Ultra-high molecular weight polyethylene. 2. 1.4.2. High density polyethylene. 3. 1.4.3. Cross linked polyethylene. 3. 1.4.4. Medium density polyethylene. 4. 1.4.5.
SCREENING OF LOW DENSITY POLYETHYLENE DEGRADINGBACTERIAL ISOLATES FROM PLASTIC RECYCLING PLANT: A STEP TOWARDS PLASTIC FREE ENVIRONMENT
Muhammad Ovais Shagufta Naz July, 2015
A thesis submitted to the University of Peshawar in partial fulfillment of the degree of bachelors in Biotechnology CENTRE OF BIOTECHNOLOGY AND MICROBIOLOGY UNIVERSITY OF PESHAWAR
IN THE NAME OF ALLAH, THE BENEFICENT THE MERCIFUL
Are those who know and those who do not know alike? Only the men of understanding are mindful. (Surah Al Zumar 39:9) (Al-Quran)
CERTIFICATE OF APPROVAL This thesis titled “Screening of low density polyethylene degrading bacterial isolates from plastic recycling plant: A step towards plastic free environment” submitted by Muhammad Ovais and Shagufta Naz is hereby approved and recommended as partial fulfillment for the award of Degree of bachelors in Biotechnology
1. Supervisor
___________________________
2. External Examiner
___________________________
3. Director
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Center of Biotechnology and Microbiology University of Peshawar 2015
Dedication We dedicate all our efforts for this scientific work in the name of Holy Prophet Muhammad (P.B.U.H) and his pious companions, to our parents, teachers and to all those who taught us even a single word, we will also love to dedicate this effort to all thoseinnocent Muslims who were killed in the name of “war against terror” plus all those Palestinian childrenwhowere brutally killed byIsraelianArmy. Muslim scientists who are striving for the revival of Muslims dignity, and the era of glory also deserves all of our dedication.
M.Ovais.Q.Khan
CONTENTS
Page Number
List of Tables
I
List of Figures
II
List of Schemes
III
Acknowledgments
IV
Abstract
V
Pictures
VI
CHAPTER 1: INTRODUCTION & LITERATURE REVIEW 1.1
History of polyethylene
1
1.2
What is polyethylene?
1
1.3
Structure of polyethylene
1
1.4
Classification of polyethylene
2
1.4.1
Ultra-high molecular weight polyethylene
2
1.4.2
High density polyethylene
3
1.4.3
Cross linked polyethylene
3
1.4.4
Medium density polyethylene
4
1.4.5
Linear low density polyethylene
4
1.4.6
Low density polyethylene
4
1.4.7
Very low density polyethylene
5
1.5
Applications
5
1.6
Problems associated with polyethylene
6
1.7
Degradation of polyethylene
8
1.7.1
Photodegradation
9
1.7.2
Thermal degradation
10
1.7.3
Oxo-biodegradation
10
1.7.4
Biodegradation
10
Factors affecting biodegradation
11
1.7.4.1
1.7.4.2
End products of biodegradation
11
1.7.4.3
Mechanism of biodegradation
13
Methods used to determine biodegradation
15
1.8.1
Scanning electron microscopy
15
1.8.2
Fourier transformed infrared spectroscopy (FTIR)
15
1.8.3
Chromatography
15
1.8.4
Percent weight loss
16
1.8
1.9
AIMS & OBJECTIVES
17
CHAPTER 2: MATERIAL& METHODS General experimental information
18
2.1.1
Sample location
18
2.1.2
Sample collection
18
2.1.3
Study design
18
2.2
Serial dilution
20
2.3
Media preparation
21
2.4
Inoculation
21
2.5
Incubation
22
2.6
Total heterotrophic Count
22
2.7
Sub-Culturing
22
2.8
Identification
23
Morphological Identification
23
2.8.1.1
Gram staining
23
2.8.1.2
Colony morphology
24
Biochemical tests
24
2.8.2.1
Catalase test
24
2.8.2.2
Motility test
25
2.8.2.3
Endospore test
25
2.1
2.8.1
2.8.2
2.8.2.4
Methyl red test
26
2.8.2.5
Carbohydrate fermentation test
27
2.8.2.6
Triple sugar iron test (TSI)
28
2.8.2.7
Urease test
28
2.8.2.8
Citrate utilization test
29
2.8.2.9
Tube coagulase test
29
2.8.2.10
Oxidase test
30
2.8.3
Bacterial Identification
31
2.8.4
Biodegradation testing
32
2.8.4.1
Pretreatment of the sample
32
2.8.4.2
Film culturing and harvesting
32
2.8.4.3
Method for monitoring biodegradation
32
Percent weight lossdetermination
33
2.8.4.3.1
CHAPTER 3: RESULTS & DISCUSSION 3.1
Heterotrophic bacterial count
34
3.2
Morphological tests
35
3.3
Biochemical tests
39
3.4
Bacterial identification via PIBWin bacterial identification software
55
3.5
Biodegradation estimation via % weight loss
57
3.6
Conclusion and Recommendations
59
REFERENCES
60-64
LIST OF TABLES Number
Tables
Page number
Table 1
Composition of Mineral salt media used in screening of polyethylene
33
degrading microbes. Table 2
Total Heterotrophic bacterial count in Sample A, B, & C.
34
Table 3
Colony Morphology of Bacterial strains showing various variables.
35
Table 4
Gram staining results showing highly diverse Morphologies.
38
Table 5
Results of catalase tests are indicated, diverse results have been shown
40
by the bacterial isolate. Table 6
Bacterial isolates are separated on the basis of their Motile and Non-
41
Motile Nature. Table 7
Endospore positive and negative cases for the bacterial isolates.
42
Table 8
Methyl red test result of Bacterial species.
43
Table 9
Carbohydrate fermentation test result of bacterial species.
45
Table 10
TSI results of Bacterial species.
47
Table 11
Urease test results of bacterial species.
49
Table 12
Citrate utilization test results of bacterial isolates.
50
Table 13
Oxidase test results of bacterial isolates.
52
Table 14
Coagulase test results of bacterial isolates.
54
Table 15
Result of bacterial identification via PIBWin bacterial identification
56
software. Table 16
Result of degradation of plastic sample by bacterial isolates after 1
58
month.
i
LIST OF FIGURES Number
Figures
Page number
Figure 1
Low density polyethylene beads.
VIII
Figure 2
Low density polyethylene plastic.
VIII
Figure 3
Chemical structure of pure polyethylene.
2
Figure 4
General mechanism and end products of plastic biodegradation under
12
aerobic conditions. Figure 5
Samples collected from recycling plants and their respective solutions.
20
Figure 6
Serially diluted test tubes for Sample A, B, C respectively (from left to
21
right) Figure 7
Properly packed and inoculated nutrient media plates.
22
Figure 8
Slides of gram staining at the end of experiment.
22
Figure 9
Conversion of Hydrogen per oxide from 37% to 3 % working solution.
25
Figure 10
Malachite green stain solution.
26
Figure 11
Methyl red indicator.
27
Figure 12
PIBWin programme showing result of A1 sample.
31
Figure 13
Pure cultures of bacterial isolates on nutrient agar media.
36
Figure 14
100x image of Gram –ve Cocci.
37
Figure 15
Catalase +ve and catalase –ve results of bacterial isolates.
39
Figure 16
Methyl red -ve result.
44
Figure 17
Methyl red +ve result.
44
Figure 18
-ve result of carbohydrate fermentation (upper) +ve result of
46
carbohydrate fermentation (lower) Figure 19
Multiple results of TSI Test.
48
Figure 20
Positive result of urease test.
50
Figure 21
Positive result of citrate test.
51
Figure 22
Various results of oxidase test.
53
ii
LIST OF SCHEME Number
Scheme
Page number
Scheme 1
Various mechanisms of plastic degradation.
9
Scheme 2
Study design of the project.
19
iii
ACKNOWLEDGMENTS All praises are for Allah Almighty, the most merciful and beneficent, the one and only who bestowed us with the creative mind to think, sight to see and endowed us with the courage to work more and more with creative moves. Blessings and salutations are upon Prophet Muhammad ﷺwhoisa role model for each and every human.“There has certainly been for you in the Messenger of Allah an excellent pattern for anyone whose hope is in Allah and the Last Day and [who] remembers Allah often” (Surat Al-Ahzab 33:21). It’s our honor to work under the supervision of Dr.Ibrar Khan (Assistant Professor) Center of Biotechnology and Microbiology University of Peshawar,weexpress our deepest gratitude for his genuine cooperation, dedicated efforts and continuous support throughout our research tenure due to his dedicated character we consider him among the top mentors of our department. We will also love to acknowledge Prof.Dr.Ghousia Lautfullah (Director) Center of Biotechnology and Microbiology University of for her strong support interms of practical advices and parental character, her humble personality and dedicated work for her department had nurtured many seedlings into potent scientists. We will never forget the support of Lalina Maroof (Ph.D. Scholar) for her guidance and support at each and every step of our project. How we can forget our genuine friends who supported us at every moment of our social life and research carrier, and whom support is one of the main reasons we glitter today, Muhammad Khalid Afghan, Usman Ghani, Abdu’s Samad, Naila Aziz, Noreen Malik, Rabia Tareen, Asad Ali and Rafiq Malik. We will also like to thank Dr.Kafeel Ahmed Assistant Professor at COBAM, Dr.Zakiullah, Dr.Fazli Khuda and Dr.Gowhar Ali Lecturers at Department of Pharmacy, Dr.Khishroon (Post.doc USA) Assistant Professor, Department of Zoology University of Peshawar and Dr.Sajid Ali, Assistant Professor Biotechnology Department, Abdul Wali Khan University Mardan for their love and guidance. Thanks to all those who bestowed upon us the light of education. Shagufta Naz
Muhammad Ovais
iv
