,93 8,48 8,02 7,60 6,67 5,74 4,75

49,4 50,3 53,2 55,7 60,1 64,2 67,7 74,1 78,2 77,7

nq

nss

10,81 10,34 9,576 9,370 8,522 8,284

52,9 54,0 58,3 60,0 64,8 66,7


7 8 9 10

9,58 8,24 6,53 4,19

N o 1 2 3 4 5 6 7 8 9 10

Q [l min–1] 14,79 13,78 12,99 10,98 10,05 8,58 7,22 6,55 4,96 3,7

8,3 8,5 8,5 8,7

13,0 11,5 9,14 6,00

H [m] 6,84 7,0 7,0 7,2 7,1 7,2 7,4 7,3 7,5 7,5

Pw [W] 16,5 15,8 14,9 12,9 11,7 10,1 8,78 7,86 6,11 4,56

275, 269 266 272,

4,7 4,2 3,4 2,2

Pf [W]

η%

228, 223, 231, 234, 239 212, 212 215, 212, 225,

7,2 7,0 6,4 5,5 4,8 4,7 4,1 3,6 2,8 2,0

0,225 0,427 0,194 0,362 0,153 0,293 0,098 0,224 n = 2600 [r m-1] NPSHR W [m s–1] [m] 0,348 0,773 0,324 0,694 0,306 0,637 0,258 0,505 0,236 0,452 0,202 0,377 0,170 0,319 0,154 0,293 0,116 0,244 0,087 0,214

100 127 198 492,

23,5 27,7 34,2 44,6

Es [W h m– 3 ] 35,6 38,0 46,0 64,0 81,1 86,2 118 149 247 504

NSPHA/ NPSHR 12,9 14,4 15,7 19,8 22,2 26,6 31,4 34,1 41,1 46,8

7,697 7,010 6,241 4,911

71,7 75,3 78,5 76,7

nq

nss

11,12 10,50 10,20 9,180 8,877 8,115 7,291 7,016 5,981 5,166

57,1 59,7 61,8 67,6 70,3 74,4 77,5 78,5 78,5 74,7

In the ideal case for a VSD pump in six steps, the system curve consists mainly of the dynamic head, and the pump typically operates at its best efficiency point. If the system curve has a notable share of static head, a change in the rotational speed can change the operating point into a location with a lower efficiency. Efficiency decreases affected pumps reliability, the resulting operating point locations with two different system curve shapes is given in (Figure 1). Pump and system curves of centrifugal pump 9

12

8

10

7

8

5

6

4

4

3 2

2

1 0

0

5

10

15

20

η [%]

H [m]

6

2000 3000 2800 3000 2800 2600 2400 2200

0

Flow [l min-1] Figure 1. Resulting operating points, when the system curve shape varies and the pump is driven at different rotational speeds.

Results and discussion In this research the effect of rotational speed on the pump energy consumption and reliability is studied and laboratory test results are introduced. The results show that the recommendable operating region of a VSD centrifugal pump can be determined with energy efficiency, reliability-based criteria, and the limits of this operating region are strongly dependent on the rotational speed of the pump. Each measured values calculated only three steps for the number of rotation speed (2600 ÷ 3000 [r m-1]). In 3000 [r m-1] each measured value of Q in (Table II) calculated the net head, output power, input power and pumping efficiency applying equations (1, 2, 3 and 5). In (Table II) the calculation results show the decreases of efficiency in three steps of rotation from (3000 – 2600 [r m-1]) have negative effects on pump’s reliability, especially whenever the pump driven at 2600 [r m-1] or slow revolutions per minute. NPSHR is a function of the pump and it calculated via equation (7) 1,072 m. For calculating of velocity w, [m s-1] and for circular surface area A we used equations (8), (9), then we obtain 0,427 [m s-1] and 0, 0007 [m2] respectively. Calculating specific energy consumption Es, equation (10) which is


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For calculating of velocity w, [m s-1] and for circular surface area A we used equations (8), (9), then we obtain 0,427 [m s-1] and 0, 0007 [m2] respectively. Calculating specific energy consumption Es, equation (10) which is especially useful to determine the energy efficiency is 44.1 [Wh m-3]. Ratio of (required and available) NPSHR/NPSHA net suction position of head, specific speed nq and suction specific speed nss is calculated by applying equations (15), (16), All above measured values for 3000 [r m-1] is shown in (Table II) the same process of measurement is done for different rotational set of frequency converter, for (2800, and 2600 [r m-1]) until ten steps revolutions per minute respectively. The similarity rules are calculated using equations (11, 12, 13, and 14) and using experimental data are shown in (Table I) that can be used to estimate the effect of fluid change, efficiency, speed, or size of any dynamic pump or turbine from a geometrically similar family. If the pump is driven outside this operating region, the pump efficiency decreases and it may be susceptible to harmful phenomena.

Conclusion The issue of study was the reliability of a centrifugal pump at different rotational speeds and flow rates. The use of common energy efficiency and reliability based criteria in the determination of the recommendable operating region of a VSD centrifugal pump was investigated. And the monitoring of a centrifugal pump operation utilizing the information available from a frequency converter. Variable-speed-driven centrifugal pumps are widely used in industrial and municipal applications, and often the pump may be driven in adverse operating conditions, which can lead to an increase in the energy and maintenance costs for the pumping system. Although the study focuses on radial flow end suction centrifugal pumps, the studied methods can also be applied to other centrifugal pump types, such as gear flow, peripheral flow and axial flow pumps, if allowed by their characteristics. The research as well discussed the estimation of the pump operating point location, which can be performed by a frequency converter. The obtained results showed the applicability of typical-based methods to the operating point estimation without additional measurements.

Acknowledgments This paper was prepared within the work on a research project KEGA MŠ SR 003TU Z-4/2016: Research and education laboratory for robotics.

References 1.

Ahonen, T.: Monitoring of centrifugal pump operation by a frequency converter. Finland 2011. ISSN: 1456– 4491 2. Bloch, H. P.: Improving Machinery Reliability. USA, 1998. ISBN 0–88415–661–3 3. Bloch, H. P.: & Allan, R. B.: Pump User’s Handbook Life Extension 3rd Edition. Indian Trail 2010. ISBN–10: 0– 88173–627–9 4. Blišt'an, P. & Pacaiova, H.: Modelling environmental influence on the pipelines integrity. 11th International Multidisciplinary Scientific Geo conference and EXPO, Varna; Bulgaria 2011. Volume 2, Pages 645–652. Code 101584 5. Benra, F. K.: Measurement of the characteristics of a centrifugal pump. Rhine–Ruhr 2013. available in https://www.uni–due.de/sm/Downloads/Praktika/ Centrifugal_Pump.pdf 6. Chalghoum, I.: Transient behaviour of a centrifugal pump during starting period. Tunisia 2016. available from http://ac.els–cdn.com/S0003682X16300238/1–s2.0–S0003682X16300238–main.pdf?_tid= 0ce4b 116–1824–11e6–99c8–00000aab0f01&acdnat=1463045559_ba8aef040e0a29877c1ea1fe568ac0d8 7. Euro, P. & Hydraulic I.: Variable Speed Pumping. U.S. Department of Energy Industrial Technologies Program Washington, D.C 2004. EERE Information Center. Available in http://www. pumpfundamentals.com/variable_speed_pumping %20Hyd%20Inst%20summary%20guide.pdf 8. EDIBON: Technical Teaching Equipment. Computer Controlled Multi pump Testing Bench with SCADA. Spain 2014. 9. Gülich, J. F.: Centrifugal Pumps. Springer–Verlag. Berlin. Germany 2008. 10. Haidary, J.: MANUAL. Chemical engineering laboratory. Kabul University Publisher (2013) 11. Hovstadius, G., Tutterow, V. & Bolles, S.: Getting it Right. Applying a Systems Approach to Variable Speed Pumping. In Proceedings of the 4th International Conference on Energy Efficiency in Motor Driven Systems (EEMODS ‘05). Heidelberg. Germany 2005. 12. Karassik, I. J. & Mcguire, T.: Centrifugal Pumps, second edition. Chapman & Hall, New York, USA 1998.

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13. Larralde, E. & Ocampo, R.: Centrifugal pump selection process: Pages 24–28. Vol. 2. (2010). Available in: http://ac.els–cdn.com/S0262176210700298/1–s2.0–S0262176210700298–main.pdf?_tid=bb4f322c– 1823–11e6–b16b–00000aab0f02 &acdnat=1463045422_feccae9ea7c5efa40934b952b18f95de 14. Qazizada, M.E., Sviatskii,V., & Božek, P.: Analysis performance characteristics of centrifugal pump, Zvolen, Slovakia 2016 15. Sentyakov, B. A., Sviatskii, V. M., & Sentyakov, K. B.: Calculation of the average velocity and modelling the air flow in the working area of the blow head, Automation and modern technologies, № 6, Moscow 2013. Pages 20–24. ISSN 1585–1558 16. Wang, D.: Application of the two–phase three–component computational model to predict activating flow in a centrifugal pump and its validation. Xuefu Road, Zhenjiang China 2016. Available in: http://www.sciencedirect.com/science/ article/pii/S0045793016300834


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FATTY ACIDS PROFILE ANALYSIS IN FRESH SUSPENSION OF JAPONOCHYTRIUM SP. Rouskova M.1, Maleterova Y.1, Hurkova K.2, Kastanek P.3, Hanika J.1, Solcova O.1 Institute of Chemical Process Fundamentals, CAS, Rozvojova 135, 165 02 Prague 6 University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague 6 3 Ecofuel Labs., Ocelarska 9, 190 00 Prague 9 [email protected] 1 2

Abstract Japonochytrium sp. represents the unicellular marine fungi, which doesn’t contain chlorophylls in its biomass. It accumulates high proportion of lipids, containing a significant amount of omega-3 polyunsaturated fatty acids, especially DHA (docosahexaenoic acid, C22:6n3). These valued components should be isolated and applied as components in food supplements, cosmetics or pharmaceutical products. The study is focused on concentration of a lipid content and determination of the extractable part oriented on included fatty acids in separated phases of centrifuged wet biomass. The main attention was focused on the DHA concentration from the upper lipid layer, containing obviously concentrated proportion of fatty acids. Water fungi suspension was submitted to centrifugation with subsequent separation of individual liquid/solid fractions and extracted by two solvent systems. Dichloromethane/methanol 1/1 and 1-hexane/ethanol 2/3 was applied. Both solvent systems afforded comparable fatty acids profiles. Dominant acids C16:0 (in the range of 43-47%) and DHA C22:6n3 (43-47%) were detected. Biomass residue can be applied e. g. as an animal food supplement due to high protein content. Also level of pigments with antioxidant effect, particularly carotenoids, can be dietary significant.

Introduction Japonochytrium sp. belongs to the unicellular marine fungi, which is characterized by lipid content with a relatively high proportion of omega-3 unsaturated fatty acid triglycerides1. They have a high nutritional potential with the perspective application in the food industry as a valued dietary supplement. This microorganism is not investigated in detail, yet. The existing studies were focused on morphology and ultrastructure definition2 of a Japonochytrium sp., optimal cultivation conditions3, suitable nutrition4,5 and/or proper strain selection or modification for DHA yield maximisation. The aim of study was focused on optimisation of operating conditions for extractive separation of lipids from fresh biomass suspension of Japonochytrium sp. (produced by the Ecofuel Labs. Ltd. company). The extraction experiments were conducted with regard to the selection of extraction system for maximization of fatty acid production.

Materials and methods Sample preparation and processing A sample of the Japonochytrium sp. water suspension (dry matter 16 g.l-1) was centrifuged to separate three phases: a - an upper lipid layer, b - bottom centrifuged biomass and c- middle aqueous solution. Individual phases were processed as demonstrated in the diagram in figure 1. Biomass was not dried in order to avoid undesirable losses of unstable substances. Two extraction systems were chosen for the extraction experiments. The proportions of components in the extraction solvents were as follows: dichloromethane/methanol 1/1 (v/v), 1-hexane/ethanol 2/3 (v/v). The presence of alcohol was necessary for the processing of wet biomass to limit the possible formation of emulsions or foams. All extraction experiments were carried out in a sealed stirred Erlenmeyer flask protected from light under an inert nitrogen atmosphere at ambient temperature of 23 °C for 4 h. This procedure was suitable for the separation of thermally unstable materials susceptible to oxidation by atmospheric oxygen. Extraction of both, the upper lipid layer and the lower sediment biomass, was performed with the ratio of a raw material:solvent 1:15, whereas in the case of the aqueous phase, the ratio 1:5 was selected. Original suspension of fungi Japonochytrium sp. was separated into individual fractions to identify the phase with concentrated lipid proportion.

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Figure 1.

Scheme of three phases of Japonochytrium sp. centrifuged suspension processing

Analysis of fatty acids The profile of fatty acids contained in lipids was analysed at the Department of Food Analysis and Nutrition of the University of Chemistry and Technology Prague. The obtained extracts were transformed applying saponification to fatty acids methyl esters; and, based on accredited ISO 17025 method, they were analysed using gas chromatography. The presence of fatty acids in the range of C4 to C24 string lengths was examined.

Results and discussion From the mass balance and microscopic observation of the individual phases was noticeable, that centrifugation of 1,000 grams (about 1 litre) of fresh culture Japonochytrium sp. arose three phases in the following proportions: 19.4 g of the upper lipid phase, 941.9 g of aqueous phase and 38.7 g of moist bottom biomass. Extraction of upper lipid layer In this study, the main attention was focused on the extraction of the upper lipid layer, containing obviously the largest proportion of fatty acids. It consists of wet biomass of cells and lipid bodies. Figure 2 shows the weight proportional representation of fatty acids in the lipid layer and in homogeneous extract and/or raffinate after extraction with a mixture of dichloromethane/methanol.

Figure 2.

Proportion of selected fatty acids in lipid layer. Dichloromethane/methanol extraction system was used. a1 – original lipid layer, a2a – organic extract, a2b aqueous raffinate


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It is evident that the lipid layer and extraction products contain in addition to the dominant saturated palmitic acid (C16:0) also omega-3 unsaturated docosahexaenoic acid (C22: 6n3), referred to as DHA. The second valued omega-3 eicosapentaenoic acid (C20:5n3), referred to as EPA, is contained in lipid layer in an insignificant amount (0.5%). In the lipid layer there were further identified 3% of myristic acid (C14: 0), 7-8% of unknown long chain acid and other unidentified components ( 95 %), hexanal (98 %), isobutanal (98 %), isopentanal (98 %), pentanal (97 %), phenylacetylene (98 %) and propanal (97 %) were purchased from Sigma Aldrich, acetone (p.a.), dimethylformamid (p.a.), methylalcohol (p.a.), tetrahydrofuran (p.a.) from Penta, syringaldehyde (98 %) from Acros organics, 4iso-butylbenzaldehyde (> 95 %) from TCI, 4-terc-butylbenzaldehyde (p.a.) from Chemical point, methyl(isobutyl)ketone (p.a.) from Ubichem, ethyl(methyl)ketone (p.a.) from Lachner. Ethylvanillin and cinnamaldehyde were kindly supplied by Aroma a.s. Anisaldehyde, crotonaldehyde and diethylketone were taken from UCT sources.

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PETROCHEMICALS AND ORGANIC TECHNOLOGY

Catalytic test Two types of reaction were chosen for testing catalytic activity of phenylacetylene; acetalization and acylation. Acetalization/ketalization Aldehyde/ketone (100 mg) was added to the flask with 5 mL of methanol and appropriate amount of phenylacetylene (0 or 10 wt. % calculated to the mass of aldehyde/ketone). The flask was equipped with condenser and the reaction mixture was stirred in a preheated oil bath (60 °C) for 4 or 24 hours. Acylation Aldehyde (syringaldehyde or ethylvanillin) (100 mg) was added to the flask with acetanhydride (2x molar mass of aldehyde),2 mL of tetrahydrofurane (THF) and appropriate amount of phenylacetylene (10 wt.% calculated to the mass of aldehyde). The flask was equipped with condenser and the reaction mixture was stirred in a preheated oil bath (90 °C) for 48 h. Analytical In above mentioned reactions the taken samples were analysed using gas chromatography (GC) and gas chromatography with mass spectrometer (GC/MS). GC/MS was used for confirmation of the product structures.

Discussion and result analysis Acetalization/ketalization Acetalization is acid catalysed reaction which proceeds without catalyst in some cases. Due to this fact the reaction without phenylacetylene addition was tested at first. The mechanism of acetalization and ketalization is depicted on Figure 1. In the following picture (Figure 2) the results of non-catalysed reactions are summarized.

Figure 1. The mechanism of acetalization or ketalization


417

Conversion 4 h (%)

80

60

40

20

0

Aldehyde Figure 2. Acetalization; 100 mg aldehyde, 5 mL methanol, 60 °C, 4 h Acetalization resulted in relatively high yields of products, acetals (more than 50 %), in the case of reactions of aromatic aldehydes. In comparison to these results, the reactions of aliphatic aldehydes did not lead to the yields over 10 % (penatanal, hexanal, isopentanal) or did not take place at all (propanal, heptanal, isobutanal) except octanal, where the conversion was 24 %. The presence of aromatic ring increased the reactivity of aldehydic group (activation of aldehydic function groups by electrodonor aromatic system). The reaction of ketones (acetone, ethyl(methyl)ketone, methyl(isobutyl)ketone, diethylketone) also did not occur without catalyst. Based on these results the acetalization of aliphatic aldehydes and ketones were performed using phenylacetylene as a catalyst (Figure 3).

Conversion (%)

80

4h

24 h

60

40

20

0

propanal

pentanal

hexanal

heptanal

isobutanal

isopentanal

Aldehyde Figure 3. Acetalization; 100 mg aldehyde, 10 mg phenylacetylene, 5 mL methanol, 60 °C, 24 h

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The addition of catalyst increased the reaction rate in the case of acetalization of aliphatic aldehydes and enabled the reaction of aldehydes that did not react without catalyst (propanal, heptanal, isobutanal). Only acetals were detected in the reaction mixture, hemiacetals were not detected due to their low stability in the reaction mixture. The ketalization did not also take place despite the catalyst addition (acetone, ethyl(methyl)ketone, methyl(isobutyl)ketone, diethylketone). The lower reactivity of carbonyl group is in the case of ketones enabled by the positive induction effect given by the presence of two alkyl groups. The strict relationship between neither the chain length nor the structure of the aldehyde and the reaction rate could be found because of small differences in aldehyde conversions which were in the range of measurement error in some cases (e.g. hexanal, heptanal, Figure 3 ). The initial reaction rate of the linear aldehydes was similar and lower in comparison to the branched aldehydes (isobutanal and isopentanal). However, the yields of acetals were comparable after 24 hours of reaction. The highest reaction rate in the case of propanal and the highest achieved conversion after 24 hours could be explained by its low boiling point (50 °C). Acylation Acylation of aldehydic function groups can be used as well as acetalization for protection of aldehydic groups. Two substrates were chosen for testing the reaction course, syringaldehyde and ethylvanilin, reaction scheme is shown on Figure 4. The obtained results were summarized in Figure 5.

Figure 4. Reaction scheme of acylation/esterification of syringaldehyde and ethylvanillin

Conversion of aldehyde (%)

100 80 60 40

syringaldehyde ethylvanillin

20 0

0

12

24

36

48

Time (h) Figure 5. Esterification; 100 mg aldehyde, 2xmol mass of acetanhydride, 10 wt. % phenylacetylene, 2 mL THF


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Although the reaction of benzaldehyde with acetanhydride forming 1,1-diacetate as the main product was 7 described in some papers , the reaction of syringaldehyde or ethylvanillin with acetic anhydride catalysed by phenylacetylene did not lead to the formation of this type of product. The product of esterification was detected (by GC/MS) in a reaction mixture using both syringaldehyde and ethylvanillin, moreover, esterification took place with 100% selectivity. Reaction rate was higher in the case of syringaldehyde, total conversion was achieved, in comparison with ethylvanilin, where the conversion was only 82% after 48 hours.

Conclusions The possible application of phenylacetylene as an acid catalyst was tested in our work. Two reaction types were chosen for testing its catalytic activity, acetalization of aldehydes and ketones and acylation of syringaldehyde and ethylvanilin with acetanhydride. Because of possible spontaneous acetalization of some substrates with methanol, the non-catalysed acetalization was tested. The reactions with aromatic aldehydes resulted in relatively high yields of acetals (higher than 50 %) due to the activation of aldehyde group for reaction by negative mesomeric effect (of electrodonor aromatic system). The catalysed acetalization was performed using aliphatic aldehydes and ketones as substrates. It the case of aliphatic aldehydes the reaction rate increased with the catalyst addition, on the other site the reactions with ketones did not follow up this trend probably because of deactivation of carbonyl group by positive induction effect of alkyl groups. Any strict relation between aldehyde structure or chain length and reaction rate cannot be found because of the slight differences between used aldehydes. Acylation of neither syringaldehyde nor ethylvanilin resulted in the formation of 1,1-diacetal, because the competitive reaction, esterification, was preferred reaching total conversion of syringaldehyde or 82 % conversion of ethylvanilin and 100% selectivity after 48 hours of reaction. Phenylacetylene can be effectively used as a catalyst for acetalization of aliphatic aldehydes and also for esterification of acetanhydride by aromatic alcohols bearing aldehydic function group on benzene ring. Phenylacetylene can be simply separated from reaction mixture by distillation.

Acknowledgements This work was realized within the Operational Programme Prague – Competitiveness (CZ.2.16/3.1.00/24501) and “National Program of Sustainability” ((NPU I LO1613) MSMT-43760/2015). We also acknowledge grant project GACR 17-03474S and Specific University Research (MSMT NO 20-SVV/2017).

References 1. Öztürk B. Ö., Şehitoğlu S. K.: Appl. Organometal. Chem. 30, 367 (2016). 2. Piacenti F., Bianchi M., Frediani P., Menchi G.: J. Mol. Catal. 83, 83 (1993). 3. Roy R., Rajasekaran P., Mallick A., Vankar Y. D.: Eur. J. Org. Chem. 25, 5564 (2014). 4. Krasnyakova T. V., Zhikharev I. V., Mitchenko R. S., Burkhovetski V. I., Korduban А. M., Kryshchuk T. V., Mitchenko S. A.: J. Catal. 288, 33 (2012). 5. Mitchenko S. A., Khomutov E. V., Shubin A. A., Shul’ga Y. M.: J. Mol. Catal. A: Chem. 212, 345 (2004). 6. Mitchenko S. A., Krasnyakova T. V., Mitchenko R. S., Korduban A. N.: J. Mol. Catal. A: Chem. 275, 101 (2007). 7. Luštická I., Vrbková E., Vyskočilová E., Paterová I., Červený L.: Reac. Kinet. Mech. Cat. 108, 205 (2013).

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ETHANOL SEPARATION BY SOLVENT EXTRACTION USING [TDTHP][NTf2] IONIC LIQUID: ALTERNATIVES OF EXTRACTION SOLVENT REGENERATION Steltenpohl P., Graczová E. STU in Bratislava, FCHPT, Institute of Chemical and Environmental Engineering, Radlinského 9, 812 37 Bratislava, Slovakia [email protected]

Abstract Assuming large worldwide ethanol production, minimization of expenditures connected with the separation of this commodity is of great interest. Nowadays, solvent extraction or membrane-based separation processes are replacing distillation in ethanol separation from its diluted aqueous solutions. Here, solvent extraction was considered for ethanol–water mixture separation in the presence of ionic liquid tetradecyltrihexylphosphonium bis(trifluoromethylsulfonyl)imide used as the extraction solvent. Simulations of a counter-current extraction column show that raffinate phase is free of IL, while the extract phase comprises all components considered: IL, ethanol, and water. Then, a further separation step is required to obtain practically pure ethanol and to regenerate the extraction solvent for its repeated use. Three strategies of IL regeneration were assumed: extracted components separation from the extract phase by distillation, evaporation and stripping into an inert gas. Performance of these procedures was compared based on their respective operational costs that were approximated by the equipment heating and cooling demands. Large differences in the heat and cooling duties were identified comparing the chosen IL regeneration strategies. As the most economic strategy, stripping of the volatile mixture components to a stream of inert gas was evaluated. Due to the high values of molar heat capacity and molar vaporization heat of the IL used as the extraction solvent, any change of temperature of the streams comprising this component causes significant heat duty influencing the process overall costs.

Introduction Ethanol is considered a clean energy source produced from industrial crops (sugar cane and corn). It is obtained by the conversion of saccharides as a product of yeast metabolism (fermentation) in aqueous media. Separation of ethanol from this mixture can be achieved by distillation; however, purity of the product is equilibrium-limited. Consequently, further separation has to be used to obtain pure ethanol. Considering typical composition of an ethanol-containing fermentation broth, purification of ethanol by distillation is an energy-intensive process1,2. In order to lower expenditures on ethanol separation from its diluted aqueous mixtures, several alternatives were proposed1,2, among which extraction shows very good performance3. In our previous work3, ionic liquid tetradecyltrihexylphosphonium bis(trifluoromethylsulfonyl)imide ([TDTHP][NTf2]) was considered as the extraction solvent. Due to its unique properties, negligible mutual solubility with water, low density and viscosity, and fair extraction capacity, [TDTHP][NTf2] is a promising candidate for ethanol extraction from its water solutions. Compared to the feed composition, ethanol-to-water mole ratio in the extract phase was considerably increased without the necessity of water evaporation3. On the other hand, further separation step(s) had to be considered in order to obtain pre-concentrated ethanol and to regenerate the solvent. Jongmans and coworkers4 studied the separation of an ethylbenzene–styrene mixture by extraction distillation in the presence of a 4-methyl-N-butylpyridinium tetrafluoroborate IL. The authors proposed and, based on the minimization of capital and operational expenditures, evaluated several strategies of IL regeneration4. In the present study, selection of an extraction solvent regeneration method was carried out considering the operational costs. Simulations were carried out for the [TDTHP][NTf2] regeneration by the extracted components (ethanol and water) removal using distillation, evaporation, and stripping by nitrogen.

Simulation Results of a counter-current extractor simulation for water (1)–ethanol (2) mixture separation in the presence of [TDTHP][NTf2] (3) at the temperature of 25 °C were presented earlier3,5. It was found that extraction allows increasing the ethanol-to-water mole ratio from 0.075 (feed) to 10.58 (extract phase) as shown in Table I. Liquid–liquid equilibrium was described using the isoactivity condition5 where the components activity coefficients were computed using the NRTL excess Gibbs energy model6. Parameters for the liquid-phase activity coefficients calculation were found in literature7. These data were also used for the prediction of vapor–liquid equilibria in down-stream separation equipment for the extraction solvent regeneration.


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Table I Water (1)–ethanol (2) –[TDTHP][NTf2] (3) separation at 25 °C in an extractor with three theoretical stages3 Feed Solvent Raffinate Extract Component mole fraction 1 0.93 0 1.000 0.010 2 0.07 0 0.000 0.106 3 0 1 0.000 0.884 Molar flow, kmol h–1 10 5.85 9.25 6.60 Considered separation strategies In total, three strategies for the extract phase components separation and extracted components recovery were considered: a) distillation, b) evaporation, and c) stripping into an inert gas stream. Simplified flow sheets of the respective separation strategies are given in Figure 1.

a

extract phase

3 4

1

5

2

distillate 6

b

extract phase

7

regenerated solvent

7

regenerated solvent

5

condensed vapors

1 8

c

10

extract phase stripping gas cycle 9

regenerated solvent

11

4

condensed vapors

Figure 1. Flow sheet of the IL regeneration via volatile components separation by distillation (a), evaporation (b) and stripping into inert gas (c): 1 – throttle valve, 2 – vacuum distillation column, 3 – partial condenser, 4 – phase separator, 5 – total condenser/sub-cooler, 6 – reboiler, 7 – cooler, 8 – single-effect vacuum evaporator, 9 – stripping column, 10 – system of heat exchangers, 11 – gaseous phase heater.

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The simulations were carried out using proprietary simulation programs developed in MS Excel. For each individual separation apparatus, a set of MESH equations (Material, Equilibrium, Summation, and entHalpy balances) was written and solved. Equilibrium conditions and continuous operation of the separation unit was assumed. Economic aspects connected with the separation unit operation were evaluated based on the heat/cooling duties of the respective thermal equipment shown in Figure 1. Compression cooling was considered in case that the material stream has to be cooled below the ambient temperature. In such case cooling demand costs were rated twice the costs of cooling to or heating above the ambient temperature. Estimation of the components’ properties Prior to the simulation of the above regeneration strategies, relevant properties of pure components were estimated based on literature data. Properties such as normal boiling point, molar heat capacity in both liquid and vapor phases, saturated vapor pressure, and vaporization heat as well as their variation with temperature (if applicable) for components 1 and 2 (water and ethanol) can be found in databases of pure components’ 8–10 properties . In case of [TDTHP][NTf2] IL used as the extraction solvent, limited information on the component properties is available in literature. Missing data were predicted. Variation of the [TDTHP][NTf2] molar heat capacity with temperature is given in several papers11–13, from which also the polynomial dependence of heat capacity on temperature can be deduced. Normal boiling point (Tb = 1311 K) and critical parameters (Tc = 1587 K, Pc = 0.85 MPa) of this ionic liquid were predicted based on the group contribution method as presented by Valderrama and Rojas14. These data, temperature–pressure pairs, were used to predict the Antoine equation parameters for [TDTHP][NTf2] as well as the temperature variation of the molar vaporization heat of this IL. In order to assess the three-parameter Antoine equation, at least three pairs of temperature–pressure data are necessary. As in open literature, no experimental data on saturated vapor pressure of [TDTHP][NTf2] are available, we used information on the saturated vapor pressure of an ionic liquid with the same anion, 1-ethyl3-methylimidazolium bis(trifluoromethylsulfonyl)imide15. Then, using vapor pressures of 0.03 Pa, 101325 Pa, and 850000 Pa, corresponding to temperatures of 168 °C (similarity to that reported for imidazolium IL), 1037 °C (normal boiling temperature of [TDTHP][NTf2]), and 1314 °C (critical temperature of [TDTHP][NTf2]), respectively, Antoine equation parameters for [TDTHP][NTf2] IL were obtained (see Table II). By combining Antoine equation with the Clausius–Clapeyron equation, vaporization heat of [TDTHP][NTf2] was predicted. Summary of selected properties of the mixture components is given in Table II. Table II Selected properties of mixture components Antoine equation* M, tb , Component kg mol–1 °C A B C 1 0.018 100.0 9.580571 1396.58 204.8497 2 0.046 78.2 10.85867 1948.88 254.3902 3 0.764 1037 10.72240 8743.05 492.0861 *log(P°/Pa) = A – B/(C + (t/°C)); ** at 298 K.

cP,l**, J K–1 mol–1 75.32 116.9 1366

Δvh**, J mol–1 44005 42403 55646

Discussion [TDTHP][NTf2] regeneration by distillation It is expected that bottom product from the distillation column shown in Figure 1a is pure [TDTHP][NTf2] IL. Consequently, pressure in this column should be decreased as the predicted normal boiling point of this IL exceeds 1000 °C. According to Ferreira and coworkers13, decomposition temperature of [TDTHP][NTf2] is 612 K (339 °C). Therefore, based on the predicted parameters of the Antoine equation given in Table II, pressure in the distillation column was set to 1250 Pa. Table III Results of [TDTHP][NTf2] regeneration by distillation for N = 1, partial condenser, q = 0.997, and R = 0.05 Component molar fraction P, t, n, Stream Pa °C kmol h–1 1 2 3 Extract phase 101325 25.0 0.0100 0.1059 0.8841 6.597 Feed 1250 24.9 0.0100 0.1059 0.8841 6.597 Distillate 1250 –3.2 0.0863 0.9137 0.0000 0.764 Bottom product 1250 297.6 0.0000 0.0000 1.0000 5.833


423

Reduction of the pressure of feed entering the distillation column was carried out at isoenthalpy conditions in a throttle valve (Figure 1a). Calculations of state of the feed entering the distillation column were essentially the same as those carried out in our previous study16. Due to the very large difference of the components boiling temperatures, column with only one theoretical plate, partial condenser, and a reboiler provided the required separation extent. Results of the simulation of [TDTHP][NTf2] regeneration by distillation are given in Table III for the computed state of the feed (q = 0.997) and the reflux ratio of R = 0.05. As the feed to the distillation column, extract phase from the counter-current extractor (Table I) was considered. The measure of ethanol overall pre-concentration, the ethanol-to-water mole ratio in the distillate stream of 10.58, remained unchanged compared to the value obtained for the extract phase. Results of the simulation of distillation column operation were used to assess the operational costs in the proposed separation unit. For this purpose, heat and cooling demands of the respective thermal equipment were computed as shown in Table IV. All calculations are related to the reference state chosen as: pure component in the liquid phase at the temperature of 25 °C. Effect of pressure on the components’ heat capacity and vaporization heat values was neglected. Table IV Results of heat duty calculations for thermal equipment involved in the flow sheet for [TDTHP][NTf2] regeneration by distillation (Figure 1a) partial condenser condenser/sub-cooler reboiler cooler Overall* Equipment 3 5 6 7 Heat duty, kW –9.3 –0.5 618.6 –608.8 1247.0 *Unit costs of cooling duty related to condensation and sub-cooling at temperatures lower than the ambient temperature were rated two times the unit costs of heating above and cooling to the ambient temperature. [TDTHP][NTf2] regeneration by evaporation In case of IL regeneration by evaporation (Figure 1b), similar experimental conditions were chosen compared to the IL regeneration by distillation. Again, throttling of the extract phase to the pressure of 1250 Pa was considered to avoid the IL thermal decomposition. Further, a minimum content of [TDTHP][NTf2], molar fraction of 0.9995, in the stream of regenerated IL was pre-set to keep unchanged the amount of extraction solvent used in the counter-current extractor. Extractor simulations showed no appreciable effect of impurities at the IL content in the regenerated extraction solvent higher than 0.999. Results of the single-effect evaporator simulation are given in Table V. Table V Results of [TDTHP][NTf2] regeneration by evaporation P, t, Stream Pa °C Extract phase 101325 25.0 Feed 1250 24.9 Condensed vapors 1250 25.0 Regenerated solvent 1250 197.6

Component molar fraction 1 2 3 0.0100 0.1059 0.8841 0.0100 0.1059 0.8841 0.0865 0.9135 0.0000 0.0000 0.0003 0.9997

n, kmol h–1 6.597 6.597 0.764 5.833

It was found that regeneration of the extraction solvent by evaporation has no important effect on the ethanolto-water molar ratio. In the stream of condensed vapors, the ethanol mole fraction was on the level of 0.914. Taking into account only a negligible content of [TDTHP][NTf2] in this stream, the ethanol-to-water mole ratio was 10.56, which is close to the value observed in the extract phase (Table I). Information given in Table V was used to compute the heat and cooling demands of the equipment shown in Figure 1b. Results of these calculations are summarized in Table VI. Table VI Results of heat duty calculations for thermal equipment involved in the flow sheet for [TDTHP][NTf2] regeneration by evaporation (Figure 1b) condenser/sub-cooler cooler evaporator Overall* Equipment 5 7 8 Heat duty, kW –6.0 –382.1 384.9 779.0 *Unit costs of cooling duty related to condensation and sub-cooling at temperatures lower than the ambient temperature were rated two times the unit costs of heating above and cooling to the ambient temperature.

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[TDTHP][NTf2] regeneration by stripping using inert gas Due to the high molar heat capacity of [TDTHP][NTf2] IL, stripping of ethanol and water from the extract phase was carried out at ambient temperature to minimize the overall operation costs. At these conditions, a stripping column with ten theoretical stages was considered for the [TDTHP][NTf2] regeneration. Nitrogen was selected as the stripping gas. At each theoretical stage, gas–liquid phase equilibrium was simulated using the Raoult law modified for ideal behavior of the gas phase and real behavior of the liquid phase. At equilibrium, the gaseous phase is saturated with the liquid mixture components. In real conditions, this goal is not reached. Kossaczký and Surový17 reported that the common extent of the gas phase saturation in desorption/stripping columns ranges between 60 % and 70 %. In order to calculate the inert gas flow through the stripper, a medium value of the gas phase saturation with volatiles from the above range (65 %) was considered. Ethanol (and water) recovery from the gaseous phase by cooling/freezing the gaseous phase to –50 °C in a series of heat exchangers was further assumed. It was considered that the cooling equipment parameters allow reaching phase equilibrium at the given temperature. Condensed liquid phase containing concentrated ethanol with a small amount of water was considered the final product. The gas phase from a set of coolers was recycled back to the stripper thus closing the loop of stripping gas utilization. Due to the limits of stripping gas freeze-drying, separation of ethanol and water from the extraction solvent in stripping column was not complete. Especially, ethanol content in the regenerated [TDTHP][NTf2] IL increased to 0.6 mole %. Therefore, new simulation of aqueous ethanol mixture separation by extraction was carried out applying the regenerated extraction solvent composition computed by the stripping column simulation. No substantial differences compared to the base simulation of the counter-current extractor were observed. Results of simultaneous solution of the respective MESH equations for the separation unit shown in Figure 1c are given in Table VII. Table VII Results of [TDTHP][NTf2] regeneration by stripping into inert gas Component molar fraction P, t, n, Stream Pa °C kmol h–1 1 2 3 inert Extract phase* 101325 25.0 0.0104 0.1102 0.8794 – 6.613 (Recirculated) stripping gas 101325 25.0 3.6×10–5 20.8×10–5 0.0×10–5 0.9998 222.157 Stripper gaseous effluent 101325 25.0 0.0003 0.0031 0.0000 0.9966 222.893 Regenerated solvent 101325 –50.0 0.0000 0.0060 0.9940 – 5.877 Condensed vapors 101325 –50.0 0.0790 0.9210 0.0000 – 0.736 *Results of the extractor simulation based on the regenerated solvent composition found in this table. Considering the composition of the stripper gaseous effluent phase and condensed vapors from the separator situated just after the freeze-drying of the stripper gaseous effluent phase, the ethanol-to-water mole ratio in these streams further increased; in the extract phase, the value was 10.58, while in the gas phase from the stripper it was 10.94 and finally reached the value of 11.65 in the liquid phase leaving the phase separator. Data presented in Table VII served as the base for the selected operational costs calculation. It was assumed that in the heater for the gas phase (see Figure 1c), enough heat is introduced to keep the stripping column working at isothermal conditions. The other thermal equipment involved in this flow scheme was a set of coolers in which also volatiles condensation was accomplished. Results of the heat/cooling demand computation are given in Table VIII. Table VIII Results of heat duty calculations for thermal equipment involved in the flow sheet for [TDTHP][NTf2] regeneration by stripping of volatiles into inert gas (Figure 1c) set of coolers gas phase heather Overall* Equipment 10 11 Heat duty, kW –10.5 11.1 53.1 *Unit costs of cooling duty related to condensation and sub-cooling at temperatures lower than the ambient temperature were rated four times the unit costs of heating (necessity of two-stage compression cooling).

Conclusions Comparing results obtained by the simulation of separation equipment based on material balances and vapor– liquid equilibrium, the ethanol pre-concentration achieved by liquid extraction is preserved. A minor decrease of the ethanol-to-water molar ratio was observed only in case of the extraction solvent regeneration by


425

volatiles evaporation. On the other hand, a slight increase in this parameter was observed when the [TDTHP][NTf2] regeneration was accomplished by stripping volatiles into inert gas. In this case, n2/n1 = 11.65 which corresponds to the ethanol mole fraction in the stream of condensed vapors of 0.921. Enthalpy balance of the proposed strategies for [TDTHP][NTf2] IL regeneration revealed that any change of temperature or state of the streams comprising this ionic liquid is connected with high energy demand. This conclusion is related to enormous values of the IL molar heat capacity and molar vaporization heat. When comparing the IL regeneration by distillation and evaporation, heat duty of the equipment is clearly linked to the temperature to which the originally extract phase is heated to achieve its separation. It can be concluded that very high separation efficiency is reached by evaporation. Purity of the gaseous and liquid phases is not as high as in case of the IL regeneration by distillation; however, the savings connected with a decrease of the heat and cooling demand are substantial (about 37 %). At the given conditions, composition of the regenerated extraction solvent has practically no effect on the results of the counter-current extractor operation. Operational costs computed for the second strategy (solvent regeneration by evaporation) can be further decreased; but, at the price of lower ethanol pre-concentration in the final product. In comparison with the previous strategies, the [TDTHP][NTf2] regeneration by stripping of volatiles into an inert gas showed overwhelming priority in terms of operational costs. Compared to the calculated heat duties for the extraction solvent regeneration by distillation and evaporation, the expenditures obtained for the third separation strategy were on the level of 4.3 % and 6.1 %, respectively. For the IL regeneration by stripping into inert gas, the computed overall heat consumption usage expressed in terms of the overall heat demand per treated amount of ethanol was about 185 kJ kg–1. However, one should bear in mind that simplified economy balance was considered in this study. Separation efficiency in case of the IL regeneration by stripping using inert gas is also equilibrium limited. Fortunately, favorable properties of the [TDTHP][NTf2] IL, e.g. high selectivity and appropriate capacity, mitigate the adverse effect of the relatively high ethanol content in the regenerated extraction solvent. Based on the simulation results it can be concluded that for the ionic liquids regeneration, separation procedures not requiring heating/cooling are preferential. Properties of ionic liquids, especially high values of heat capacity and vaporization heat provoke elevated heat/cooling demands if a change of temperature and/or state of streams containing the IL is necessary.

Acknowledgement This study was supported by the Research and Development Assistance Agency (APVV-0858-12).

References 1 Sattler K., Feindt H.J.: Thermal Separation Processes: Principles and Design. VCH, Weinheim, Germany, 1995. 2 Friedl A.: FEMS Microbiol. Lett. 363, fnw073 (2016). 3 Steltenpohl P., Graczová E.: Chem. Eng. Res. Des., in press. 4 Jongmans M.T.G., Trampé J., Schuur B., de Haan A.B.: Chem. Eng. Proc. Process Intens. 70, 148 (2013). 5 Steltenpohl P., Graczová E.: Chem. Eng. Trans. 39, 187 (2014). 6 Renon H., Prausnitz J.M.: AIChE J. 14, 135 (1968). 7 Neves C.M.S.S., Granjo J.F.O., Freire M.G., Robertson A., Oliveira N.M.C., Coutinho J.A.P.: Green Chem. 13, 1517 (2011). 8 Poling B.E., Prausnitz J.M., O’Connell J. P.: The Properties of Gases and Liquids (5th ed.). McGraw-Hill, New York, 2001. 9 Majer V., Svoboda V.: Enthalpies of Vaporization of Organic Compounds: A Critical Review and Data Compilation. Blackwell Scientific Publications, Oxford, 1985. 10 NIST: NIST Chemistry Webbook. Retrieved January, 2016, from http://webbook.nist.gov/chemistry/ 11 Ge R.L., Hardacre C., Jacquemin J., Nancarrow P., Rooney D.W.: J. Chem. Eng. Data 53, 2148 (2008). 12 Ferreira A.F., Simões P.N., Ferreira A.G.M.: J. Chem. Thermodynamics 45, 16 (2012). 13 Ferreira A.G.M., Simões P.N., Ferreira A.F., Fonseca M.A., Oliveira M.S.A., Trino A.S.M.: J. Chem. Thermodyn. 64, 80 (2013). 14 Valderrama J.O., Rojas R.E.: Ind. Eng. Chem. Res. 48, 6890 (2009). 15 Zaitsau D.H., Kabo G.J., Strechan A.A., Paulechka Y.U., Tschersich A., Verevkin S.P., Heintz A.: J. Phys. Chem. A 110, 7303 (2006). 16 Graczová E., Dobcsányi D., Steltenpohl P.: Chem. Eng. Trans., submitted. 17 Kossaczký E., Surový J.: Chemical Engineering 2 (5th ed., pp. 85). ALFA, Bratislava, Czechoslovakia, 1987. (in Slovak)

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Mg-Al-Zn MIXED OXIDES AS EFFECTIVE CATALYSTS IN ALDOL CONDENSATIONS 1,2

1

2

1

Vrbková E. , Varlamov A. , Tišler Z. , Vyskočilová E. , Červený L.

1

1

Vysoká škola chemicko-technologická v Praze, Technická 5, 160 00, Praha 6 Unipetrol Centre of Research and Education, Inc., Areál Chempark 2838, Záluží 1, 436 70, Litvínov [email protected] 2

Abstract A mixed oxide with Mg:Al ratio 3:1 was prepared by co-precipitation at constant pH value. This material was mixed with different amounts of zinc oxide. Content of zinc oxide in prepared materials varied from 4.9 wt.% to 47.7 wt. %. Prepared Mg-Al-Zn mixed oxides were analyzed using different techniques (XRF, ICP) to determine Mg:Al:Zn molar ratio. These materials were used as catalysts in aldol condensation of heptanal with substituted benzaldehydes Activity and selectivity in these reactions were evaluated with accent to influence of zinc amount on reaction course. The reaction of heptanal with substituted benzaldehydes gives a desired product of cross-aldol condensation and undesired product of heptanal autocondensation. Experiments showed that adding the zinc oxide to the catalyst caused the increase of reaction rate. Influence of zinc oxide amount in catalyst to selectivity to cross-aldol and autocondensation product was negligible.

Introduction Aldol condensation between substituted 4-alkylbenzaldehyde and heptanal is a way to produce different fragrances. This type of substances is frequently used in perfumery industry due to its stability in basic environment, which makes them perfect to be used in number of detergents, shampoos and soaps. In industry these aldol condensations are usually catalyzed by inorganic hydroxides, but this way suffers from using corrosive mixtures and producing large amounts of waste salts. The use of heterogeneous basic catalysts is promising because it offers simple catalyst separation or even possibility of catalyst reuse. In this work Mg-AlZn oxides were chosen to perform aldol condensation of substituted benzaldehydes with heptanal. Mixed MgAl oxides were already successfully used as catalysts in different aldol condensations [1-6].

Experimental Mg–Al mixed oxides with Mg:Al molar ratio 3:1 were synthesized by co-precipitation method at constant pH value (pH = 9.5, T = 60 °C). The preparation procedure involves mixing of an aqueous solution 1 consisting of magnesium nitrate Mg(NO3)2·6H2O (Lachner, p.a.) and aluminum nitrate Al(NO 3)3·9H2O (Lachner, p.a.) (both Mg 3 and Al concentration 1 mol/dm ) and a basic solution 2 of potassium carbonate K 2CO3 (Penta, p.a.) and 3 3 potassium hydroxide KOH (Penta, p.a.) (3 mol/dm KOH + 0.5 mol/dm K2CO3). After precipitation the solids were isolated by press-filtration using S15N filters (Hobra), washed until the solution was neutral and dried in an oven at 60 °C overnight. The wet filter cake was 15 minutes kneaded with powdered ZnO (Lachner), and prepared sample was thereafter dried overnight at 65 °C. For the studied aldol condensation, substituted benzaldehydes (4-methylbenzaldehyde - Sigma Aldrich, 4-isopropylbenzaldehyde - Acros, 4isobutylbenzadehyde - TCI, 4-tertbutylbenzadehyde - Chemical Point) and heptanal (Sigma Aldrich) were used. The catalysts were activated before the reaction by calcination (air, 450 °C, 15 h). Thermo-gravimetric analysis (TGA) of dried LDH catalysts were obtained using TGA Discovery series (TA Instruments) operating at heating ramp 10 °C/min from temperature 40 °C to 900 °C in flow of nitrogen (20 ml/min, Linde 5.0).The chemical composition was verified by using an ICP-EOS Agilent 725 (Agilent 3 Technologies Inc.). Before analysis, a 200 mg sample was dissolved in 10 cm of H2SO4 (1:1) and heated. After dissolution, the sample was cooled down, diluted by demineralized water and heated to 100 °C for a few minutes. Finally, the solution of sample was transported to volumetric flask and measured. The crystallographic structure of the precursors and catalysts was determined by examining the X-ray diffraction (XRD) patterns of the powder samples obtained by using a D8 Advance ECO (Bruker) applying CuKα radiation (λ = 1.5406 Å). In a typical experiment, 25 ml round-bottomed flask was equipped with condenser and filled up with catalyst and substituted benzaldehyde, the reaction mixture was stirred vigorously and heated to desired temperature th th (120 °C). Then the heptanal was added in three portions – in 0, 30 and 60 minute of the reaction (to suppress its autocondensation). The reaction conditions were as follows: 20 wt. % of catalyst calculated to heptanal amount, heptanal:substituted benzaldehyde molar ratio 1:2. Typical experiment involved: substituted benzaldehyde (8.50 mmol), heptanal (4.25 mmol) and 0.097 g of catalyst.


427

Prior to analysis, samples (4 x 0.05 ml) were centrifuged, diluted with ethanol, and then analyzed using Shimadzu GC 17A chromatograph fitted with nonpolar column ZB-5 (60 m, 0.32 mm diameter, 0.25 μm film) and FID.

Discussion Material characterization Determination of material composition was performed using ICP analysis (Table I). Table I Composition of catalysts (ICP analysis) Molar ratio from ICP Catalyst Zn Mg Al CMA CZMA-11 CZMA-12 CZMA-13 CZMA-14 CZMA-15 CZMA-16

0.11 0.34 0.84 1.30 1.68 2.46

3.22 3.18 3.23 2.94 2.95 2.94 2.95

1 1 1 1 1 1 1

All catalysts were analyzed using X-ray diffraction (Figure 1). In this analysis, bands corresponding to zinc oxide occurred (32, 34, 36, 48, 57, 63, 68 a 69 °2ϴ). Intensity of these bands increased with the increasing amount of zinc oxide in catalyst. The bands corresponding to Mg-Al oxide were also present (12, 23 a 39 °2ϴ), which intensity decreased with increasing content of zinc oxide.

Intensity (a.u)

CMA CZMA-11 CZMA-12 CZMA-13 CZMA-14 CZMA-15 CZMA-16

5

15

25

35

°2ϴ

45

55

65

Figure 1. X-ray diffractograms of pure Mg-Al oxide (CMA) and Zn-Mg-Al oxides with different zinc content Materials were also analysed using thermogravimetry (Figure 2). This analysis showed that with increasing amount of zinc oxide in catalyst the decrease of weight with time was less intensive. From this it can be concluded that weight decrease is caused probably by water desorption from Mg-Al oxide and by the loss of interlayer ions. Weight loss in the range 50 - 200 °C corresponds to loss of physisorbed water and weight loss in the range 200 - 500 °C probably corresponds to loss of interlayered carbonate ions.

428


100

Weight (%)

90 CMA CZMA-11 CZMA-12 CZMA-13 CZMA-14 CZMA-15 CZMA-16

80 70 60 50

0

100

200

300

400 500 Temperature (°C)

600

700

800

Figure 2. TG analysis of prepared materials Aldol condensation Prepared materials were tested in aldol condensation of substituted benzaldehydes (subsBZ) with heptanal (HP, Figure 3).

a R = Me, iPr, iBu, tBu

b

Figure 3. Aldol condensation of substituted benzaldehydes with heptanal: product of mixed aldol condensation (a), product of heptanal autocondensation (b) These aldol condensations give two possible products - product of mixed aldol condensation (MIX) and product of autocondensation of two heptanal molecules (PN). Chosen catalysts were tested in aldol condensation of substituted benzadehyde with heptanal (Table II) to determine influence of zinc oxide amount in catalyst on catalyst´s activity. Achieved heptanal conversions were in all cases higher than 90 % (7 h). Generally a slightly increased selectivity to mixed aldol condensation product in case of substrates with longer and branched substituent in paraposition was observed. The higher selectivity to the mixed aldol condensation was observed in the case of catalyst CZMA-13 in all cases, but no straight trend concerning zinc amount to reaction result was observed. Nevertheless the selectivity to mixed aldol condensation using catalyst with zinc was higher comparing the catalyst without any zinc in the structure.


429

Table II Reaction results using different catalysts and substrates (HP:subsBZ = 1:2 molar, 20 wt. % cat. to heptanal, no solvent, 120 °C, 7 h) Catalyst

430

Substrate

Heptanal conversion (%)

MIX/PN ratio

CMA

97

1.6

CMZA-11

99

1.9

CMZA-12

98

1.8

CMZA-13

99

2.0

CMZA-14

99

1.8

CMZA-15

99

1.7

CMZA-16

99

1.9

CMA

97

1.6

CMZA-11

97

1.7

CMZA-12

96

2.1

CMZA-13

99

2.4

CMZA-14

99

2.2

CMZA-15

99

2.1

CMZA-16

99

2.4

CMA

96

1.7

CMZA-11

93

1.7

CMZA-12

94

1.8

CMZA-13

96

2.3

CMZA-14

97

1.8

CMZA-15

99

2.2

CMZA-16

94

1.8

CMA

94

1.8

CMZA-11

95

2.0

CMZA-12

97

2.1

CMZA-13

97

2.2

CMZA-14

98

2.1

CMZA-15

99

2.0

CMZA-16

98

2.2


Conclusion Mg-Al mixed oxide with molar ratio Mg:Al = 3:1was prepared using coprecipitation method. This material was mixed with different amounts of zinc oxide and six catalysts with increasing zinc amount were prepared. These materials were characterized using X-ray diffraction, ICP analysis and thermogravimetry. Prepared materials were used as catalyst in aldol condensation of substituted benzaldehydes with heptanal. Higher catalyst activity was observed with increasing content of zinc oxide in catalyst. Influence on selectivity to reaction products was negligible. Substituent in p-position had slight influence on selectivity.


References 1. 2. 3. 4. 5. 6.

Chunxiang M., Gang L., Zhenlu W., Yufei L., Jing Z., Wenxiang Z., Mingjun J.: React. Kinet., Mech. Catal. 98, 149 (2009). Abelló S., Medina F., Tichit D., Pérez-Ramírez J., Groen J., Sueiras J., Salagre P., Cesteros Y.: Chemistry 11, 728 (2005). Hora L., Kikhtyanin O., Čapek L., Bortnovskiy O., Kubička D.: Cat. Today 241, 221 (2015). Paterová I., Vyskočilová E.,Červený L.: Top. Catal. 55, 873 (2012). Xu J., Cao Y., Ma Q., Peng X.: Asian J. Chem. 25, 3847 (2013). Shiauo S., Ko A.: J. Chin. Chem. Soc.: 54, 1539 (2006).


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PHARMACEUTICAL TECHNOLOGY

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EFFECT OF ALCOHOL ON TRAMADOL HYDROCHLORIDE RELEASE FROM CONTROLLED RELEASE FORMULATIONS CONTAINING CO-PROCESSED DRY BINDERS Myslíková K.1, Komersová A.1, Lochař V.1, Mužíková J.2 Department of Physical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 95, 532 10 Pardubice, Czech Republic 2 Department of Pharmaceutical Technology, Charles University, Faculty of Pharmacy in Hradec Králové, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic [email protected] 1

Abstract

The effect of alcohol on tramadol hydrochloride (TH) release from hydrophilic matrix tablets containing coprocessed dry binder DisintequikTM MCC 25 and hypromellose in different viscosity degrees (as retarding component) was studied due to possibility of dose dumping effect. The release of TH from prepared drug formulations was studied by the dissolution test method using rotating basket apparatus. Dissolution test was performed in acidic medium (pH 1.2), in simulated gastric fluid (SGF) and in SGF containing 5% (v/v) of alcohol. The released amount of TH was determined using UV VIS spectroscopy at wavelength of 271 nm. Obtained dissolution profiles was evaluated by non-linear regression analysis. It was found that the presence of alcohol influeces the release rate of TH from studied formulations only in pepsin presence. The cumulative amount of TH released in 24 h is lower in comparison with acidic medium. Keywords: co-processed dry binders, matrix tablets, alcohol, pepsin, simulated gastric fluid

Introduction

Controlled release drug formulations contain significant amounts of an active substance which, if released as a single bolus dose, could cause severe adverse events. Therefore, the release mechanism of these drugs must be sufficiently robust to prevent of any possibility of uncontrolled release of the active substance leading to dose dumping. Dose dumping (DD) effect can be caused e.g. by change of pH and/or ionic strength1 or by alcohol presence2 (ADD). The evaluation of the drug release rate in vitro based on the dissolution testing allows to some extent to predict the drug behaviour in vivo. For the correlation in vitro/in vivo it is crucial to conduct the dissolution experiments under conditions that resemble the key parameters of gastrointestinal (GI) tract3 therefore the dissolution medium should mimic gastric or intestinal fluid. Simulated gastric fluid (SGF) is a synthetic form of the gastric fluid which simulates the gastric conditions in the fasted state. Preparation of this artificial dissolution medium containing sodium chloride, purified pepsin (from porcine stomach mucosa) and hydrochloric acid (pH of about 1.2) is described in European Pharmacopoeia4 and United States Pharmacopoeia5. More accurate simulation of GI conditions than simulated gastric fluid and simulated intestinal fluid can provide the biorelevant media simulating the fasted and fed states 3. GI fluids affect the drug release rate from hydrophilic matrices particularly by varying pH value and ionic strength. In man under both fasted and fed states and various physiological pH conditions, the ionic stregth of GI fluids covers a range of 0-0.4 M6. The ionic strength of fasted stomach was estimated approximately 0.11 M 7 (Lindahl et al., 1997). It was also reported7 that the gastric emptying is inhibited by pure ethanol. Mean half-emptying time of 27.8 min with 40% solution of ethanol is significantly longer than after ingestion of water (14.7 min). Presence of ethanol influences not only gastric emptying but it was described that some oral extended release dosage forms exhibit more rapid drug release in the presence of ingested ethanol due to a higher solubility of the drug and/or excipients in hydro-alcoholic media2. The higher release rate of the drug and after dose dumping effect may be an issue for patient safety. The extended release formulations contain a larger unit dose than immediate release tablets (as was mentioned above) and therefore their dissolution kinetics have to be tighly controlled. Accordingly, the dissolution testing used for in vitro evaluation of the drug release rate should be performed in media containg different amount of ethanol and effect of ethanol on release kinetics should be described quantitatively. The aim of this in vitro study was to evaluate the effect of alcohol on the release rate of tramadol hydrochloride from hydrophilic matrix tablets containing microcrystalline cellulose in co-processed dry binder due to possibility of dose dumping effect.


433

Experimental

Materials Hypromelloses MethocelTM K4M Premium CR or MethocelTM K100M Premium CR (both from Colorcon GmbH, Germany) were used as controlled release agents forming a hydrophilic matrix system. Disintequik TM MCC 25 (Kerry, USA) was used as co-processed dry binder and magnesium stearate (Acros Organics, USA) was used as a lubricant. Tramadol hydrochloride (TH, European Pharmacopoeia Reference Standard, Sigma Aldrich Chemie GmbH, Germany) was chosen as a model water soluble drug. Pepsin from porcine gastric mucosa (Sigma Aldrich Chemie GmbH, Germany) was used for the preparation of simulated gastric fluid (SGF). Ethanol 96% (alcohol) according to specification ph. eur. (Lach-Ner s.r.o., Neratovice, Czech Republic) was used to prepare the hydroalcoholic dissolution medium (5% (v/v)). For the preparation of dissolution media and standard solution of tramadol hydrochloride, redistilled water and chemicals of analytical grade (Lach-Ner s.r.o., Neratovice, Czech Republic) were used. Preparation of tablets Composition of studied formulations is described in Table I. Tablets were prepared by direct compression method using a material testing equipment T1-FRO 50 TH.A1K Zwick/Roell (Zwick GmbH&Co, Germany) by means of a special die with a lower and an upper punch. The rate of compaction was 40 mm/min, pre-load was 2 N, and the rate of pre-load 2 mm/s. The tablets were of cylindrical shape without facets of a diameter of 13 mm and weight of 0.5 ± 0.0010 g. Preparation of tablets and tableting materials is described in detail in previous paper11. For one dissolution test, 6 tablets with the active ingredient and 1 tablet without the active ingredient as the blind sample (69% of Prosolv® SMCC 90 or DisintequikTM MCC 25, 30% of MethocelTM K4M (or K100M) Premium CR and 1% of magnesium stearate) were compressed. Table I Tablets composition (%). Formulation F1

Formulation F2

Disintequik™MCC 25

49

49

Methocel™K4M Premium CR

30

-

Methocel™ K100M Premium CR

-

30

Tramadol hydrochloride

20

20

Magnesium stearate

1

1

In vitro dissolution studies The release of TH from prepared drug formulations was studied by the dissolution test method according to the European Pharmacopoeia4 using rotating basket apparatus (Sotax AT 7 Smart, Allschwil, Switzerland). Dissolution test was performed in different dissolution media:1) acidic medium (2.0 g of NaCl was dissolved in 80 mL of 1M HCl and added water to a volume of 1 L, pH adjusted to 1,2 with HCl); 2) simulated gastric fluid (SGF, 2.0 g of NaCl and 3.2 g of pepsin powder was dissolved in 7 mL of HCl (420 g/l) and added water to a volume of 1 L); 3) acidic medium with 5% (v/v) of alcohol (855 ml of acidic medium (1) was mixed with 45 ml of alcohol); 4) simulated gastric fluid with 5% (v/v) of alcohol (SGF was prepared as described above and 855 ml of this solution was mixed with 45 ml of alcohol). Six tablets with TH and one blank tablet were placed to the baskets and immersed in the dissolution medium (900 mL). All tests were carried for 24 hours at a stirring rate of 125 rpm. Temperature was maintained at 37 ± 0.5°C. At predetermined times, 3 mL of the dissolution medium was automatically withdrawn, samples were filtered and the TH concentration was determined using UV VIS spectroscopy at wavelength of 271 nm. The corresponding cumulative amount of TH released was determined using calibration curve method. Each dissolution experiment was performed once (with six tablets) and the mean values of the released amount of TH with their standard deviations were calculated. Kinetics of drug release The kinetics of drug release was evaluated using the first-order kinetic model9,10 (Eq. (1)) and Weibull model (Eq. (2)): 𝐴𝐴𝑡𝑡(𝑙𝑙) = 𝐴𝐴∞ (1 − 𝑒𝑒 −𝑘𝑘𝑘𝑘 )

𝐴𝐴𝑡𝑡(𝑙𝑙) = 𝐴𝐴∞ (1 − 𝑒𝑒

434

−𝜆𝜆(𝑡𝑡−𝑇𝑇𝑖𝑖 )𝑏𝑏

)

(1)

(2)


where 𝐴𝐴𝑡𝑡(𝑙𝑙) is the amount of drug released in time t, k is the first order release rate constant in units of time-1, A∞ is the maximum releasable amount of drug, λ represents reciprocal value of time scale of the process, the location parameter Ti represents the lag time before the onset of the dissolution (in most case is zero) and b describes the shape of the dissolution curve progression. All experimental data were mathematically processed and statistically evaluated by means of the computer programmes Graph Pad Prism and Origin 9 Pro. Statistical significance was tested using Student’s t test for unpaired samples, at a significance level of P80 %) and in high purity (>95%). Final products and intermediates were characterized by means of FTIR spectroscopy, XRD powder diffraction and ESI-MS spectrometry in the negative ionization mode.

Main body Introduction Cancer diseases are after cardiovascular diseases the second most serious diseases causing death of over 8 million people in the world (over 1.3 million in EU) annually. Cancer treatment is based on the individual patient’s state, stage of disease and type of cancer. Current treatment methods are usually based on a combination of several methods such as chemotherapy, radiotherapy, surgery, etc. One of the most frequent 1 and successful treatment is chemotherapy . One of the biggest group of chemotherapeutics in clinical use are 2 platinum based drugs. These drugs are coordination compounds where the central atom is Pt(II) . The first platinum cytostatic was complex called Cisplatin (1978) followed by complexes Carboplatin (1980), Oxaliplatin (1986), etc. (Figure 1). Although hundreds of complexes were synthesized only these complexes used are currently in clinical use. Platinum complexes exhibit high efficiency against many solid tumors but their use is limited due to their gradual initiation of cell resistance and many side effects such as neurotoxicity, 3 th nephrotoxicity, vomiting, etc. . Current research is focused on the 4 generation of platinum complexes of 4 octahedral geometry with central atom of Pt(IV) . This structural type gives the complex new chemical and physical properties such as improved solubility and balanced lipophilicity. Also, new octahedral structure provides steric hindrance of platinum atom preventing the inactivation of complex by proteins in blood 5 plasma . These complexes also exhibit different mechanism of action because they are in prodrug form and inside of cell they are reduced to Pt(II). The first clinically tested compound has been Satraplatin followed by 6, 7 experimental drug known under code name LA-12 . These complexes exhibit higher cytotoxicity against many 8 tumors (ovarian, prostate, colon) and less side effects than currently used platinum cytostatics . Both 9, 10 complexes have successfully passed Phase II clinical trials . The synthesis of Pt(IV) complexes consist of three individual steps (condensation, oxidation and esterification). Final product and each intermediate have been isolated in pure form and analytically characterized. For this purpose, the new analytical method has been developed for LC-MS system. For the determination of purity there has been employed liquid chromatography with various reversed stationary phases (strong and weak anion exchanger and C18) and conditions. The biologic activity of new complexes was tested against tumor cell lines and one epithelial cell line in MTS cytotoxicity assay.

Figure 1. Clinically used platinum cytostatics (Cisplatin, Carboplatin and Oxaliplatin)


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Experimental Chemicals The platinum precursor K[PtNH3Cl3] used for synthesis of complexes was obtained from VUAB Pharma, a.s. (Czech Republic). Ligands used in synthesis: 1-adamantylamine, 2-adamantylamine, 1-(3,5® dimethyladamantanyl)amine and acetic anhydride were purchased from Sigma-Aldrich (Germany). All other solvents and reagents used during a synthesis – methanol, water, dioxane were obtained from Lach-Ner (Czech Republic) in reagent quality. Hydrogen peroxide (30 %) was purchased from Penta, s.r.o. (Czech Republic). Prior to use, all solvents were filtered through 0.22 µm nylon membrane filter. Instrumental methods All prepared complexes (final products and intermediates) were analytically characterized by ESI-MS and FTIR. Analyses were recorded on system consisting of Accela 1200 pump, Accela AS and TSQ Vantage mass spectrometer equipped with ESI ionization (Thermo Scientific, USA). The spectra were obtained in negative full scan mode in 100-1000 Da mass range. Infrared spectra were recorded on FT-IR Nicolet 6700 -1 spectrophotometer (Thermo Scientific, USA) using ATR crystal in the 400-4000 cm wavelength range. The XRay powder diffraction analyses were performed on XRD 3000 P (Seifert, Germany) in the 0-60° (2Theta) range. For measurements, there was used Cu anode lamp and collected data were compared to PDF-2 crystallography database. Results were evaluated in X’Pert Data Viewer. Synthesis of complexes The complexes were synthetized according to general procedure described in patent of company PLIVA11 Lachema, a.s. . This process comprises three individual steps (condensation, oxidation and esterification). The starting material for all complexes was platinum complex K[PtNH3Cl3].

Figure 2. Ligands used for synthesis of platinum complexes; from left: 1-adamantylamine a, 2-adamantylamine b, 1-(3,5-dimethyladamantanyl)amine c Synthesis of condensation product The mixture of the chosen adamantylamine ligand (Figure 2; a, b or c) in 0.6 mL methanol was added at once to the mixture of K[PtNH3Cl3] in 0.4 mL DMF preheated at 55 °C. The mixture was stirred without presence of light for 2 hours. After the mixture was cooled to 25 °C, 1 mL of methanol was added and mixture was stirred for half a hour. Then, the mixture was filtered and yellow solid (1a-1c) was obtained. Synthesis of Amminedichloro(adamantan-1-amine)-platinum (II) complex (1a) -

K[PtNH3Cl3] (0.10 g; 0.28 mmol), a (0.04 g; 0.28 mmol). Yield 85 % (0.10 g). ESI-MS [M+HCL] expected mass: -1 434.269; Mass founded: 469.986. FT-IR (ATR, cm ) ν: 3221 (γNH2), 2905, 2848 (CH2), 1452, 1382, 1112 (Pt complex). Synthesis of Amminedichloro(adamantan-2-amine)-platinum (II) complex (1b) -

K[PtNH3Cl3] (0.10 g; 0.28 mmol), a (0.04 g; 0.28 mmol). Yield 98 % (0.12 g). ESI-MS [M+HCL] expected mass: -1 434.269; Mass founded: 469.974. FT-IR (ATR, cm ) ν: 3234 (γNH2), 2921, 2834 (CH2), 1476, 1385, 1114 (Pt complex). Synthesis of Amminedichloro(3,5-dimethyladamantan-1-amine)-platinum (II) complex (1c) -

K[PtNH3Cl3] (0.10 g; 0.28 mmol), a (0.04 g; 0.28 mmol). Yield 98 % (0.12 g). ESI-MS [M+HCL] expected mass: -1 462.322; Mass founded: 496.974. FT-IR (ATR, cm ) ν: 3234 (γNH2), 2921, 2834 (CH2), 1476, 1385, 1114 (Pt complex). Synthesis of oxidation product The second step of the synthesis is based on oxidation of condensation product with hydrogen peroxide. The 0.1 g of condensation product 1a-1c was suspended in 1.5 mL water and then the mixture was heated to 70 °C

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while stirring. Subsequently, the solution of 30% hydrogen peroxide was slowly added to the mixture. The mixture was stirred for 1 hour. Then, the mixture was filtered and pale yellow solid for 2a-2c was obtained. Synthesis of Amminedichlorodihydroxy(adamantan-1-amine)-platinum (IV) complex (2a) -

1a (0.10 g; 0.23 mmol), H2O2 (0.5 ml; 4.6 mmol). Yield 98 % (0.11 g). ESI-MS [M+HCL] expected mass: 467.071; -1 Mass founded: 503.151 FT-IR (ATR, cm ) ν: 3580 (OH), 3254 (γNH2), 2906, 2848 (CH2), 1452, 1362, 1103 (Pt complex). Synthesis of Amminedichlorodihydroxy(adamantan-2-amine)-platinum (IV) complex (2b) -

1b (0.10 g; 0.23 mmol), H2O2 (0.5 ml; 4.6 mmol). Yield 96 % (0.10 g). ESI-MS [M+HCL] expected mass: 467.071; -1 Mass founded: 503.151. FT-IR (ATR, cm ) ν: 3582 (OH) 3278 (γNH2), 2906, 2834 (CH2), 1453, 1349, 1105 (Pt complex). Synthesis of Amminedichlorodihydroxy(3,5-dimethyladamantan-1-amine)-platinum (IV) complex (2c) -

1c (0.10 g; 0.22 mmol), H2O2 (0.5 ml; 4,4 mmol). Yield 94 % (0.10 g). ESI-MS [M+HCL] expected mass: 495.102; -1 Mass founded: 531.152. FT-IR (ATR, cm ) ν: 3397 (OH), 3234 (γNH2), 2921, 2834 (CH2), 1476, 1385, 1114 (Pt complex). Synthesis of crude product The final stage of synthesis was esterification step. In this step, the 0.1 g of product 2a-2c was dissolved in 5 mL of dioxane and mixture was heated at 60 °C with intensive stirring. Then, the acetic anhydride was slowly added to the solution. The reaction time was 12 hours. The mixture was filtered, filter cake was dried at 50 °C and 45 torr and slightly yellow product 3a-3c was obtained. Synthesis of Bis(acetato-κO)amminedichlorodihydroxy(adamantan-1-amine)-platinum (IV) complex (3a) -

2a (0.10 g; 0.21 mmol), acetic anhydride (0.4 ml; 4.2 mmol). Yield 93 % (0.11 g). ESI-MS [M-H] expected mass: -1 552.357; Mass founded: 550.986. FT-IR (ATR, cm ) ν: 3452 (γNH2), 2908, 2848 (CH2), 1704, 1619 (C=O), 1362, 1102 (Pt complex). Synthesis of Bis(acetato-κO)amminedichlorodihydroxy(adamantan-2-amine)-platinum (IV) complex (3b) -

2b (0.10 g; 0.21 mmol), acetic anhydride (0.4 ml; 4.2 mmol). Yield 91 % (0.11 g). ESI-MS [M-H] expected mass: 552.357; Mass founded: 550.988. FT-IR (ATR, cm-1) ν: 3482 (γNH2), 2906, 2834 (CH2), 1705, 1620 (C=O), 1361, 1103 (Pt complex). Synthesis of Bis(acetato-κO)amminedichlorodihydroxy(3,5-dimethyladamantan-1-amine)-platinum (IV) complex (3c) -

2c (0.10 g; 0.20 mmol), acetic anhydride (0.4 ml; 4.0 mmol). Yield 85 % (0.10 g). ESI-MS [M-H] expected mass: -1 580.410; Mass founded: 579.016. FT-IR (ATR, cm ) ν: 3222 (γNH2), 2961, 2878 (CH2), 1748, 1660 (C=O), 1449, 1342, 1108 (Pt complex). Biological methods Each of final products was tested against breast cancer cell line MCF-7 and colorectal carcinoma CaCo-2 cell line. Tests were also performed on healthy epithelial cell line MRC-5 where the toxicity of prepared compounds against healthy tissue was monitored. The tests were performed at the Faculty of Medicine and Dentistry of the Palacky University in Olomouc, Department of Molecular and Translation Medicine. Each test was performed twice to clarify the results and to avoid possible mistakes. The assay was performed in the 368 well plates where the cell were cultivated. Then, the solution of sample in DMSO was added to the cells at five concentration levels in two copies. Together with tested substances, positive and negative controls were created and blank sample was created to verify the authenticity of the test. Positive controls included two known cytostatics Actinomycin D and Mitomycin C. Negative controls were DMSO and blank containing only the culture medium. Then, the cells were exposed. Subsequently the MTS contrast agents were added to the cells. The results were acquired after 2 hours of exposition using a spectrometer at a wavelength of 490 nm. Results were evaluated by software. The required parameter of these assays was the IC 50 value, the inhibitory concentration.

Discussion and results Synthesis and characterization


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known cytostatics Actinomycin D and Mitomycin C. Negative controls were DMSO and blank containing only the culture medium. Then, the cells were exposed. Subsequently the MTS contrast agents were added to the cells. The results were acquired after 2 hours of exposition using a spectrometer at a wavelength of 490 nm. Results were evaluated by software. The required parameter of these assays was the IC50 value, the inhibitory concentration.

Discussion and results Synthesis and characterization

Intensity (counts)

The complexes 3a-3c were synthetized by multistep reaction of K[PtNH 3Cl3] with ligand a-c followed by oxidation with hydrogen peroxide and esterification with acetic anhydride. All complexes were obtained as yellow solids and were stable on air. Complexes were prepared with high yield and high purity. Final complexes 3a-3b are slightly soluble in methanol and soluble in the DMSO and DMF. All products and intermediates were analytically characterized using of FT-IR spectroscopy, ESI-MS -1 -1 spectrometry. Infrared spectra in the 4000-400 cm region for ligands a-c contain bands in the 2906-2834 cm range; these bands are associated with ν(CH2) absorptions of adamantyl ligands. Typical bands for platinum -1 complex were found in the 1452, 1382, 1294, 1156, 1112 and 1036 cm . For complexes 2a-2c were found -1 bands in the 3580-3394 cm ; this bands are associated with ν(OH) absorption of hydroxyl group. For complexes -1 3a-3c were typical bands in range of 1600-1700 cm which are associated with ν(C=O) and ν(COO ) absorption of carboxyl groups from axial ligands. ESI-MS analyses were performed for each complex. The data were acquired in negative scan mode in the range of masses 100-1000 Da. Each platinum cluster corresponded with respective theoretical calculation. X-ray powder diffraction was measured for final complexes. All complexes were crystalline and diffractograms were obtained as seen in Figure 3, Figure 4 and Figure 5.

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Figure 5. Diffractogram of complex 3c Biological activity The cytotoxicity activity of prepared complexes was investigated using in vitro MTS assays on human cell lines. The cell lines used for tests were human line of colorectal carcinoma - CaCo-2 and breast cancer cell line MCF7. The samples were tested in DMSO. The determined IC50 values, the concentration of complex needed to inhibit 50 % of cell growth, are summarized in Table I. The values were compared to approve platinum cytostatic Oxaliplatin. The prepared platinum complexes exhibit promising activity towards various cell lines, however the best results were obtained for the colorectal carcinoma cell line. Table I: Results of MTS assays of platinum complexes with comparison to clinically used Oxaliplatin IC50 [µM ] 3a 3b 3c Oxaliplatin

MCF-7 5.63 18.84 3.54 3.69

CaCo-2 4.63 13.25 2.23 2.93

MRC-5 8.02 18.74 7.21 >50

Conclusion The series of three platinum complexes with ligands: 1-adamantylamine a, 2-adamantylamine b, 1-(3,5-dimethyladamantanyl)amine c was prepared. The biologic activity was tested on the human cell lines including MCF-7 (Carcinoma of breast) and CaCo-2 as cell line of colorectal carcinoma. The results were compared to healthy epithelial cell line MRC-5. The best result showed complex 3a for all tested cell lines. The promising results were obtained for complex 3c on the both lines CaCo-2 (2.23 µM) and MCF-7 (3.54 µM). This complex exhibits better cytotoxicity than commercial cytostatic Oxaliplatin. All complexes demonstrate correlation between structure of horizontal ligand and final cytotoxicity.

Acknowledgement This work was supported from specific university research (MSMT No 20-SVV/2017), “Operational Programme Prague – Competitiveness” (CZ.2.16/3.1.00/21537) and the “National Programme of Sustainability I” - NPU I (LO1601 - No.: MSMT-43760/2015).

References 1. 2 3 4 5 6

Eitel A., Scherrer M., Kümmerer K.: Handling cytostatic drugs a practical guide. Bristol-Myers Squibb, Bristol, 1999. Gibson D., Wexselblatt E. J.: Inorg. Biochem. 117, 220 (2012). Enzo A., Biorganic Medicinal Chemistry. WILEY, Germany 2011. Foltinová V., Švihálková Šindlerová L., Horváth V., Sova P., Hofmanová J., Janisch R., Kozubík A.: Spisy Lek. Fak. Masaryk. Univ. 81, 105 (2008). Ryvolova M., Smerkova K., Chomoucka J., Hubalek J., Adam V., Kizek R.: Electrophoresis. 34, 801 (2013). Graf N., Lippard S. J.: Adv. Drug Delivery Rev. 64, 993 (2012).


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7 8 9 10 11

Boulikas T., Pantos A., Bellis E., Christofis P.: Cancer Therapy. 5, 537 (2007). Wexselblatt E., Gibson D.: J. Inorg. Biochem. 117, 220 (2012). Turánek J.; Hofmanová J., Vaculová A., Souček K., Vondráček J., Kozubík A.: Met.-Based Drugs 2008, (2008). Švihálková-Šindlerová L., Foltinová V., Vaculová A., Horváth V., SoučekK., Sova P., Hofmanová J., Kozubík A.: Anticancer Res. 30, 1183 (2010). Zak F., Mistr A., Poulova A., Melka M., Turanek J., Zaluska D.: Platinum complex its preparation and therapeutic application. PCT Intl. Patent Appl. Publ. WO 9961451.

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EFFECT OF HYDROPHILIC POLYMERS ON DISSOLUTION PROFILES OF BINARY MIXTURES Školáková T., Slámová M., Patera J., Zámostný P. University of Chemistry and Technology, Prague, Department of Organic Technology, Technická 5, Prague 6, 166 28, Czech Republic [email protected]

Abstract Solubility and dissolution rate are limiting step in absorption of an active pharmaceutical ingredient (API), especially for a poorly water-soluble drug substance. This study is aimed at determining the effect of hydrophilic polymers (Kollidon® 12 PF, Kollidon® VA 64 or Soluplus®) on dissolution profiles of acetaminophen binary mixtures: physical mixtures of API and polymer and corresponding solid dispersions which were prepared by melt method. An erosion-diffusion mechanism of acetaminophen release was also evaluated. The Wood apparatus was used to determine the intrinsic dissolution rate of acetaminophen and its dissolution profiles of release were measured using a flow-through cell apparatus in an open-loop configuration (USP4). It was found that all of hydrophilic polymers formed gel layer. Kollidon® VA 64 and Soluplus® retarded the acetaminophen release and thus, the diffusion mechanism dominated. On the other hand, Kollidon® 12 PF accelerated the acetaminophen release and in this case, the erosion mechanism dominated.

Introduction The dissolution rate of API from the solid dosage form is one of the key parameters which can be affected by various factors, e.g. the physicochemical properties of the drug substance or drug product1,2. Most solid dosage forms contain API and a number of excipients leading to better oral administration, manufacture, stability or identification3,4. Moreover, hydrophilic polymers are commonly used as oral drug delivery systems5. It is generally known that drug release is controlled by dissolution of polymeric carrier which is dependent on two processes, i.e. diffusion of solvent into the polymer and the complete dispersion of the polymer in the medium. Moreover, some polymers can swell after diffusion of solvent into the their structure and the swollen layer forms a barrier which can critically influence the drug release by opposing penetration of solvent into the solid dosage form and also movement of dissolved solutes out of the form5-8. Thus, the release of drug substance is controlled by combination of erosion-diffusion mechanism9. The solid dispersions are mainly drug-polymer two-component systems10. Therefore, the aim of this study was to investigate the impact of hydrophilic polymers on drug release from solid dispersion and to compare them with corresponding physical mixtures and to evaluate the erosiondiffusion mechanism of drug release.

Materials and Methods Materials Acetaminophen (A) was obtained from Zentiva, k.s. (Prague, Czech Republic). Kollidon® 12 PF (K12), Kollidon® VA 64 (K64) and Soluplus® (Sol) were donated by BASF Pharma (Ludwigshafen, Germany). Hydrochlorid acid and phosphoric acid were purchased from Penta (Prague, Czech Republic), and diammonium phosphate and methanol were obtained from Sigma-Aldrich (Prague, Czech Republic). Preparation of physical mixture (PM) and solid dispersion (SD) The API was mixed with polymers in 50:50 or 25:75 (w/w) ratios to prepare PMs of 2 g each. Solid dispersions were prepared by melt method. The PMs were melted in an evaporating dish using a special metal holder which was placed on a magnetic stirrer with heating. The temperature for a mixtures was selected based on the thermal data (i.e. melting and glass transition temperature) of components. The melted liquid was cooled and milled using pestle and mortar. Wood intrinsic dissolution apparatus For intrinsic dissolution determination measurements, the PMs were compressed by 15 kN force for 1 minute into the die which was attached to the rotor shaft. Dissolution testing of tablets was performed in dissolution apparatus Sotax AT7 Smart (50 rpm). The tablets were dissolved in 0.1M hydrochloric acid (37 °C). The samples were taken in following times: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 minutes. The concentration of acetaminophen was measured by UV-VIS Specord® 200 PLUS.


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Flow-through methods for dissolution The dissolution tests were carried out in the USP apparatus 4 with flow-through cell in an open-loop configuration (Sotax Dissotest CE1) and piston pump (Sotax CY 1). The cell with a 12-mm diameter and a 32-mm height was used for powders. The cell was equipped by placing a 5-mm diameter glass bead into the apex of the cone to protect the inlet tube and a 1-mm diameter glass beads into the conical part to ensure laminar flow profile. Approximately 0.2 g of powder was placed on the two sieves which were positioned on the glass bead bed. The cell was closed by filter to prevent undissolved material from escaping. The flow-through cell apparatus and degassed dissolution medium (0.1M HCl) were placed into the 37 °C water bath. The flow rate of dissolution medium was 25 mL/min and the samples were taken into the vials in following times: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 180, 240, 360, 480, 600 and 900 seconds. The samples were analysed by high performance liquid chromatography (Shimadzu Prominence) with photodiode detector (column: LiChrospher 60, RP-select B, 5 µm, Merck). The injection volume of the sample was 2 µL. The flow rate of mobile phase was 1 mL·min-1 and the temperature of oven was 30 °C. Acetaminophen was monitored at 280 nm. The mobile phase consists of phosphate buffer pH 2.5 and methanol (3:2, v/v). Phosphate buffer was prepared by dissolving 4 g diammonium phosphate in 900 mL distilled water. This solution was adjusted to pH 2.5 ± 0.1 with hydrochlorid acid-distilled water (1:1, v/v). The volume was added to 1000 mL with distilled water. The mobile phase was degassed.

Results and discussion All dissolution profiles of PMs and SDs obtained by the Wood apparatus were linear and therefore, the results are summarized in the form of slopes and calculated intrinsic dissolution rates (IDRs) in Table 1 and 2. IDRs were calculated using the following Equation (1). 𝐼𝐼𝐼𝐼𝐼𝐼 =

𝑉𝑉 𝑑𝑑𝑑𝑑 𝐴𝐴 𝑑𝑑𝑑𝑑

(1)

where V is the volume of the dissolution medium (L), A is the surface area of the sample (cm2), c is the concentration of dissolved API in the dissolution medium (mg/L), and t is time (s). Table 1: Intrinsic dissolution rate of acetaminophen from PMs PM

Ratio (w/w)

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K12-A K64-A Sol-A

Table 2: Intrinsic dissolution rate of acetaminophen from SDs SD

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1:1 3:1 1:1 3:1 1:1 3:1

K12-A K64-A Sol-A

Slope (mg·L-1·min-1) 0.77 1.17 1.28 0.37 0.36 0.27 0.04

IDR (mg·cm-2·min-1) 1.53 2.32 2.54 0.74 0.71 0.54 0.07

Slope (mg·L-1·min-1) 0.77 0.95 1.20 0.33 0.26 0.05 0.03

IDR (mg·cm-2·min-1) 1.53 1.88 2.39 0.65 0.52 0.10 0.06

Results clearly show that formulations containing hydrophilic polymer displayed a constant drug release rate which corresponds zero-order type release kinetics. Moreover, the release rate can be controlled by the type and properties of the polymer. As can be seen in Table 1 and 2, K12 accelerated the drug release in all cases. IDR was about 52 % to 66 % (with increasing amounts of K12) higher for PMs than for acetaminophen (A) itself. For

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SDs, IDR was about 23 % to 57 % higher in comparison to API. Therefore, the erosion was probably the predominant mechanism and K12 provided a faster drug release. In contrary, the retardation effect of K64 and Sol was observed. In this case, the formed gel layer was thicker and acted as a diffusion barrier and as a result they delayed drug dissolution and release. Therefore, the drug-diffusion probably was the predominant mechanism. IDRs were even over 90 % lower for PMs and SDs containing Sol in compare to acetaminophen. In Figure 1, the appearance of tablets after dissolution test is illustrated. A viscous gel mass is evident on some tablets. The presence of some grooves and lamination was also observed. The tablets containing K12 were completely dissolved, apart from SD K12-A 1:1. This tablet was also significantly deformed which can be caused by polymer erosion which was, however, far slower in comparison to other mixtures containing K12.

Figure 1: The appearance of tablets after dissolution In the case of SDs, it was also shown that as the molecular weight of the polymer increased, the gel layer on the surface of sample increased too and in contrary, the dissolution decreased. It proves that K12 with the lowest the molecular weight formed the weaker the gel layer. Therefore, these tablets displayed faster release due to its less swelling and the erosion was the main mechanism. These facts were in compliance with release of A which


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was measured using flow-through apparatus (Figure 2). On the other hand, the dissolution of A from PMs (Figure 3) was effected by many factors, such as swelling out of die (especially for Sol) or possible interactions between A and K64 which caused an uneven dissolution.

Figure 2: The release profiles of acetaminophen from SDs a) 1:1, b) 3:1 K12-A,

K64-A,

Sol-A

Figure 3: The release profiles of acetaminophen from PMs a) 1:1, b) 3:1 K12-A,

K64-A,

Sol-A

Although, the retardation effect of Sol was observed, from the Figure 3 a) is obvious that PMs containing K12 or Sol had very similar the release profiles of acetaminophen. This difference from the results obtained by the Wood apparatus can be explained by the role of compression which is an important factor in the releasing of API. Therefore, in the case of tablets, the swelling greatly influence the drug release due to the external layers which absorb the dissolution medium and form a swellable barrier in comparison to powder.

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Conclusions We have shown the effect of hydrophilic polymers (Kollidon® 12 PF, Kollidon® VA 64 and Soluplus®) on the release profiles of acetaminophen from different binary mixtures (physical mixtures or solid dispersion). The Wood apparatus was found to be suitable for determining the intrinsic dissolution rate and for assessing the retardation effect of each specific polymer. Moreover, the flow-through cell apparatus in an open-loop configuration was able to characterize the dissolution behaviours. Our results indicated that the type of hydrophilic polymer used as a matrix in the formulation can control the dissolution rate which, in turn, can lead to control of the release either immediate or sustained. All three polymers which were used in this study are suitable for the modification of release. Moreover, our results suggest that the concentration of active substance in the mixtures does not play a key role in its release.

Acknowledgement This research was financially supported by specific university research MSMT No. 20-SVV/2017. The authors would also like to acknowledge the BASF Pharma for providing polymers.

References 1. 2. 3. 4. 5. 6.

Petrů J., Zámostný P.: Procedia Eng., 42, 1463 (2012). Petrů J., Zámostný P.: Dissolut. Technol., 21, 40 (2014). Gavatur R., Vernuri M. N., Chrzan Z.: J. Therm. Anal. Calorim., 78, 63 (2004). Jackson K., Young D., Pant S.: Pharm. Sci. Technol. Today, 3, 336 (2000). Sriamornsak P., Thirawong N., Weerapol Y., Sungthougjeen S.: Eur. J. Pharm. Biopharm., 67, 211 (2007). Knopp M. M., Chourak N., Khan F., Wendelboe J., Langguth P., Rades T., Holm R.: Eur. J. Pharm. Biopharm., 205, 106 (2016). 7. Craig D. Q. M.: Int. J. Pharm., 231, 131 (2002). 8. Harland R. S., Gazzaniga A., Sangalli M. E., Colombo P., Peppas N. A.: Pharm. Res., 5, 488 (1988). 9. Raval A., Parikh J., Engineer C.: Braz. J. Chem. Eng., 27, 211 (2010). 10. Huang Y., Dai, W.: Acta Pharm. Sin. B, 4, 18 (2014).


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EFFECT OF DILUENT PARTICLE SIZE ON FLOW PROPERTIES AND HOMOGENITY OF BLENDS FOR DIRECT TABLET COMPRESSION Zámostný P.1, Majerová D.1,2, Bartáková M.1,2 University of Chemistry and Technology Prague, Technická 5, 166 28 Prague 6 – Dejvice, Czech Republic Zentiva k.s., U Kabelovny, Prague, Czech Republic [email protected]

1 2

Abstract The study is focused on composition optimization of the blend for direct tablet compression, which exhibits poor flow characteristics leading to less weight uniformity of compressed tablets and to other technological problems. The aim of this study was to modify the composition of the blend by partial or complete substitution of current excipient with the same one with coarser grade, so that the flow properties would improve. The impact on segregation was also investigated. Several blend batches were made, using varying proportions of the fine and the coarse grade of the excipient. Flow characteristics of the individual excipients and blends were measured, as well as the blend homogeneity and segregation indices. It was found that blends using partial substitution of excipients show a better combination of segregation and flow characteristics than the original composition.

Introduction Direct tablet compression represents a preferable and cost-effective approach, adopted to manufacture pharmaceutical tablet. Favorable flow properties, required for uniform filling of the compression die and thus the tablet weight uniformity, are achieved by blending the active pharmaceutical ingredient (API) with excipients having good flow properties by themselves1. However, the excipients must fulfill other goals as well, so that tradeoffs are a commonplace in direct-compression formulations2. Diluent-binder systems based on calcium hydrogen phosphate dihydrate and microcrystalline cellulose belong among the more common direct-compression formulations used in direct tablet compression. Commonly available forms of CaHPO4 dihydrate include the fine powder constituted by individual crystallites (d90 < 5 μm) and the Emcompress® constituted by aggregates rather than individual particles. Emcompres® exhibits superior flow properties, but the larger particle size may cause problems associated with the blend homogeneity when combined with fine API particles3. The study is focused on composition optimization of the blend for direct tablet compression, which exhibits poor flow characteristics leading to less weight uniformity of compressed tablets and to other technological problems due to the small mean particle size of the filler calcium hydrogen phosphate dihydrate. The aim of this study was to modify the composition of the blend by partial or complete substitution of current excipient with the same one with coarser grade, so that the flow properties would improve. Since the substitution may be accompanied by a deterioration of the segregation characteristics with subsequent negative impact on the homogeneity of blend, investigating this substitution impact on segregation was also an important goal.

Sample preparation Direct-compression formulation for amlodipine besylate was used as a model formulation in this study. Since amlodipine besylate cannot be formulated using lactose, this type of formulation is typical for the component and it is employed by many manufacturers. The exact composition of the model formulation is summarized in the Table I. All materials present in the mixture were provided by courtesy of Zentiva company. Calcium hydrogen phosphate dihydrate is used as a generic name. Five batches representing alternative formulations were prepared containing either the “fine” or the “coarse” grade of the excipient, or a combination of both grades. Table II shows the proportion of the two grades of calcium hydrogen phosphate dihydrate used for preparing specific batches of the formulation. The more detailed particle size distribution (PSD) is shown in Figure 1.

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Table I Basic composition of the tablet formulation and mean particle sizes of the components obtained by static light scattering Component w, wt. % d50, μm Amlodipine besylate 5.8 20 Calcium hydrogen phosphate dihydrate 31.3 10/100* 50 MCC Avicel® PH101 10.1 100 MCC Avicel® PH102 49.3 Sodium starch glycolate 2.0 Magnesium stearate 1.5 *The sizes are valid for the “fine” and “coarse” type of the excipient respectively Table II Distribution of CaHPO4.2H2O grades in prepared batches CaHPO4.2H2O grade fine Batch code wt. % T0 100 T25 75 T50 50 T75 25 T100 0

coarse wt. % 0 25 50 75 100

Figure 1. Particle size distribution of “fine” (▪) and “coarse” (▪) grade of calcium hydrogen phosphate dihydrate by sieve analysis The mixtures were prepared by premixing the API with a portion of calcium hydrogen phosphate dihydrate in 3:2 weight ratio, using the Turbula T10B (Willy A. Bachofen AG Maschinenfabrik, Switzerland) for 3 min at 25 rpm and then blending the whole mixture using a tumbling blender Hotic (Tico, Czech Republic) for 20 min at 28 rpm.

Flow properties The prepared mixtures were evaluated for their flow properties using pharmacopoeia methods. The angle of repose α was determined by pouring the mixture through the funnel to a substrate to form a cone. The angle of repose was then determined by measuring the height and the radius of the cone and calculating the angle using following equation


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tg  

h r

The flow though orifice was measured using Pharmatest PTG-S3 device and the results are reported as time required for the 100 g of material to pass the funnel. Hausner ration calculation was based on measuring the bulk ρB and the tapped density ρT using SOTAX TD2 Tap Density Tester. Hausner ratio (HR) was then calculated as 𝜌𝜌𝑇𝑇 𝐻𝐻𝐻𝐻 = 𝜌𝜌𝐵𝐵 The results of measured flow properties are summarized in Table III. All three criteria indicate the flow properties improve as more of the “coarse” grade excipient is added. Especially the flow through the orifice accelerates as more of the coarse-grade excipient is added. Thus, the flow properties of the blend may be significantly improved by altering the particle size distribution of added calcium phosphate dihydrate. Table III Flow properties of the prepared batches determined by pharmacopoeia methods Batch T0 T25 T50 Sample 1 22,99 18,39 18,07 α, ° Sample 2 21,05 17,87 17,53 Average 22,02 18,13 17,80 Sample 1 17,9 17,5 10,8 t, s.100g-1 Sample 2 10,3 9,2 8,4 Average 14,08 13,34 9,60 Sample 1 1,59 1,40 1,40 HR, Sample 2 1,40 1,40 1,40 Average 1,50 1,40 1,40

T75 17,85 16,32 17,09 7,5 4,6 6,03 1,39 1,34 1,36

T100 15,98 14,96 15,47 5,8 4,2 4,98 1,36 1,35 1,36

Figure 2. Uniformity of the different batches of the formulation using different PSD of the excipient

Uniformity and segregation Homogeneity of the mixture was verified by withdraving 9 samples from the blender and assaying the API content using a standardized HPLC method. The relative standard deviation (RSD) of the API content in the samples was determined and the results are shown in Fig. 2. Since the particle size of API is small, the addition of coarse grade excipient is likely to cause deterioration of the uniformity, due to increasing segregation propensity caused by the size difference between the API and the excipient particles. However, this anticipated trend was only partially observed as there was a local RSD minimum around the 75 % portion of the fine calcium hydrogen phosphate dihydrate replaced by the coarse grade. This finding is very interesting from the formulation development point

452


of view as the T75 blend presents substantial improvement in flow properties at only moderately decreased uniformity, which may lead to overall improved performance of the blend in the manufacturing process. The causes of this local minimum was further investigated by measuring the segregating propensity of the individual mixtures. The segregation behavior of prepared mixtures was carried out in an in-house segregation device4. The device consisted of two glass tubes (250 x 25 mm) equipped by a connector, enabling the powder to flow from one tube to another and taking samples during the process. The operation of the segregation device was as indicated in Fig. 1. The mixture to be tested was placed into the upper tube and the connector was opened, so as to allow the powder to flow gravitationally from the upper cell into the bottom cell. After the transfer was completed, the tubes switched their position and next cycle of the test was started. The test was finished after 30 cycles. The samples of the mixture were taken during cycles no 1, 5, 10, 15, and 30. The sampling cycles involved taking five samples per cycle during the powder flow from one tube to another. The position of the sample taken was recorded. The samples were then analyzed using HPLC, so as to determine API content.

Figure 2. Scheme of segregation device operation (1. mixture is placed into the top cell; 2. mixture is flowed to the bottom cell, samples are taken during the process; 3. finished flow cycle; 4. the top and the bottom cells are swapped and next cycle begins) Powder mixtures may generally segregate by several mechanisms, but during the gravitational flow, the ‘sifting’ and ‘fluidization’ are the most important ones. The term sifting means percolation of finer particles through the voids between more coarse particles, leading to fines being accumulated downstream the powder flow direction. The fluidization may lead to finer particles being slowed down more than coarse ones due to the aerodynamic resistance, and hence they accumulate upstream the powder flow direction. Using the segregation device described above, the 30 cycles of the segregation test were carried out for each formulation. Illustration of the test evaluation id shown in Fig. 3. In each sampling cycle, five samples were taken along the segregation tube and the API content was determined using HPLC. Figure shows, that the concentration of API is not constant, but increases in the upwards direction of the segregation tube, after the first cycle. This trend becomes more pronounced in further sampling cycles. So as to obtain single-number overall measure of segregation the profiles are approximated by linear regression. The slopes of regression profiles (K) were plotted vs the sampling cycle number and again processed by linear regression to provide segregation index KK. Although the linear approximation may not seem justified in some cases, it provides good overall trend indicator. The segregation index value is a good measure of the mixture tendency to segregate and positive or negative value may indicate the general direction of segregation trend. The results of the segregation tests are summarized in Fig. 4. The segregation index values change from positive to negative as the proportion of coarse excipient increase. The development of the segregation mechanism and its change due to changing particle size of the excipients is indicated in Fig. 5. In T0 batch, the API is one of the coarser components and it tends to remain in the upper parts due to the percolation of the fine excipient. In batches containing some coarse filler this mechanism works less efficiently. The consolidated bed of particles gets formed in mixtures having broad particle size distribution and it hinders the percolation of the particles. This phenomenon is known from the literature for other formulations 5. As the content of coarse filler increases the aggregates between the large filler particles and the small particles of API start forming and partially ordered mixture develops. The flow of the mixture then occurs more in the form of the aggregates than individual


453

particles, so that the segregation is suppressed. This trend is most significant in blend T75 showing minimum segregation. When all the fine filler is replaced by the coarse one, the API becomes the fine component which segregate by percolation down the segregation tube in T100.

Figure 3. Illustration of the segregation test evaluation

Figure 4. Segregation indices for different batches of the formulation

Figure 5. Schematic illustration of the powder flow and segregation depending on the particle size of the filler

454


Conclusions It was found that blends using the substitution of the fine calcium hydrogen phosphate dihydrate filler by coarse grade of the same excipient exhibit significantly better flow properties. The uniformity of the mixture deteriorated in the general direction of this composition change, but local minimum of the composition variability occurred for the blend having the coarse to fine calcium hydrogen phosphate dihydrate ratio of 3:1. Using the segregation test, the cause of this local minima development was identified as being due to the partial ordered mixture formation. Therefore, using the blend having the coarse to fine calcium hydrogen phosphate dihydrate ratio of 3:1 was recommended as good formulation candidate for the direct tablets compression.

Acknowledgement This work was supported by Ministry of Education, Youth and Sports specific university research (MSMT No 20SVV/2017)


Mehta R., Farina J.A., Madhu K., Deorkar N.: Pharm. Technol. 35, 98 (2011). McCormick D.: Pharm. Technol., 5, 52 (2005). Majerova D., Kulaviak L., Ruzicka M., Stepanek F., Zamostny P.: Eur. J. Pharm. Biopharm. 106, 2 (2016). Zamostny P., Kreibichova B., Hofmanova D., Kulaviak L., Zelenkova K., Ruzicka M.: Proceedings of the 3rd International Conference on Chemical Technology, 478 (2015). Liu L.X., Marziano I., Bentham A.C., Litster J.D., White E.T., Howes T.: Int. J. Pharm. 362, 109 (2008).


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POLYMERS, COMPOSITES

456


VANADYL COMPLEX WITH TETRADENTATE MACROCYCLIC LIGAND AS A DRIER FOR SOLVENT-BORNE ALKYD PAINTS 1

1

Charamzová I. , Honzíček J. , Vinklárek J.

2

1

Institute of Chemistry and Technology of Macromolecular Materials, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic 2 Department of General and Inorganic Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic [email protected]

Abstract This work is focused on study drying effects of vanadyl complex with Geodken’s macrocyclic ligand in solventborne alkyds of different oil length modified with soybean oil. Tested coatings, treated by the vanadium-based drier, were characterized by film drying time, relative hardness, EPR and IR spectroscopy. The results revealed its very good drying activity in short and medium-length alkyd resin compared to commercial cobalt(II) 2ethylhexanoate. Study of kinetics revealed a negligible induction period of the autoxidation process in the test coatings threated by vanadyl complex. EPR spectroscopy showed the presence of vanadium(IV) species in alkyd films even after 100 days of curing.

Introduction Alkyd resins are synthetic binders nowadays used in paint-producing industry due to their ability to form various modifications. Their application depends on resin’s composition, mainly on the content and type of modifying fatty acids. Alkyd resins are usually prepared from phthalic anhydride, glycerol and vegetable oil (e.g. soybean and linseed oil). Generally, alkyds and oil-based paints form protective coatings with a high surface 1 resistance. High gloss of these coatings determines their good visual properties . Special application uses alkyd binders for preparation of sand moulds to casting metals with positive environmental effect (non-toxic gas is 2 released) . Alkyd resins belong to the group of air-drying paints. The crosslinking of unsaturated fatty acid chains is caused by autoxidation process, which is generally very slow without additives. Therefore, catalysts (so-called driers) are usually used in industry in order to accelerate the curing process at ambient temperature. Cobalt(II) carboxylates (e.g. cobalt(II) 2-ethylhexanoate; Co-Nuodex) are currently the most commonly used alkyd driers. 3,4 Nevertheless, treir use starts to be restricted owing to genotoxic properties . Our scrutiny to new non-toxic alkyd driers led us to vanadyl complexes. These compounds show promising 5,6 activity at lower concentrations than usual for cobalt(II) carboxylates . This work deals with vanadyl complex with tetradentate Geodken’s macrocyclic ligand (VOGM; see Fig. 1). Activity of the complex was established in alkyd resins of different oil length and detailed study of autoxidation process was done by infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR).

N O N V N N

Figure 1. Structure of vanadium-based drier (VOGM)

Experiment Materials and chemical 7,8

Vanadyl compound VOGM was prepared as described elsewhere . Alkyd resins modified by soybean oil CHSALKYD S 401 X 55 (short oil length, AV = 7 mg KOH/g), CHS-ALKYD S 471 X 60 (medium oil length, AV = 6 mg KOH/g) and CHS-ALKYD S 621 W 60 (long oil length, AV = 7 mg KOH/g) were supplied by Spolchemie. The commercial available drier cobalt(II) 2-ethylhexanoate (Octa-Soligen Cobalt 10 in D60, Co-Nuodex) was supplied by Borchers.


457

Film preparation VOGM was grinded in a mortar to the desired particle size immediately before use. Co-Nuodex was used as obtained without further modification. The calculated amount of the drier was weighted on analytical balance, treated with alkyd resin (5 g), dispersed for 5 min and sonicated by ultrasound for 3 min to give clear formulation. The test films were prepared by frame applicators with different slots (76 µm for measurements of drying time, 150 μm for measurement film hardness, and 100 μm for kinetic experiments).

Film drying time Measurements of drying time were performed on a BYK Drying Time Recorder according to ASTM D5895-03 and under standard laboratory conditions (T = 23 °C, rel. humidity = 50%). Films on glass test strips (305 × 25 × 2 mm) were placed into holders of the device. Holder with hemispherical-ended needle (D = 2 mm) was placed to beginning of wet film clamped in horizontal direction with 5 g weight. The straight-line groove resulting during 24 h gives tack free time (τ1) and total drying time of film curing (τ2). During the first stage of film drying (t = 0 – τ1) the solvents are evaporated and sol-gel transition proceeds. The needle gives bold and uninterrupted line. During the second stage (t = τ1 – τ2) the needle starts to climb over the film. It tears formed layer and lefts ragged groove. After τ2, no visible mark is observed on the film.

Determination of film hardness Film hardness development was monitored using a Persoz type pendulum (Elcometer Pendulum Hardness Tester, UK). The method is based on registering the number of pendulum swings, the amplitude of oscillation of a pendulum placed on examined film decreases more rapidly the softer the surface is. All measurements were carried out in conformity with ISO 1522 and under standard laboratory conditions (T = 23 °C, rel. humidity = 50%). Films were prepared on glass plates (100 × 200 × 4 mm) and their properties were measured within 100 days. The measured values were related to the hardness of a glass standard and expressed as relative hardness.

Time-resolved FTIR spectroscopy The kinetics of autoxidation process was characterized by development of the infrared spectra in time. All data –1 were collected on FTIR spectrometer Nicolet iS50 (32 scans per spectrum, resolution of 2 cm ) in the range of –1 4000–500 cm . Alkyd resin treated by VOGM was applied on NaCl crystal using film applicator with 100 μm slot and placed into the spectrometer. Transmission infrared spectra were registered every 5 min (T = 23 °C, rel. humidity = 50%). Collected series of the spectra were integrated using fixed two-point baseline in the −1 −1 bounds 3014–2997 cm [νs(cis-CH=CH), isolated double bond] and 1011–947 cm (cis-trans conjugated C=C–H wagging). Rate coefficient (kCH,max) was obtained from logarithmic plot of integrated area of νs(cis-CH=CH) as the steepest slope.

EPR spectroscopy EPR spectra were measured on Miniscope MS 300 spectrometer in microwave X-band (9.5 GHz). The apparatus was gauged on DPPH value (giso = 2.0036 ± 2). Solution spectrum was measured in dichloromethane in glass capillary (ID = 0.5 mm) at room temperature (293 K). Frozen spectrum was measured in mixture dichloromethane/ethanol (1:1) in quartz tube (ID = 3 mm) at 123 K. Solid spectra of cured film were measured in quartz tubes (ID = 5 mm) at 293 K. The films were scratched off after 100 days of curing from glass plates (100 × 200 × 4 mm).

Results and discussion Vanadyl complex with tetradentate ligand VOGM (Fig. 1) was prepared from VO(acac)2 by ligand exchange 7,8 reaction according to literature procedure . The complex was characterized by EPR spectroscopy in fluid and frozen solutions (see Fig. 2). Isotropic spectrum obtained from dichloromethane solution at ambient 51 temperature shows hyperfine splitting typical for interaction of unpaired electron with one nucleus of V (I = 7/2), which natural abundance is 99.8%. The observed isotropic hyperfine coupling constant (Aiso = 88.7 G) as 9 well as the isotropic g-factor (giso = 1.978) near the values reported in literature . Full conversion of the reaction is proved by absence of starting VO(acac)2, which EPR parameters differ considerably (Aiso = 108.7 G, giso 5 =1.969) . Anisotropic spectrum of VOGM, obtained from frozen solution in mixture of dichloromethane/methanol, is axial symmetric (A‖ = 165.0 G, AꞱ = 52.5 G, g‖ = 1.959, gꞱ = 1.969) that is in accordance with square-pyramidal structure.

458


Figure 2. EPR spectra of VOGM: CH2Cl2 solution (left), frozen solution in 1:1 mixture CH2Cl2/ethanol (right).

Drying activity of VOGM Solvent-borne alkyd resins of short (S 401 X 55), medium (S 471 X 60) and long oil length (S 621 W 60) were used for examination of drying activity of the complex VOGM. The measurements were performed at concentration range 0.1–0.005 wt.% of the drier in the dry matter content of given alkyd resin and compared with commercial Co-Nuodex (0.1–0.03 wt.%). The tack free time (τ1), total dry time (τ2) and relative hardness obtained after 10 days (Hrel;10d) and 100 days (Hrel;100d) are summarized in Table I. In the alkyd resins of short and medium oil length, the compound VOGM shows considerably shorter tack free times (τ1) than Co-Nuodex at the same dosage (Table I). The total dry times (τ1) are also satisfactory up to concentration 0.03 wt.% not exceeding 12 h. The main advantage of the vanadium-based drier is very fast solgel transition even at low concentration as evidenced from low values of τ1. Hence, the short and medium length alkyds at 0.005 wt.% give the tack-free film already within 2.7 h and 13.8 h, respectively. Although cured films, treated with VOGM, show generally lower final hardness than those treated by Co-Nuodex, the values obtained for short and medium-length alkyd are suitable for common applications. Unfortunately, the drying effect of VOGM on long-length alkyd binder was not satisfactory. The total dry films were not obtained within 24 h even at high metal concentrations. Final hardness of these coatings stays considerably lower than in case of Co-Nuodex.

Time-resolved FTIR spectroscopy The kinetics of autoxidation process was investigated by time resolved infrared spectroscopy. Such method –1 enables to follow disappearance of moieties with isolated cis-double bonds [ν(CH) at 3008 cm ] that is related 6 to initial phase of the autoxidation process (hydroperoxide formation) . Decreasing of the ν(CH) band in intensity is shown in Fig. 3 for short and medium-length alkyd formulations treated with 0.03 wt.% of VOGM and Co-Nuodex. The formulations of the long-length alkyd were rejected from such investigation due to aforementioned low drying activity. The formulations under the scrutiny show behavior typical for reaction of pseudo-first order up to about 50% conversion when the system is saturated with air oxygen. It is apparent form linear part of logarithmic plot shown in the Fig. 3. The vanadium based drier VOGM exhibits very different kinetic parameters from CoNuodex (Table II). In short-length alkyd, VOGM shows much smaller rate coefficient (–kCH,max) and its better drying performance is due to absence of induction time (IT). The autoxidation of medium-length alkyd formulations is generally slower. Both dries show similar induction time exceeding ten hours but the rate coefficients near the values obtained in short-length alkyd formulations. Table II further gives the values of –1 tconjug estimated as a maximum on the time-dependent integral plot of the CH wagging band at 989 cm that was assigned to conjugated double bond moiety. This point can be considered as the end of extensive alkyd oxidation and the addition of the radicals on the conjugated double bond system predominates.


459

Table I a,b c,d Drying times and relative hardness (Hrel) of alkyd films dried with VOGM at various concentrations Metal conc. [wt.%] 0.1 0.06 0.03 0.01

Formulation

VOGM/ S 401 X 55

0.005 0.1 0.06 0.03 –

a

τ2 [h] 6.2

b

c

d

Hrel;10d [%] 32.1

Hrel;100d [%] 58.0

1.5

6.5

22.2

50.8

1.5

10.8

29.8

55.4

2.3

12.3

32.1

54.0

2.7

16.9

34.1

53.7

Co-Nuodex/ S 401 X 55

5.5

7.6

25.9

61.2

2.7

6.9

35.3

58.5

5.5

15.4

35.8

57.4

S 401 X 55

>24

>24

6.8

41.9

0.1

4.5

8.1

23.9

53.1

0.06

5.7

9.3

19.5

53.7

5.8

10.6

21.1

52.4

7.7

17.7

20.5

51.4

13.8

>24

35.7

50.6

0.03 0.01

VOGM/ S 471 X 60

0.005 0.1 0.06 0.03 –

Co-Nuodex/ S 471 X 60 S 471 X 60

0.1 0.06 0.03 0.01

VOGM/ S 621 W 60

0.005 0.1 0.06 0.03 a

τ1 [h] 0.6

Co-Nuodex/ S 621 W 60

5.2

6.7

20.7

54.1

7.7

9.5

20.6

53.7

19.2

>24

22.4

49.8

>24

>24

2.4

33.8

18.1

>24

9.1

36.1

20.8

>24

9.1

35.3

>24

>24

9.4

35.7

>24

>24

10.3

36.1

>24

>24

10.5

35.2

3.4

13.2

26.3

47.9

5.0

12.7

28.3

46.4

18.5

19.6

25.5

42.2

– S 621 W 60 >24 >24 3.2 14.5 b c d Tack free time, Total dry time, Relative hardness after 10 days, Relative hardness after 100 days

Figure 3. Time-dependent integral plots of the ν(CH) in alkyd films in linear (left) and logarithmic scale (right) of following formulation: 0.03 wt.% VOGM/S 401 X 55 (A), 0.03 wt.% Co-Nuodex/S 401 X 55 (B), 0.03 wt.% CoNuodex/S 471 X 60 (C), 0.03 wt.% VOGM/S 471 X 60 (D).

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EPR spectra of cured films EPR spectra of the cured coatings treated with VOGM were measured after 100 days of drying. The formulation of short (S 401 X 55) and medium-length alkyds (S 471 X 60) have anisotropic character with clearly resolved hyperfine structure of axial symmetry (A‖ = 193.2 G, AꞱ = 73.6 G, g‖ = 1.934, gꞱ = 1.980). The observed species has considerably higher values of the hyperfine tensor components than aforementioned spectrum in inert solvent. Such observation implies that VOGM works as a precursor of the catalytically active species, which composition was not fully elucidated. EPR parameters of this species near the values previously observed in 6 films cured with vanadyl 2-ethylhexanoate (A‖ = 193.7 G, AꞱ = 74.6 G, g‖ = 1.935, gꞱ = 1.981) . In case of longlength alkyd formulation, broad band without apparent hyperfine structure was obtained. The absence of resolved hyperfine structure is due to interactions between undissolved paramagnetic species. The partial dissolution is probably the main reason for low activity of VOGM in long-length alkyd binder. Table II Kinetic data of the autoxidation process. IT –kCH,max Formulation –1 [h] [h ]

tconjug [h]

VOGM/S 401 X 55

–

0.17

9.1

Co-Nuodex/S 401 X 55

6.2

0.43

10.5

VOGM/S 471 X 60

10.5

0.09

26.6

Co-Nuodex/S 471 X 60

12.0

0.43

15.8

Conclusion This study revealed a very good drying activity of VOGM in solvent-borne alkyd resins modified with soybean oil of short and medium oil length. This drier shows optimal performance at metal concentration 0.03 wt. % that is considerably lower than in case of commercial Co-Nuodex. Kinetic study, performed on formulations treated with VOGM, revealed relatively low rate coefficient of the autoxidation process. The high activity of VOGM is due to short induction time. EPR spectroscopic measurements proved the necessity of full dissolution of the vanadium-based driers in the alkyd binder. The spectra measured after 100 days of drying show presence of vanadium species in oxidation state IV that is not consumed during the autoxidation reaction and thus works as a true catalyst.

Acknowledgement This work was supported by Ministry of Education, Youth and Sports of the Czech Republic No. UPA SG370006.

References 1. Hofland A.: Prog. Org. Coat., 73, 274 (2012). 2. Sosa A. D., Echeverría M. D.: Procedia Mater. Sci., 8, 155 (2015). 3. Lison D., De Boeck M., Verougstraete V., Kirsch-Volders M.: Occup. Environ. Med., 58, 619 (2001). 4. Magaye R., Zhao J., Bowman L., Ding M.: Exp. Ther. Med., 4, 551 (2012). 5. Preininger O., Vinklárek J., Honzíček J., Mikysek T., Erben M.: Prog. Org. Coat., 88, 191 (2015). 6. Preininger O., Honzíček, J., Kalenda P., Vinklárek J.: J. Coat. Technol. Res. 13, 479 (2016). 7. Niewahner J. H., Walters A. K., Wagner, A.: J. Chem. Educ., 84, 477 (2007). 8. Lee S., Floriani C., Chiesi-Villa A., Guastini C.: J. Chem. Soc., Dalton Trans., 145 (1989). 9. Schumann, H.: Z. Naturforsch., 50, 1494 (1995).


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PREPARATION OF NANOSTRUCTURED SURFACES USING UV RADIATION AND THEIR ANALYSIS Knapová T., Neubertová V., Kolská Z. Faculty of Science, J. E. Purkinje University in Usti nad Labem, 400 96 Usti nad Labem, Czech Republic [email protected]

Abstract Surfaces of solid polymer foils were modified by photochemical method. Surfaces were firstly activated by UV radiation of different wavelengths and then grafted with cysteamine. Surface properties of samples under study were studied and their changed significantly before and after individual modification steps. They were studied using various methods to characterize changes in surface chemistry, polarity and wettability. The surface wettability was determined by goniometry from static contact angle measurement. Surface chemistry, polarity and charge were studied by electrokinetic analysis and by X-ray photoelectron spectroscopy.

Introduction Polymers are widely used in industry and play an important role due to the many benefits. Polymers are in fact mechanically and chemically resistant materials. However for certain applications, the finish is also a problem due to a low wettability of the surface. The wettability affects the surface properties of polymers important in many applications such as cytocompatibility and adhesion. There are various methods for surface treatments and change of surface properties e.g. chemical grafting, plasma treatment or UV radiation without affecting the bulk properties of the polymer. When the surface of polymers is exposed to of UV radiation there may be formed reactive sites on the surface and consequently can lead to chemical grafting of polymers. Modification of UV radiation may also affect the optical properties of polymers. In this work different polymer foils and nanofibres were characterized before and after modification by several available methods. Polymer foils were firstly exposed under UV light and then grafted with chemical compounds. Wettability of the surface was determined by static contact angle by goniometry and changes in chemistry, charge and polarity surface by electrokinetic analysis.

Experiment Materials As the substrates were employed various polymeric foils of different polarity from GoodFellow, UK: polytetrafluorethylene (PTFE, thickness 50 μm, ultra-high-molecular-weight polyethylene (UHMWPE, thickness 75 μm, polylactic acid (PLLA, thickness 50 μm), polyethylene terephthalate (PET, thickness 50 μm), polystyrene (PS, thickness 50 μm), polyvinylidene chloride (PVDC, thickness 43 μm), polyetheretherketone (PEEK, thickness 50 μm), polyoxymethylene (POMH, thickness 500 μm). Another substrate was employed nanofiber of polyvinylidenefluoride (PVDF) from Nanovia Litvínov. The activated surfaces were thereafter grafted by cysteamine (HS(CH2)2NH2, 98%, Sigma Aldrich, CYS). Surface modification When the polymer surfaces are exposed to UV radiation, reactive sites may form on the surface which can become functional groups for the initiation of the grafting of new substances to the polymer surface. This method has the ability to adjust the depth of surface reactivity by using different wavelengths and thus absorption coefficient. Modifications by UV radiation can also affect the optical properties of polymers.1

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Figure 1. Principle of photochemical modification of polymer surface by UV radiation. The basic processes of photochemical modification of the polymer are: (1) surface reactions, (2)atmospheric reactions, (3) polymer reactions.2 Polymer foils (PEEK, POMH, PMMA, UPVC, PVDC, UHMWPE, PET) and polymer nanofibre (PVDF), were firstly activated with UV radiation. The samples were irradiated for 5 minutes, 30 minutes and 60 minutes. Samples were taken immediately after irradiation introduced into the aqueous solution of cysteamine (concentration 10%) for 30 minutes. The samples were then dried for several minutes in Petri dishes. Analytical methods used Surface properties of the samples (before modification, after exposure to UV radiation for 5, 30 and 60 minutes and subsequent grafting with cysteamine) were determined by goniometry and electrokinetic analysis. Surface wettability was determined by assessing the contact angle at room temperature using the SEE system (Surface Energy Evolution System) immediately after surface activation or chemical modification. Drop of water of volume 8.0 µl was applied using automatic pipette, drop of water was photographed and evaluated.3,4 Electrokinetic analysis (zeta potential determination) was performed for the planar samples of polymer foil on the device SurPass (Anton Paar, Austria). Two samples of the same surface were fixed on two brackets, size of the samples was 2x1 cm2. Measurements were carried out in a cell with adjustable gap about 100 µm, at room temperature, atmospheric pressure and constant pH 6.5. For determination of zeta potential, the streaming current and streaming potential methods were used and the Helmholtz-Smoluchowski (HS) and FaitbrotherMastins (FM) equations to calculate zeta potential.5-7

Discussion and result analysis All analytical methods were performed on the surfaces before and after their activation by UV radiation, after subsequent grafting of amino- compound (cysteamine, CYS). Hydrophobicity of the surface plays an important role in the study of polymers. After UV irradiation, they have been grafted onto the activated surface of the amino compound, e.g. cysteamine. Wettability changes step by step modifications are evident from Figure 2. All values of contact angle of polymer foils PEEK and UPVC are in Table I. The average value and error is presented from 10 measurements for all samples.

Figure 2. The contact angle of the unmodified PEEK polymer film (left), PEEK after activation with UV irradiation for 30 minutes (middle) and after activation of PEEK to UV irradiation for 30 min and subsequent grafting cysteamine for 30 minutes (right).


463

Table I Values of contact angle of polymer foils before and after UV radiation and grafting. PEEK and PVC unmodified (Pristine), irradiated for 5 min (5m), 30 min (30m), 60 min (60m) and subsequently grafted with cysteamine (CYS). Polymer [°] Pristine UV5m UV30m UV60m UV5m/CYS UV30m/CYS UV60m/CYS

PEEK 68.7  0.4 68.0  1.2 37.5  1.0 23.2  0.7 40.3  1.7 17.2  1.6 10.8  1.4

UPVC 96.8  0.3 90.9  0.7 90.5  0.9 80.1  1.7 76.6  1.9 68.9  2.1 60.8  0.8

Determination of the zeta potential of the planar samples was performed using electrokinetic analysis unit SURPASS (Anton Paar, Austria). Measurement was done in the cell with the adjustable slit at room temperature and atmospheric pressure, and a constant pH of 6.5. Individual samples were measured 4 times with a relative deviation of 5%. To determine the zeta potential were determined by two methods, current potential and current (streaming potential, streaming current) and to calculate the zeta potential were used Helmholtz Smoluchowskiho (HS) and Fairbrother-Mastinsova (FM) equations. Values of zeta potential of the PEEK are presented in graph of Figure 3. From this graph it is evident that the action of UV radiation on the surface leads to a change of surface chemistry and surface charge and zeta potential changes due to presence of polar groups created on the surface. Also grafting of cysteamine leads to other zeta potential changes to less negative values due to presence of amino- groups on the surface.

Figure 3. The values of the zeta potential of the PEEK unmodified surface (Pristine) surface UV irradiation for 5 min and subsequently grafted with cysteamine (CYS).

Conclusion Analytical methods showed the successful activation of polymer surfaces by UV radiation, the successful grafting of the amino- compound to the surface of the materials under study, and also confirmed the changes in the surface properties of polymeric substrates studied. Activating the surface with UV radiation causes the formation of reactive sites on the polymer surface, changing the surface charge, surface chemistry as well as

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changes and polarity and wettability. The biggest changes were observed only after grafting cysteamine due to presence of amino- groups on the surface. These materials with modified surfaces could be of potential use in, e.g. the tissue engineering field.

Acknowledgement This work was supported by the Grant Agency of CR under project No. 13-06609S, grant Agency of Health Ministry No. 15-33018A and by the Grant Agency of J. E. Purkyně University in Usti nad Labem under project No. 5351815000801. The authors acknowledge the assistance provided by the Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2015073.

References 1. 2. 3. 4. 5. 6. 7.

Goddard J.M., Hotchkiss J.H.: Prog. Polym. Sci. 32, 698 (2007). Bačáková L., Švorčík V.: Nova Sci. Publish 5, 56 (2008). Benkocká M., Knapová T., Braborec J., Matoušek J., Černá H., Londesborough M.G.S., Švorčík V., Kolská Z.: Chem. Listy 109, 960 (2015). Lupínková S., Benkocká M., Braborec J., Matoušek J., Kolářová K., Londesborough M.G.S., Švorčík V., Kolská Z.: Czech. Chem. Soc. Symp. Ser. 14, 19 (2016). Kolská Z., Makajová Z., Kolářová K., Kasálková Slepičková N., Trostová S., Řezníčková A., Siegel J., Švorčík V.: Polymer Science (Ed. Dr. Faris Yılmaz), In Tech d.o.o., Rijeka, Croatia, 203 (2013). Kolská Z., Slepičková Kasálková N., Siegel J., Švorčík V.: J. Nano. Res. 25, 31 (2013) Kolská Z., Řezníčková A., Nagyová M., Slepičková Kasálková N., Sajdl P., Slepička P., Švorčík V.: Polym. Degrad. Stabil. 101, 1 (2014).


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REMOVAL OF CAESIUM FROM A SOLUTION OF BORIC ACID BY USING ZEOLITE Kůs P.1, Foubíková A.1,2, Skala M.1, Koloušek D.2, Kotowski J.1 Centrum výzkumu Řež, Hlavní 130, Husinec – Řež, CZ -250 68, Czech Republic VŠCHT, Technická 5, 16000 Praha [email protected]

1 2

Abstract This work was dealing with separation of caesium ions from a solution of boric acid, which is used in nuclear power plants types PWR (Pressurised Water Reactor) or VVER (Water-Water Energetic Reactor) as an absorber of neutrons originating from nuclear fission. The sorption on lab-made nanozeolite and natural clinoptilolite (Zeocem) was studied and all the experiments have been carried out in a batch process arrangement. The concentration of caesium has been determined by using AAS (atomic absorption spectrometer) with flame and electro-thermal ionization of the sample. The influence on sorption properties of nanozeolite and clinoptilolite has been examined with respect to the various parameters such as: pH, stirring intensity and presence of other (potassium) ions (which are used for pH control). Furthermore, the experimental data have been fitted by Langmuir isotherm and the values of maximal Cs saturation of zeolites have been evaluated.

Introduction Artificial radioisotopes 134Cs a 137Cs are mainly produced by anthropogenic activity and have been released into the environment continuously. In the past, one of the major sources of this radioisotope was nuclear operation in the United Kingdom, with a release of 41,300 TBq 137Cs between years 1952 and 2014 (source: http://www.environment.no). Another and the greatest source was nuclear weapons testing, with 940,000 TBq 137 Cs released. The caesium isotope is presumed to leak from the nuclear fuel into the reactor cooling medium via the failure of the fuel rod cover. Released caesium may according to its high radioactivity increase the collective dose and therefore it is need to be removed.

Experiments In the first phase of the experiments, the zeolites were analysed by SEM (scanning electron microscope), by using powder X-ray diffraction and the porosity of zeolites was determined. Then, sorption tests were carried out in a 1 g.L-1 boric acid solution. Subsequently, in most of the experimental loads, the ratio of Caesium removal was followed by the equation:

A

co  c r 100 co

where A is the ratio of removal [%], c0 is the analyte input concentration [mg.L-1], cr the analyte output concentration [mg.L-1]. In addition, the maximum saturation of the sorbent was monitored by Langmuir's isotherm:

a

V (c0  cr ) m

a  amax

bcr 1  bcr

where amax - maximum absorption capacity [mg.g-1], b – equilibrium constant [L.mg-1 ], cr - equilibrium concentration [mg.L ], V – volume [L], m – mass of absorbent [g], c0 - input concentration [mg.L-1], a – absorption capacity [mg.g-1]. 1

Results and discussion Two zeolites - natural (Zeocem) and synthetic (laboratory made at the VSCHT) were tested in this work. Firstly, zeolites were determined by image analysis on a scanning microscope (Figure 1 and 2).

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Figure 1. Natural zeolite – Clinoptilolite

Figure 2. Synthetic zeolite The surface of zeolites was then measured by sorption and subsequent desorption of N 2. By this method, it was found out that both zeolites contain certain amounts of micro pores.

Figure 3. Measurement of surfaces (on the left - synthetic zeolite, on the right – natural clinoptilolite) Table I Surfaces of zeolites

Specific surface area [m2.g-1]

Specific volume [cm3.g-1]

Pore radius [nm]

Clinoptilolite

34.72

0.1198

1.923

Synthetic zeolite

70.33

0.08457

1.921

After measurement of porosity, the tests of pH value influence on Cs and K sorption on synthetic zeolite were POLYMERS, COMPOSITES 467 carried out (Figure 4).

Synthetic zeolite

70.33

0.08457

1.921

After measurement of porosity, the tests of pH value influence on Cs and K sorption on synthetic zeolite were carried out (Figure 4).

Figure 4. Sorption of Cs (left) and K (right) on synthetic zeolite The same experiment was done with natural zeolite - Clinoptilolite. (Figure 5).

Figure 5. Sorption of Cs (left) and K (right) on natural zeolite It is clear from the figures that the optimal pH for sorption is in the range of 3-7, for both zeolites, whereas the natural zeolite captured less potassium ions.

Conclusion The optimal pH value for sorption on both zeolites is very similar and ranges in a slightly acidic area (pH = 3-7). From the experiments, it was found out that the synthetic zeolite less captured potassium cations, which are in this case the competitive cations to Caesium cations. For both zeolites, the Cs+ removal efficiency was greater than 75% at all tested pH values.

Acknowledgement The presented work was financially supported by the Ministry of Education, Youth and Sport Czech Republic Project LQ1603 (Research for SUSEN). This work has been realized within the SUSEN Project realized in the framework of the European Regional Development Fund (ERDF) in project CZ.1.05/2.1.00/03.0108 We also thank Zeocem producing company for providing sorption material.

References 1. Radioactive decay. Radioactivity and radioaction [online]. Buckten - Switzerland [cit. 2016-11-15]. Dostupné z: http://www.geigercounter.org/radioactivity/decay.htm 2. Wu, J. J.; Li, B.; Liao, J. L.; Feng, Y.; Zhang, D.; Zhao, J.; Wen, W.; Yang, Y. Y.; Liu, N. Behavior and analysis of Cesium adsorption on montmorillonite mineral. Journal of Environmental Radioactivity 2009, 100, 914920. 3. Kim, G. N.; Kim, S. S.; Choi, J. W. Development of an agent suited for adsorbing Cs-137 from ash and soil waste solutions. Separation and Purification Technology 2017, 173, 193-199.

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4. 5. 6. 7. 8.

Lee, K. Y.; Park, M.; Kim, J.; Oh, M.; Lee, E. H.; Kim, K. W.; Chung, D. Y.; Moon, J. K. Equilibrium, kinetic and thermodynamic study of cesium adsorption onto nanocrystalline mordenite from high-salt solution. Chemosphere 2016, 150, 765-771. Jelínek, L. Desalinační a separační metody v úpravě vody. Vyd. 1. Praha: Vydavatelství VŠCHT, 2008, ISBN 978-80-7080-705-7. Zeocem [online]. Bystré: Zeocem, 2017 [cit. 2017-03-16]. Dostupné z: http://www.zeocem.sk/ LIMA-DE-FARIA, J. Structural classification of minerals. Dordrecht: Springer, 2011. ISBN 978-904-8156-801. Johan, E.; Yamada, T.; Munthali, M. W.; Kabwadza-Corner, P.; Aono, H.; Matsue, N.: Natural zeolites as potential materials for decontamination of radioactive cesium. In 5th Sustainable Future for Human Security; Trihartono, A., McLellan, B., Eds.; Procedia Environmental Sciences, 2015; Vol. 28; pp 52-56.


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CHLORINE DIOXIDE BLEACHING OF SODA RAPESEED PULP Potůček F., Říhová M. University of Pardubice, Faculty of Chemical Technology, Institute of Chemistry and Technology of Macromolecular Materials, 532 10 Pardubice, Czech Republic [email protected]

Abstract Soda pulp cooked from rapeseed straw (Brassica napus L. convar. napus, winter line genotype Labrador) was subjected to a four-stage elemental chlorine-free bleaching under laboratory conditions. The bleaching sequence comprised three chlorine dioxide stages. The alkali extraction enhanced with hydrogen peroxide addition followed the first chlorine dioxide delignification. For comparison, kraft pulp cooked from a blend of spruce and pine was subjected to the same bleaching sequence D0EPD1D2. After each bleaching step, the optical and strength properties were measured. The preliminary results showed that bleachability of soda rapeseed pulp was lower in comparison with kraft softwood pulp for D 0EPD1P bleaching sequence. The final brightness of 83.5 % ISO and 87.8 % ISO was achieved for soda and kraft pulps, respectively. However, the bleaching had a negative impact on the strength of soda rapeseed fibres. The zero-span breaking length decreased from 4.0 km to 3.3 km for unbleached and bleached soda pulps, respectively, while, for kraft softwood pulp, a decrease in fibre strength was not found. Keywords: soda rapeseed pulp, ECF bleaching, brightness, zero-span breaking length

Introduction Bleaching is a chemical purification and modification process in which the optical properties of pulp fibres are changed either by removing components capable of absorbing visible light or by reducing their light absorption capability. Bleaching reactions can be grouped according to their principal mode of operation. Electrophilic reactions (oxidative) typically initiate lignin-degrading bleaching processes, frequently in acidic conditions, and involve cations and also radicals generated from used bleaching chemicals. Nucleophilic reactions (reductive) typically occur in lignin-retaining bleaching. These reactions generally take place in alkaline media and involve 1 anions and, to a much lesser extent, radicals . Chlorine dioxide is a typical delignifying chemical and reacts primarily with free phenolic hydroxyl groups under acidic conditions. Chlorine dioxide is reduced through a series of steps involving several intermediated. Hypochlorous acid and chlorine are among these intermediates, and they are capable of forming organochlorine compounds just as molecular chlorine. Another unwanted intermediate is the chlorate ion, which is not reactive. Hence, it is important, when using chlorine dioxide for delignification, to manipulate reaction conditions to minimize the formation of chlorate and free chlorine or hypochlorous acid so as to improve delignification efficiency and minimize the formation of chlorinated organic matter. This implies that 1 pH should be low when chlorine dioxide is used in the first stage of bleaching . Since elemental chlorine is no longer used in modern pulp mills because of environmental reasons, chlorine dioxide has become the most important bleaching chemical. Chlorine dioxide is a multi-purpose bleaching agent. It is efficient in delignification, but it is still more important in brightening pulp by reducing or eliminating residual lignin content without significant carbohydrate losses and by reducing chromophores in pulp. At first, chlorine dioxide in combination with subsequent alkaline extraction was used as a first bleaching stage after cooking or oxygen delignification. Later, owing to high selectivity towards the oxidation of chromophoric structures, chlorine dioxide was applied not only for delignification in the first bleaching stage but also for its capability for pulp brightness in the final bleaching stage of elemental chlorine-free sequences 2–6 to produce chemical pulps with sufficient strength properties . Usually, the alkaline extraction stage follows chlorine oxide delignification. The purpose of an alkaline extraction stage is to dissolve and then remove compounds made alkali-soluble in the preceding acidic delignification treatment. Extraction can be enhanced by adding oxidants such as oxygen and/or hydrogen 1 peroxide . In this paper, soda pulp cooked from rapeseed straw was subjected to a four-stage elemental chlorine-free bleaching sequence D0EPD1D2 under laboratory conditions. Bleaching sequence comprised three chlorine

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dioxide stages in combination with an alkaline extraction which followed the first delignification stage. Optical and strength properties of pulp measured after each bleaching step were compared with those obtained for kraft softwood pulp produced in industrial scale.

Experimental Rapeseed straw (Brassica napus L. convar. napus, in our case winter line genotype Labrador) collected from the field in Polabian lowlands near the city of Pardubice (Czech Republic) was used for the pulping process. Raw materials consisted mainly of stalks, but approximately one third of total amount were valves of siliques. After removing natural dirt and silique valves, the stalks were manually cut to 1 to 2 cm pieces which were used for laboratory soda pulping. Chemical composition of both basic components of rapeseed straw, stalks and silique 7 valves, was reported in our previous paper . Batch soda-AQ pulping of rapeseed straw was carried out in a laboratory rotary digester comprising six 3 autoclaves of 750 cm capacity, immersed in an oil bath. Batch cooks were performed at the liquor-to-raw material ratio of 5:1, alkali charge of 19 % expressed as Na2O per oven-dried raw material, and the anthraquinone charge of 0.1 %, based on oven-dried raw material. 7 On the basis of pulping experiments performed earlier , the temperature regime consisted of four periods, i. e., o o at first heating from a room temperature to 105 C for 45 min, then dwelling at 105 C for 30 min, followed by o heating to 160 C for 30 min, and finally dwelling at cooking temperature. The batch cooks were ended as soon as the H-factor reached a value of 1,600 h. After the cooking process, the cooked pulp was refined, thoroughly washed with tap water, and screened to remove rejects using 10 mesh sieve. The soda pulp was stored cold at a temperature of 6 °C before bleaching experiments. The kappa number of unbleached soda pulp determined according to standard method ISO 302 had a value of 17.9. Unbleached pulps were subjected to a D0EPD1D2 bleaching sequence. Chlorine dioxide (ClO 2) solution was applied as bleaching agent. The chlorine dioxide solution was prepared by acidification of a sodium chlorite (NaClO2) solution under laboratory conditions. Commercial product of hydrogen peroxide (H2O2) having a concentration of 30 mass % was used for enhancing the alkali extraction. Water solutions of sodium hydroxide and/or sulphuric acid were added to pulp samples to achieve a desired pH value. A solution of magnesium sulphate in the amount corresponding to 0.5 kg of MgSO 4 per oven-dried tonne of pulp to protect cellulose in the pulp samples from degradation was added in the alkaline extraction stage. The bleaching stages were performed in sealed polyethylene bags immersed in a tap water bath preheated to the required temperatures. The pulp samples were hand-kneaded before and during bleaching steps. The pulp consistency, i. e., mass fraction of moisture-free fibres in suspension expressed in mass %, in each stage was maintained at a value of 10 %. The bleaching sequence D0EPD1 D2 is illustrated in Fig. 1 in which one can find the charge of bleaching agents, for chlorine dioxide expressed as active chlorine aCl 2, retention time (τ), pH value, and temperature (t) which characterize operating conditions of each bleaching stage. After each bleaching stage a multi-stage washing based on dilution at 4 % pulp consistency followed by thickening was performed with distilled water until neutral effluent was achieved. For comparison, the once-dried kraft softwood pulp having the kappa number of 18.8 was undergone elemental chlorine-free bleaching under the same conditions. 2 Pulp handsheets of 80 g/m were prepared using a standard handsheet former as described in TAPPI test method T 205 sp-2. Using an L&W Elrepho SE 071/070R instrument, the brightness of soda pulp was measured for handsheet samples obtained in each bleaching stage. The zero-span breaking length was determined according to TAPPI test method T273 by means of a TIRA test instrument. Before strength measuring, the handsheets were air-conditioned in the conditioning room under a constant temperature of (23±1) °C and relative humidity of (50±2) %. All the strength measurements were performed at least on 20 replicates per each tested sample.

Results and discussion The soda pulp cooked from the stalks of rapeseed straw under laboratory conditions was undergone a fourstage bleaching sequence using chlorine dioxide as bleaching chemical. A simplified flowsheet of the D 0EPD1D2 sequence is illustrated in Fig. 1. The kappa number of unbleached soda pulp cooked up to H-factor of 1,600 h had a value of 17.9. For comparison, the once-dried unbleached kraft pulp cooked industrially from a blend of spruce and pine having the kappa number of 18.8 was undergone the same bleaching steps simultaneously. The initial brightness of soda and kraft pulps was 28.6 % ISO and 33.6 % ISO, respectively.

2


471

Figure 1. Simplified flowsheet of D0EPD1D2 bleaching sequence. The pulp brightness attained after bleaching steps is shown in Fig. 2. After last bleaching stage, the final brightness of soda and kraft pulps was 83.5 % ISO and 87.8 % ISO, respectively. The difference in brightness of 5.0 % ISO for unbleached pulps slightly decreases to 4.3 % ISO for bleached pulps. Thus, the total brightness increments of 54.9 % ISO and 54.2 % ISO were achieved for soda and kraft pulps, respectively. The results obtained showed that chlorine dioxide provides pulp of high brightness. It should be noted also that no chlorine dioxide residuals were found after completion of chlorine dioxide bleaching stages for both pulps. Figure 3 illustrates the brightness increments attained in each bleaching step for soda and kraft pulps. The brightness increment in the D0 stage, where delignification is the primary goal, was too low for soda pulp comparing with kraft pulp. While the brightness increment of 12.3 % ISO was reached for soda pulp, an increase in brightness was 25.1 % ISO for kraft pulp. It was confirmed that the D 0 chlorine dioxide bleaching step has predominant influence upon the final brightness of kraft pulp treated by the ECF bleaching. However, for soda rapeseed pulp, the alkaline extraction stage is clearly beneficial from a brightness point of view. Chlorine dioxide oxidizes lignin via a number of reaction pathways, highly depending on pH value. The optimum 4 pH for hardwood pulps is between 2.8 and 3.5 (ref. ). Moreover, the pH governs the proportion of each 6 reactive component (ClO2, HClO2, HClO/Cl2) present in the solution . However, during the bleaching of pulp with chlorine dioxide, part of the chlorine dioxide is converted into chlorate. Since chlorate is an ineffective 5 delignification chemical, its formation represents waste of the oxidizing power of chlorine dioxide . It was found that a lower pH results in less chlorate formation. Thus, lower pH of 2.5 in the D 0 stage may result in a slight loss in delignification efficiency, but, on the other hand, may result in a substantial removal of nonprocess metals. This improved metals removal may reduce peroxide decomposition in the subsequent E P 4 stage . Nevertheless, the difference in bleachability between various pulps is not easy rationalised particularly when they have approximately the same kappa numbers and brightnesses. In the following steps, alkaline EP and both chlorine dioxide D1 and D2, the brightness increment of soda pulp was greater than that of kraft pulp so that the difference in brightness decreased. It is worth mentioning that the final brightness measured for soda and kraft pulps was much greater than that reached for oxygen-

3

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predelignified kraft softwood pulp with the initial kappa number of 9.7 when the final brightness did not 8 exceed 65 % ISO in the case of totally chlorine-free bleaching with hydrogen peroxide and peracetic acid .

Figure 2. Pulp brightness after bleaching stages for soda and kraft pulps.

Figure 3. Brightness increments in bleaching stages for soda and kraft pulps. 9

For comparison, Enayati et al. report the bleaching results of canola stalks soda pulp with the initial kappa number of 23.8 and brightness of 36.5 % ISO. Using the three-stage bleaching sequence D0EPD1, the final brightness was found to be 78.4 % ISO. For this three-stage bleaching sequence, the final brightness reported 10 11 for wheat straw pulp by Jimenéz et al. and Tschirner et al. was about 80 % ISO and 87.5 % ISO, respectively, 11 while for corn stalks the brightness of 88.9 and 85.4 % ISO was achieved by Tschirner et al. and Jahan and 12 Rahman , respectively. During sequential bleaching operations, pulp fibre properties are gradually changed due to mechanical and chemical treatment. Hence, besides brightness, the strength of pulp was measured as well. The evaluation of pulp strength properties by conventional methods is not suitable for detailed specifications of pulps or fibre line, as the measured tensile strength is a combination of tensile strength of fibres and fibre-to-fibre bond strength. Therefore, the zero-span tensile test is a widely used method for evaluating the average strength of

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individual fibre rather than the strength of the paper itself. In the zero-span test, the tested sheet strips and, 13 consequently, a given fibre is clamped at zero span of the tester jaws . The influence of the bleaching steps on the zero-span breaking length (ZSBL) of soda and kraft pulps is shown in Fig. 4. The D0EPD1D2 bleaching sequence had a negative impact on the fibre strength of soda pulp. It is evident that the all bleaching stages contributed to a strength decrease. However, the final zero-span breaking length of 3.3 km seems to be acceptable for using soda rapeseed bleached pulp to paper production. For comparison, 9 Enayati et al. report for unrefined unbleached and bleached canola stalks soda pulps, the tensile index of 24 N m/g and 23.1 N m/g, respectively, measured by a convectional tensile strength method. In contrast to soda pulp, the bleaching stages, excepting the D 0 stage, had no substantial effect on the zerospan breaking length of the kraft pulp fibres. Thus, the final zero-span breaking length of once-dried kraft softwood pulp was around 4 km. The results obtained for kraft pulp showed that chlorine dioxide reactions with carbohydrates are minimal so there is very little pulp strength deterioration in chlorine dioxide bleaching. For comparison, the zero-span breaking length of 3.92 km was determined for never-dried unbleached kraft 8 pulp cooked from a blend of spruce and pine, having the kappa number of 9.7 after oxygen bleaching .

Figure 4. Influence of bleaching steps on zero-span breaking length for soda and kraft pulps. Error bars – 95% confidence limits. Legend: Type of pulp, kappa number

Conclusions In conclusion, the final brightness of soda pulp with the initial kappa number of 17.9 and the initial brightness of 28.6 % ISO subjected to the four-stage D0EPD1D2 sequence was found to be 83.5 % ISO. The total brightness increment of 54.9 % ISO was slightly greater than that of 54.2 % ISO attained for once-dried kraft softwood pulp having the initial kappa number of 18.8. For kraft softwood pulp, the D0EPD1D2 bleaching sequence did not have a negative impact on fibre strength. However, for never-dried soda rapeseed pulp, the initial zero-span breaking length of 4.0 km decreased to 3.3 km after completion of bleaching. The final the zero-span breaking length seems to be acceptable for paper production from bleached soda rapeseed pulp. 14 In comparison with the D0EPD1P bleaching sequence mentioned in our preceding paper , the D0EPD1D2 sequence comprising three chlorine dioxide stages brought an increase in the final brightness of soda rapeseed pulp by 11.5 % ISO. This fact confirms the important role of chlorine dioxide in bleaching process and its impact on the pulp brightness in elemental chlorine-free bleaching. It can be concluded that chlorine dioxide is certainly the most important bleaching chemical with high oxidation power mainly in combination with a subsequent alkaline extraction. Acknowledgements This work was supported by the Internal Grant Agency of University of Pardubice under the research project SGSFCHT_2017_006.

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Chemical Pulping, Book 6, Eds. J. Gullichsen and C.-J. Fogelholm. Fapet Oy 2000. Costa M. M., Colodette J. L.: Braz. J. Chem. Eng. 24, 61 (2007). Fišerová M., Gigac J.: Wood Research 60, 451 (2015). Hart P., Connell D.: TAPPI J. 7, 3 (2008). Ni Y., Kubes G. J., Van Heiningen A. R. P.: J. Pulp Paper Sci. 19, J1 (1993). Sevastyanova O., Forsström A., Wackerberg E., Lindström M. E.: TAPPI J. 11, 44 (2012). Potůček F., Gurung B., Hájková K.: Cellul. Chem. Technol. 48, 683 (2014). Potůček F., Milichovský M.: Chem. Pap. 54, 406 (2000). Enayati A. A., Hamzeh Y., Mirshokraie S. A., Molaii M.: BioResources 4, 245 (2009). Jiménez L., Martínez C., Pérez I., López F.: Process Biochem. 32, 297 (1997). Tschirner U., Barsness J., Keeler T.: Bioresources 2, 536 (2007). Jahan M. S., Rahman M. M.: Carbohydr. Polym. 88, 583 (2012). Lin B., He B., Liu Y., Ma L.: Bioresources 9, 5024 (2014). th Potůček F., Říhová M.: Proc. 4 Int. Conf. on Chem. Technol., pp. 354 – 359, Mikulov 2016.

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DISPLACEMENT WASHING OF KRAFT SPRUCE PULP COOKED TO LOW KAPPA NUMBER Potůček F., Rahman M. M. University of Pardubice, Faculty of Chemical Technology, Institute of Chemistry and Technology of Macromolecular Materials, 532 10 Pardubice, Czech Republic [email protected]

Abstract The paper deals with the displacement washing of unbleached pulp cooked from spruce wood by kraft pulping process under laboratory conditions. Using the step function input change method, the washing breakthrough curves measured for alkali lignin as a tracer were described by the dispersed plug flow model, containing dimensionless criterion, the Péclet number. Besides the traditional wash yield, the relationship between the Péclet and Reynolds numbers was investigated as well. The results obtained for spruce pulp were compared with those for kraft beech pulp published earlier. The pulp yield achieved for spruce pulp was found to be lower than that for beech pulp. Key words: displacement washing, kraft softwood pulp, wash yield, dispersed flow model

Introduction The kraft pulping industry is the first known to combine pulp washing with the recovery of materials used and produced in the wood cooking process. The initial motivation behind materials recovery is the higher cost of chemicals used in the kraft process compared to those used in the sulphite process. For the kraft process to be economically viable, it is imperative that a very high amount of the cooking chemicals be recovered and 1 reused . Hence, brownstock washing is an important and critical step in processing lignocellulosic materials for making paper and related products. The imperative objective of brownstock washing is to remove cellulose fibres from the black liquor, which mainly include alkali, sodium salts, lignin, and other chemically degraded wood 2 components, while using a minimal amount of wash water . The digester discharged cooked pulp mixture consist of a pulp-liquor suspension containing two main phases: free liquor phase and fibre phase. The fibre phase includes wood fibres and the liquor entrained insides the fibres. The entrained liquor is in close contact with the fibres and can be assumed to behave as immobile liquor phase connected to the free liquor through mass transfer. The free liquor is quite easily removed during 2 washing whereas the immobile liquor can only be removed by diffusion and capillary force . Brownstock washing can be carried out in four basic processes known as dilution, dewatering, diffusion, and 1 displacement . Displacement washing process requires the lowest of amount of wash liquor among the four washing processes. The displacement washing is affected by many process variables: consistency of pulp bed, height of pulp bed, wash liquor velocity, temperature of wash liquor, variation of pulp, and heterogeneity of pulp bed. Because of complexity of the relationships between these process variables and the washing efficiency, as well as the use of different kinds of pulp and experimental techniques, the results so far reported 3 disagree with each other . Therefore, the present paper is aimed to investigate the displacement washing of unbleached pulp cooked to relatively low kappa number from spruce wood by the kraft pulping process.

Experimental Wood chips were collected from the Mondi Pulp and Paper Mill. The composition of collected wood chips sample was heterogeneous which contain many undesirable particles such as oversized chips, bark, and knots. These unwanted particles were sorted out by hand to an almost unified chip composition in accordance with TAPPI Test Method T 265. Before starting batch cooking experiments, the chips were stored in the laboratory. Kraft cooking experiments were carried out on a multiple bomb assembly consisting of six 0.75 L stainless steel bomb digesters of 90 g spruce wood capacity. Cooking conditions, which were held constant, were as follows: 18 % active alkali

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charge, 4:1 liquor to wood ratio, and 168 °C cooking temperature. Industrial white liquor at a sulfidity of 28 % was used. The temperature regimes of cooking procedure were as follows: 45 min heating to 110 °C, 45 min dwelling at 110 °C, 60 min heating to 168 °C, and then dwelling at cooking temperature, depending on the desired H-factor of 3 190 h. As soon as the H-factor of the cooking was achieved, the bombs were brought out from the microdigester as quickly as possible and cooled down under cold water to stop down further delignification. Then the pulp was disintegrated with laboratory disintegrator and washed up with tap water by dilution/thickening way, screened manually using 10-mesh sieve, and dewatered to consistency of approximately 30 %, using a laboratory centrifuge machine. The kappa number of spruce kraft pulp was determined according to TAPPI Test Method T 236 om-99. The displacement washing was simulated under laboratory conditions. The stimulus-response experiments, using a step input, were performed in the displacement washing cell consisting of a vertical glass cylinder 110 mm high, having 35 mm inner diameter. The fibre bed occupied the volume between the permeable septum 4 and piston. The experimental apparatus was similar to that used by Lee . For each experiment, the slurry of unbeaten unbleached kraft pulp in black liquor was used. After compressing it to the desired thickness of 30 mm, the consistency, i. e., mass concentration of moisture-free pulp fibres in -3 -3 the bed was maintained within the limits from 127.1 to 131.5 kg m , the mean value being 128.2 kg m . Using a Kajaani analyser, the length of softwood fibres in the wet state was characterized by the weighted average of -1 2.77 mm, as well as the numerical average of 1.91 mm. Estimated coarseness of fibres was about 0.103 mg m . The degree of pulp delignification, expressed in terms of the kappa number, was found to be 18.1. The pulp beds were not mechanically conditioned and were used as prepared. -3 Concentration of alkali lignin in the black liquor taken from SuperBatch cooking plant was 61 g dm . Further properties of sulphate liquor were as follows: solids content of 21.4 %, of which the ash made up 64 %, and -3 o organics 36 %, density of 1097 kg m at 25 C, and pH value of 12.0. o Distilled water at a temperature maintained at 25 C was employed as wash liquid. The superficial wash liquid -1 velocity, based on empty cross-sectional area, varied in the range from 0.05 to 0.2 mm s . Samples of the washing effluent leaving the pulp bed were analysed for alkali lignin, using an ultraviolet spectrophotometer Cintra 10e operating at the wavelength of 295 nm. More detailed description of experiments can be found 3 elsewhere . Analogous measurements at various consistencies of the bed were focused on the determination 5 of the effective specific volume and surface of pulp fibres according to Ingmanson .

Results and Discussion Washing curves A response to step change in concentration provided time dependences called washing or also breakthrough curves. In order to compare displacement washing process for various wash liquid velocity, the washing curves were plotted as the dependence of dimensionless concentration of a tracer, in our case alkali lignin, in the outlet stream expressed as ρe/ρ0, against to the wash liquor ratio, RW, defined as the mass of wash liquid passed through the bed at that time divided by the mass of liquor originally present in the bed. A typical breakthrough curve measured for spruce pulp is shown in Fig. 1. The first portions of the liquor discharged from the pulp bed are of the same concentration as the mother liquor. Then, the concentration of a solute in the outlet stream drops very rapidly. In this washing period, it can be supposed that the major part of the mother liquor in interparticle voids is removed and replaced by the wash liquid. At the end of the washing, the solute adsorbed on the surface of the fibres, as well as the solute inside the fibre walls is being transferred by diffusion to the wash liquid surrounding the fibres. In this last period, the leaching operation prevails over displacement mechanism. For comparison, a breakthrough curve measured for displacement of black liquor from pulp bed formed from 6 kraft beech pulp fibres cooked to the kappa number of 14.1 (ref. ) is also demonstrated in Fig. 1. Our results showed that both systems differ markedly from one another. From Fig. 1 follows that the breakthrough curves for spruce and beech pulp fibres approach that of plug flow. The flat profile of the breakthrough curve obtained for spruce pulp fibres having a kappa number of 18.1 is probably the result of the nature of pulp bed which can be characterised as an unmovable packing dumped randomly into the washing cell, along with black liquor occurring both in the void spaces and inside porous compressible particles. The ratio of the weighted to arithmetic average length, which is a measure of the polydispersity of fibre length, was roughly the same for the both pulps, 1.43 and 1.45, for hardwood and softwood fibres, respectively.

2


477

Figure 1. Typical displacement washing curves: 6 1 – spruce pulp (Pe = 10.2), 2 – beech pulp (Pe = 42.9), 3 – plug flow, 4 – perfectly mixed flow

Péclet number – Reynolds number relationship The shape of the washing curve can be characterised in terms of the dimensionless Péclet number, derived from the mass balance of the tracer for a given system in unsteady state, in the following form

Pe 

hu

.

Dε

(1)

The determination of the Péclet number from the residence time distribution measured for displacement 3 washing can be found in the previous paper . The Péclet number obtained on the basis of breakthrough curve characterises not only the degree of backmixing of fluid flowing through a packed bed, but also, particularly, the ratio of the bulk mass flow to the dispersion mass flow. In accordance with the shape of washing curves, the Péclet number evaluated for spruce pulp beds varied in the limits of 10 to 20, while the values of the Péclet 6 number obtained for beech pulp beds were in the range of 25 to 41 (ref. ). Presumably, the lower values of the Péclet number measured for pulp fibre bed represent more bridgings between particles and greater variations in local voidage which promote a channelling phenomenon. Of course, because of fibre swelling the mass transfer from within the fibre walls to the wash liquid must be taken into account. The type of flow in a pore of given geometry may be characterized by the Reynolds number. For wash liquid flowing through an individual pore in a packed bed, the Reynolds number can be written in the form

Re 

dm u ρ εμ

(2)

where dm = 4 ε/(av (1-ε)) is the hydraulic mean diameter defined as four times the cross-sectional area divided by the wetted perimeter of the pore. The average porosity of pulp bed was evaluated on the basis of permeability determined experimentally. -12 -12 Depending on the consistency of pulp bed, the permeability varied within the limits of 1.2 × 10 to 3.0 × 10 2 -11 2 m . For comparison, the permeability of the bed of glass beads was found to be 9 × 10 m . More details can 7 be found in the previous paper . In Fig. 2, the Péclet number is plotted as a function of the Reynolds number. From Fig. 2 it seems that the -3 -3 Péclet number is independent upon the Reynolds number ranging from 2.73  10 to 1.04  10 . The Péclet number varied within the 95 % confidence limits from 13.1 to 16.1, with an average value of 14.6. The values of the Reynolds number indicate that the assumption of the laminar character of wash liquid motion was fulfilled. Owing to low number of measurements, it is necessary to verify the relationship between the Péclet number and Reynolds number on other pulp fibres for greater interval of the wash liquid velocity.

3

478


Figure 2. Péclet number as a function of Reynolds number for kraft spruce pulp fibre bed 8

Our results can be compared with those obtained by the other authors. Miller and King who investigated axial dispersion in laminar flow through beds of 0.051, 0.099, and 0.47 mm microspheres, and 1.4 mm glass spheres in the range of Reynolds numbers between 0.003 and 40 found that the dependence of the Péclet number on the Reynolds number at first drops, passes through a minimum near Re  10, and then rises. For glass spheres, 9 Raschig rings, Berl saddles, and Intalox saddles as packing, Ebach and White found that the Péclet number varied from 0.3 to 0.8 for the range of Reynolds numbers from 0.01 to 150. On the other hand, using the 10 frequency response technique, Strang and Geankoplis report that the Péclet number is approximately constant at a value of 0.88 over the range of Reynolds numbers of 12 to 42 for the glass beads which had an 11 average diameter of 6 mm. Montillet et al. characterized the hydrodynamic behaviour of a liquid flowing through various nickel foams, as well as packed beds and proposed a single correlation in the form Pe = 0.43  0.10 in the range 0.1 < Re < 100 for fixed beds packed with cylinders, spheres, and nickel foams. Using pulse 12 and step response techniques, Leitao et al. report a systematic study of liquid phase axial dispersion in columns packed randomly with granular sand. For the Reynolds number in the range from 1 to 50, the 12 0.02 authors derived the correlation between the Péclet and Reynolds number in the form Pe = 0.508 Re . It 8-10, 12 , in contrast to our work, considered the Péclet number and Reynolds should be stressed that the authors 11 number based on the particle diameter, while the authors used these dimensionless numbers based on the mean pore diameter. Wash yield The displacement washing curve area (Fig. 1) is directly proportional to the amount of alkali lignin removed from the bed. It must be emphasised that the washing experiments were finished at the wash liquor ratio equal to about 7 when the lignin concentration in the exit stream was lower than one thousandth of the initial lignin concentration in the pulp fibre bed. Quality of the displacement washing can be characterised by the wash yield. The traditional displacement wash yield, WYRW=1, is defined as the amount of solute washed out at the wash liquor ratio equal to unity divided by the total amount of solute present in the bed at time equal to zero. This yield may be expressed as RW1

ρe

RW0

ρ0



WYRW1 

RW ρ e



RW0

ρ0

d (RW) .

(3)

d (RW)

The influence of the Péclet number on the wash yield is shown in Fig. 3. In spite of the scatter in the data, in the case of beech pulp fibres covering a range of the Péclet number from 25 to 41, it is evident that the wash yield slightly increases with increasing Péclet number for both softwood and hardwood pulps. The experimental

4


479

13

points are located below the curve derived for the packed bed of non-porous particles by Brenner . In contrast 7 to the packed bed of non-porous particles , when the washing process is reduced to the displacement mechanism and interfacial mixing between the displaced and displacing fluids, leaching may play a significant role in the case of compressible porous fibres in the swollen state. Comparing the spruce and beech pulp fibres, the greater values of the wash yield were achieved for beech pulp. Presumably, lower values of the Péclet number covering the range from 10 to 20 measured for softwood spruce pulp fibre bed can be ascribed to fingering or channelling phenomena typical for packed beds randomly formed from heterogenous particles. Moreover, the high Klason lignin content of 30.4 mass % found in spruce is comparable with that of pine (29.5 6 mass %), while beech evidences the lower lignin content of 24.5 mass % (ref. ). Besides the degree of delignification, the initial alkali lignin concentration of the black liquor is affected by the lignin content in the wood samples. Lower values of the wash yield reported for spruce pulp can be also attributed to the high initial -3 -3 6 alkali lignin concentration of 61 kg m in contrast to 27 kg m in the case of beech pulp in the previous paper . It was confirmed that hardwoods having generally lower lignin content, which is advantageous for delignification and following washing process, produce kraft pulps with better washability for lower alkali lignin 3 concentration in the mother liquor is usually achieved. As previously reported (ref. ), the displacement wash yield decreases with increasing initial lignin concentration in the mother liquor. As for the values of the wash -3 14 yield obtained in our work for the initial lignin concentration of 61 kg m , Trinh et al. reported the wash yield -3 varying from 0.84 to 0.87 for initial lignin concentration of 25 kg m in the bed of softwood pulp fibres.

Figure 3. Displacement wash yield as a function of the Péclet number: 6 13 ○ spruce pulp, □ beech pulp , 1 – Eq. (4), 2 – Eq. (5), 3 – according to Brenner On the basis of our own data measured for beech pulp bed, the effect of the Péclet number on the wash yield can also be expressed by the following correlation equation

WYRW1  0.656 Pe

0.0885

(4)

with a mean relative quadratic deviation equal to 0.6 %. Since the values of regression coefficients, which were evaluated by the least square method, represent an estimate of the real values, the 95% confidence intervals were calculated as well. They are (0.647; 0.664) and (0.0837; 0.0932) for the coefficient and the power of the Péclet number, respectively. For the qualitative evaluation of the effect of the Péclet number on the wash yield, the following correlation equation

WYRW1  0.732 Pe

0.0503

(5)

6

was developed for beech pulp beds in our previous work with the mean relative deviation of 1.4 %.

5

480


Conclusions The preliminary results obtained for spruce kraft pulp with the kappa number of 18.1 enabled that some conclusions valid within the framework of our study can be made.

(i) The displacement washing of softwood spruce pulp fibres is characterised by greater values of the Péclet number in comparison with hardwood beech pulp with kappa number of 14.1. (ii) The Péclet number based on the pulp bed thickness seems to be independent upon the Reynolds number -3 -3 ranging within the limits of 2.73  10 to 1.04  10 . (iii) The efficiency of the displacement washing increases with increasing the Péclet number. The wash yield for kraft spruce pulp was found to be lower in comparison with that published for beech pulp in our preceding papers. Acknowledgements This work was supported by the Internal Grant Agency of University of Pardubice under the research project SGSFCHT_2017_006.

Symbols av D dm h Pe Re RW u WYRW=1

-1

specific surface of pulp fibres, m 2 -1 axial dispersion coefficient, m s hydraulic mean diameter (= 4 ε/(av (1-ε)), m thickness of bed, m Péclet number based on bed thickness defined by Eq. (1), dimensionless Reynolds number (= 4 u ρ/(av μ (1-ε)), dimensionless wash liquor ratio, dimensionless –1 wash liquid superficial velocity, m s wash yield at RW = 1 defined by Eq. (3), dimensionless

Greek letters  average porosity of packed bed, dimensionless μ wash liquid viscosity, Pa s –3 ρ wash liquid density, kg m –3 exit solute (in our case alkali lignin) concentration from bed, kg m ρe –3 initial solute (in our case alkali lignin) concentration in bed at t = 0, kg m ρ0

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Santos R. B., Hart P. W.: TAPPI J. 13, 9 (2014). Lobo L. A., Bolton T. S.: TAPPI J. 12, 69 (2013). Potůček F.: Collect. Czech. Chem. Commun. 62, 626 (1997). Lee P. F.: TAPPI J. 62, 75 (1979). Ingmanson W. L.: Chem. Eng. Prog. 49, 577 (1953). Potůček F., Miklík J.: Acta Facultatis Xylologiae Zvolen 53, 49 (2011). Potůček F.: Pap. Celul. 56, 49 (2001). Miller S. F., King C. J.: AIChE J. 12, 767 (1966). Ebach E. A., White R. R.: AIChE J. 4, 161 (1958). Strang D. A., Geankoplis C. J.: Ind. Eng. Chem. 50, 1305 (1958). Montillet A., Comiti J., Legrand J.: Chem. Eng. J. 52, 63 (1993). Leitao A., Carlos P., Santos S., Rodrigues A.: Chem. Eng. J. 53, 193 (1994). Brenner H.: Chem. Eng. Sci. 17, 229 (1962). Trinh D. T., Poirier N. A., Crotogino R. H., Douglas W. J. M.: J. Pulp Paper Sci. 15, 28 (1989).

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481

ECO FRIENDLY POLYMER SYSTEMS BASED ON POLYVINYL ACETATE AND SACCHARIDES Puková K.1, Machotová J.2, Mikulášek P.1, Rückerová A.2 Institute of Environmental and Chemical Engineering FChT, University of Pardubice Studentská 573, 532 10 Pardubice 2 Institute of Chemistry and Technology of Macromolecular Materials FChT, University of Pardubice Studentská 573, 532 10 Pardubice *[email protected] 1

Abstract The work is focused on the synthesis of aqueous polymeric dispersions based on polyvinyl acetate and Dglucose by emulsion polymerization technique. These polymeric latexes can be used in the field of sustained release fertilizer encapsulation or as an ecological paper glue with subsequent low biological disposal. The main objective of this work was to bond covalently D-glucose into polyvinyl acetate ensure intended feature of these materials is their expected biodegradation of the polymer mainly in the soil environment. Was searched for optimal ratio between the polymer and the simple sugar in the alkaline medium has been investigated so that the resulting latex exhibits good water solubility, sufficient bond strength, and atmospheric humidity stability. Using infrared spectroscopy, binding of D-glucose to the polymer chain of vinyl acetate has been demonstrated. Keywords: polyvinyl acetate, D-glucose, eco-friendly polymer

Introduction The consumption of polymeric materials is constantly increasing, that is why the emphasis is on using organic raw materials and also on the recyclability and biodegradability of the final polymeric products. Film production from conventional latex coatings is primarily based on the coalescence of thermoplastic polymer particles. The quality of coalescence determines the final mechanical properties of the film. Coalescence is the result of physical coupling of polymer particles.[1] This work focuses on the development of eco-friendly latexes based on polyvinyl acetate (PVAc) and Dglucose.[2] The potential application can be e.g., encapsulation[3] of progressive release fertilizers or ecologically gluing of paper. Biodegradable polymeric fertilizer packaging can be subdivided on the basis of the nature of the natural or synthetic polymer, into hydrolyzed hydrophilic polymers and hydrolyzed hydrophobic polymers.[4] There is a general consensus that hydrolyzed polymers are more biodegradable than the nonhydrolyzed ones due to differences in biodegradation and nutrient release mechanisms.[5] Based on the distribution mentioned above, D-glucose and polyvinyl acetate-based latexes from the portion saponified to polyvinyl alcohol can be included among the hydrolyzed polymers. It is evident from the literature survey that emulsion polymers with covalently bonded sugar units applied as encapsulation materials for controlledrelease fertilizers have not been published so far.[6] The common application of polyvinyl acetate latex is gluing of wood and paper substrates. There is one problem with wastepaper recycling − the presence of sticky compounds that are predominantly made up mostly of organic adhesives, including styrene-butadiene rubbers, acrylates, and polyvinyl acetate. When wastepaper containing such adhesives is defibered, the “stickies” are broken down into 0.05- to 0.5-mm particles. These particles in recycled paper reduce its quality and cause paper machine downtime as well. To clean the paper machines, environmentally hazardous solvents are used. [7] In the case of using polyvinyl acetate polymer adhesive comprising covalently linked D-glucose units, this problem may be avoided. This kind of paper glue exhibits good solubility in water, therefore the glued joints are supposed to be dissolved completely in the washing water during the paper recycling process without the risk of coagulum formation or effluents pollution.[8] The synthesis of latexes has been carried out using an emulsion polymerization technique[9] in the alkaline medium. Because of the alkaline environment, sugars are able to pass into the reactive endiol form[10,11] which is capable of participating in radical polymerization with vinyl acetate monomer due to the presence of a carbon-carbon double bond (Figure 1). During the polymerization, alkaline hydrolysis of acetate groups also results in the formation of vinyl alcohol units in the polyvinylacetate chain (Figure 2) which promote the solubility of the resulting polymer in water.[12,13]

482


OH CH2 HO

CH

CH

OH

OH

CH

H CH OH

C

-OH -

O

OH CH2 CH HO

open string glucose

HO HO H

CH2

H C

C H

C

CH

CH

OH

OH

C

C

OH

OH H

endiol glucose

OH O

H

C

C

HO

OH

H

cyclical glucose

Figure 1. Scheme of formation of endiol form of D-glucose

O O

C

OH CH3

CH CH2

+

H2O

-

OH

CH CH2

n

+

H3C COOH

n

Figure 2. Alkaline hydrolysis of polyvinyl acetate

Experimental Materials Vinyl acetate (VAc) purchased from Sigma-Aldrich (Czech Republic) was used as the major monomer for the preparation of aqueous polymer dispersions. Polyvinyl alcohol, commercially available as Mowiol 4-88 (SigmaAldrich, Czech Republic), was used as a protective colloid in the preparation of latexes that led to stability of the formed polymer particles. Anhydrous D-glucose obtained from the company Lach-Ner (Czech Republic) was used to increase the degradation by microorganisms. To adjust the pH during polymerization, sodium carbonate was purchased from Lach-Ner (Czech Republic). Preparation of aqueous polymer dispersions Polyvinyl acetate latex with a copolymerized variable content of D-glucose were prepared by emulsion polymerization technique.[14] D-glucose is soluble in water and is introduced as an aqueous solution into the polymer system. The aqueous medium during the polymerization was adjusted to pH 8-9 with sodium carbonate. Simple saccharides are able to form an enol form in an alkaline environment that can then react with monomers of vinyl acetate in an aqueous medium by a radical mechanism. However, this is merely a simplified idea of the ongoing reaction, since it also offers the possibility of creating double bonds in sugar degradation in an alkaline environment and subsequently the possibility of reaction these degraded products with vinyl acetate. To ensure colloidal stability of latexes during polymerization, polyvinyl alcohol (PVA) was used to prevent coagulation of latex particles instead of a conventional anionic emulsifier.[9,15] The latexes were prepared under an inert atmosphere of nitrogen in a 700 ml glass reactor at a polymerization temperature 50 °C using a hydrogen peroxide initiator. The 1.2 g of hydrogen peroxide initiator, 2.5 g of sodium carbonate, 10 g of polyvinyl alcohol and the selected amount of D-glucose (Table I) were dissolved in 150 g of water. Upon heating the reactor at the polymerization temperature, an aqueous monomer emulsion consisting of 150 g of water, 1.2 g of the hydrogen peroxide initiator and 66 g of vinyl acetate was added drop-wise into


483

the reactor at the rate 1.29 ml/min. The polymerization proceeded at 50 °C for 5 hours. The solid polymer content was around 40 % by weight. The D-glucose content of the starting monomer is shown in Table I. Table I Representation of D-glucose to the initial monomer content. Sample

Vinyl Acetate (g)

D-glucose (g)

P0

66

0.0

PG 10

66

6.6

PG 25

66

16.5

PG 50

66

33.0

PG 100

66

66.0

Characterization of aqueous polymer dispersions The average size of the polymer particles in the aqueous phase was obtained by dynamic light scattering (DLS). DLS experiments were performed using a Brookhaven 90 Plus Partical Size (Brookhaven Instruments, USA). DLS measurements were performed at room temperature. The concentration of the polymer dispersion was about 0.05 wt. %, the Zeta-potential was measured using the same apparatus. The minimum film formation temperature (MFFT) was measured using the MFFT-60 instrument (Rhopoint Instruments, UK) in accordance with ISO 2115. The MFFT is defined as the minimum temperature at which the casting from the polymeric dispersion creates a continuous and clear film. The content of polyvinyl acetate in the emulsion copolymer, which is a water-insoluble polymeric fraction, was determined by the extraction in distilled water for 24 hours in a Soxhlet extractor. Approximately 1 g of the dried dispersion sample was transferred to the extraction thimble. After extraction, the thimble was dried in an oven at 75 °C for 6 hours, cooled in a desiccator overnight and the content of insoluble polyvinyl acetate was calculated from the initial and final weight of the thimble, assuming the polymer remained in the thimble. For the confirmation of covalently linked D-glucose units in the polyvinyl acetate chain, infrared spectroscopy with Fourier Transform (ATR) with diamond crystal was scanned on a FTIR Nicolet iS50 instrument (Thermo Scientific, USA) 32 scans with a spectral resolution of 0.09 cm-1 with unlimited use in the wavelength range 4000 - 500 cm-1. Before these measurements, samples were precipitated in acetic acid and repeatedly washed with distilled water to ensure the isolation of the polymer with a predominant content of vinyl acetate units. Results and discussions Polyvinyl acetate latexes with a copolymerized variable content of D-glucose having a minimum amount of coagulum (0.4  2%) were prepared by emulsion polymerization technique. All prepared polymer dispersions were stable for only about two weeks, therefore these dispersions are suitable for fast consumption. The latexes were evaluated for their particle size in the aqueous phase, Zeta-potential, viscosity and polyvinyl acetate content relative to the D-glucose concentration. These characteristics are shown in Table II. The DLS results showed that the size of the latex particles was affected by the amount of D-glucose in the latex, increasing the particle size with increasing amounts of D-glucose. The measured Zeta-potential values ranged from -6.7  -3.2 mV. These results show low stability of the dispersions and thus explain the relatively rapid coagulation of the system during storage under laboratory conditions. On the other hand, the polymeric systems containing carbohydrate had lower viscosity. All synthesized latexes exhibited MFFT values close to 0 °C, indicating good film-forming properties. This can be explained by the presence of higher amounts of lowmolecular polymer fractions that plasticize the polymer in the coalescence phase of latex particles. The content of polyvinyl acetate in the emulsion polymer decreased significantly by increasing amounts of D-glucose in the latex. It can be assumed that during the emulsion polymerization in the presence of D-glucose there is a significant saponification of the acetate groups as well as a decrease in the molecular weight appears leading to the formation of low molecular weight polymers and oligomeric products (Figure 3). This phenomenon was described by Takasu et al.[2] who discussed the biodegradability of emulsion copolymers based on polyvinyl acetate and sugar derivatives.

484


HO CH2 HO CH O O

C

HO CH CH3 HO CH OH

CH CH2

C

CH

OH CH CH2

OH Figure 3. Incorporation of D-glucose into the polymer chain formed by vinyl acetate and vinyl alcohol units.

Table II Composition and characteristics of biodegradable latexes based on polyvinyl acetate and D-glucose Sample P0 PG 10 PG 25 PG 50 PG 100

Particle size in the water phase (nm) 189.2 205.6 465.4 587.5 802.3

Zeta potentional (mV) -4.8 -5.9 -6.7 -5.4 -3.2

Viscosity (mPa.s)* 101.2 54.6 39.5 31.2 25.7

PVAc content (%) 51.9 35.8 11.5 3.8 4.9

MFFT (°C) 0.9 0.6 0.7 0.3 1.1

The infrared spectrum of a D-glucose-containing polyvinyl acetate sample is shown in Figure 4. The spectrum shows the absorption band at 1060 cm-1 belonging to the carbohydrate units which is characteristic for the glycosidic bond (C-O-C). This band is superimpose in part by a band corresponding to the plane deformation vibration of the C-OH band which in this case is located in the side substitutes of the D-glucose molecule, in the region of 1 290 - 1 240 cm-1. In the spectra, the band corresponding to the carbonyl group (C=O) can also be found in the wavelength range of 1 732 – 1 729 cm-1. It can be argued that vinyl acetate groups have been confirmed in this case since groups whose valence vibration is at the wavelength of 1 248 cm-1 correspond to an absorption band of the methyl acetate bond (C-O). The polyvinyl alcohol building units are proved because their deuterated alcohol groups exhibit a band in the area of 820 cm-1 corresponding to the plane deformation vibration of bond (C-O-D), as deuterated alcohol derivatives that can be found in the spectrum at the wavelength of 820 cm-1. Deuteration shifting the vibration frequency into the region of very low wavelengths and interrupts the interactions with hydrocarbon residue vibrations.[16] If the polymer system does not contain D-glucose units, the of deformation vibration band at 1060 cm-1 disappears (Figure 5).


485

1,2 1,0

1 240 cm-1

Absorbance

0,8

1 729 cm-1 0,6

1 060 cm-1

0,4

820 cm-1

0,2 0,0 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumbres [cm ]

Figure 4. Infrared spectrum for a sample PG 100

1,2

1 240 cm-1

1,0

1 729 cm-1

Absorbance

0,8 0,6 0,4

820 cm-1

0,2 0,0 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumbers [cm ]

Figure 5. Infrared spectrum for a sample P 0

Conclusion Latexes based on polyvinyl acetate and D-glucose were prepared by emulsion polymerization as an aqueous dispersion that can be expected to result in faster microbial decomposition. During the synthesis, simple and eco-friendly starting materials were used. The presence of covalently bonded sugar units in polyvinyl acetate polymer chains was confirmed by infrared spectroscopy. Furthermore, the Zeta-potential and size of the polymer particles in the dispersion were measured by dynamic light scattering. These methods confirmed the low latex stability, therefore rapid processing is appropriate this latex. These aqueous dispersions may find their application as adhesives for paper, but also as controlled-release fertilizer packs.

486


References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Chern, Chorng-Shyan; Principles and Applications of Emulsion Polymerization, Chapter 1, John Wiley & Sons, Inc.,New York (2008) ISBN: 9-7804-7037-7949 Takasu,M. Baba, T. Hirabayashi; Macromolecular Bioscience, 8, 193, (2008) K. Patra, S. Dahiya; Man-made in India, 52, 224 (2009) D. W. Davidson, M. S. Verma, F. X. Gu; SpringerPlus, 2, 4, (2013) Azeem, K. Kushaari, Z. B. Man, A. Basit, T. H. Than; Journal of Controlled Release, 181, 11, (2014) Z. Majeed, N. K. Ramli, N. Mansor, Z. Man; Reviews in Chemical Engineering, 31, 69, (2015) A. Blaney, S. U. Hossain; ChemTech, 27, 48, (1997) H. Lange; Emulsion polymerization of vinyl acetate with renewable raw materials as protective colloids, 2nd edition, Degree project in Coating Technology, Sweden (2011) J. Machotová, J. Šňupárek, L. Prokůpek, T. Rychlý, P. Vlasák; Progress in Organic Coatings, 63, 175 (2008) H. S. Isbell, H. L. Frush, C. W. R. Wade, C. E. Hunter; Carbohydrate Research, 9, 163 (1969) De Wit, A. P. G. Kieboom, H. Van Bekkum; Carbohydrate Research, 74, 157 (1979) Sakurada; Polyvinyl Alcohol Fibers, Chapter 7, Marcel Dekker, Inc., New York (1985) ISBN: 0-8247-7434-5 S. Carra, A. Sliepcevich, A. Canevarolo; Polymer, 46, 1379 (2005) Machotová, L. Zárybnická, R. Bačovská, J. Vraštil, M. Hudáková, J. Šňupárek; Progress in Organic Coatings, 101, 322 (2016) M. Okubo; Polymer Particles, Chapter 3, Springer, Berlin (2005) ISBN: 3-540-22923-X M. Horák, D. Papoušek; Infračervená spektra a struktura molekul, Chapter 2.11, Academia, Praha, (1976)


487

ANIONIC POLYMERIZATION AND PHYSICAL PROPERTIES OF THE POLYMERS MADE FROM TRANS-ΒFARNESENE Trnka T.1, Pleska A.1, Yoo T.2, Henning S.K.2 1

Cray Valley Czech, O. Wichterleho 816, 278 01, Kralupy nad Vltavou Total Cray Valley, Stockton drive 665, 19341, Exton (PA) [email protected]

2

Abstract For several decades, commercially relevant monomers used in anionic polymerization technology were limited to dienes (butadiene and isoprene) and vinyl aromatics (styrene). Industrial manufacturers are still largely limited to these monomers when introducing new products to the market. Recently Amyris developed a new molecule, trans-β-farnesene (-farnesene, or BioFene™) using a biotechnology platform, and its conjugated diene structure has been shown to be a drop-in monomer for a range of polymerization chemistries, including anionic polymerization. Total Cray Valley has partnered with Amyris to develop farnesene-based anionic polymers, including mono- and di-functionalized oligomers. Due to the highly branched structure, farnesenebased polymers show interesting rheological properties compared with linear analogs. The anionic polymerization of trans-β-farnesene, the functionalization and modification of the oligomers, and the resulting physical properties will be discussed.

Introduction Current commercially used monomers for liquid resins made by anionic polymerization were limited to dienes and vinyl aromatics. During the last decade, papers describing the use of new types of monomer can be found. The majority of them focus on finding a suitable monomer made from renewable resources (e.g. ocimene, myrcene, etc.). Recently, Amyris has introduced a new process to produce β-farnesene (BioFene™) in highpurity1. The Total group has established a partnership with Amyris in 2010. Thanks to this partnership, Total Cray Valley was able to develop a process to polymerize the BioFene producing mono or di-hydroxyl (-OH) terminated polymers. This work describes the principal of synthesis, polymer properties as well as properties of hydrogenated polymers.

Experiment Syntheses of OH functionalized polymers based on BioFene were done using batch anionic polymerization2. Organo-lithium based initiators were used for the reactions. Polymerizations were done in non-polar or polar solvents (ethers). Optimal reaction temperature was found at 30-50 °C. Copolymers with other monomers (butadiene, styrene) were prepared by the similar process or synthesizing the homopolymers. Molar mass of polymers was controlled by the initiator/monomer ratio. After the consumption of all monomer from the mixture, the functionalization step was carried out by reacting the polymer living ends with a cyclic ether3. Final polymer was obtained by removing alkalinity using water washing and solvent evaporation. Hydrogenation of the polymer was made on Nickel catalyst in non-polar solvent solution. Both the non-hydrogenated and hydrogenated grades of polymer were analyzed by GPC, HPLC, DSC and Brookfield viscometry.

Discussion and result analysis The polymerization process of BioFene was developed with respect to the industrial production capabilities of Total Cray Valley. The selection of initiator was limited to the organolithium compounds. For mono functionalized polymers, the standard solution of organolithium compound in toluene was used. For difunctional polymers, unique proprietary dilithio-organic based initiator was used4. Solvent of suitable purity for anionic polymerization was used. The reaction was kept under nitrogen atmosphere. Temperature was monitored during the reaction. After the temperature drop-off, the functionalization using a cyclic ether was done. The functionalization was followed by water washing to remove the alkalinity coming from lithium. Organic layer (polymer solution) was heated above the boiling point of water and the final product was isolated by residual water and solvent evaporation. For hydrogenated grades, the polymer was diluted in non-polar solvent and hydrogenated using Nickel based catalyst at elevated temperature and pressure. Degree of 5 hydrogenation was monitored using FTIR . Molecular weights of all polymers were analyzed using GPC and a polystyrene calibration was used as a standard. Very good correlation of theoretical and measured molar mass was achieved for all samples. Efficiency of the functionalization step was evaluated using HPLC method. OH value for each polymer obtained from the analyses was close to the calculated value. Dependence between

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molar mass and Tg of prepared polymers was investigated on DSC. Glass transition temperature of polyfarnesene diols decreased with increasing molecular weight. -60 PF

PFM

PFD

-65

Tg, °C

-70 -75 -80 -85 1 000

10 000 100 000 Mn, g mol-1 Figure 1. Glass transition temperature of nonfunctionalized (PF), monofunctionalized (PFM) and difunctionalized (PFD) polymers. This phenomenon can be explained by the predominant effect of H-bonds on terminal groups over the effect of chain length6. Viscosity curve was measured on Brookfield viscometer. Obtained viscosity curves of polyfarnesenes were compared to the viscosities of polybutadienes having the same molar mass. All prepared polyfarnesenes have much lower viscosity than butadiene based polymers. Table I Theoretical and measured values for prepared non-hydrogenated polyfarnesenes Polyfarnesene Parameter Monol 1 Monol 2 Monol 3 Diol 1 Target Mn, g mol–1 2000 5000 10000 2200 Meas. Mn, g mol–1 2200 5000 13000 2200 Đ 1.110 1.060 1.030 1.381 Theor. OH value, mmol g-1 0.457 0.199 0.078 0.917 Meas. OH value, mmol g-1 0.452 0.195 0.077 0.902 Viscosity at 25 °C, mPa s 500 1400 4500 1500

Diol 2 4900 5100 1.165 0.390 0.385 2400

Diol 3 9200 9000 1.120 0.223 0.221 12000

Table II Glass transition temperature and viscosity dependence on copolymer composition Polyfarnesene copolymer Parameter Copo 1 Copo 2 Copo 3 Copo 4 Mn, g mol–1 2000 2000 2000 2000 BioFene, % 90 80 70 75 Butadiene, % 25 Styrene, % 10 20 30 Tg, °C -54.8 -48.0 -37.3 -58.4 Viscosity at 25 °C, mPa s 2708 5735 17954 2260

Copo 5 2000 50 50 -50.6 3152

Copo 6 2000 25 75 -48.6 4087


489

30000 PFD

PBD

25000

Viscosity mPa s

20000 15000 10000 5000 0

20

30

40

50

60

70

80

90

Temperature °C Figure 2. Viscosity curves of polyfarnesene diol (PFD) and polybutadiene diol (PBD).

Conclusion Farnesene can be successfully polymerized using organolithium initiators in polar solvent. Quantitative functionalization was done by introducing the OH groups at the chain-ends. In comparison with mono or di functionalized polybutadienes, the viscosity of prepared polyfarnesenes is much lower at the same molecular weight. Farnesene can be copolymerized with butadiene or styrene. Increasing comonomer content increases the viscosity and Tg of polymer.

References 1. 2. 3. 4. 5. 6.

http://investors.amyris.com/releasedetail.cfm?releaseid=950471 T. Yoo and S. K. Henning, Paper #118, Presented at the Fall 176th Technical Meeting of Rubber Division, ACS, Pittsburgh, PAH, October 13-15, 2009. N. Hadjichristidis, M. Pitsikalis, S. Pispas, and H. Iatrou, Chem. Rev., 101, 3747, (2001). J. Pytela and M. Sufcak, Proceedings of the Polyurethanes World Congress 1997, Amsterdam, The Netherlands, 704 (1997). K. Bouchal, M. Ilavsky, and E. Zurkova, Die Angewandte Makromolekulare Chemie, 165, 165 (1989). K. Xing, S. Chatterjee, T. Saito, C. Gainaru, and A. P. Sokolov, Macromolecules, 49(8), 3138 (2016).

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SECURE MANAGEMENT PROCESSES, ACCIDENTS PREVENTION


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INHERENTLY SAFER DESIGN OF A NOVEL INDUSTRIAL SCALE REACTOR FOR ALKYLPYRIDINE DERIVATIVES PRODUCTION Janošovský J., Kačmárová A., Danko M., Labovský J., Jelemenský Ľ. Institute of Chemical and Environmental Engineering, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia [email protected]

Abstract Alkylpyridines and their derivatives are chemical compounds widely used in pharmaceutical industry and agriculture. In recent years, alkylpyridine-N-oxides have received attention due to their increased reactivity provided by the N-oxide group. In our paper, design of an industrial scale continuously stirred tank reactor for production of 3-methylpyridine-N-oxide with the focus on process safety was discussed. 3-methylpyridine was converted into 3-methylpyridine-N-oxide by homogeneously catalysed reaction in the presence of hydrogen peroxide as the oxidizing agent and phosphotungstic acid as the catalyst. Reactor dimensions were proposed based on a scale-up of a laboratory unit. Sensitivity and uncertainty analyses of selected key process parameters were performed to determine optimal operating point within the safety constraints. The proposed continuous process presents a suitable inherently safer alternative to conventional semi-batch production.

Introduction With the recent development in process intensification activities, detection of possible hazardous events and operability problems has become more difficult. One of the basic concepts of process intensification is the transition towards continuous productions. Continuous reactor is a preferred alternative to batch or semibatch reactor not only from the economic point of view (size reduction), but also because of its inherently safer character1,2. Inherent safety principles have been applied to the N-oxidation process and design of continuous production of 3-methylpyridine-N-oxide (3-MPNOX) in a continuous stirred-tank reactor (CSTR) as an alternative to the conventional semi-batch process is presented. 3-MPNOX belongs to alkylpyridine derivatives that are frequently used in pharmaceutical industry due to their increased reactivity provided by the N-oxide group. This paper compiles necessary activities for safe reactor operation and optimization of the reaction conditions towards higher product yield utilizing computer simulations. Key operating parameters and construction dimensions of CSTR were proposed based on a scale-up of a laboratory unit and consequently optimized based on sensitivity analyses and a complex process hazard identification procedure. Mathematical model for process simulation was built in the MATLAB® environment. Impact of model parameters’ uncertainties on the optimization results and proposed operating points of reactor was also studied.

Case study In pharmaceutical industry, 3-MPNOX (C6H7NO) is produced by the N-oxidation of 3-methylpyridine (C6H7N, 3MP) in the presence of phosphotungstic acid (H3PW12O40) as a metal catalyst and aqueous hydrogen peroxide (H2O2) solution as an oxidizing agent (Equation 1)3. N-oxidation is carried out at temperatures close to the boiling point of the reaction mixture in an open semi-batch reactor to allow discharge of oxygen generated by competitive decomposition of hydrogen peroxide (Equation 2)4. Recent research proposed transition from a semi-batch to a continuous reactor at elevated pressure (200 – 300 kPa) and temperature (110 – 125 °C) to achieve more efficient N-oxidation with inherently safer operation5. A scheme of the proposed manufacturing process is depicted in Figure 1. For the proposed reactor configuration, hydrogen peroxide decomposition reaction is significantly reduced and can be neglected5,6. Although N-oxidation is a complex reaction system, reaction rate for the given range of pressures and temperatures can be calculated from Equation 3 representing simplified reaction kinetic model where C represents the molar concentration of the corresponding component. Values of kinetic parameters used in this case study are summarized in Table I3,5. Constant reaction enthalpy of N-oxidation of – 160 103 J.mol-1 was considered. Key operating parameters were taken from a laboratory unit model with the reaction mixture volume of 1 L6. After the appropriate scaleup (Tables II and III), further process intensification and hazard identification were performed. Mathematical modeling of products’ separation and purification steps is not discussed in this paper. →

492

(1)


(2)

→

(3)

Figure 1. Simplified process scheme of 3-MPNOX production Table I Reaction kinetic data Ai [L.mol-1.s-1]

Arrhenius equation (

(

(

)

)

)

3.23 10 1.66 10 8.12 10

3

12 10

[K] 3 952 12 989 7 927

Table II Reactor inlet and outlet streams after the scale-up of a laboratory unit Feed 1

Feed 2

Products

Mass flow [kg.h-1]

219.9

151.3

371.2

Temperature [°C]

50

50

118.9

3-MP

100

-

1.6

H2 O 2

-

55.9

1.6

H2 O

-

44.1

29.1

3-MPNOX

-

-

67.7

Mass composition [%]

Process variable

Table III Selected parameters of CSTR after the scale-up of a laboratory unit Reaction Agitator Cooling water Molar ratio of Cooling water mass mixture speed inlet temperature H2O2 : 3-MP [-] flow [103 kg.h-1] volume [L] [rpm] [°C] 1 000

1.05

180

25

12.3

Overall heat transfer coefficient [W.K-1.m-1] 255

Process intensification, hazard identification and results analysis The goal of process intensification is to maximize the production rate of 3-MPNOX. The effect of feed temperature, molar ratio of H2O2 : 3-MP, catalyst concentration and cooling medium inlet temperature was


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studied. Process variable „feed temperature“ represents the temperature of streams Feed 1 and Feed 2 leaving the heat exchanger (e.g. operating setpoint for both feed streams). Figure 2 represents one of the obtained results from two-parametric optimization of reaction conditions where the production rate of 3-MPNOX as a function of feed temperature and molar ratio of H2O2 : 3-MP is depicted. As it is shown, increase of feed temperature and decrease of molar ratio of H2O2 : 3-MP leads to gradual increase of 3-MPNOX production rate.

Figure 2. Effect of feed temperature and molar ratio of H2O2 : 3-MP on 3-MPNOX production rate However, process safety limitations have to be considered. As previously mentioned, operating regime of the reactor was determined in the temperature range from 110 °C to 125 °C. If the reactor temperature exceeds 125 °C, vaporization of the reaction mixture can occur, which leads to possible over-pressurization of the reactor. Below 110 °C, secondary reaction of H2O2 decomposition is triggered and thermal runaway occurs. Therefore, safe operating regime has to be monitored. Possible application of these safety constraints is depicted in Figure 3, where the temperature in reactor was analyzed. Red zone represents hazardous operating regime and yellow-to-green zone represents the safe one.

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Figure 3. Effect of feed temperature and molar ratio of H2O2 : 3-MP on the temperature in the reactor after safety constrictions’ application (red zone – hazardous operating regime) As it can be seen in Figure 3, only a limited number of simulated steady states of CSTR can be considered safe. Numerical algorithm for finding maximum production rate of 3-MPNOX (in the matrix visualized in Figure 2) considering temperature limitations (Figure 3) was developed. User-dependent parameter in the searching procedure was maximum allowed operating temperature in the reactor. For the parameter uncertainty analysis, six different operating points are proposed (Table IV). For every operating point, optimal molar ratio of H2O2 : 3-MP was found in the region of ca. 0.91. Table IV Proposed operating points after safety constrictions’ application Process variable

Operating points A

B

C

D

E

F

Maximum allowed operating temperature in the reactor [°C]

125

124

123

122

121

120

Feed temperature [°C]

55.0

50.9

46.1

42.4

37.6

33.9

Production rate of 3-MPNOX [kg.h-1]

267.8

267.6

267.3

267.1

266.8

266.5

Parameter uncertainty analysis Most model parameters involved in the prediction of reactor behavior are uncertain. The influence of uncertainties in the reaction kinetic parameters (Table I) and reaction enthalpy on the proposed reactor operating points was analyzed. Reactor behavior was found to be most sensitive to changes in the reaction enthalpy. Therefore, reaction enthalpy uncertainty was further examined. The original value of the reaction enthalpy in this case study was – 160 103 J.mol-1. However, the reaction enthalpy value for 3-MP N-oxidation varies in literature significantly (from ca. – 120 103 to – 190 103 J.mol-1)6. For the purposes of parameter uncertainty analysis, the range of reaction enthalpy from – 10 % to + 10 % was studied. The position of the proposed operating points (Table IV) for various values of the reaction enthalpy is depicted in Figure 4.

Figure 4. Location of the proposed operating points as a function of relative change of reaction enthalpy value from the original value of – 160 103 J.mol-1


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As it can be seen in Figure 4, none of the proposed operating points is located in the safety operating regime for every uncertainty of the reaction enthalpy in the studied value range. However, if the reaction enthalpy was decreased only by 5 %, all proposed operating points ae still satisfactory. In case of a reaction enthalpy decrease by 10 %, operating point F was shifted to the hazardous operating regime because of the reactor temperature decreased below 110 °C and the consequent decomposition of hydrogen peroxide leading to a runaway would take place. An increase of the reaction enthalpy had a more significant impact on the position of the proposed operating points. In case of a reaction enthalpy increase by only 5 %, all but one (F) operating points were shifted to the hazardous operating regime because of the temperature in the reactor exceeded the upper safety constraint of 125 °C, which leads to possible over-pressurization of the reactor. If the reaction enthalpy was increased by 10 %, every proposed operating point was in the hazardous operating regime.

Conclusion Simulation-based approach for process intensification and hazard identification combination was proposed. As a case study, the production process of 3-methylpyridine-N-oxide was selected. First, a mathematical model suitable for scale-up and reaction conditions optimization of the CSTR for 3-methylpyridine-N-oxide production was developed in the MATLAB® modelling environment. In the next step, reaction conditions were optimized towards maximizing the production rate of 3-methylpyridine-N-oxide with process safety constraints’ implementation. Six different reactor operating points with the production rate increased by ca. 6 % were proposed based on process simulation and multi-parametric optimization. Consequent model parameter uncertainty analysis was performed. For the studied range of reaction enthalpy relative change by 5 % from the original value, only one from the proposed operating points was found to be satisfactory for safe operation. If the range of reaction enthalpy relative change was increased (relative change by 10 % from the original value), none of the proposed operating points was satisfactory for safe operation. This study has shown that an appropriate safety analysis is always required prior to the implementation of an intensified process. The need for model parameter uncertainty analysis in the simulation-based process intensification and hazard identification was underlined. In our future work, implementation of the presented procedures into smart software solution for supporting hazard identification techniques will be studied. Such software tool can be used to design inherently safer processes and also to train operators and process engineers in existing industrial plants.

Acknowledgment This work was supported by the Slovak Scientific Agency, Grant No. VEGA 1/0749/15 and the Slovak Research and Development Agency APP-14-0317 and the AXA Endowment Trust at the Pontis Foundation.

Literature 1. 2. 3. 4. 5. 6.

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Kletz T. A.: Chem. Ind. 9124, 287 (1978). Mannan S.: Lees’ Loss Prevention in the Process Industries: Hazard Identification, Assessment and Control. Elsevier Science, Oxford 2012. Sempere J., Nomen R., Rodriguez J. L., Papadaki M.: Chem. Eng. Process. 37, 33 (1998). Pineda-Solano A., Saenz L. R., Carreto V., Papadaki M., Mannan S.: J. Loss Prev. Process Ind. 25, 797 (2012). Pineda-Solano A., Saenz-Noval L., Nayak S., Waldram S., Papadaki M., Mannan S.: Process Saf. Environ. Prot. 90, 404 (2012). Cui X., Mannan S., Wilhite B. A.: Chem. Eng. Sci. 137, 487 (2015).


MULTILEVEL DATA ANALYSIS IN COMPUTER AIDED HAZARD IDENTIFICATION Janošovský J., Danko M., Labovský J., Jelemenský Ľ. Institute of Chemical and Environmental Engineering, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia [email protected]

Abstract Hazard identification techniques in chemical industry are constantly being improved by advanced computer simulations of chemical plants. Output from computer simulations is a large set of simulation data containing relevant information about individual streams and units involved in the simulated plant such as flow, temperature, pressure, composition, etc. This data is frequently used in process intensification activities but can be also exploited for process safety improvement. Software structure and simulation data analysis methods appropriate for computer aided hazard identification are discussed in this paper. The HAZOP (HAZard and OPerability study) methodology was adopted to generate process variables deviations and to evaluate their consequences via multilevel analysis comprising optimised numerical procedures as well as several graphical interpretations. Software features are previewed in application to two case studies. Presented approach allows investigating complex chemical processes from the safety engineering point of view in more depth and provides more effective analysis of complex fault propagation paths.

Introduction The constant growth of chemical industry led to the increase of manufacturing processes complexity to achieve higher product yields and purity at lower costs. Therefore, appropriate process safety analysis has become one of the most important aspects in plant design and operation. With the development of CAPE/PSE (computer aided production engineering/process systems engineering) tools, the demand for computer aided hazard identification has also increased. Several hazard identification techniques are well established in industrial companies’ policy such as What-If analysis, Checklist, FMEA (Failure modes and analysis) and HAZOP (HAZard and OPerability study)1,2. Structural and systematic approach of these techniques qualify them as potential candidates for computer aided hazard identification 3,4. In our work, HAZOP principles were chosen to be implemented in software solution because of its robustness and wide application in chemical industry5. HAZOP methodology is based on generation of process variable deviations from design intent and analysis of their consequences. Process variable deviations are created by the combination of standardised guide words (No, More, Less, As Well As, Part Of, Reverse, and Other Than) and appropriate process variables (temperature, pressure, flow, level …)6. Although conventional HAZOP is considered as the most comprehensive hazard identification procedure, various drawbacks of this method have been noticed by experienced practitioners, e.g. uncommon hazards overlooking, significant time-consuming character, insufficient design intent definition, considerations of redundant deviations not leading to scenarios of concern, etc. 7 Some conventional HAZOP drawbacks can be reduced or fully eliminated by implementing simulation-based approach. In this paper, a smart software system utilizing HAZOP principles and mathematical modelling of common chemical processes is introduced. The presented software was tested in combination with the simulation platform represented by Aspen HYSYS – a commercial process simulator widely used in chemical industry, particularly in oil and gas industry. The HAZOP methodology served as a tool for the generation of simulation inputs (HAZOP deviations). Severity of the simulated process states after HAZOP deviation occurrence (HAZOP consequences) was determined by multilevel simulation data analysis comprising optimised numerical procedures. Examples of software graphical user interface (GUI) are also provided.

Case studies Mathematical models of examined processes prepared in the corresponding simulation platform are necessary for computer aided hazard identification using the proposed software solution. For the demonstration of software application variability, two manufacturing processes employing different reactor types were analysed. Mathematical models of an ammonia synthesis reactor (Figure 1) and a glycerol nitration process (Figure 2) built in Aspen HYSYS were selected as case studies. Detailed overview of their model parameters and design intent conditions considered for HAZOP can be found in our previous works8,9. Implemented reaction kinetic models in both case studies were verified by experimental results and by comparison with data from industrial operation10,11.


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Figure 1. Model of ammonia synthesis reactor built in Aspen HYSYS environment

Figure 2. Model of glycerol nitration process built in Aspen HYSYS environment

Software application – simulation phase After the reliability and validity of mathematical models were confirmed, the proposed software tool could initiate the HAZOP analysis. At first, connection with the corresponding Aspen HYSYS case file was established and information about individual HAZOP nodes was accessed. The user can browse through three different types of HAZOP nodes: material streams, energy streams and unit operations (Figure 3). As shown, the software tool found six unit operations (Figure 3a) suitable for HAZOP analysis of the ammonia synthesis reactor (unit V-100 represented phase separator which text label in Aspen HYSYS flowsheet (Figure 1) was hidden) and seven material streams (Figure 3b) suitable for HAZOP analysis of the glycerol nitration process.

Figure 3. Example of HAZOP nodes’ parameters display in GUI of the proposed software tool for ammonia synthesis reactor (a) and glycerol nitration process (b) After successful access to Aspen HYSYS data, generation of HAZOP deviations was allowed. The user can apply logic guide words to any of the permitted process variables to create a list of HAZOP deviations. Example of the HAZOP deviation list for the ammonia synthesis reactor is provided in Figure 4 where HAZOP deviations “higher/lower pressure of fresh feed” and “higher/lower content of nitrogen in fresh feed” were created and stored. Currently, only the application of quantitative guide words is implemented. The correct use of qualitative guide words in computer aided approach is very limited because of their imprecise definition and therefore

498


practically infinite possibilities of their interpretation. In the next step, the user can assign a value range to selected HAZOP deviations. When the final HAZOP deviation list is completed, the proposed software tool proceeds into the process simulation phase. Stored HAZOP deviations are selected one-by-one and inserted to Aspen HYSYS. Once the simulated process correctly converged to a new state, configuration of the Aspen HYSYS simulation case file, i.e. HAZOP consequence, is assigned to the corresponding HAZOP deviation and stored for severity determination via multilevel simulation data analysis.

Figure 4. Example of HAZOP deviation list in GUI of the proposed software tool for ammonia synthesis reactor

Software application – data analysis Evaluation of HAZOP consequences’ severity is performed in a simulation data analysis module of the proposed software tool. It employs advanced numerical algorithms for the automated HAZOP analysis in optional combination with the analysis and monitoring of process- or unit-specific safety restrictions provided by the user. These two approaches are implemented for partial automation of the investigation procedure. When the investigation procedure is completed, the identified hazards and operability problems are assigned to a HAZOP consequence and stored. In GUI of the presented software tool, the user can browse through several types of analysis. An example of such analyses is depicted in Figures 5 and 6. Figure 5 represents the hazard identification procedure for a process exhibiting nonlinear behaviour with steady state multiplicity and Figure 6 represents the hazard identification procedure for a process exhibiting nonlinear behaviour without steady state multiplicity. The first type of analysis monitors the effect of the HAZOP deviation value on one parameter of one HAZOP node. This analysis consists of three supplementary methods – analysis of absolute parameter change from the design intent, analysis of relative parameter change from the design intent and parametric sensitivity analysis. Parametric sensitivity analysis allows capturing a sudden change of the process parameter that indicates e.g. the presence of steady state multiplicity in examined system. Figure 5a and Figure 6a show the difference in the parametric sensitivity analysis outputs for a system with and without steady state multiplicity. The second type of analysis monitors the effect of one HAZOP deviation value on selected parameters of one HAZOP node. The default mode of this type of analysis for graphical interpretation depicts relative change of selected parameters from the design intent (Figure 5b and Figure 6b). This analysis allows a more detailed overview of the overall response of one HAZOP node for particular HAZOP deviation and thus reduces the possibility of hazardous parameter change overlooking. The third possible analysis is focused on monitoring a change of one parameter of the selected HAZOP nodes for one HAZOP deviation value (Figure 5c and Figure 6c). This method provides indepth analysis of the deviation propagation path through the examined system. Identified hazardous events and significant operability problems are formulated in a simplified HAZOP-like report that can serve as a preliminary analysis and supporting material for human expert HAZOP teams to detect complicated deviation propagation paths in modern complex production systems and thus to reduce time requirements of hazard identification in modern industrial manufactures.


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Figure 5 – Example of multilevel simulation data analysis in GUI of the proposed software tool for ammonia synthesis reactor (a – parametric sensitivity analysis, b – relative change of selected parameters for one HAZOP node, c – relative change of one parameter for selected HAZOP nodes)

500


Figure 6 – Example of multilevel simulation data analysis in GUI of the proposed software tool for glycerol nitration process (a – parametric sensitivity analysis, b – relative change of selected parameters for one HAZOP node, c – relative change of one parameter for selected HAZOP nodes)


501

Conclusion Application of a smart software tool for automated model-based hazard identification in two case studies was presented. Aspen HYSYS was used as the simulation engine and HAZOP was used as the hazard identification method because of their frequent and successful application in chemical industry. The proposed simulationbased approach enables identification of process hazards and operability problems considering the HAZOP deviation size and represents an upgrade to the conventional HAZOP study where usually only the existence of a deviation is considered. A set of presented graphical interpretations of deviation propagation in systems with and without the presence of multiple steady states phenomena demonstrated the variability and robustness of the hazard identification procedure performed by the proposed software tool. The developed tool can be easily adapted for other chemical plants using the general modelling environment of Aspen HYSYS. Future research will be focused on the development of HAZOP consequences ranking system for more effective hazard assessment and on the proposal of a new simulation engine optimised for the purposes of hazard identification.

Acknowledgment This work was supported by the Slovak Scientific Agency, Grant No. VEGA 1/0749/15 and the Slovak Research and Development Agency APP-14-0317.

Literature 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

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Mannan S.: Lees’ Loss Prevention in the Process Industries: Hazard Identification, Assessment and Control. Elsevier Science, Oxford 2012. Occupational Safety and Health Administration: Process Safety Management (OSHA 3132). U.S. Department of Labor, Washington 2000. Dunjó J., Fthenakis V., Vílchez J. A., Arnaldos J.: J. Hazard. Mater. 173, 21 (2010). Khan F., Rathnayaka S., Ahmed S.: Process Saf. Environ. Prot. 98, 116 (2015). Crawley F., Tyler B.: HAZOP: Guide to Best Practice, 3rd edition. Elsevier Science, Oxford 2015. Kletz T. A.: Reliab. Eng. Syst. Saf. 55, 263 (1997). Baybutt, P.: J. Loss Prev. Process Ind. 33, 52 (2015). Janošovský J., Labovský J., Jelemenský Ľ.: Acta Chim. Slovaca 8, 5 (2015). Janošovský J., Danko M., Labovský J., Jelemenský Ľ.: Process Saf. Environ. Prot. 107, 12 (2017). Morud J., Skogestad S.: AIChE J. 44, 889 (1998). Lu K., Luo K., Yeh T., Lin P.: Process Saf. Environ. Prot. 86, 37 (2008).


CERIUM DIOXIDE NANOPARTICLES – ECOLOGICAL RISK ASSESSMENT Kobetičová K., Krejsová J. Department of Materials Engineering and Chemistry, Faculty of Civil Engineering, Czech Technical University in Prague, Thákurova 7, 166 29, Prague 6 [email protected]

Abstract Cerium oxide in nanoform has been rapidly used in many types of industrial applications. These particles can be also found in building materials based on stone, plaster, cement, glass or ceramic. From these reasons, we should know the nano Ce02 potential for environmental and human health. The current ecotoxicological data do not allow clear risk assessment conclusion, because the literature survey on aquatic and terrestrial ecotoxicology showed a high variability of the ecotoxic data for nano-CeO2. Due to the lack of knowledge on terestrial and sea ecotoxicity, predictive no effect concentration (PNEC) was expressed only for freshwater environment on the base of present ecotoxicity data.

Introduction The production and use of engineered nanomaterials (ENMs) in consumer products is increasing rapidly. The number of published articles revealed that estimation based on a survey sent to companies producing and using ENMs that CeO2 is produced in 5.5–550 t.year-1. CeO2 is used in many types of applications as in the coating products and metal surface treatment products, for the manufacture of chemicals, mineral products, electrical, electronic and optical equipment and fabricated metal products, surfactants. Using of this particles has been also intensively studied in processes of environment remediation2. They can be also found in building materials based on stone, plaster, cement, glass or ceramic (about 10% of application)3. CeO2 is incorporated into building materials for better capture and catalytic decomposition of pollutants such e.g. nitrogen oxides, e.g. 4, 5. We can suppose that these particles should not relax into environment from building materials (with exception of building waste production or disasters) but their manufacturing and application can potent risk for human and environment. From this reason, in order to relate the toxic effects of nano-CeO2, the existing data have to be carefully collected and analysed. The search in Thomson Reuters WoS using the time span of “all years” indicated that all the papers about ecotoxicity of CeO2 particles have been published within the last ten years3. Very helpful database containing the information about NMs ecotoxicity and experimental designs of individual studies was published in 2015 by Juganson et al.3. The data from this database and data from the newer studies are used for assessment in this paper.

Experiment Aquatic freshwater environment The most scientist papers interested in NMs have been totally listed in the database of Juganson et al.3. The numbers of articles divided according to used organism groups are mentioned: crustacean (8), algae (3), fish (4), plants (7), bacteria (5), the others (5). CeO2 of various sizes were tested on different trophic levels including fish, crustaceans, algae, cynobacteria, insect, frogs (embryo) and microorganisms in sludge in short as well as long-lasting tests. Mortality, immobilisation, reproduction, fish/insect embryo development, behaviour changes, respiration of sewage sludge and growth were studied. Effective concentrations EC/LC/IC10-EC100 were analysed. The values were ranged from several thousands of nanograms to thousands of miligrams. All experiments were performed in standardized aquatic media or in tap water, not in sediment. Aquatic sea environment Only one test was performed with saline organisms, especially microorganism Vibrio fischeri (EC50 > 100 mg.l-1). The results of this study did not indicate toxicity. CeO2 nano-particles have never been tested on organisms living in sea sediment3. Terrestrial environment More than scientific articles interested in soil and ecotoxicity are mentioned in WoS (downloaded on May 24th 2017). Most of them is focussed on agricultural plants (13), microorganisms (3) and some papers on earthworms, isopods and nematodes (5). These organisms were exposed nano-CeO2 in short as well as longlasting tests with various media (water medium, agar, soils). Effective concentration EC/LC/IC 10-EC100 were


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analysed. The values were ranged from several hundreds of miligrams to thousands of miligrams. Seed germination, bioaccumulation, feed activity and behaviour, weight changes, mortality and growth of model organisms were studied. The most used model organisms were higher plants, mainly agriculture crops - cucumbers, tomatoes, corn, barley, lettuce, sunflower, red clover, rice, sweet potatoes, Arabidopsis thaliana. In treated plants the number of tillers, leaf area and the number of spikes per plant were reduced respectively. Although morphological effects were not visible, changes in biomass and oxidative stress response of sunflower were also observed 6. Plant biomass (red clover) and ist symbiotic microorganisms root colonization were not negatively affected by NP exposure. Generally, root elongation of various plant species provided very different results. Poštulka et al. did not described effects on prolongation of L. sativa2 but the same test species was affected in the other study in aquatic medium (EC100 = 0.32 mg.l-1)3. The effect on elongation was also described for tomatoe (EC94 = 0.13 mg.l-1) 3. Discrepancies between these results are possible to explain by used media (artificial soil versus various aquatic media) because the medium properties affect bioavailabilty of chemicals and their toxicity7. Effects were described for nematode species Caenorhabditis elegans in acute aquatic (EC11.28 = 1 mg.l-1) or chronic 18-day reproduction test (1nM) on agar medium3. On the other hand, study of Carbone et al. 8 has been shown no effect on earthworm species Eisenia fetida after nano-CeO2 dietary uptake. Earthworms were able eliminate particles from their bodies without any health consequences (2016). The toxic effects of CeO2 on the isopod Porcelio scaber were evaluated. Nanotoxicity was assessed by monitoring the lipid peroxidation level and feeding rate after 14-days exposure to food amended with nano-CeO2. At exposure doses of 2,000 and 5,000 mg of CeO2 per g-1 of dry weight food, cerium particles significantly decreased the feeding rate and increased the LP level9. This has aggravated the risk of contaminating agricultural fields, potentially threatening associated food webs. To assess possible ENM trophic transfer, cerium accumulation from cerium oxide nanoparticles and their bulk equivalent terrestrial food chain. Kidney bean plants (Phaseolus vulgaris) grown in soil contaminated with 1,000-2,000 mg.kg-1 nano-CeO2 or 1000 mg.kg-1 bulk. CeO2 were presented to Mexican bean beetles (Epilachna varivestis), which were then consumed by spined soldier bugs (Podisus maculiventris). Cerium content in tissues of bugs was found and from this reason this is a proof of nano-cerium biomagnificience10. Ecological risk assessment (ERA) a) ERA - introduction Briefly, ecological risk quotient is based on ecotoxicological indexes (NOEC or LOEC or EC/LC/IC1-100) and environmental levels of interest chemical. These values can be adjusted and expressed by following relation: 𝐻𝐻𝐻𝐻 =

𝑃𝑃𝑃𝑃𝑃𝑃

𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃

, where

PEC is Predicted Environmental Concentration and PNEC is Predictive No Effect Concentration. PEC value is modelled from the relevant analytical environmental or manufacturing data and overestimated on levels in tunes.year-1. PNEC value is calculated from an ecotoxicological index from the bio-test with the most sensitive relevant organisms. This value is then divided by uncertainty factor according to number of used species and trophic levels. The uncertainty factors have been usually ranged from 1 to 1,000. The value “one” is used in the case that the ecotoxicological data were scored from many tests including all trophic levels (destruents, producers, consumers) and on the results from microcosms and in situ-studies (biomonitoring). The less relevant data we have, the higher value of the factor is used. When HQ value is higher than 1, the probability of ecological risk is relevant and there is necessary to review and analyse more in details all aspects of interested chemical manufacturing, distribution and using. b) Estimation of PNEC for freshwater environment (with exception of sediment) The EC50 index from the test with the most sensitive organisms Ceriodaphnia affinis (crustacean species) in chronic reproduction test was used as the enter value (0.79 mg.l-1)11 per PNEC calculation. This value was divided by 100 (uncertainty factor) because we have relatively enough ecotoxicity results on pelagic organisms (crustacean, fish, algal species) compared to other environmental compartments, but the ecotoxicological data are relevantly affect by nanoparticle manufacturing, distribution and size properties. PNEC value can such be assumed as 79 x 10-4 mg.l-1 in the present study.

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Discussion and result analysis Generally, ecological risk assessment of NMs is not easy because they dispose varied properties in contrast to bulk chemicals. Nanoparticles can aggregate, accumulate and react with other chemicals in the environment. In addition, the ecotoxicity has been always affected by their size and surrounding medium properties. This paper in focussed on ecotoxicity of cerium nanoparticles and discussion about ecological risk evaluation on the base of available published data. As we can read above, most data were measured for the freshwater ecosystem 3. Ecological risk assessment for Ce02 in sea and terrestrial environment is not presently possible because we have lack of relevant results. Agriculture plans were dashingly used as model organisms but in aquatic media and the results are focussed on CeO2 translocation and distribution in plant bodies predominantly, not on growth and fertility endpoints in solid matrices. From this reason, the PNEC value was calculated only for plankton freshwater organisms. To explain, ecotoxicological results are expressed from acute or long-lasting (chronic) bioassays. From an ecological point of view, chronic data are more relevant for risk assessment than lethality because they characterize effect on growth and fertility. From this reason, the EC 50 = 0.79 mg.l-1 was used for PNEC calculation prior to others lower values expressing ecotoxicity in acute tests, where the most sensitive organisms was algal species Pseudokirchneriella subcapitata: EC10 = 0.0058 mg.l-1 12. In addition, particles used in this study on algae were stabilised by polyacrilic acid and so, the EC50 value from C. afinis test was preferred (study characterisation: 7d-exposition in 15 ml of nanoparticles solution, crustacean were fed by yeast suspension, medium pH ranged from 7.5 to 8.0, size of CeO2 nano-particles was 10-100 nm, fertility was a studied endpoint)11.

Conclusion The understanding of the underlying mechanisms relating to the potentially adverse effects of NMs on aquatic and terrestrial organisms is important for determining appropriate hazard assessment strategies. Unfortunately, we have lack of knowledge concerning behaviour in the environment, parameters determining bioavailability, mechanisms of toxicity, and dependency of size. Current data does not allow to obtain relevant PEC values for interest environments but we can try to calculate PNEC value for freshwaters (not freshwater sediments) at least. The aim of this study was to calculate the PEC of nano-CeO2 for freshwater ecotoxicity on base of results in the scientific database WoS. Naturally, the present PEC value should be subject to revise after addition of each the other relevant scientist data and knowledges in various databases in the future.

Acknowledgement This work was supported by grant SGS16/199/OHK1/3T/11.

References 1. 2. 3. 4. 5. 6.

Piccinno F., Gottschalk F., Seeger S., Nowack B.: J. Nanopart. Res. 14, 1109 (2012). Poštulka V.: Diploma work, UCT in Prague, Prague 2014. Juganson K., Ivask A., Blinova I., Mortimer M., Kahru A.: Beilstein J. Nanotechnol. 6, 1788 (2015). Richerson D. W.: Modern Ceramic Engineering: Properties, Processing, and Use in Design. New York 1992. Kaštyl M.: Bachelor work, BUT in Brno, Brno 2009. Tassi E., Giorgetti L., Morelli E., Peralta-Videa J. R., Gardea-Torresdey J. L., Barbafieri M.: Plant Physilol. Biochem. SI 110, 50 (2017). 7. Maňáková B., Kuta J., Svobodová M., Hofman, J.: J. Hazard. Mater. 280, 544 (2014). 8. Carbone S., Turid H. A., Joner E. J., Oughton D. H.: Chemosphere 162, 16 (2016). 9. Malev O., Trebse P., Piecha M., Novak S., Budic B., Dramicanin M. D., Drobne D.: Arch. Environ. Contam. Toxicol. 72 (2), 303 (2017). 10. Majumdar S., Trujillo-Reyes J., Hernandez-Viezcas J. A., White J. C., Peralta-Videa J. R., Gardea-Torresdey J. L.: Environ. Sci. Technol. 50 (13), 6782 (2016). 11. Tomilina I. I., Gremyachikh V. A., Myl'nikov A. P., Komov V. T.: Inland Water Biology 4 (4), 475, (2011). 12. Booth A., Storseth T., Altin D., Fornara A., Ahniyaz A., Jungnickel H., Laux P., Luch A., Sorensen L.: Sci. Tot. Environ. 505, 596 (2015).


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RISK ANALYSIS BASED ON THE CRITICALITY CLASSES AND THEIR DETERMINATION USING ACCELERATING RATE CALORIMETRY Mašín J., Ferjenčík M., Šelešovský J. Institute of Energetic Materials, University of Pardubice, Doubravice 41, 53210, Pardubice, Czech Republic.

Abstract The scope of this paper is to incorporate thermokinetic parameters determined by Accelerating rate calorimetry (ARC) into risk analysis framework. This approach provides a reliable way for identification and consequent prevention of the risks resulting from the thermal hazards of the assessed processes. ARC is used to determine adiabatic temperature rise (ΔTad), self-accelerating decomposition temperature (SADT) and time to maximum rate (TMR). The other two temperatures are known from the process characteristics: process temperature (Tp) and maximal temperature for technical reasons (MTT). The relation among these temperatures determines the critical class of an assessed process. Event tree analysis (ETA) was used to construct the prefinished event tree for every particular class, which universally describes developments of thermal runaway scenarios. The critical scenarios are consequently analysed using Layer of protection analysis (LOPA) in order to obtain probability of a particular scenario. New method is demonstrated on phenol plant explosion, where the explosion of cumene hydroperoxide (CHP) occurred.

Introduction Some processes performed on the industrial scale use the chemical reactions associated with a thermal hazards. Major accidents like Seveso1 or T2 laboratories2 served like motivation to fully comprehend the thermokinetic behavior of reactions with a runaway potential. Although the field of thermal hazard prevention registered great advancements in the past decades, we believe there is need for a practical hazard analysis method, which would incorporate the results of a thermal analysis into the existing hazard analysis framework. New proposed approach uses generalized event trees to identify critical scenarios which are consequently evaluated by layer of protection analysis (LOPA). Event tree analysis (ETA) and LOPA deploy systematic approach which should be the key to avoid the omission of any credible accident scenario. However none of these methods takes implicitly into account the results of the calorimetric measurements of the reactions possessing a thermal hazard. Conversely the calorimetric measurements yield results, which are used to develop more or less complicated mathematical models of the examined reactions. These models are difficult to be applied in a practical risk analysis. The aim of this work is to overcome the disadvantages of both traditional risk analysis methods and thermal hazard analysis. The solution of disadvantages connected with both approaches is to combine them. Or more precisely to use only basic parameters defining thermal hazard of the assessed reaction and use them in the risk analysis. This paper describes how to use the results obtained from the ARC for dividing the examined processes into the criticality classes. Consequently ETA is used to derive the generalized event tree for the each criticality class. Once all the credible scenarios are identified, their likelihood is evaluated with LOPA.

Accelerating rate calorimetry In accelerating rate calorimeters a tested sample is measured in an enclosed bomb allowing monitoring the temperature and pressure changes. A bomb is placed in the environment, where temperature can be regulated precisely. Usually so called “heat-wait-search” mode is used for determination of thermokinetic behavior of a sample. In this mode a tested sample is kept at certain temperature for some time interval (e.g. 15 min). If the instrument does not detect any temperature change caused by samples decomposition, the temperature is elevated (e.g. by 5 °C) and this procedure is repeated untill a sample begins to decompose. Once decomposition starts, the calorimeter switches to the adiabatic mode, maintaining zero temperature gradient between the bomb and its surroundings. The decomposition characteristics are recorded in the terms of the temperature and pressure rise. For the scope of this work three parameters were calculated from the ARC measurements using the calorimeter software: self-accelerating decomposition temperature (SADT), adiabatic temperature rise (ΔTad) and time to maximum rate (TMR).

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Since the adiabatic conditions in the calorimeter are not ideal, the obtained parameters must be corrected with so called Φ-factor, which is the measure of the adiabaticity. The Φ-factor is given by relation between thermal masses of the sample and its surroundings: Φ=1+

mB c B mS cS

Where m are masses, c are specific heats, indexes S are for the sample and indexes B are for the surroundings (bomb). In the case of ideal adiabaticity Φ-factor would be equal to 1, but the bomb serves as a cooler, absorbing a part of the released heat, increasing Φ-factor. That is why the measurements conducted with higher Φ-factor values have to be recalculated to achieve real adiabatic values.

Dividing processes with runaway potential into criticality classes According to Stoessel3 all the processes with a thermal risk can be divided into five criticality classes. The advantage of this approach is that it uses four temperatures and their relation to characterize the assessed process, so every particular process can be incorporated into one of the classes. The first temperature is the temperature of the process (Tp), the temperature at which process normally operates. The second temperature is the maximum temperature for technical reasons (MTT), for opened systems it is the boiling point, for enclosed systems it is the point when pressure reaches the highest acceptable pressure. These two temperatures are given by the process technology, while remaining two temperatures can be obtained by ARC measurement. The third temperature is the maximum temperature of synthesis reaction (MTSR). This temperature is equal to sum of Tp and adiabatic temperature rise (ΔTad), which determines how much the reaction mass can heat itself when cooling fails. The last self-accelerating decomposition temperature (TD) represents the thermal stability limit of reaction mass, at which runaway commences. Relation among the temperatures upon which particular classes are based is depicted in Fig. 1. The first two classes represent low risk, because their ΔTad cannot heat the system to the TD without an external source of heat. The third class represents the case where evaporative cooling can prevent reaching T D level in an opened system. Last two classes are the most critical, since they possess enough energy to heat up to decomposition levels, despite evaporative cooling in the fourth class case. As the temperatures reflect the dynamic character of an analyzed process, time to maximum rate (TMR) simply describes its kinetics. This temperature-dependent parameter is used as the measure of the examined reaction controllability. It can be calculated from TD, the calculation is based on measured data and thus provide quite reliable information of time, at which the reaction can be controlled before it reaches its peak. However Stoessel3 uses TD24; this is the temperature at which TMR is 24 hours. For the calculation of this temperature TMR must be extrapolated. The extrapolated values do not represent exact TMR values. Besides TD24 can be overly conservative for some processes.

Figure 1. Dividing scenarios into classes according to relation among Tp, MTSR, MTT and TD.3

ETA This scenario-based qualitative method of risk analysis is used to identify how an initiating event can develop into an accident. The possible development of a scenario is expressed in the form of the graphical tree describing successes/failures of the protective measures. The technique is good for analyzing the complex cases, where more layers of protection plays role in the scenario development. This is also the reason why ETA


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was chosen as a tool for constructing prefinished event trees (ET), which describe possible runaway development for each criticality class.4

LOPA LOPA is the semi-quantitative risk assessment method used in the process industries. Once a scenario of interest is identified (in our case using prefinished ETs), it is analyzed like the initiating event – consequence pair. The analysis is focused on identification of so called independent protection layers (IPLs) which should lower a risk to tolerable level and whether they are sufficient. The technique assigns order-of-magnitude values to both initiating event to quantify its occurrence and to each IPL to quantify its reliability. This allows an analyst to decide whether the frequency of the risk is acceptable. 5

Results and discussion Example: Phenol plant explosion In order to test how ARC results can be incorporated into the existing risk analysis framework, synthesis of phenol and acetone using Hock process was chosen as the testing example. Although the examined accident occurred almost thirty years ago, ongoing research concerning determination of safety parameters for cumene oxidation from the past decade proves the topic is still actual - typically thermal activity monitor III is used to determine CHP decomposition parameters.6 Or DSC combined with other calorimetric methods such as vent sizing package 2.7 Hock process is based on oxidation of cumene to cumene hydroperoxide. The resulting organic peroxide is cleaved with catalytic quantity of mineral acid to yield phenol and acetone. Despite the simplicity of the involved chemical reactions, the peroxidic bond cleavage is highly exothermic, which is the source of serious thermal hazard. On March 9, 1982 there was a great explosion in the phenol plant, during which 100 cubic meters of 50 % w/w cumene hydroperoxide exploded and consequently started fire in the plant. The explosion preceded problems with fueling the boilers, so the process was switched to standby mode. When the fueling problems were solved, another problem with vacuum for the distillation column emerged. While operators tried to reestablish vacuum, columns content was pumped into the remote tank. The steam regulation valve for the column was leaking steam, heating the columns content, but operators did not noticed the temperature rise. High temperature alarm was disconnected, so it did not warn operators either. These circumstances allowed temperature in the tank to rise from normal 70 - 80˚C to 149˚C. The solution in the tank started to boil and relief valves opened, leaking the vapors. Operators switched off the boiler, but it was too late. Vapors ignited first and the explosion of the tank followed.8

ARC measurements The thermal decomposition of CHP was studied with accelerating rate calorimetry. For this ARC-es, manufactured by Thermal Hazard Technology, UK, was used. The experiments were performed in heat-waitsearch mode with starting temperature 50 ˚C, wait time 15 minutes and temperature step 5 ˚C. Temperature rate 0.02 ˚C/min was set as the detection limit for the decomposition of the sample. The experiments were carried out in the Ti-LCQ titanium bomb with following parameters: (8 g, 0.54 J/g°C) with the diameter 25.4 mm and the volume 9.8 ml. 200 mg of 50 % w/w CHP solution in cumene was measured; Φ = 17,74.

Assigning the criticality class to the examined process According to description of the phenol plant explosion,8 Tp was given by interval 70 - 80˚C. ΔTad was measured to be 344 ˚C, which means, that MTSR as the sum of ΔTad and Tp equals 524 ˚C. Boiling point of the solution is 152˚C at normal pressure, concerning that runaway occurred in the enclosed system, MTT is significantly higher than TD, which was estimated to be 121 ˚C. The relation among the given and calculated temperature values indicates the evaluated process belongs to the fifth criticality class. TMR at TD equals 8.5 min; such low value only confirms that once decomposition starts control of the reaction is impossible. ARC results are illustrated in Figure 2.

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Figure 2. The first graph depicts temperature vs time dependency obtained by ARC measurement. The second graph represents TMR dependency on temperature. Full line is based on measured data, dashed line represents values extrapolated to Tp.

Generalized event trees ETA was used in order to obtain the prefinished event trees which would serve as a tool systematically describing possible development of thermal runaway scenarios, taking into account results of ARC measurements. The analysis was focused on the criticality classes instead on particular systems. Resulting event trees serve as a list of all the credible scenarios caused by any undesired temperature rise in an analyzed system. Applying the event trees allows an analyst to address the critical scenarios and the factors influencing their development. This qualitative analysis serves like a basis for LOPA, which deals with particular scenarios and facilitates quantitative estimation of their likelihood. For the fifth class decomposition and thermal explosion scenarios are imminent, since both MTSR and MTT are above TD.

Application of generalized event trees to the phenol plant explosion example As described above, the assessed example of the phenol plant explosion belongs to the fifth criticality class. Prefinished ET for this class includes overall six possible scenarios; four of them are critical. At the beginning of the ET it has to be decided whether an external heating of the system is possible, although in the fifth class the reaction mass possess enough energy to heat to Tdec by itself, but external heating increases probability of the critical scenarios to develop. It means although the runaway in the solved example was caused by the unintended external heating, the critical scenarios without external heating remain credible, even though less probable. In the next step we decide whether the system reaches TD, which depends mainly on the amount of the unreacted reaction mass. From the accident description it is known, that the runaway involved almost 100 m 3 of 50 % w/w of cumene hydroperoxide solution. Because Tp is 80 ˚C, ΔTad is 344˚C and SADT is 121 ˚C, we can exclude any non-critical scenario, in which a temperature rise would terminate by itself. At the end of ETA there are two possibilities of the scenario severity: either decomposition or thermal explosion. Like in the preceding decision, it depends mainly on the amount of the unreacted reaction mass and the system parameters which scenario applies. If we take into account the amount of unreacted CHP and its decomposition properties, it is highly probable, that any decomposition would develop into thermal explosion as highlighted in Fig. 3.


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1aA1

Figure 3. 2. Generalized event tree of the fifth class highlighting the thermal explosion scenario.

LOPA Thermal explosion scenario was identified as logical result of any temperature rise, use of LOPA is demonstrated and summarized in the Tab. I. Order of magnitude estimations of the probabilities are usually used for the analysis and our example is not an exception. At the beginning the risk tolerance is set; for accidents with catastrophic consequences value 10-4 is usually used. Frequency of initiating event (steam leakage in our case) was estimated to be 0.1. The analysis does not take into account any enabling events or conditional modifiers, because more information about the plant operation conditions would be necessary to estimate these parameters. It was assumed that frequency of unmitigated consequence has the same value as the initiating event, because it is probable that every steam leakage in the assessed point leads to decomposition and consequent thermal explosion. In next phase the particular IPLs are evaluated. Probability of failure on demand (PFD) for the basic control process system (BCPS) alarm and human action was assumed to be 0.01. The pressure relief device is completely ineffective in the thermal explosion scenario, its PFD is 1. Taking into account these values, frequency of mitigated scenario 1aA1 is still ten times higher than tolerance criteria. Conversely scenario 1aA2 demonstrates, that tolerance criteria could be met e.g. by introducing a safety instrumented function (SIF) with PFD 0.1. Table I Simplified LOPA sheet Scenario title Risk tolerance criteria Initiating event frequency (/yr) Frequency of unmitigated consequence BCPS alarm and human action Pressure relief device Safety instrumented function Total IPLs Total PFD for all IPLs Frequency of mitigated consequence

PFD

0.01 1 N/A 0.01 0.01

1aA1 Frequency [/yr] 0.0001 0.1 0.1

0.001

PFD

0.01 1 0.1 0.01 0.01

1aA2 Frequency [/yr] 0.0001 0.1 0.1

0.0001

Conclusion

A new approach for evaluation of thermal hazard was proposed. This approach was applied on the risk analysis of the phenol plant explosion example. Accelerating rate calorimetry was used to analyze thermokinetic behavior of CHP decomposition. Results of the ARC measurements were incorporated into existing risk analysis methods. Risk analysis results proved its applicability for the practical thermal hazard evaluation.

References 1.

Prerna, J. (2016), Did we learn about risk control since Seveso? Yes, we surely did, but is it enough? An historical brief and problem analysis, Journal of Loss Prevention in the Process Industries, In Press.

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2. 3. 4. 5. 6. 7. 8.

Theis, A. E. (2014), Case study: T2 Laboratories explosion, Journal of Loss Prevention in the Process Industries 30, (296-300). Stoessel, F. (2009), Planning protection measures against runaway reactions using criticality classes, Process Safety and Environmental Protection 87, (105-112). AIChE (2008), Guidelines for Hazard Evaluation Procedures, Center for Chemical Process Safety and John Wiley &Sons, New York, (158). AIChE (2001), Layer of Protection Analysis: Simplified Process Risk Assessment, Center for Chemical Process Safety and John Wiley &Sons, New York, (11-12). Sheng-Hung, W. (2013), Explosion evaluation and safety storage analyses of cumene hydroperoxide using various calorimeters, Journal of Thermal Analysis and Calorimetry 111, (669–675). Kun-Yue, Ch. (2008), Runaway reaction and thermal hazards simulation of cumene hydroperoxide by DSC, Journal of Loss Prevention in the Process Industries 21, (101–109). Schwab, R. F. (1982), EXPLOSION & FIRE AT A PHENOL PLANT, 1.CHEM.E. SYMPOSIUM SERIES NO. 110, (683-685).


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WASTE PROCESSING, AIR AND WATER PROTECTION, TECHNOLOGIES FOR THE DECONTAMINATION OF SOILS

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WASTE PROCESSING, AIR AND WATER PROTECTION, ...

DIRECT NO DECOMPOSITION OVER K-PROMOTED Co-Mn-Al MIXED OXIDES Bílková T., Pacultová K., Obalová L.

1

1

Institute of Environmental Technology- VŠB- Technical University of Ostrava, 17. listopadu 15/2172, 708 33, Ostrava- Poruba [email protected]

Abstract The effect of two different preparation methods (bulk promotion and impregnation) and calcination temperature of potassium promoted cobalt-based mixed oxide catalysts was investigated for understanding their role in direct NO decomposition. The catalysts were characterised by XRF and N2 physisorption techniques and tested for direct NO decomposition in inert gas at temperatures of 560-650 °C. Long term experiments showed slow deactivation at the initial period (up to 10-20 h) of measurements. It was found out that the preparation method and catalyst pretreatment strongly influence final catalyst activity. Catalysts prepared by bulk promotion were more active than catalysts prepared by impregnation method.

Introduction Nitric oxide is one of the oxides from a group known as NOx (NO, NO2). Nowadays, the selective catalytic reduction or selective non-catalytic reduction is used in commercial applications for NO x emission abatement. The main drawback is a necessity of reducing agent adding. This problem could be eliminated by application of 1 direct catalytic decomposition of NO. Although many catalysts have been already tested (noble metals, metal oxides, zeolites), the development of active and stable catalyst still remains a challenging subject of basic research. During the study of direct NO catalytic decomposition, it was found out that the catalytically less active oxides 1 can be activated by the alkali promoters . It is also known that the alkali metals can volatilize at temperatures 2 about 500 °C and higher . Since the instability of the catalysts prepared by impregnation of Co-Mn-Al mixed 3 oxide with potassium nitrate was found out earlier , the aim of the present paper was to test the preparation process based on the bulk potassium promotion and evaluate resulting catalysts activity and stability. The 3 effect of alkali addition was shown on the basis of comparison with non-promoted sample .

Experiment The Co-Mn-Al layered double hydroxide precursor with Co:Mn:Al molar ratio of 4:1:1 was prepared by coprecipitation of corresponding nitrates in an alkaline Na 2CO3/NaOH solution. The resulted mixed oxide was calcined at 500 °C, formed into tablets and denoted as Co-Mn-Al. Samples modified with potassium promoter were laboratory prepared by two methods- impregnation and bulk promotion. The Co-Mn-Al mixed oxide was impregnated by pore filling method in solution containing the promoter (aqueous solutions of KNO3), dried and calcined at 500 °C or 800 °C for 4 h. The catalysts were denoted as IMP-500 °C and IMP-800 °C. For the bulk promotion the washed precipitate of Co-Mn-Al layered double hydroxide precursor was dispersed in an aqueous solution of KNO 3 and the dried filtration cake was calcined in air at 500 °C or 800 °C for 4 h and denoted as BP-500 °C and BP-800 °C. Catalytic measurements of direct NO decomposition were performed in an integral fixed bed stainless steel reactor of 5 mm internal diameter. At the beginning the stability at 650 °C was measured for 50 h. After this period, when stable performance was observed, the temperature dependence of conversion was launched -1 with cooling rate of 5 °C min and the catalysts activity was measured for 3 h at each temperature (640 °C, 620 °C, 600 °C, 580 °C and 560 °C). After the conversion curve measurement, the reactor was heated to 650 °C again and the stability after 80 h was evaluated. The catalyst bed contained 0.5 g of the sample with a particle -1 size of 0.16 - 0.315 mm. Total flow was kept at 50 ml min (20 °C, atmospheric pressure). The catalysts were -1 activated 1 h in 50 ml min of N2. Infrared spectrometer (Ultramat 6, Siemens) was used for NO online analysis. The low-temperature NO2/NO converter (TESO Ltd.) was connected at the bypassed line before NO analyser and the gas for analysis was periodically switched through the convertor in order to analyse the sum of NOx and thus control the amount of NO2. Presence of N2O species was controlled on FTIR spectrometer (Antaris IGS, Nicolet). Energy dispersive X-ray fluorescence (ED XRF) spectrometer Elva X Light IV (Elvatech Ltd., Ukraine) was used for evaluation of Co, Mn and Al contents. X-ray tube with a Pd anode operated at 35 kV and 20 μA; primary Al filter


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was used for excitation beam pretreatment, integration time was 30 s. For specific surface area evaluating, N2 physisorption at -196 °C performed on AutoChem II(Micromeritics) and one point BET method were used.

Discussion and result analysis Characterisation of fresh and used catalysts The chemical composition of fresh catalysts and specific surface areas of fresh and used catalysts are shown in Tab. I. The amount of Co, Mn and Al was similar in all samples and reached target value of molar ratio Co:MnAl 4:1:1. The theoretical amount of potassium in all samples was 2 wt. %. The specific surface area of fresh samples decreases with increasing calcination temperature indication sintering of the catalysts. More pronounced decrease was observed for sample prepared by bulk promotion method (decrease of 83 %). This result corresponds with the “re-calcination” process (during long term stability measurement) of BP-500 °C at 650 °C, when surface area decreased of 65 % from the value of the fresh sample. Table I Chemical composition of fresh catalysts and specific surface area of fresh and used catalysts. 2 -1 2 -1 Sample Co (wt. %) Mn (wt. %) Al (wt. %) Co:Mn:Al SBET fresh (m g ) SBET used (m g ) molar ratio BP-500 °C 55 14 5 4: 1.1: 0.9 94 33 BP-800 °C 56 14 5 4: 1.1: 0.8 16 16 IMP-500 °C 56 15 5 4: 1.2: 0.8 45 31 IMP-800 °C 57 15 5 4: 1.2: 0.8 28 23 Long term stability Time dependences of NO conversion over BP-500 °C, BP-800 °C, IMP-500 °C and IMP-800 °C are depicted in Fig. 1. NO conversions show the same trends. Decrease of NO conversion was observed during the initial 10-20 h period. Bulk promoted catalysts revealed faster stabilization of catalytic performance than impregnated catalysts, however, all the samples were stable up to 80 h of on time measurements. The value of conversion after 50 h was the same as the value after 80 h despite the fact that the catalysts were cooled to 560 °C and heated back to 650 °C during this period. BP-500 °C conversion was stabilized as the first one; the steady state conversion was reached already after 10 h. According to obtained results of catalysts characterisation, the decrease of specific surface area observed from N2 physisorption (Tab. I) could not be the possible reason for the observed initial gradual deactivation, since both samples calcined at 800 °C showed initial conversion decrease period, however, the specific surface area did not change after reaction. According to literature, another explanation for time dependent activity 4 decrease could be reoxidation of the catalyst by the oxygen formed during NO decomposition and the alkali 1 metal volatilization or rearrangement of surface species .

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Figure 1: Long term stability of 2wt% K/Co4MnAlOx prepared by impregnation (IMP) and bulk promotion (BP) -1 -1 calcined at 500 °C or 800 °C. Conditions: 1000 ppm NO in N 2, GHSV= 6 l g h , T=650 °C. Catalytic activity Two calcination temperatures (500 °C and 800 °C) were used during preparation of potassium promoted Cobased mixed oxide catalysts. However, during stability measurements at 650 °C for 50 h, which were done at first, the samples calcined at 500 °C were thus “re-calcined” at 650°C. The temperature dependences of NO conversion are shown in Fig. 2. Catalysts activity of bulk promoted samples slightly decreased with increasing calcination temperature. The differences in conversion increased with reaction temperature for bulk promoted samples. Impregnated samples conversions were almost identical. Bulk promoted catalysts are significantly more active than impregnated ones. In order to evaluate the effect of surface area on the catalytic performance, the specific activity was calculated, where the activity of each sample is related to the unit amount of surface area (Tab. II). They were calculated in relation to the surface area determined for fresh as well as for used samples in order to cover the effect of surface area decrease. The obtained values are almost identical, which confirms results of long term stability test and it can be said that the higher catalytic activity of the catalysts at the initial 10-20 h period is not closely related to the specific surface area and that higher catalytic activity of BP-500 °C sample is not the feature of its specific surface area. 1 Since it is believed that alkali species are responsible for cobalt based mixed oxides activity , the higher activity 5 can be caused by different state or location of potassium in bulk promoted and impregnated samples , which can be connected with its amount remaining in the catalysts after high temperature treatment.


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Figure 2: Temperature dependence of NO conversion over 2wt% K/Co 4MnAlOx prepared by impregnation (IMP) -1 -1 and bulk promotion (BP) calcined at 500 °C or 800 °C. Conditions: 1000 ppm NO in N2, GHSV= 6 l g h . Table II Specific activities of fresh and used catalysts. Catalyst BP-500 °C BP-800 °C IMP-500 °C IMP-800 °C

-2 -1

Specific activity (mol NO m s ) Fresh Used -6 -6 1.3 x 10 3.35 x 10 -6 -6 7.7 x 10 7. 7 x 10 -6 -6 8.7 x 10 3.3 x 10 -6 -6 2.9 x 10 1.9 x 10

Conclusion In summary, it was found that preparation method and calcination treatment are crucial for the resulting catalyst activity. Catalysts prepared by bulk promotion method showed higher activity and faster stabilization of catalyst performance than catalysts prepared by impregnation method. The effect of calcination temperature on catalyst properties was observed - higher calcination temperature caused specific surface area drop, however, the specific surface area of catalysts did not influence obtained catalytic deNO activity.

Acknowledgement This work was supported by Ministry of Education, Youth and Sports, project LO1208 TEWEP.


Haneda M., Hamada H.: C. R. Chim., 19, 1254 (2016). Niemantsverdriet J.W.: Spectroscopy in Catalysis: An Introduction, Willey, 2007. Pacultová K., Draštíková V., Chromčáková Ž., Bílková T., Mamulová Kutláková K., Kotarba A., Obalová L.: J. Mol. Cat. A, In press Amirnazmi A., Benson J.E., Boudart M.: J. Catal., 30, 55 (1973). Grzybek G., Wójcik S., Legutko P., Grybós J., Indyka P., Leszczyński B., Kotarba A., Sojka Z.: Appl. Catal. B, 205, 597 (2017).

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UTILIZATION SORBENT ON LIGNOCELLULOSE MATERIALS BASE FOR REMOVES HALOGENATED DYES INCREASING PARAMETER AOX FROM MODEL EFFLUENT WATER Filipi M. Institute of Chemistry and Technology of Macromolecular Materials, Studentska 95, 530 10University of Pardubice, Czech Republic [email protected]

Abstrakt Abstract

Dyes are a class of organic most visible pollutants. In the current study, the structural and adsorptive properties of the oxycellulose and cultivated flax as well as the possibility to use them as adsorbents for removal of reactive dyes Procion Blue H-5R, Reactive Blue 4 and Reactive Red 120 from aqueous solutions have been investigated. Determining the amount of dye removed from the solution is performed by spectroscopy prepared solutions of model dye.

Introduction

Dyes Removal of textile dyes from wastewater constitutes an important topic of study for environmental protection specialists, considering the impact of these pollutants on the waters where they are discharged 1. Thus, the presence of dyes in surface waters leads to problems related to aesthetics, inhibition of aquatic flora and fauna development, occurrence of different by-products with carcinogenic effect formed by dyes degradation 1. Different techniques have been developed and applied for treatment of textile wastewater, which is characterized by colour, high values of pH, considerable amounts of suspended solids, different and unacceptable COD levels, and the presence of non-biodegradable chemical compounds2-5. Physical methods, such as mechanic separation (coagulation, flocculation, precipitation) or membrane processes, physic-chemical processes (adsorption, chemical precipitation, coagulation-flocculation, and ionic exchange), chemical process (advanced oxidation with ozone, H2O2, UV), biological process (biological processes in connection with the activated sludge processes and membrane bioreactors) or combination of those can be applied in order to ensure the efficiency of dye containing wastewater treatment process2, 5-17. Adsorption remains one of the techniques that have been successfully applied for dyes removal1, 8. This is the result of the fact that adsorption is an easy and feasible technology that can use a variety of materials as adsorbents. Oxycellulose and cellulose are carbohydrate polymer consisting of β-D-glucose repeating units is considered the most abundant renewable polymer resource available on Earth1. Depending on the technological process used to produce them, celluloses may be found in many forms and types ranging from fibres, linters, microcrystalline powders, softwood pulp, bacterial cellulose and many others. Dyes were selected for experimental studies.

Figure 1. Procion Blue H-5R (PB H5R), Alfa-Aesar Co., dye content 100 wt. %


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Figure 2. Reactive Blue 4 (RB4), Sigma-Aldrich Co., dye content 35 wt. %, Amax= 595 nm

Figure 3. Reactive Red 120 Table I Specification of dyes Colours

c [mol l-1]

m [g]

Procion Blue H-5R

0.01

4.33

Reactive Blue 4

0.01

7.97

Reactive Red 120

0.01

7.35

Experiment

A stock solution of 0,01mol/l was prepared by dissolving the appropriate amount of dyes in 100ml and completing to 1000ml with distilled water. Calibration was performed in a set of 50-ml graduated flasks with various initial concentrations (6.47x10-7- 3.85x10-5). For equilibrium studies, the experiment was carried out for 90min to ensure equilibrium was reached. The adsorption experiments were performed through batch method by contacting different amounts of adsorbent with 250 ml of solution containing 10ml of dye. Preparation of materials for experiment Wood chips were milled a special laboratory mill for 30 seconds. Cultivated flax was cutting first into short fibres (0.5 cm). Stems were sorted on the sorting sieves with a mesh size of 5 mm and 3.5 mm. oxycellulose were also cut into small pieces of 0.5 × 0.5 cm. Work The batch device used for the model wastewater treatment consisted of electromagnetic stirrer with equipped heating. The chemisorption process was carried out applying rapid agitation at 90oC of alkaline reaction suspension or solution for complete nucleophilic substitution of chlorine in chlorotriazine reactive group or nucleophilic addition on vinylsulfone reactive group. The pH was adjusted using 10.6 g/l Na2CO3. After the nucleophilic reaction followed by neutralization or acidifying of the reaction mixture, the used sorbent was removed by subsequent filtration and dye concentration of treated wastewater was determined using absorbance determination. The batch device used for the model wastewater treatment consisted of electromagnetic stirrer with equipped heating. The pH was adjusted using altered by a 50% NaOH aq solution to 12 - 12.5.

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The mixture was intensively stirred for 2 hours at the temperature of 90 or 25 0C. After a period of 2 hours, the pH was adjusted using 16% H2SO4 on value 1-2. The mixture was filtered through filter-paper. The dye concentrations in filtrates were determined using spectrophotometry18.

Results analysis

The effects of sorbent type of (PB H5R) dye were presented in Figures 4-6.

Figure 4. Procion Blue H-5R 25oC

Figure 5. Reactive Blue 4, 90oC

Figure 6. Reactive Red 120, 25oC


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Of the results obtained is apparent that adsorption capacity of the investigated materials for removal of dyes from aqueous environment strongly depends on the structure of dyes and the working conditions Adsorption at room temperature was more efficient method for removing the reactive dyes from waste water before the removal of selected organic dyes by heat treatment. Use of stems of annual plants better than the use of wood chips and sawdust from trees because they contain accompanying substances (mainly tannins and pigments extractable by water) that affect the reactivity and subsequent evaluation. Oxycellulose were decomposed, gelling adsorbent material. Further research regarding the study of adsorption equilibrium is encouraged in order to gain useful information. It was observed that the optimum values of experimental parameters and the maximum amount of dye adsorbed onto oxycellulose were dependent on the type of dye. Conclusion Different biomass based materials was tested for simple removal of dyes. Biomaterials were indicated as the most effective bio sorbent for the sorption of dye. For comparison, effect of temperature for the decolourization yield was compared and as could be seen surprisingly, slightly better yield at room temperature rather than reaction conditions suitable for reactive dyeing (temperature 900C).

Acknowledgement

This work supported by TH02030200. References 1. Suteu D., Biliuta G., Rusu L., Coseri S., Nacu G.: Environ. Eng. Manag. J. 14, 525 (2015). 2. Ding S., Li Z., Rui W.: Water Resour. Pro. 26, 73 (2010). 3. Pereira L., Alves M.: Dyes – Environmental Impact and Remediation, In: Environmental Protection Strategies for Sustainable Development, Malik ., Grohmann E. (Eds.), Springer Science Business, 111 (2012). 4. Sulak M.T., Yamaz H.G.: Desalin. Water. Treat. 37, 169 (2012). 5. Zaharia C., Suteu D., Textile Organic Dyes – Characteristics, Polluting Effects, and Separation/Elimination Procedures from Industrial Effluents. A Critical Overview, In: Organic Pollutants Ten Years after the Stockholm Convention – Environmental and Analytical Update, INTECH Publisher, Rijeka, Croatia, 55 (2012). 6. Allen S.J., Koumanova B., J.: Univ. Chem. Technol. Met. 40, 175 (2005). 7. Anjaneyulu Y., Sreedhara Chary N., Raj S. S. D.: Environ. Sci. Bio/Technol. 4, 245 (2005). 8. Kanawade S.M., IJCMS 2, 059 (2014). 9. Kasperchik V.P., Yaskevich A.L. : Pet. Chem. 52, 545 (2012). 10. Kharub M.: JERAD 6, 879 (2012). 11. Latif A., Noor S., Sharif Q.M., Najeebullah M.: JCSP, 32 115 (2010). 12. Mohamed R.M.S.R., Nanyan N.Mt., Rahman N.A., Kutty M.A.I., Kassim A.H.M.: Asian J. Applied. Sci. 2, 650 (2014). 13. Ozdemir C., Oden M.K., Sahinkaya S., Kalipci E.: Water, 39, 60 (2011). 14. Ramesh Babu B., Parande A.K., Raghu S., Prem Kumar T.: J. Cotton. Sci. 11, 141 (2007). 15. Saraswathy T., Singh A., Ramesh S.T.: Enviromen. Eng. Sci. 30, 333 (2013). 16. Suteu D., Zaharia C., Bilba D., Muresan R., Popescu A.: Industria. Textila. 60, 254 (2009a). 17. Zaharia C., Suteu D., Muresan A., Muresan R., Popescu A.: Eng. Manag. J. 8, 1359 (2009). 18. Filipi, M., Milichovsky, M.: JEAS, 4, 105 (2014).

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SORPTION OF SELECTED HEAVY METALS FROM OXALIC ACID SOLUTION USING DIFFERENT TYPES OF SORBENTS Chlupáčová M.

, Kůs P.1, Parschová H.2

1,2

Research center Řež, Husinec - Řež, 25068, Czech Republic UCT Prague, Technická 5, 16628 , Prague, Czech Republic [email protected], [email protected], [email protected]

1 2

Abstract Possibility of adsorption of following metals: zinc, cobalt, nickel, copper and manganese from very acidic solution of oxalic acid was observed by six different types of commercial sorbents: Purolite SST65;Lewatit MonoPlus SP112; Purolite A932; Lewatit Mono plus TP 207; Lewatit CNP80 and Dowex M4195. Metal ions were separated from the acidic solutions in batch experiments. The aqueous samples were analyzed by atomic absorption spectroscopy. Tested sorbents were compared on the basis of percentage adsorption. The best adsorption efficiencies related to the number of selected metals were achieved by Dowex M4195 with bispykolylamine functional group (Co: 72%, Ni: 75%, Cu: 86% Zn: 84%). Two of the tested sorbents had high adsorption efficiency of manganese removal (Purolite SST65: 59% and Lewatit MonoPlus SP112: 52%). We have screened several commercial resins for their suitability for metals separation at low pH. The chelating resin Dowex M-4195 appears to be the most promising. The operating conditions of sorption process were discussed in this paper.

Introduction Metal pollution is not just one of the most serious environmental problems but also the problem of power plant safety1. It is necessary to use decontamination methods to decontaminate corroded parts of the reactor. This is realized by the use of decontamination solutions. The decontamination method used in VVER-type pressurized water reactor is called AP-CITROX2. The used decontamination solutions contain metals (zinc, cobalt, nickel, copper, iron and manganese) in different quantities and with different amount of radioactivity. Minimizing critical path time, reduction of workers exposure and maintenance activities are important economic considerations 3. Thus it is convenient to remove metals from acidic solution, moreover to remove isotopes selectively. Oxalic acid is frequently used additive for the removal of rust. Specifically, this acid is common decontamination agent (alone or in combination with other complexing species) for water cooled nuclear power plants4. Various techniques can be used for removal of metals: precipitation-filtration, ion exchange, reverse osmosis, oxidation-reduction, solvent extraction and membrane separation. Ion exchange resins have been widely used due to their many advantages, such as high treatment capacity, high selectivity and also storage properties 5. According to previous studies various types of sorbents (weakly acidic cation exchanger, strongly acidic cation exchanger, chelating sorbents, weakly basic anion exchanger) can be used for removal of heavy metal ions from water and industrial wastewaters using ion exchange technique6,7,8. Chelating sorbents are produced by chloromethylation of polystyrene with divinylbenzene and then with reactions with ammonia and chloroacetic acid. Chelating ion exchange resin apart from conventional ion exchange resin has the ability to form coordinating bonds. In general, chelating exchangers are coordinating copolymers with covalently bound side chains (functional group), which contain one, two or more donor atoms9. These donor atoms (mostly N, O or S) act as a Lewis base, thus are able to form coordinating bonds with Lewis acids such as heavy metal ions. Due to coordination-type interactions, all such chelating exchangers offer extremely high selectivity toward metal cations, even in highly acidic solutions10. Several commercial types of chelating exchangers with iminodiacetate, carboxylate, aminophosphonate and bis-pikolylamine functional groups are currently available on the market. The aim of this study is to provide experiments with chosen sorbents for the separation of metals from mixed aqueous solutions of oxalic acid.


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Experimental In order to obtain the ion exchange equilibrium data, batch experiments were carried out in polymetallic aqueous solutions of oxalic acid (10 g.L-1) with the metal concentration (copper, nickel, zinc, cobalt and manganese) of 0.5 mmol.L-1. A specified amount of sorbent in protonated form (1 mL) was added into a beaker with stirrer (500 rpm) containing 1000 ml of aqueous solution. Batch experiments were placed in constant temperature (23°C) to reach equilibrium. Sorption efficiency was observed in following time intervals: 1h, 2h, 4h, 24h, 48h, 72h, 97h and 146h. Aqueous samples were analyzed by atomic absorption spectrometry (Atomic Absorption Spectrometer SpectraAA 220, VARIAN). Table I. Basic specifications of selected sorbents11 Lewatit Purolite SST Lewatit Lewatit MonoPlus SP 65 CNP 80 MonoPlus TP 112 207 Matrix polystyrene polystyrene polyacrylate polystyrene functional carboxylic iminodiacetic sulfonic acid sulfonic acid group acid acid Ionic form as Na+ Na+ H+ Na+ shipped Total exchange 1.7 eq/L 3.7 eq/L 4.3 eq/L 2.0 eq/L capacity

Dowex M4195

Purolite A 830

polystyrene bispicolylamine weak base/partial H2SO4 salt

polyacrylate complex amine TEPA

1.0 eq/L

2.75 eg/L

free base

Figure 1. Functional groups of tested sorbents

Discussion and result analysis Tested sorbents were compared on the basis of percentage adsorption. Percentage adsorption was calculated using equation (1) as follows: 𝑐𝑐 ∗𝑐𝑐 ∗100 (1) [%] (1) 𝐴𝐴 = 0 𝑖𝑖 𝑐𝑐𝑜𝑜

Where: A is sorption efficiency [%], c0 is inlet concentration [mg.L-1], ci is the outlet concentration of ions in the sampling time [mg.L-1]

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Equilibrium adsorption experiment Based on the obtained results in table II. the metal sorption from very acidic solution is possible onto selected sorbents. Adsorption efficiency vary according to the functional group of sorbent and the affinity for selected metal. The lowest value was obtained for manganese removal. In most experiments high adsorption efficiency was proven for nickel, zinc and cobalt removal. Table II. Percentage adsorption of selected sorbent Cu Ni Lewatit MonoPlus 1% 72% SP 112 Purolite SST 65 43% 68% Lewatit CNP 80 50% 73% Lewatit MonoPlus TP 39% 67% 207 Dowex M4195 86% 75% Purolite A830 54% 74%

Mn

Zn

Co

52%

90%

73%

59% 1%

86% 95%

76% 76%

44%

91%

72%

1% 35%

84% 90%

72% 87%

In figure 2. results from experiments with strongly acidic cation exchangers (SAC) with sulfonic acid functional group can be seen. The most significant difference in sorption process was observed in copper removal. Conventional SAC exchanger, Lewatit TP 207 reached only 1% adsorption efficiency, on the other hand Purolite SST 65, with the inactive core reached 43% adsorption efficiency. However, it did not reached over 50%. Both cation exchangers showed very good properties for zinc removal.

SAC Lewatit MonoPlus SP 112

Purolite SST 65 90%

72%

73% 76%

68% 52%

43%

86%

59%

1% Cu

Ni

Mn

Zn

Co

Figure 2. adsorption efficiencies of strongly acidic cation exchangers In figure 3. results from experiments with chelating sorbents can be seen. Dowex M4195 showed very good properties for selected metals removal with the highest adsorption efficiency of copper, conversely manganese removal was unsuccessful (1%). However, Dowex M4195 was considered as the most promising sorbent for selected metals removal. Lewatit TP 207 had very good adsorption efficiency of zinc removal. Sorption efficiencies of copper and manganese did not reach over 50%, thus could be considered as unsuccessful.


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CHELATING SORBENTS Lewatit MonoPlus TP 207

Dowex M4195 91%

86% 67%

75%

84% 72% 72%

44%

39%

1% Cu

Ni

Mn

Zn

Co

Figure 3. adsorption efficiencies of chelating sorbents pH effect on adsorption Sorbent with TEPA functional group, Purolite A830 showed good results for zinc and cobalt removal. In experiments with weakly acidic cation exchanger Lewatit CNP 80 pH was adjusted to 5. In these conditions sorbent reached high adsorption efficiency for zinc removal. Based on results it could be also considered for cobalt and nickel removal.

Conclusion Based on our results it is possible to use tested sorbents for metal removal even from acidic solutions with pH = 1, except weakly acidic cation exchanger Lewatit CNP 80 (pH adjusted to 5). Nevertheless it is important to consider the affinity for selected metal. In most experiments high adsorption efficiency was proven for nickel, zinc and cobalt removal. Manganese removal was considered as the most problematic sorption process for all tested sorbents. Chelating sorbent, Dowex M4195 with the combination of ionic and co-ordination interactions showed high adsorption efficiency for nickel, zinc and cobalt removal, moreover it reached the highest value of adsorption efficiency for copper removal. Thus, we considered this sorbent as the most promising for technological processes of metal removal from polymetallic solution.

Acknowledgement The presented work was financially supported by the Ministry of Education, Youth and Sport Czech Republic Project LQ1603 (Research for SUSEN). This work has been realized within the SUSEN Project realized in the framework of the European Regional Development Fund (ERDF) in project CZ.1.05/2.1.00/03.0108. The authors also gratefully acknowledge „Financial sup-port from specific university research (MSMT No 20-SVV/2017)“.

Nomenclature SAC A c0 ci

strongly acidic cation exchanger sorption efficiency [%] inlet concentration [mg.L-1] concentration of ions at the output in the sampling time [mg.L-1]

References 1. 2. 3.

Fenglian Fu, Qi Wang: Journal of Environmental Management, Volume 92, 407-418 (2011) Varga K., Németh Z., Somlai J., Varga I., Szánthó R., Borszéki J., Halmos P., Schunk J., Tilky P.: Journal of Radioanalytical and Nuclear Chemistry, Volume 254, No. 3 589–596 (2002). Vargaa K., Németha Z., Szabóa A., Radóa K., Oravetzb D., Homonnayc Z., Schunkd J., Tilkyd P., Kőrösie F.: Journal of Nuclear Materials, Volume 348, 181–190 (2006).

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4. 5.

Zhang Y., Kallay S.N., Matijevi M., Langmuir, 1, 201-206 (1985). Jelínek L., Parschová H., Paidar M., Mištová E., Desalinační a separační metody v úpravě vody, Praha: VŠCHT, ISBN: 978-80-7080-705-7 (2009). 6. Hubicki Z., Kołodyńska D., Intech., Chapter 8, 193-194 (2012). 7. Fenglian Fu, Qi Wang: Journal of Environmental Management, Volume 92, 407-418 (2011). 8. Njikam E., Schiewer S., Journal of Hazardous Materials, Volume 213-214, 242-248 (2012). 9. Sengupta A.K., Zhu Y., Hauze D., Environ. Sci. Technol., Volume 25, 481-488 (1991). 10. Zhao D., Sengupta A.K., Stewart L., Ind. Eng. Chem. Res., Volume 37, 4383-4387 (1998). 11. Production information, Rohm and Haas Company, LanXess Company and Dowex Company


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APPLICATION OF ION EXCHANGE RESINS TO RECOVERY OF METALS FROM DECONTAMINATION SOLUTIONS Chlupáčová M.

1,2

, Kůs P.1, Parschová H.2

1

Research centre Řež, Husinec - Řež, 25068, Czech Republic UCT Prague, Technická 5, 16628 , Prague, Czech Republic [email protected], [email protected], [email protected]

2

Abstract Three ion exchange resins were used for the selective removal of nickel (II), copper (II), cobalt (II), zinc (II), manganese (II) and iron (III+II) from synthetic solutions simulating AP-CITROX decontamination solution. In this study, we tested two chelating resins; Dowex M4195, Lewatit MonoPlus TP207 and strongly acidic cation exchange resin Lewatit MonoPlus SP112, all promising in acidic solutions. The efficiencies of resins were tested in column experiments. The aqueous samples were analyzed by atomic absorption spectroscopy. The chelating resin, Dowex M4195 showed the best results for nickel, cobalt, copper and iron selective sorption from model solution. On the other hand the metal removal by Lewatit MonoPlus TP207 was not efficient. The zinc and manganese sorption results onto Lewatit MonoPlus SP112 were very promising. Thus, to achieve high selectivity of extracting tested metals we tried the series column circuits with DowexM4195 and Lewatit MonoPlus SP112. Based on this study, it would appear that treatment of mixed metal wastes, generated by APCITROX decontamination process, by ion exchange resins is a technically feasible process for removal and separation of metals, even in lower pH, such as pH=1.

Introduction Metal ions such as iron, copper, nickel, manganese, zinc and cobalt are present in the waste streams from tanneries, electronics, mining and decontamination operations. Metal precipitation, reverse osmosis, ion exchange resins, biosorbents and electrochemical processes are generally used for the removal of metals in wastewater. Ion exchange resins have been widely used due to their many advantages, such as high treatment 1 capacity, high selectivity and also storage properties . According to previous studies various types of sorbents (weakly acidic cation exchanger, strongly acidic cation exchanger, chelating sorbents, weakly basic anion exchanger) can be used for removal of heavy metal ions from water and industrial wastewaters using ion exchange technique(2-4). Special type of ion exchange resin is chelating exchange resin. This type of resin is selective to metal ions in appropriate pH value. A chelating sorbent essentially consists of two components: the chelate forming functional group and the polymeric matrix. Several commercial types of chelating exchangers with iminodiacetate, carboxylate, aminophosphonate and bis-pikolylamine functional groups are currently available on the market. The most common chelating sorbents have IDA functional group. Chelating sorbents are produced by chloromethylation of polystyrene with divinylbenzene and then with reactions with ammonia and chloroacetic acid5. Chelating ion exchange resin apart from conventional ion exchange resin has the ability to form coordinating bonds. Generally, chelating exchangers are coordinating copolymers with covalently bound functional group, which contain one, two or more donor atoms6. These donor atoms, mostly N, O or S act as a Lewis base, thus are able to form coordinating bonds with Lewis acids such as metal ions. Due to coordinationtype interactions, all such chelating exchangers offer extremely high selectivity toward metal cations, even in highly acidic solutions6,7. The decontamination method used in VVER-type pressurized water reactor is called AP-CITROX8. The used decontamination solutions is mixture of citric and oxalic acid containing metals (zinc, cobalt, nickel, copper, iron and manganese) in different quantities and with different amount of radioactivity. Minimizing critical path time, reduction of workers exposure and maintenance activities are important economic considerations9. Thus it is convenient to remove metals from acidic solution, moreover to remove isotopes selectively.

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In our study we explored the possibility of using Dowex M4195 (bis-picolylamine functional group), Lewatit MonoPlus TP 207 (iminodiacetate functional group) and Lewatit MonoPlus SP 112 (sulphonic acid functional group) (Figure 1-3.) for the selective removal of metals (nickel, copper, cobalt, zinc, manganese and iron) from highly acidic AP-CITROX decontamination technology wastewater.

Experimental In this study, for the separation of metals from aqueous solutions of citric and oxalic acid three different types of exchange resins: Dowex M4195; Lewatit MonoPlus TP 207 and Lewatit MonoPlus SP 112 (Table I) were used in dynamic column experiments. Table I Basic specification of selected exchange resins10 Lewatit MonoPlus SP 112 Matrix polystyrene Functional group sulfonic acid Ionic form as shipped Na+ Total exchange capacity 1.7 eq/L

Figure 1. bispicolylamine functional group

Lewatit MonoPlus TP 207 polystyrene iminodiacetic acid Na+ 2.0 eq/L

Figure 2. iminodiacetate functional group

Dowex M4195 polystyrene bis-picolylamine base/partial H2SO4 salt 1.0 eq/L

Figure 3. sulphonic acid functional group

The polymetallic model solution simulating the used decontamination solution with the concentration of 10 g.L-1 oxalic and citric acid was prepared. The concentration of selected metals: nickel, copper, cobalt, zinc, manganese and iron) was 0.5 mmol.L-1. All solutions were prepared from analytical grade chemicals: FeSO4(NH4)2SO4.6H2O, CuSO4.5H2O, NiSO4.7H2O, ZnSO4.7H2O, MnSO4.H2O and CoSO4.7H2O. For all solutions demineralized water with conductivity value less than 0.1 µS.cm-1 was used. Before the sorption process all types of exchange resins were conditioned by following the standard procedure to protonated form. All the equilibrium data in this study were generated by column runs where the model solution (10 L) of fixed composition and pH (1) were passed through glass column (1 cm diameter) containing 15 mL of the resin at room temperature (23°C). The model solution was flowed with the flow rate of 6 BV.h-1 until the breakthrough of the limit concentration of 0.5 mg.L-1. Aqueous samples were analyzed by atomic absorption spectrometry (Atomic Absorption Spectrometer SpectraAA 220, VARIAN).

Discussion and result analysis Tested exchange resins were compared on the basis of breakthrough capacity q0 of exchange resin expressing amount of captured ions during column experiments until the breakthrough of the limit concentration (0.5 mg.L-1). The breakthrough capacity q0 was calculated using equation (1) as follows:

(1) Where: q0 breakthrough capacity of ion exchanger [mg.L-1], ρ0 concentration of ions in the inlet solution [mg.L], ρi concentration of ions at the outlet in the sampling time [mg.L-1], V volume of the flowed solution [L], V0 volume of the cation exchanger [L]

1


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The efficiencies of the resins were tested in column experiments and calculated on the basis of the breakthrough capacity. Figure 4. shows the breakthrough capacities of Dowex M4195 converted to mmol.L-1 for appropriate comparison of metal ions. The chelating resin, Dowex M4195, demonstrated an unusual ability to adsorb many of these metals, most notably copper (q0 = 521 mmol.L-1), even at very low pH. The amount of captured iron and nickel was also considered as satisfactory for our purpose. The selectivity order measured from the experiments is: Cu > Fe > Ni > Co > Zn > Mn, in accordance with the literature data. The efficiency of manganese and zinc removal was insignificant. It did not reach the limit concentration during the experiment.

Figure 4. The breakthrough capacities q0 of chelating exchange resin with bispicolylamine functional group in mmol.L-1 On the other hand the zinc and manganese sorption results onto Lewatit MonoPlus SP112 were very promising. Figure 5. shows the breakthrough capacities of Lewatit MonoPlus SP112. The strongly acidic cation resin, Lewatit MonoPlus SP112, demonstrated strong ability to adsorb manganese (q0 = 261 mmol.L-1). The adsorption of zinc, cobalt and nickel was also observed. The selectivity order measured from the experiments is: Mn > Zn > Ni > Co > Cu > Fe, the selectivity order from the literature data is: Ni > Cu > Zn > Fe > Mn > Co. The sorption of metal in solution with the presence of organic acid is affected by the oxalic and citrate complex constant of stability. Due to this fact, the selectivity order in aqueous solution can be different from the selectivity order in the model solution. Our results indicate that Lewatit MonoPlus SP112 could be used for the manganese and zinc removal even in highly acidic solution (pH 1).

Figure 5. The breakthrough capacities q0 of strongly acidic exchange resin with sulphonic acid functional group in mmol.L-1

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The metal removal by Lewatit MonoPlus TP207 was insignificant. It did not reach the limit concentration during the experiment. On the other hand it can be clearly seen in Figure 6., that the sorption of selected metals occurred in similar trend without considerable difference. Thus could be considered as a possible exchange resin for the operations with higher limit concentration. The selectivity order measured from the experiments is: Cu > Ni > Zn > Co > Fe > Mn, in accordance with the literature data: Cu > Ni > Zn > Fe > Mn > Co, except from cobalt.

Lewatit TP 207 50

ρ[mg/L]

40 30 20 10 0

0

100

200

300

400

500

600

V/V0 Ni

Fe

Cu

Mn

Zn

Co

Figure 6. The sorption process of Lewatit MonoPlus TP 207 with iminodiacetate functional group

Conclusion Based on our results, it would appear that it is technically feasible to use chelating exchange resin and strongly cation exchange resin for the removal of mixed metal wastes generated by AP-CITROX decontamination technology, even in the highly acidic streams with pH=1. Because the concentration of the metals did not reach the limit concentration during the experiments with Lewatit MonoPlus TP207, chelating resin with iminodiacetate functional group was not considered as efficient. The chelating resin with bis-picolylamine functional group, Dowex M4195 showed high affinity for copper, iron and nickel ions. However, zinc and manganese concentration decrease was negligible. The zinc and manganese sorption results onto Lewatit MonoPlus SP112 were very promising. Thus, to achieve high selectivity of extracting tested metals we tried the series column circuits with Dowex M4195 and Lewatit MonoPlus SP112.

Acknowledgement The presented work was financially supported by the Ministry of Education, Youth and Sport Czech Republic Project LQ1603 (Research for SUSEN). This work has been realized within the SUSEN Project realized in the framework of the European Regional Development Fund (ERDF) in project CZ.1.05/2.1.00/03.0108. The authors also gratefully acknowledge „Financial sup-port from specific university research (MSMT No 20SVV/2017)“.


529

Nomenclature ρ0 ρi q V V0

concentration of ions in the inlet solution [mg.L-1] concentration of ions at the outlet in the sampling time [mg.L-1] amount of captured ions by cation exchanger [mg.L-1] volume of solution [L] volume of cation exchanger [L]

References 1. Patterson J.W., Passino R.: Metals Speciation, Separations and Recovery, Lewis Publishers, Chelsea, MI, 1987. 2. Hubicki Z., Kołodyoska D., Intech., Chapter 8, 193-194 (2012). 3. Fenglian Fu, Qi Wang: Journal of Environmental Management, Volume 92, 407-418 (2011). 4. Njikam E., Schiewer S., Journal of Hazardous Materials, Volume 213-214, 242-248 (2012). 5. Jelínek L., Parschová H., Paidar M., Mištová E., Desalinační a separační metody v úpravě vody, Praha: VŠCHT, ISBN: 978-80-7080-705-7 (2009). 6. Sengupta A.K., Zhu Y., Hauze D., Environ. Sci. Technol., Volume 25, 481-488 (1991). 7. Zhao D., Sengupta A.K., Stewart L., Ind. Eng. Chem. Res., Volume 37, 4383-4387 (1998). 8. Varga K., Németh Z., Somlai J., Varga I., Szánthó R., Borszéki J., Halmos P., Schunk J., Tilky P.: Journal of Radioanalytical and Nuclear Chemistry, Volume 254, No. 3 589–596 (2002). 9. Vargaa K., Németha Z., Szabóa A., Radóa K., Oravetzb D., Homonnayc Z., Schunkd J., Tilkyd P., Kőrösie F.: Journal of Nuclear Materials, Volume 348, 181–190 (2006). 10. Dowex M4195, Production information, Lewatit Mono plus TP 207, Production information, LanXess, (2011)Lewatit Mono plus SP 112, Production information, LanXess, (2011).

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MULTISPECIES AQUATIC MICROCOSM AS A TOOL FOR CHEMICAL ASSESSMENT Kobetičová K., Krejsová J. Department of Materials Engineering and Chemistry, Faculty of Civil Engineering, Czech Technical University in Prague, Thákurova 7, 166 29, Prague 6 [email protected]

Abstract Multispecies tests belong to modern analytical methods of anthropogenic stress on living organisms in aquatic environment. This bio-assay is usually performed including organisms from varied trophic levels (producers, consumers, destruents) simultaneously. Results of the microcosm test might be more likely to be indicative of natural ecosystem responses to chemicals than single species toxicity tests because microcosm test can indicate the more relevant effects that might occur in a community. The aim of this paper is an introduction of a simple microcosm in which species duckweed Lemna minor, freshwater green alga Desmodesmus subspicatus and water-fly Daphnia magna are present. The response of model organisms can be observed after 7 days. The method has been successfully introduced by reference chemical substance (K2Cr2O7) and can be used for ecotoxicity determination in waste waters or eluates of solid wastes as well for testing of individual chemicals.

Introduction Briefly, environmentally relevant information on the exposure by varied pollutants and their effects on aquatic ecosystems can be feasibly obtained from standardized microcosm studies simulating selected environmental conditions1, 2, 3. Consequently, various aquatic microcosm systems have been developed whose are designed to meet the following requirements: performance of experiments under standardized environmental conditions, flexibility to cover relevant environmental situations, suitability to study fate and effects in one single experimental set up, reasonable cost efficiency. Such methods have been validated by international organisations as ASTM 4, OECD5 or US EPA6. Presently, the multispecies aquatic assays can be divided into: -

Benthic or pelagic microcosm

-

Sub-divided lake

-

Flask mixture culture

-

Pond microcosm

-

Microcosm with sediment

-

Ecomicrocosm

-

Standard aquatic microcosm

-

River microcosm

-

Wastewater treatment plant microcosm7.

It is evident that microcosms include indoor (laboratory) and outdoor (natural) systems. However, all of them are quite complicated because we have to perform them with many algal species, several benthic and pelagic crustacean species, periphyton. Some types of bio-cosmos can also contain fishes8. The evaluation of data from such systems is time-consuming and very professional. They can be performed at research institutes (Academies of Science, Universities) but very difficult in private companies dealing with ecotoxicity of pollutants for legislative purposes and as students graduate works. Present study therefore deals with introduction of simple aquatic microcosm with the three often used model ecotoxicological species - duckweed Lemna minor, freshwater green alga Desmodesmus subspicatus and water-fly Daphnia magna in a microplate arrangement.


531

Experiment Chemicals and model organisms Pottasium dichromate was purchased from Lach-Ner (Czech Republic) (Figure 1). Stock solutions were prepared in pure destilled water for all experiments. Duckweed community (Lemna minor) was donated from Federal Environment Agency (Berlin, Germany), daphnids (Daphnia magna) from MicroBioTests Inc. (Gent, Belgium) and alga species (Desmodesmus subspicatus) from Botanical Institute of the Academy of Sciences in Třeboň (Czech Republic).

Figure 1. Potassium dichromate Toxicity experiment The tested concentrations for K2Cr2O7 were 0.1, 1 and 10 mg.l−1. This chemical was dilluted in Steinberg medium with changed salts concentrations according to previous studies9. Experiment was performed in triplicates in three plastic microplates, containing 10 ml medium with 5 healthy fronds of Lemna minor, 3 daphnids not old order than 24 h and algal concentration (1,000,000 cells.ml-1) per one hole. Stock cultures and experiment were incubated in a temperature − controlled chamber at 24 ± 0.5 °C under artificial illumination (light period 16/8 h – light/dark). All species were introduced into experiment according to appropriate test quidelines10, 11, 12. The duration of the experiment was 7 days and the estimation of inhibition was based on calculation of specific growth rate (duckweed), on chlorophyll concentration (alga) and immobilisation (daphnids). The number of Lemna fronds and crustacean immobilisations were made visually and chlorophyll concentrations were analysed spectrophotometrically (VIS, Hach Lange DR 3900). The specific growth rate of alga and duckweed was calculated according to appropriate norms 10, 12. MS Excel 2013 (MS Office) was used for the construction of all graphs and expression of inhibitions in the current study.

Discussion and result analysis The results from microcosm study can be considered valid because they match the validity criteria (80% daphnids survival, sufficient specific growth rate of algae and duckweed) of standard single tests according to the appropriate norms. The results can be Potassium dichromate has proven effect on aquatic organisms13. According to the results of toxicity experiment, fronds were green but also with chlorosis effects (1 and 10 mg.l−1), while no statistical differences (p > 0.05) were observed on specific growth rates of Lemna minor. Daphnids immobilisation was observed after 24 h of exposition with exception to the lowest tested concentration (0.1 mg.l−1). All daphnids died after 48 h of exposure. The alga and duckweed were affected from the lowest tested concentration (see Figure 2, 3, 4). The results from microcosm experiment were compared to results from standard single species tests (un-publicated data). We can see that organisms in microcosm assay were similarly (alga, daphnids) or more (duckweed) sensitive to reference substance than in standard tests (see Figure 2, 3, 4). The inhibitions and effects from the current study are orderly similar to data from literature (legislative norms, safety data sheets) 10,11,12,13. It is clear that they cannot be the exactly the same values as the data from standard acute tests because the test periods and test designs are not adequate. In the current study, we used two microplates, each with 6 holes (see Figure 5). Control and all concentrations were made in three replicates. Of course, there is possible used more microplates and concentration replicates. We also used an initial algal concentration of 1,000,000 cells.ml-1 to feed the daphnia and to be able to measure absorbance after 7 days. However, this algal concentration made daphnids observation harder. The use of this bio-assay is also for test of colour samples because the colouring can disturb spectrophotometrical analyse. The use of light microscope can be a solution of this problem but it is more time-consuming method.

532


120

Inhibition, %

100 24 h

80

48 h

60 40 20 0

0,1 0.1

1

10

Concentration K2Cr2O7, mg.l-1

Figure 2. Results of daphnia test for microcosm and standard single test after 24 and 48 hours of exposure (the same course in both the experiments).

120 standard test

Inhibition, %

100

microcosm

80 60 40 20 0

0.1 0,1

1 Concentration K2Cr2O7, mg.l-1

10

Figure 3. Results of duckweed test for microcosm and standard single test after 168 hours of exposure.


533

120 standard test

Inhibition, %

100

microcosm

80 60 40 20 0

0,1 0.1

..

1 Concentration K2Cr2O7, mg.l-1

10

Figure 4. Results of algal test for microcosm and standard single test after 168 hours of exposure.

Conclusion Effects of K2Cr2O7 were noticed for all studied parameters of model organisms. The measured inhibitions were similar to effects in single species tests from our previous experiment as well for data from literature. The suggested bio-assay is relatively simple and fast, we can recommend it for the testing of common samples but there is important to decide case by case, if this method is suitable for using in respect to sample properties as acidity, colour or water turbidity.

Figure 5. The pattern of multispecies microcosm in a microplate arrangement (photo: K. Kobetičová).

Acknowledgement This work was supported by grant SGS16/199/OHK1/3T/11.


Caquet T.: Radioprotection-Colloquie 37, 173 (2002). Taub F. B.: Environ. Sci. Technol. 23, 1064 (1989). Boxal A., Brown C., Barrett K.: Higher Tier Laboratory Aquatic Toxicity Testing. Cranfield Centre for EcoChemistry, Silsoe, UK, 2001. ASTM: Standard Practice for Standardized Aquatic Microcosms: Fresh Water. ASTM E1366, 2016. OECD: Guidance document on simulated freshwater lentic field tests (outdoor microcosms and mesocosms). OECD Series Testing and Assessment No. 53 ENV/JM/MONO17, 2006.

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6. 7. 8. 9. 10. 11. 12. 13.

U.S. EPA: Ecological Effects Tests Guidelines. OPPTS 850, 1900 Generic Freshwater Microcosm Test, Laboratory. EPA 712-C-96-134, 1996. Fargašová A.: Sborník konference Vodárenská biologie 2017, 122 (2017). Petchey O. L., Downing A. L., Mittelbach G. G., Persson L., Steiner CH. F., Warren P. H., Woodward G.: OIKOS 104, 467 (2004). Kasperová A.: Bachelor work, UCT in Prague, 2015. OECD: Guideline document Freshwater Alga and Cyanobacteria, Growth Inhibition Test. OECD Series Testing and Assessment No. 201, 2011. OECD: Guideline document Daphnia sp. Acute Immobilisation Test. OECD Series Testing and Assessment No. for testing of chemicals No. 202, 2004. OECD: Guideline document Lemna sp. Growth Inhibition Test. OECD Series Testing and Assessment No. 221, 2006. https://echa.europa.eu/cs/information-on-chemicals (available May 1st, 2017).


535

CESIUM DOPED Co3O4 SPINEL FOR N2O DECOMPOSITION Michalik S.2, Pacultová K.1, Obalová L.1 VŠB – Technical University of Ostrava, Institute of Environmental Technology, 17. listopadu 2172/15, 708 00 Ostrava 2 BorsodChem MCHZ, Chemická 2039/1,709 00 Ostrava [email protected] , [email protected] 1

Abstract A series of cesium doped Co mixed oxide was prepared from cobalt nitrate using different precipitating agents and tested for low temperature N2O decomposition. Evaluation of catalytic efficiency for the low temperature N2O catalytic decomposition in simulated waste gas from nitric acid production showed that the highest N 2O conversion was obtained over catalyst prepared by calcination of precursor containing Co(II)-Co(III) hydrotalcite with traces of β-Co(OH)2 which was prepared by using NH3.H2O as precipitation agent. The strong beneficial effect of cesium was observed; the optimum cesium amount was 1 wt. % regardless precipitation agent used.

Introduction Nitrous oxide has attracted a great attention as one of the atmospheric pollutants. The negative effect of nitrous oxide on the environment follows from its long durability (about 130 years), and its ability to absorb infrared radiation from the earth [1]. It is estimated that the overall effect of nitrous oxide is about 6 % of the anthropogenic contribution to the greenhouse effect [2]. Nitrous oxide molecule goes up to the stratosphere, where it is photochemically oxidized to nitric oxide which is harmful to the ozone layer. Chemical processes associated with the production and use of nitric acid and fluidized bed combustion are two main sources of nitrous oxide and their contribution to the total emissions of nitrous oxide is 20 % [2]. Catalytic decomposition of N2O belongs to the best available technologies for N2O abatement from nitric acid production [3]. Our recent work [5] showed that among the catalysts tested for N2O decomposition, cobalt oxide Co 3O4 with spinel structure is very promising. Since the reaction of N 2O catalytic decomposition is generally considered as a charge donation from the catalyst into the antibonding orbital of N 2O, weakening the N–O bond and leading to scission, a transition metal oxide like Co 3O4 is active for the decomposition of N2O because of its relatively high redox properties. In presented contribution, Co3O4 mixed oxide was prepared in three different ways and modified by certain amount of Cs promoter. AAS, physical adsorption of nitrogen, TPR-H2 were used for characterization of the prepared materials. Evaluation of catalytic efficiency for the low temperature N 2O catalytic decomposition in simulated waste gas from nitric acid production (in the presence of O 2, H2O) is provided. The aim is determination of effect of precipitation agent and Cs content on physico-chemical properties and catalytic activity.

Experimental Catalyst preparation Co3O4 was prepared by three types of methods: 1st method: Co(NO3)2 · 6H2O with Na2CO3 was mixed in a stirred reactor at 60 °C and pH 9.5 ± 0.1. The formed suspension was mixed for another 30 minutes and subsequently the solid product was filtered, washed with distilled water and dried at 30 °C. The dried product was calcined for 4 h at 500 °C in air and sieved to obtain a fraction with particle size of 0.160–0.315 mm. Catalyst prepared by this method was assigned as UCC. 2nd and 3rd method: Co(NO3)2 . 6H2O was added to NaOH solution (2nd method) or ammonia in water (3rd method) at room temperature while stirring. The concentration of the alkaline solution was chosen in order to maintain the required molar ratio OH/Co in the reaction mixture. The resulting suspension was maintained under stirring and bubbling gas at room temperature for the specified time; subsequently the solid product was filtered, washed with distilled water and dried at 30°C. The dried products were calcined for 4 h at 500°C in air and sieved to obtain a fraction with particle size of 0.160–0.315 mm. The sample prepared by precipitation with NaOH was assigned as BCC and sample prepared by precipitation with ammonia in water was assigned as CC. Next step in preparation was impregnation by the pore ﬁlling method by aqueous solutions of Cs2CO3 to obtain Cs content of 1-4 wt.% Cs. The dried products were calcined for 4 h at 500°C in air and sieved to obtain a fraction with particle size of 0.160–0.315 mm.

536


Description of characterization methods were described in our recent work [5]. Catalytic decomposition of N2O Catalytic measurements of N2O decomposition were performed in an integral fixed bed stainless steel reactor of 5 mm internal diameter in the temperature range of 300-450 °C and under atmospheric pressure. Total flow rate was 100 ml min-1 (NTP). The catalyst bed contained 0.1 g of sample with the particle size 0.160-0.315 mm. The space velocity (SV) 60 l g-1 h-1 was applied. Inlet gas contained 0.1 mol% N2O balanced by nitrogen or mixture of other gases: 5 mol% oxygen, 3 mol% water vapour and 0.02 mol% NO in nitrogen which were added to some catalytic runs in order to simulate waste gas from nitric acid plant. The reactor was heated by temperature programmed furnace and before each run; the catalyst was pre-treated in N2 flow at 450 °C for 1 h. Then N2O was added to the reaction mixture and the steady state of N 2O concentration level was measured. Temperature regime started from 450 °C with the step of temperature decrease of 30 °C to the final temperature of 300 °C. Infrared spectrometer (GMS 810 Series, Sick) was used for N 2O analysis. The content of the water vapor was determined from measurements of temperature and relative humidity.

Results and discussion Characterization of the catalysts The physicochemical properties of the prepared catalysts are summarized in Table I. XRD of prepared precursors were published recently [5]. Alkaline cobalt carbonate was formed by precipitation of cobalt nitrate by Na2CO3 while the cobalt hydrotalcite with hydroxide traces and cobalt hydroxide were formed using NH4OH and NaOH precipitation agents, respectively. After calcination, Co3O4 was the only identified phase in all prepared catalysts (not shown). Surface areas of the un-promoted samples were comparable with literature [5]. The highest SBET was obtained for CC, followed by BCC and UCC. Modification of Cs caused decrease in specific surface areas due to pore blocking by large cesium atoms. Temperature programmed reduction of catalysts with hydrogen was carried out at temperatures ranging from 25 to 500 °C. The amount of hydrogen consumed for the reduction and the temperatures of the reduction peaks maxima are shown in Table I. The reduction proceeds in two major temperature regions (not shown). The lower temperature peak in the range of 200–400 °C can be ascribed to the reduction Co3+ ions to Co2+ with the subsequent structural change to CoO. The higher temperature peak in the range of 400–500 °C is associated with reduction of CoO to metallic cobalt. The presented results clearly indicate that preparation methods and Cs content significantly affect reducibility i.e. bond strength of Co3+ as well as Co2+ with oxygen. N2O catalytic decomposition Figure 1 shows the steady-state N2O conversions over Co spinel modified with different amount of Cs at different reaction temperatures in the range from 300 to 450 °C. The increase of N2O conversions with increasing temperature is relatively steep over all catalysts. The catalyst obtained from precursors precipitated with sodium hydroxide was more active in comparison with those precipitated with aqueous ammonia solution and Na2CO3. The samples containing about 1 % Cs reached higher N 2O conversions than other samples with higher Cs content regardless of the method of preparation. The sample containing 1 wt% Cs (CC1Cs) reached the highest conversion of N2O compared to the two other samples with the same content of Cs (UCC1Cs, BCC1Cs). The dependence of the N2O conversion on the amount of cesium promoter at 420 °C is shown in Figure 2. It can be seen that with the increase of Cs content in the catalyst has a positive effect only to a certain limit, and then further increase of Cs content has a negative effect on the catalysts efficiency.


537

Table I The physical–chemical properties of cobalt oxide catalysts prepared at various reaction conditions.

Sample

Precipitation agent

Structure of the precursor

H2-TPR temperature maxima (°C)

Cs content (wt.%)

SBET (m2.g-1)

H2-TPR 25- 500°C (mmol g-1)

-

19

17.5

n.d.

n.d.

1

14

14.3

n.d.

n.d.

3

17

19.87

n.d.

n.d.

-

14

16.2

n.d.

405

C

NH4OH

CC 1Cs

NH4OH

CC 3Cs

NH4OH

BCC

NaOH

CoII-CoIII (HT) with traces of β-Co(OH)2 CoII-CoIII (HT) with traces of β-Co(OH)2 CoII-CoIII (HT) with traces of β-Co(OH)2 β-Co(OH)2

BCC 1Cs

NaOH

β-Co(OH)2

1

13

13.4

370

409

BCC 2Cs

NaOH

β-Co(OH)2

2

11

13.7

337

406

BCC 3Cs

NaOH

β-Co(OH)2

3

11

14.2

349

412

BCC 4Cs

NaOH

β-Co(OH)2

4

11

13.8

353

425

UCC

Na2CO3

Co(CO3)0.5 . 0.11 H2O

-

14

14.7

415

n.d.

UCC 1Cs

Na2CO3

Co(CO3)0.5 . 0.11 H2O

1

12

14.9

350

424

UCC 2Cs

Na2CO3

Co(CO3)0.5 . 0.11 H2O

2

10

14.5

343

420

UCC 3Cs

Na2CO3

Co(CO3)0.5 . 0.11 H2O

3

12

13.71

342

414

UCC 4Cs

Na2CO3

Co(CO3)0.5 . 0.11 H2O

4

10

12.73

342

418

This effect can be ascribed to a decrease in number of available sites active in N 2O decomposition [4]. The promotional effect of Cs promoter is caused by modification of the electronic properties of the spinel by stimulation of dissociation step according to Eq. 1 and stability of surface oxygen species (Eq. 2) [6]. N2O + e- → N2 + O-

(1)

2 O → O2 + 2e

(2)

-

-

Figure 1. Temperature dependences of N2O conversion in simulated process conditions. Conditions: 1000 ppm N2O, 5% mol. O2, 3% mol. H2O in N2, SV = 60 l g−1 h−1.

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It can be seen different behaviour of sample with the same content of Cs but using various precipitation agents similarly as in the case of non-modified catalysts. The method of preparation influenced the precursor structure and ability of final catalyst to decompose N2O [5]. Comparison with literature [6] showed that the prepared catalysts are very active and can be considered as promising candidate for low temperature N2O decomposition in waste gases from HNO3 production plant.

Figure 2. Dependence of the N2O conversion on the amount of cesium promoter. Conditions: 420 °C, 1000 ppm N2O, 5% O2, 3% H2O in N2, SV = 60 l.g−1.h−1.

Conclusions The N2O catalytic decomposition over cobalt spinels prepared by precipitation of cobalt nitrate in aqueous solutions using various precipitation agents (NH4OH, NaOH, and Na2CO3) and modified with the different content of cesium promoter was examined in gas containing nitrous oxide, oxygen and water vapour. The most active catalyst was prepared by calcination of Co(OH)2 (NH4OH was used as precipitation agent) and modified by 1 wt% Cs (sample CC1Cs).

Acknowledgement This work was financially supported by Ministry of Education, Youth and Sports of the Czech Republic in the “National Feasibility Program I” (project LO1208 “TEWEP”) and by VŠB-TUO internal student project SP2017/92.

References 1. 2. 3. 4. 5. 6.

Ruiz-Martinéz E., Sánchez-Hervás J.M., Otero J.: Catalytic reduction of nitrous oxide by hydrocarbons over a Fe-zeolite monolith under fluidised bed combustion conditions. Appl. Catal. B 50 (2004) 195-206. Obalová L.: Materiály na bázi hydrotalcitu pro katalytický rozklad N 2O. VŠB-TU OSTRAVA, (2008). ISBN 978-80-248-1884-9. Perez-Ramirez J., Kapteijn F., Schoffel K., Moulijn J.A.: Formation and control of N2O in nitric acid production: Where do we stand today? Appl. Catal. B 44 (2003)117-151. Pasha N., Lingaiah N., Seshu Babu N.: Studies on cesium doped cobalt oxide catalysts for direct N2O decomposition in the presence of oxygen and steam. Catal. Commun 10(2008)132-136. Chromčáková Ž., Obalová L., Kovanda F.: Effect of precursor synthesis on catalytic activity of Co 3O4 in N2O decomposition, Catal. Today 257 (2015) 18-25. Grybek G., Stelmachowski P., Gudyka S., Duch J., Cmil K., Kotarba A., Sojka Z.: Insights into the twofold role of Cs doping on deN2O activity of cobalt spinel catalyst – towards rational optimization of the precursor and loading. Appl. Catal. B 168-169 (2015) 509-514.


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CHLORINATED AROMATICS – OCCURRENCE, USAGE AND METHODS OF DEGRADATION Pérko J, Weidlich T. Institute of Environmental and Chemical Engineering, Faculty of Chemical Technology, University of Pardubice, Studentská 95, 532 10 Pardubice, Czech Republic, E-mail: [email protected]

Abstract This paper is summarizing the occurrence, usage and available techniques for dehalogenation of chlorinated aromatics, mainly chlorinated phenols and chlorinated phenoxy- or benzylphenols and their homologues, or polychlorinated biphenyls and dibenzodioxins (PCB´s and PCDD´s). These chemicals are among the most common organic pollutants in the wastewaters from production of pesticides, solvents, paints, pharmaceuticals, wood preserving chemicals, and from pulp and paper industry. Chlorine and other chlorine based oxidants are used for the bleaching of wood pulp accompanied with the formation of toxic organochlorine water pollutants which are polluting the river effluents, its inhabitants and accumulate in the tissues of river biota. These chemicals could be removed (and/or degraded) in many different ways, e.g. there • are advanced oxidation processes (AOP´s) involving the generation of reactive hydroxyl radicals ( OH) or hydrodehalogenation processes to effect the destruction of recalcitrant pollutants in water effluents.

Occurrence Chlorinated aromatics such as chlorinated phenols, chlorinated phenoxy/benzylphenols or polychlorinated biphenyls, polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated diphenyl ethers (see Figure 1 for structures of these compounds) are among pollutants which are the subject of many papers and scientific studies from all around the world. All of above mentioned chemicals belong to category of so called persistent organic pollutants which means they have the ability to persist in the environment and their natural breakdown is either very slow or is not even possible and they could bioaccumulate and cause serious defects in living organisms and in the environment. Because of the urgent warnings from the scientific sphere about the dangers these chemicals are posing there has been a consensus of opinion regarding their production, usage, disposal and gradually total restriction of selected compounds which were found the most dangerous. This is what has become the Stockholm Convention on Persistent Organic Pollutants enlisting many 1-3 of the above mentioned and co-signatories agreeing to limit and outlaw the chemicals on the list . OH

Cl n

Cln

1 Polychlorinated phenols

Clm

2 Polychlorinated biphenyls (PCB´s)

3 Polychlorinated dibenzodioxins (PCDD´s)

4 Polychlorinated biphenyl ethers (PCDE´s )

5 Polychlorinated dibenzofurans (PCDF´s) Figure 1. Structures of selected persistent chlorinated aromatics. However the origin of chlorinated, or let´s say halogenated aromatics is quite complex. Although many of the previous mentioned chemicals are manmade there is a large majority of organohalogen compounds which are occurring naturally in the environment. For example there are thyroid hormones containing iodine (thyroxine 4 6), product of various molds which contain chlorine (griseofulvin 7) .

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6 Thyroxine

7 Griseofulvin

Figure 2. Examples of naturally occurring halogenated aromatics. With the technological development and progression in the field of analytical methods it turns out that naturally occurring organohalogens is maybe much more than expected and we can find these in fungi, bacteria, water plants and animals. In the literature5-7 we could find a large number of chlorinated compounds detected by chemical analysis in these organisms, e.g. Streptomyces sp. bacteria are producing enormous number of metabolites containing halogens such as chloropeptin I (8), an unusual chlorinated hexapeptide produced by the soil actinomycete Streptomyces sp. WK-3419.1 is able to inhibit HIV virus replication8.

8 Chloropeptin I

9 Vancomycin

Figure 3. Examples of naturally occurring chlorinated aromatics. Many organohalogens (and non-halogenated organics as well) can serve as a chemical protection of plants and animals (antibacterial, antifungal, etc.). The antibiotic vancomycin (9) produced by Amycolatopsis orientalis has been used against several bacterial infections since 1950´s5. Growth hormone 4-chloroindole-3-acetic acid (10) and its methyl ester are biosynthesized by peas, lentil, vetch, and fava bean7.

10 4-chloroindole-3-acetic acid

11 Chloramphenicol

Figure 4. Examples of naturally occurring chlorinated aromatics.

Usage Antibiotic chloramphenicol (11) discovered in 1950´s was one of the first discovered and successfully used antibiotics. It was the first massively manufactured antibiotic and it was used both in developed and developing countries for its wide range of effect. Nowadays in the western world there are used antibiotics of a new generation and its using was abandoned but the bacteria resistance is far more common (due to frequent prescription) when using the new antibiotics. An effort of coming back to in the past used antibiotics


541

(where is no bacteria resistance) is made. In developing countries to this day it is still one of the most used antibiotics for its low price and good availability. It is effective against gram-positive, gram-negative bacteria and anaerobic organisms but the biggest disadvantage is its toxicity for bone marrow and subsequent aplastic anemia which could cause death. From this reason this pharmaceutical is only prescribed in life-threatening infections where is no other alternative and where the pros overcome the risks of chloramphenicol9-11. Chlorinated phenols and their derivatives are persistent organic pollutants which are used in the manufacturing of dyes, pharmaceuticals and many other industrial (mainly more complex chlorinated) products. These compounds are highly toxic for living organisms thanks to their carcinogenic, mutagenic and cytotoxic properties. Agrochemical manufacturing takes around 80-90% of overall consumption of chlorinated phenols, e.g. herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid, 12), 2,4-dichlorophenoxyacetic acid or a pharmaceutical clofibrate (Atromid-S, Ethyl 2-(4-chlorophenoxy)-2-methylpropanoate, 13) which was used in the past for treating high blood pressure. Among biocides (herbicides, pesticide, insecticide etc.) based on chlorinated phenols are also chlorophene and pentachlorophenol which is as well used for manufacturing of wood and cellulose preservatives.12

12 Dicamba

13 Clofibrate

Figure 5. Examples of chlorinated aromatics used in agriculture and medicine. The usage of polychlorinated biphenyls was very wide – from plasticizers, heat transfer fluids plasticizers, lubricant inks, fire retardants, paint additives to sealing liquids, immersion oils, adhesives, anti-dusting agents, waxes, carbonless copy papers and mostly as dielectric fluids for capacitors and transformers. PCB´s got into the environment by using them but significant amount got there from disposals, accidents and leakage from 13-15 . On the other hand polychlorinated dibenzodioxins (PCDD´s), polychlorinated the industrial facilities dibenzofurans (PCDF´s) and polychlorinated dibenzoethers (PCDE´s) are mostly unwanted by-products in thermal systems containing chlorine such as incomplete combustion, municipal waste incineration, metal industries, bushfires and controlled burnings. These compounds are also present as impurities in chlorinated phenols and chlorinated phenoxyacetic acids preparations16.

Degradation methods There have been several ways of remediation and affords to degrade these sort of pollutants which are already in the environment. We could name so called advanced oxidation processes (AOP´s) based on the Fenton reaction which includes generation of hydroxyl radicals (•OH) which are one of the most powerful oxidants and because of their non-selectivity it is possible to degrade wide range of different chemicals17. Particular techniques are e.g. TiO2/UV photocatalysis18,19, electrochemistry20, sonoelectrochemistry21,22, ozonation23, using of chlorine dioxide24, potassium permanganate25 and Fenton based processes which are used in recent years in many different ways. We could distinguish Fenton reactions as follows: classical Fenton reaction uses H2O2 and Fe2+ to generate •OH radicals by their combination17, Fenton-like process (H2O2 and Fe3+)26, photo-Fenton (H2O2/ Fe2+ (Fe3+) / UV19 and also electro-Fenton reaction. By electro-Fenton the hydrogen peroxide could be generated from the saturated oxygen solution on the electrodes of many kinds and materials, e.g. titanium27, graphite28, aluminum, stainless steel, copper29, platinum, boron-doped diamond30 or by the direct injection of the gas to a gas diffusion electrode. There are also reductive ways of pollutant degradation such as electrochemical reduction, e.g. one study is describing electrocatalytic process where hydrogen formed on a cathode (by the reduction of water) results in the hydrodechlorination of chlorinated pollutant31. Direct using of hydrogen is also possible and was studied by several groups of scientists, the reaction could be influenced either by pressure of hydrogen gas or by using different kinds of catalysts32,33. Using zero valent metals as reducing agents has been a significant amount of interest last few years. Particularly zero valent iron has been used the reductive dechlorination of chlorinated pollutants and most investigators agree that the reduction takes place directly on the iron surface34,35. Using of metal alloys (Al-Ni36-38, Devarda´s alloy39, etc.) proofed to be working very well as the agent for reductive dehalogenation of halogenated aromatics.

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Yet there are another processes suitable for the degradation which were studied, e.g. biological processes since it is known that there have been identified reductive dehalogenases in several bacteria types40. And also important to mention is of course the incineration process which could be used as the basic method to degrade organic pollutants. But as it is a process where many side reactions are taking place and many different by-products are generated (as above mentioned PCDD´s, PCDF´s and PCDE´s are created by incineration) which could be even more toxic than the original compounds, this method seems to be not very suitable in the modern degradation chemistry. This paper is a short summary of various methods which could be used for degradation of chlorinated aromatic pollutants persisting in the environment and which are posing a threat for the food chain and generally for the living organisms. Suitable way of remediation is therefore required to minimize the amounts and effects of these chemicals in soils, waters and air.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Rodan B. D., Pennington D. W., Eckley N., Boethling R. S.: Environ. Sci. Technol. 33, 3482 (1999). Gosh A., Gupta S. S., Bartos M. J., Hangun Y., Vuocolo L. D., Steinhoff B. A., Noser C. A., Horner D., Mayer S., Inderhees K., Horwitz C. P., Spatz J., Ryabov A. D., Mondal S., Collins T. J.: Pure Appl. Chem. 73, 113 (2001). Laine D. F., Cheng I. F.: Microchem. J. 85, 183 (2007). Macháček V., Panchartek J., Večeřa M.: Organická chemie, 1. část, Univerzita Pardubice, Pardubice, 2001, ISBN: 80-7194-363-0 Gribble G. W.: Acc. Chem. Res., 31, 141 (1998). Gribble G. W.: Prog. Chem. Org. Nat. Prod. 68, 1 (1996). Gribble G. W.: Chemosphere 52, 289 (2003). Gouda H., Matsuzaki K., Tanaka H., Hirono S.: J. Am. Chem. Soc. 118, 13087 (1996). Hernandez, M., Girardet M., Ramisse F., Vidal D., Cavallo J.-D.: J. Antimicrob. Chemother. 52, 1029 (2003). O´Connor R. P.: Tetracyclines, Chloramphenicol, Macrolides and Lincosamides (online). (cit. 12.3.2017). Cited from: http://www.federaljack.com/ebooks/My%20collection%20of%20medical%20books,%20208%20Books%2 0(part%201%20of%203)/Modern%20Pharmacology%20With%20Clinical%20Applications/Ch47.pdf Falagas M. E.; Grammatikos A. P.; Michalopoulos A.; Expert Rev. Anti Infect. Ther. 6, 593 (2008). Muller F., Caillard L.: Chlorophenols. In Ullmann’s Encyclopedia of Industrial Chemistry. John Wiley & Sons, Inc; 2011. Published online - DOI: 10.1002/14356007.a07_001.pub2. Safe S.: Crit. Rev. Toxicol. 24, 87 (1994) Baker J. I., Hites R. A.: Environ. Sci. Technol. 34, 2879 (2000). De Voogt, P. and Brinkman, U. A. T., Production, properties and usage of polychlorinated biphenyls, in Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodioxins and Related Products, Kimbrough, R. D. and Jensen, A. A., Eds., Elsevier-North Holland, Amsterdam, 1989, 3. Altarawneh M., Dlugogorski B. Z., Kennedy E. M., Mackie J. C.: Prog. Energy Combust. Sci. 35, 245 (2009). Fenton H. J. H.: J. Chem. Soc. Trans. 65, 899 (1894). Latch D. E., Packer J. L., Stender B. L., Van Overbeke J.,Arnold D. A., McNeill K.: Environ. Toxicol. Chem. 24, 517 (2005). Klamerth N., Miranda N., Malato S., Aguera A., Fernández-Alba A. L., Maldonado M. I., Coronado J. M.: Catal. Today 144, 124 (2009). Butkovskyi A., Jeremiasse A. W., Hernandez Leal L., van der Zande T., Rijnaarts H., Zeeman G.: Environ. Sci. Technol. 48, 1893 (2014). Ren Y.-Z., Franke M., Anschuetz F., Ondruschka B., Ignaszak A., Braeutigam P.: Ultrason. Sonochem. 21, 2020 (2014). de Vidales M. J. M., Sáez C., Cañizares P., Rodrigo M. A.: J. Chem. Technol. Biotechnol. 88, 823 (2013). Zhang H., Yamada H., Tsuno H.: Environ. Sci. Technol. 42, 3375 (2008). van den Heuvel M. R., Leusch F. D. L., Taylor S. , Shannon N., McKague A. B.: Environ. Sci. Technol. 40, 2594 (2006). Jiang J., Pang S.-Y., Ma J., Liu H.: Environ. Sci. Technol. 46, 1774 (2012). Munoz M., de Pedro Z. M., Casas J. A., Rodriguez J. J.: Chem. Eng. J. 198-199, 275 (2012). Yang K. S., Mui G., Moulijn J. A.: Electrochim. Acta 52, 6304 (2007). Sudoh M., Kodera T., Sakai K., Zhang J. Q., Koide K: J. Chem. Eng. Jpn. 19, 513 (1986). Vyhnánková E., Kozáková Z., Krčma F., Hrdlička A.: Open Chem. 13, 218 (2015).


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30. Sirés, I.; Garrido, J. A.; Rodríguez, R. M.; Brillas, E.; Oturan, N.; Oturan, M. A.: Appl. Catal., B 72, 382 (2007). 31. Dabo P., Cyr A., Laplante F., Jean F., Menard H., Lessard J.: Environ. Sci. Technol. 34, 1265 (2000). 32. Heinrichs B., Schoebrechts J.-P., Pirard J.-P.: J. Catal. 200, 309 (2001). 33. Roy H.M., Wai C.M., Yuan T., Kim J.-K., Marshall W.D.: Appl. Catal., A 271, 137 (2004). 34. Klausen J., Ranke J., Schwarzenbach R.P.: Chemosphere 44, 511 (2001). 35. Cantrell K.J., Kaplan D.I., Wietsma T.W.: J. Hazard. Mater. 42, 201 (1995)., 36. Weidlich T., Krejčová A., Prokeš L.: Monatsh. Chem. 141, 1015 (2010). 37. Weidlich T., Prokeš L.: Cent. Eur. J. Chem. 9, 590 (2011). 38. Weidlich T., Opršal J., Krejčová A., Jašúrek B.: Monatsh. Chem. 146, 613 (2015). 39. Weidlich T., Krejčová A., Prokeš L.: Monatsh. Chem. 144, 155 (2013). 40. Janssen D.B., Oppentocht J.E., Poelarands G.J.: Curr. Opin. Biotechnol. 12, 254 (2001).

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SAFE DISPOSAL OF METALLIC MERCURY FROM PHASED-OUT MERCURY CELLS Raschman R.1, Zápotocký L.1, Říhová M. 1

DEKONTA, a.s., Dřetovice 109, 273 42, Stehelčeves, Czech Republic [email protected]

Abstract Currently the European chlor-alkali sector is progressing towards a phase-out of mercury cell technology. Under the legally binding BAT conclusions, the mercury-based production technology must be ceased before 11 December 2017. The export of metallic mercury from a member state to a non-EU country is prohibited. According to BAT Reference Document for the Production of Chlor-alkali, stabilization (reprocessing to mercury sulphide) and subsequent permanent storage at licensed facilities is the best available technique for liquid mercury disposal. The available mercury treatment capabilities in Europe are limited and insufficient (considering the approx. 6 000 t of mercury waste still waiting for disposal in chlor-alkali plants). DEKONTA together has tested mobile mercury stabilization plant developed by econ industries GmbH. The plant is designed for safe disposal of mercury directly at the sites of phased-out mercury cells, where the waste is generated. This approach eliminates risky shipment and temporarily storage of liquid mercury. Treatment capacity of the mobile plant is 2 tones of liquid mercury per shift.

Introduction Mercury - the only metal that is liquid at ambient temperature - is an indestructible chemical element that is highly toxic to humans, animals and ecosystems. Exposure to large amounts of mercury can be fatal, but relatively low doses can also have serious health effects, affecting the nervous, cardiovascular, immune and reproductive systems in particular. In the presence of bacteria, mercury can convert to methylmercury, a more complex and harmful mercury compound, which passes both the placental barrier and the blood-brain barrier and can therefore inhibit children's mental development before and after birth. Exposure of women of childbearing age and of children is therefore of greatest concern. The main risk of exposure for human beings is through food as methylmercury accumulates in fish and seafood, in particular in large predatory fish. Other significant exposure risks result from human activities, including mercury mining, the use of mercury in products and industrial processes and artisanal and small-scale gold mining.1 To address further mercury use and pollution, more and more complex prevention strategies are being adopted at all levels - global, European and national. Currently the European chlor-alkali sector is progressing towards a phase-out of mercury cell technology. The sector has for a long time proceeded on a voluntary basis towards a phase-out deadline of 2020. Meanwhile, however, under the Industrial Emissions Directive, the BAT2 conclusions (Best Available Technology) have become legally binding. The national authorities had to reconsider permit conditions and take into account the BAT conclusions, implying that four years after publication of these BAT conclusions, this means before 11 December 2017, mercury based production technology must be ceased. The EU has traditionally been a major exporter of mercury, providing about 25% of the total global supply of around 3,000 tonnes per year. The production stopped in 2003 and mercury exports have thus been significantly reduced since then. Outside the EU, the countries that still produce mercury from cinnabar are Kyrgyzstan and China. Mercury is used in a variety of applications. In the 27 EU Member States, the demand for mercury in 2007 was estimated at more than 320 tonnes. The uses include dental amalgam, measuring and control equipment and energy-efficient lamps. Mercury is also used in the chlor-alkali sector, which produces chlorine and caustic soda. Chlorine and caustic soda are essential to economic and social welfare and are used in a wide variety of products e.g. plastics, medicine, disinfectants, clothing, building materials, etc. Nearly 20 million tonnes of chlorine, caustic soda and hydrogen are produced each year by the European chlor-alkali industry. In one of the three existing chlorine production technologies liquid mercury is a cathode (negative pole) in mercury cells. The total amount of mercury still in use in chlor-alkali manufacturing is about 5 700 tonnes (end 2016). But this use is being phased out. In 2015, the mercury technology accounted for 19.7% of EU production capacity, membrane production was 63.7%.


545

Global strategies for reduction of mercury emissions from chlor-alkali industry In 2003 the United Nations Environment Programme (UNEP) Governing Council concluded that there was sufficient evidence of global detrimental impacts related to releases of mercury. According to UNEP, the chloralkali industry is a significant user of mercury but the releases are relatively small. Nevertheless for economic and environmental reasons, mercury-cell technology is not the preferred technology and existing facilities will be phased out taking into account the economic lifetime. The World Chlorine Council (WCC) entered into a partnership with the UNEP Global Mercury Programme and developed the following strategy to promote and share best practices to reduce mercury emissions from chlor-alkali manufacturing sites3:  Promotion and implementation of best techniques and practices to facilitate reductions in mercury used by and released to the environment from mercury-cell facilities around the world.  Mercury measuring and reporting and progress. Data concerning mercury use and emissions within the chlor-alkali industry representing about 90% of the global chlor-alkali production capacity using mercury cells are collected by WCC, compiled and provided to UNEP on an annual basis.  Regional / national reduction programmes. WCC member associations continue to reduce mercury use and emissions at the national and regional levels.

Figure 1. Number of chlor-alkali plants and capacity of mercury electrolysis units in USA, Canada, Mexico, Europe, Russia, India and South America The efforts contribute to the positive global results:  Both number of mercury-based plants and the mercury-cell based production capacity show a worldwide decrease.  Global mercury emissions have been substantially reduced in the period 2002-2012. They went down from 24.6 tonnes/year to about 6.2 tonnes/year, a 75 % decrease has been reported.  Today mercury based chlor-alkali electrolysis accounts for less than one percent of the total global emissions of mercury from all natural and man-made sources.

Figure 2. Total and specific mercury emissions from chlor-alkali industry in USA, Canada, Mexico, Europe, Russia, India and South America

EU mercury strategy The Community strategy concerning mercury adopted in 2005 included a comprehensive plan aimed at reduction of mercury use and pollution:

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   

Restrictions on the inclusion of mercury or mercury compounds in a number of products including measuring devices (e.g. thermometers, sphygmomanometers, barometers), as well as batteries, electrical and electronic equipment etc. Ban on exports of mercury from the EU that came into force in 2011. Rules on the safe storage of mercury. Inclusion of provisions on mercury emissions in EU legislation, in particular in BAT Conclusions adopted under the Industrial Emissions Directive.

Table I EU mercury consumption estimates (2007) Product / Activity Chlor-alkali Batteries Dental amalgam Measuring and control equipment Switches and electrical control Lighting Chemicals Other uses Total

tonnes 160-190 7-25 90-110 7-17 0-1 11-15 28-59 15-114 320-530

EU signed the Minamata Convention on Mercury in October 2013 and thereby committed to ensure its ratification and implementation across the Union. As a first step to achieve this, the Commission proposed on February 2016 the following ratification package:  proposal for a Regulation on Mercury repealing and replacing Regulation (EC) 1102/2008;  proposal for a Council Decision concerning the conclusion on behalf of the EU of the Minamata Convention on Mercury. Between February and December 2016, the proposal was discussed in the Parliament’s ENVI Committee and in the Council and the subsequent 2 rounds of trilogue discussions led to a compromise text that was adopted by the Parliament ENVI Committee on January 12, 2017. European Parliament resolution of 14 March 2017 formally approved all the goals and measures previously formulated in the Regulation (EC) No 1102/2008 of the European Parliament and of the Council on mercury including that ones related to chlor-alkali industry:  Mercury and mercury compounds, whether in pure form or in mixtures, from any of the following large sources shall be considered to be waste within the meaning of Directive 2008/98/EC and be disposed of without endangering human health or harming the environment, in accordance with that Directive: (a) the chlor-alkali industry; (b) the cleaning of natural gas; (c) non-ferrous mining and smelting operations; (d) extraction from cinnabar ore in the Union . Such disposal shall not lead to any form of reclamation of mercury.  The export of metallic mercury, whether or not qualifying as waste, from one Member State to a nonEU country is prohibited.  Given that mercury is an extremely hazardous substance in its liquid form, the permanent storage without pre-treatment of mercury waste is prohibited owing to the risks that such disposal poses. Therefore, mercury waste shall undergo appropriate conversion, and if applicable, solidification operations prior to permanent storage.  Over 6 000 metric tonnes of liquid mercury waste will have been generated in the Union by the end of 2017, mainly as a result of the mandatory decommissioning of mercury cells in the chlor-alkali industry in accordance with Commission Implementing Decision 2013/732/EU. Given the limited available capacity for undertaking the conversion of liquid mercury waste, the temporary storage of liquid mercury waste should still be allowed under this Regulation for a period of time sufficient for ensuring the conversion and, if applicable, solidification, of all such waste produced - i.e. for a maximum period of 5 years, with the possibility to extend it with 3 additional years through a delegated act.


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

 



Temporary storage of mercury shall be carried out in accordance with the requirements set out in Council Directive 1999/31/EC. Above-ground facilities for storage of metallic mercury for more than one year have to be dedicated to and equipped for such purpose. The applicant for a permit shall make adequate provision, by way of a financial security or any other equivalent, to ensure that the obligations (including after-care provision) arising under the permit are discharged and that the closure procedures are followed. Directive 2004/35/EC on environmental liability with regard to the prevention and remedying of environmental damage applies to the facilities for temporary storage of mercury. Mercury waste that underwent conversion / solidification shall only be permanently disposed of in the following permanent storage facilities licensed for disposal of hazardous waste: (a) Salt mines that are adapted for the permanent storage of mercury waste that underwent conversion, or deep underground hard rock formations providing a level of safety and confinement equivalent to or higher than that of such salt mines; or (b) above-ground facilities dedicated to and equipped for the permanent storage of mercury waste that underwent conversion and solidification and that provide a level of safety and confinement equivalent to or higher than that of the facilities referred to in point (a). The entire process (storage, transport, conversion, and disposal of liquid mercury) must be protocoled and reported.

Available technology options for stabilisation of metallic mercury At this moment, there are three companies offering services related to the conversion of mercury to mercurysulphide3:  BATREC, Wimmis / Switzerland The installation consists of two parallel reactors and a filter-press and has a capacity of approx. 1 000 tonnes/year. The process is wet and results in mercury sulphide cake with less than 5% water. The conversion rate of 99.999% of the mercury to mercury sulphide is guaranteed. Produced mercury sulphide is packed in 200 liter drums and stored in the salt mine in Herfa Neurode, Germany. The process has been operational since 2016.  MAYASA, Spain Mayasa is considering the development of a commercial plant with an alternative mercury conversion/ stabilisation process. In this process the mercury is converted into mercury sulphide, which is then converted to a polymer cement.  REMONDIS QR, Dorsten / Germany The installation has a capacity of approx. 500 tonnes/year. Liquid mercury is converted to mercury sulphide in a dry process, packed in drums and disposed of in the salt mine in Herfa Neurode, Germany. The process has been operational since 2014.  ECON INDUSTRIES, Starnberg / Germany The company is offering a mobile unit which is able to convert 6 tonnes of mercury per day (three shifts) to mercury sulphide. It is a dry process. The unit is operated on the premises of the chlor-alkali plant. Produced mercury sulphide is shipped for final disposal to licensed underground storage facilities ( salt mines). For operation of the mobile plant, the temporary environmental permit needs to be issued.

Mobile unit for stabilisation of metallic mercury The mercury stabilization process developed by a German company econ industries and tested in DEKONTA´s technological laboratory in Slany, Czech Republic is based on the chemical reaction converting liquid mercury to mercury sulphide which is performed in a mixed batch reactor under an inert atmosphere. The process Technological scheme of the process is shown in Figure 3.

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Figure 3. Technological scheme of the mercury stabilization process developed by econ industries In the beginning of a treatment cycle, the 1 200 litre horizontal mixer is filled with approx. 320 kg of dry pulverized sulphur. After that, dosing of liquid mercury to the reactor starts. Approx. 2 tons of mercury are treated in one batch. The duration of a treatment cycle is approx. 8 hours (one shift). The guaranteed conversion rate of the mercury to mercury sulphide is 99.999%. Both sulphur and mercury are fed to the reactor directly from storage tanks by means of hermetically sealed and automatically controlled dosing systems so no emissions to the surrounding environment are generated. Continuous and intense mixing guaranties ideal homogenization for the duration of the entire batch treatment period and thus prevents uncontrolled development of the exothermic chemical reaction between sulphur and mercury as well as creation of solidified conglomerates resulting in decrease of mercury conversion rate. Temperature inside the reactor is continuously measured and controlled by a heating / cooling system. The treatment process is performed under inert atmosphere at ambient pressure. Continuous nitrogen blanketing of all sealings is ensured to eliminate entering of oxygen into the reactor. Stabilized mercury converted to mercury sulphide is discharged directly into barrels and shipped for the final disposal. The equipment is installed in a 40´´ container so transportation and installation at the client´s site is simple and fast. Treatment capacity of the containerized plant is 6 t/day in 3-shift operation. Electric power supply 150 kW 2 is requested for the plant operation. The necessary operation area is 200 m . Consumption of sulphur is 160 kg/t of stabilized mercury.

Figure 4. Mobile mercury stabilization plant installed in 40" container (econ industries)

References 1. 2. 3. 4.

http://ec.europa.eu/environment/chemicals/mercury/index_en.htm Brinkmann T., Germán G. S., Schorcht F., Roudier S., Sancho L. D.: Best Available Techniques (BAT) Reference Document for the Production of Chlor-alkali. Publications Office of the European Union, Luxembourg 2014. http://www.eurochlor.org/media/9074/wcc_mercuryleaflet_14_june_2013.pdf http://www.econindustries.com/applications/mercury-wastes


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SIMULANTS OF CHEMICAL WARFARE AGENTS FOR TESTS OF THERMAL DESORPTION TECHNOLOGY Šváb M., Zápotocký L Dekonta,a.s., Dretovice 109, 273 42 Stehelceves, Czech Republic [email protected]

Abstract Chemical warfare agents (CWA) represent specific toxic species which are still stored, possibly produced and, however, its misuse cannot be excluded, what was proven by number of incidents, recently. Although this potential danger is taken into the account seriously by authorities, usually the security measures focus only on instantaneous reaction on the unwanted situation occurred. Residual solid materials (concrete, masonry, soil, etc.) affected by low volatile CWAs, then, still represent an additional source of potential danger. Such materials should be decontaminated. Thermal desorption technology is an efficient method which can be suggested for cleanup of such materials. According to the above described assumptions, low-volatile agents should be considered as of a great importance. Mainly some nerve agents and vesicants with boiling point between 150 – 300°C fit to assumed parameters. Nevertheless, it is necessary to verify an efficiency of traditional remediation technologies in application on removal of the CWAs. However, from many reasons it is highly required to exclude an experimental work with the real CWAs. Thus, the CWAs must be replaced by harmless chemicals, which, on the other hand, have similar key physical-chemical properties for the purpose of desired experiments. In our contribution, we would like to provide a brief introduction to the topic of CWAs as potential residual contaminants of solid matrixes, and discuss selection of possible simulants for the experimental work. The interaction of CWA simulants with various solid matrixes and problems caused by its hydrolysis will be discussed in experimental part.

Introduction Chemical warfare agents (CWA) are solids, liquids, or gases, which, through their chemical properties, produce lethal or damaging effects in man, animals, plants, or materials. Historically, chemical agents have been divided into categories based on the major physiological impact caused by the agent or the target organ they attack. Nerve agents disrupt the function of the nervous system and produce effects on skeletal muscles, various sensitive organs, and the nervous system. Blister agents, also known as vesicants, affect the eyes, lungs and skin by destroying cell tissue. Blood agents are compounds which affect the ability of the blood system to either carry oxygen or transfer oxygen from the blood to cells. Choking agents are compounds that can cause the lungs to become filled with fluid. Incapacitating agents produce physiological effects that inhibit concerted effort. Tear agents cause intense eye pain and tears. Vomiting agents cause regurgitation1. Thousands of chemicals widely used in industry have been evaluated by various militaries for their possible use as chemical warfare agents and many have even been employed in combat. However, with the discovery of the more toxic nerve and blister agents, the use of most of these materials has been abandoned 1. Although the CWAs represents an instant danger for human health and are usually taken into account in this way, it represent subsequent source of risk also later its use (dispersion). Thus, effective and safe remediation technology must be applied for its removal from polluted matrixes (later on the first aid for affected people and similar actions etc.). From the point of view of residual contamination, the CWAs which are liquid/solid and low-volatile are important since they are able to remain in exposed matrixes (masonry, soil etc.). The technology which can be effectively employed for the CWAs-polluted matrixes clean-up can be based on the thermal desorption under lower pressure what prevent leak of the CWAs out from the system. However, although the thermal desorption is already used for the removal of environmental pollutants from solids, it is necessary to verify and optimize the process parameters in case of CWAs removal through experiments. However, exclusion of the real CWAs from the experimental work is needed; thus, suitable non-toxic chemical surrogates must be found.

CWAs of interest “CWAs of interest” means the CWA which are low-volatile (potentially remaining in exposed solids) and can also be expected considering its misuse. In first, gaseous or high-volatile species are excluded. In Table I, typical representatives of the low-volatile CWAs are summarized.

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Table I Typical representatives of the low-volatile CWAs2 (selection): Nerve agents other name chemical name names GB. Isopropyl T-144. Sarin methylphosphonoTrilon 46. fluoridate EA 1208 GD. Soman VR-55. EA Pinacolyl methyl 1210 phosphonofluoridate

VX

Vx

Tabun

melting point (°C)

boiling point, (oC)

-56

147

-42

198

O-Ethyl-S-(2diisopropylaminoethyl) methyl phosphonothiolate

-51

298 (dec.)

Vx. R 33 (Rus.). Medemo. EDMM. EA1699

O-ethyl S-(2dimethylaminoethyl) methylphosphonothiolate

Ned.

256

GA. EA 1205. Trilon 83

Ethyl N. Ndimethylphosphoroamido cyanidate

-49

246

-30

239

formula

melting point (°C)

Cl-CH2-CH2-S-CH2-CH2-Cl

14

boiling point. (oC) 216 - 224 (dec.)

VX (USA). EA 1701

GF. Cyclohexyl Cyclosarin methylphosphono EA 1212. fluoridate T-2139. Blister agents (vesicants) other chemical name name names H. mustard Yperite Bis (2-chloroethyl) sulfide gas. HD. HS. EA 133 HN-1. Ethyl Nitrogen S. NH-Lost. mustard NSC 10873. 2.2’-Dichlorotriethylamine gas –1 TL 329. TL 1149 HN-2. dichloren. NSC 762. Nitrogen Bis-(2TL 146. mustard chloroethyl)methylamine T-1024. ENT-25294. MBA HN-3. Nitrogen TS 160. 2. 2’. 2”mustard EA 1053. Trichlorotriethylamine –3 TO. TL 145. TS 160 L. L-1. Lewisite M-1. Dichloro (2-chlorovinyl)arsine EA 1034

formula

CMPF


-34

192

-70

177

-4

257 (dec.)

-45 - -2

196 (dec)

551

PD. Pfiffikus. DJ. TL 69. FDA ED. Dick. Green TL 214. Cross 3 Yellow Cross 1 MD. TL 294. Medikus Methyl-dick Sternite

Phosgenoxime

CX

Choking agents other name names Difosgene

Chlorpicrine

DP. Perstoff. Superpalite. Surpalite

Phenyldichloroarsine

-23

>255

Ethyldichloroarsine