2006, “Assay of poly3-Hydroxybutyrate depolymerase activity and product determination”, Applied and Environmental Microbiology, 72 9, pp.. 1999, “Isolation of a microorganism capable of
Trang 1Screening and study on microorganisms degrading biopolymers in Vietnam
Trịnh Thị Vân Anh
Viện Vi sinh vật và Công nghệ Sinh học Luận văn Thạc sĩ ngành: Công nghệ sinh học; Mã số: 60 42 80
Người hướng dẫn: TS Nguyễn Quang Huy
Năm bảo vệ: 2010
Keywords Vi sinh vật; Tạo vòng phân huỷ; Biopolymers; Công nghệ sinh học
Content
Table of contents
INTRODUCTION 1
CHAPTER 1: LITERATURE REVIEW 4
1.1 PLASTIC WASTE POLLUTION 4
1.1.1 Plastic waste pollution in the world 4
1.1.2 Plastic waste pollution in Vietnam 5
1.1.3 Treatment of plastic waste 6
1.1.3.1 Landfill 6
1.1.3.2 Recycling 6
1.1.3.3 Incineration 7
1.2 BIODEGRADABLE PLASTICS 8
1.2.1 Biodegradable plastics 8
1.2.1.1 Poly(Lactic Acid) (PLA) 11
1.2.1.2 Poly(3-Hydroxybutyrate) (PHB) 12
1.2.1.3 Poly(ε-Caprolactone) (PCL) 13
1.2.2 Applications 13
1.2.2.1 Medicine and pharmacy 14
1.2.2.2 Packaging 15
Trang 21.2.2.3 Agriculture 15
1.2.2.4 Others fields 16
1.3 THE DEGRADATION OF BIOPOLYMERS 17
1.4 MICROORGANISMS DEGRADING BIODEGRADABLE POLYMERS 19
1.4.1 Microorganisms degrading PLA 19
1.4.2 Microorganisms degrading PHB 22
1.4.3 Microorganisms degrading PCL 23
CHAPTER 2: MATERIALS AND METHODS 25
2.1 MATERIALS 25
2.2 CHEMICALS 25
2.3 EQUIPMENTS 26
2.4 METHODS 26
2.4.1 Isolation of biopolymer-degrading microorganisms 26
2.4.2 Screening biopolymer-degrading microorganisms 27
2.4.3 Identification of biopolymers-degrading strains 27
2.4.3.1 Gram staining method 27
2.4.3.2 Observation under scanning electron microscopy (SEM) 28
2.4.3.3 Extraction of genomic DNA from bacteria 29
2.4.3.4 Amplification of the 16S rDNA PCR reaction 29
2.4.3.5 Agarose gel electrophoresis 30
2.4.3.6 Sequencing 30
2.4.3.7 Effect of culture conditions 31
2.4.3.8 Utilization of of sugars 31
2.4.3.9 Activity of some extracellular enzymes 31
2.4.4 Study degradation of biodegradable polymers by isolated strains 32
2.4.4.1 Growth experiment in the PLA, PHB or PCL containing media 32
2.4.4.2 Measurement of the PLA, PHB or PCL residual weight 32
2.4.4.3 Determination of TOC in culture broth 32
2.4.4.4 Degradation experiment with biopolymer film 34
2.4.4.5 Statistical analysis 34
CHAPTER 3: RESULTS AND DISCUSSION 35
3.1 ISOLATION AND SCREENING PLA, PHB, PCL-DEGRADING ORGANISMS 35
Trang 33.2 PLA-DEGRADING MICROORGANISMS 37
3.2.1 Identification of strains G5 and Cz1 37
3.2.1.1 Morphology of strain G5 and Cz1 38
3.2.1.2 16S rDNA sequencing of strain G5 39
3.2.1.3 Biochemical and physiological characteristics of strains G5 and Cz1 41
3.2.2 PLA degradation by S thermoflavus G5 and P citrinium Cz1 44
3.3 PHB-DEGRADING MICROORGANISM 47
3.3.1 Identification of strain B2 47
3.3.1.1 Morphology of strain B2 47
3.3.1.2 Sequencing 16S rDNA gene of strain B2 48
3.3.1.3 Biochemical and physiological characteristics of strain B2 49
3.3.2 PHB degradation by B gelatini B2 52
3.4 PCL-DEGRADING MICROORGANISM 54
3.4.1 Identification of strain B1 55
3.4.1.1 Morphology of strain B1 55
3.4.1.2 Sequencing 16S rDNA gene of strain B1 56
3.4.1.3 Biochemical and physiological characteristics of strain B1 57
3.4.2 PCL degradation by Br agri B1 60
3.5 DEGRADATION OF POLYMERS BY ISOLATED STRAINS 61
CONCLUSIONS 64
FURTHER STUDY 65
REFERENCES 66
WEB REFERENCES 73
References
1 Abou-Zeid D.-M., Müller R.-J., and Deckwer W.-D (2001), “Degradation of natural
and synthetic polyesters under anaerobic conditions”, Journal of Biotechnology, 86
(2), pp 113-126
2 Akutsu-Shigeno Y., Teeraphatpornchai T., Teamtisong K., Nomura N., Uchiyama H.,
Nakahara T., and Nakajima-Kambe T (2003), “Cloning and sequencing of a
poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain
Trang 4TB-13 and its functional expression in Escherichia coli”, Applied and Environmental
Microbiology, 69 (5), pp 2498-2504
3 Anson M.L (1939), “The estimation of pepsin, trypsin, papain, and cathepsin with
hemoglobin”, The Journal of General Physiology, 22, pp 79-89
4 Asano Y and Watanabe S (2001), “Isolation of poly(3-Hydroxybutyrate)
(PHB)-degrading microorganisms and characterization of PHB-depolymerase from
Arthrobacter sp strain W6”, Bioscience, Biotechnology, and Biochemistry, 65 (5), pp
1191-1194
5 Asrar J and Gruys K., Biopolymers, Wiley pubisher, 4, pp 53-68
6 Calabia B and Tokiwa Y (2004), Microbial degradation of
poly(D-3-hydroxybutyrate) by a new thermophilic Streptomyces isolate, Vol 26, Biotechnology
Letters
7 Calabia B and Tokiwa Y (2006), “A novel PHB depolymerase from a thermophilic
Streptomyces Sp.”, Biotechnology Letters, 28 (6), pp 383-388
8 Chowdhury A.A (1963), “Poly-β-hydroxybuttersaure abbauende Bakterien und
exo-enzyme”, Archives of Microbiology, 47, pp 167-200
9 Cook W.J., Cameron J.A., Bell J.P., and Huang S.J (1981), “Scanning electron
microscopic visualization of biodegradation of polycaprolactones by fungi”, Journal
of Polymer Science, Polymer Letters Edition, 19 (4), pp 159-165
10 Fan L., Sha W., Weifeng L., and Guanjun C (2008), “Purification and
characterization of poly(L-lactic acid)-degrading enzymes from Amycolatopsis
orientalis ssp orientalis”, FEMS Microbiology Letters, 282 (1), pp 52-58
11 Gebauer B and Jendrossek D (2006), “Assay of poly(3-Hydroxybutyrate)
depolymerase activity and product determination”, Applied and Environmental
Microbiology, 72 (9), pp 6094-5900
12 Gerhardt P., Murray R.G.E., Wood W.A., and Krieg N.R (1994), Methods for
general and molecular bacteriology, American Society for Microbiology, pp.791-798
13 Goodfellow M., Lacey J., and Todd C (1987), “Numerical classification of
thermophilic Streptomycetes ”, Journal of General Microbiology, 133, pp 3135-3149
Trang 514 Gross R.A and Kalra B (2002), “Biodegradable polymers for the environment”,
Science, 297 (5582), pp 803-807
15 Ikura Y and Kudo T (1999), “Isolation of a microorganism capable of degrading
poly(L-lactide)”, The Journal of General and Applied Microbiology, 45 (5), pp
247-251
16 Iwata T and Doi Y (1998), “Morphology and enzymatic degradation of poly(L-lactic
acid) single crystals”, Macromolecules, 31 (8), pp 2459-2467
17 Jarerat A and Tokiwa Y (2001), “Degradation of poly (L-lactide) by a Fungus”,
Macromolecular Bioscience, 1 (4), pp 136-140
18 Jarerat A and Tokiwa Y (2003), “Poly(L-lactide) degradation by Saccharothrix
waywayandensis”, Biotechnology Letters, 25 (5), pp 401-404
19 Jarerat A., Tokiwa Y., and Tanaka H (2003), “Poly(L-lactide) degradation by
Kibdelosporangium aridum”, Biotechnology Letters, 25 (23), pp 2035-2038
20 Jendrossek D (2001), “Microbial degradation of polyesters”, Biopolyesters, pp
293-325
21 Jendrossek D and Handrick R (2002), “Microbial degradation of
polyhydroxyalkanoates”, Annual Review of Microbiology, 56 (1), pp 403-432
22 Jendrossek D., Knoke I., Habibian R.B., Steinbüchel A., and Schlegel H.G (1993),
“Degradation of poly(3-hydroxybutyrate), PHB, by bacteria and purification of a
novel PHB depolymerase from Comamonas sp”, Journal of Polymers and the
Environment, 1 (1), pp 53-63
23 Kawata Y and Aiba S.-I (2010), “Poly(3-hydroxybutyrate) production by isolated
Halomonas sp KM-1 using waste glycerol”, Bioscience, Biotechnology, and Biochemistry, 74 (1), pp 175-177
24 Kolybaba M., Tabil L.G., Panigrahi S., Crerar W.J., Powell T., and Wang B (2003),
“Biodegradable polymers: past, present, and future”, The Society for engineering in
agricultural, food, and biological systems
25 Kongpol A., Pongtharangkul T., Kato J., Honda K., Ohtake H., and Vangnai A
(2009), “Characterization of an organic-solvent-tolerant Brevibacillus agri strain 13
Trang 6able to stabilize solvent/water emulsion”, FEMS Microbiology Letters, 297, pp
225-233
26 Lee S.Y (1996), “Bacterial polyhydroxyalkanoates”, Biotechnology and
Bioengineering, 49 (1), pp 1-14
27 Macdonald R.T., Mccarthy S.P., and Gross R.A (1996), “Enzymatic degradability of
poly(lactide): Effects of chain stereochemistry and material crystallinity”,
Macromolecules, 29 (23), pp 7356-7359
28 Malveda M., Löchner U., and Yokose K (2010), “Biodegradable Polymers”,
Chemical Economics Handbook Report
29 Matavulj M.N and Molitoris H.P (2009), “Marine fungi - degraders of
poly-3-hydroxyalkanoate based plastic materials”, Matica Srpska Proceedings for Natural
Sciences, 116, pp 253-265
30 Mayumi D., Akutsu-Shigeno Y., Uchiyama H., Nomura N., and Nakajima-Kambe T
(2008), “Identification and characterization of novel poly(DL -lactic acid)
depolymerases from metagenome”, Applied Microbiology and Biotechnology, 79 (5),
pp 743-750
31 Nakamura K., Tomita T., Abe N., and Kamio Y (2001), “Purification and
characterization of an extracellular poly(L-lactic acid) depolymerase from a soil
isolate, Amycolatopsis sp strain K104-1”, Applied and Environmental Microbiology,
67 (1), pp 345-353
32 Nguyen Q.H., Do H.D., and Le V.C (2010), “Poly(L-lactide) degradation by some
actinomyes strains isolated in Vietnam”, Journal of Sciences and Technology, 48, pp
57-65
33 Nguyen Q.H., Trinh T.V.A., and Ngo K.T (2010), "Degradation of
poly(3-hydroxybutyrate) (PHB) by Bacillus gelatini isolated from Vietnam", Journal of
Biology, 32 (3), pp 72-77
34 Nguyen Q.H and Trinh T.V.A (2010), “Degradation of polycaprolactone (PCL) at
50oC by a thermotolerant bacteria which isolated from soil”, Journal of Sciences and
Technology, Vietnam National University, 26, pp 568-574
Trang 735 Nishida H and Tokiwa Y (1993), “Distribution of poly(β-hydroxybutyrate) and
poly(ε-caprolactone) aerobic degrading microorganisms in different environments”,
Journal of Polymers and the Environment, 1 (3), pp 227-233
36 Ohkita T and Lee S.H (2006), “Thermal degradation and biodegradability of
poly(lactic acid)/corn starch biocomposites”, Journal of Applied Polymer Science,
100 (4), pp 3009-3017
37 Papaneophytou C., Pantazaki A., and Kyriakidis D (2009), “An extracellular
polyhydroxybutyrate depolymerase in Thermus thermophilus HB8”, Applied
Microbiology and Biotechnology, 83 (4), pp 659-668
38 Plastic waste management, Central Pollution Control Board pp 139-144
39 Pranamuda H., Chollakup R., and Tokiwa Y (1999), “Degradation of polycarbonate
by a polyester-degrading strain, Amycolatopsis sp strain HT-6”, Applied and
Environmental Microbiology, 65 (9), pp 4220-4222
40 Pranamuda H., Tokiwa Y., and Tanaka H (1997), “Polylactide degradation by an
Amycolatopsis sp”, Applied and Environmental Microbiology, 63 (4), pp 1637-1640
41 Reeve M.S., Mccarthy S.P., Downey M.J., and Gross R.A (1994), “Polylactide
stereochemistry: effect on enzymic degradability”, Macromolecules, 27 (3), pp
825-831
42 Romen F., Reinhardt S., and Jendrossek D (2004), “Thermotolerant
poly(3-hydroxybutyrate)-degrading bacteria from hot compost and characterization of the
PHB depolymerase of Schlegelella sp KB1a”, Archives of Microbiology, 182 (2), pp
157-164
43 Sakai K., Kawano H., Iwami A., Nakamura M., and Moriguchi M (2001), “Isolation
of a thermophilic polylactide degrading bacterium from compost and its enzymatic
characterization”, Journal of Bioscience and Bioengineering, 92 (3), pp 298-300
44 Sanchez J.G., Tsuchii A., and Tokiwa Y (2000), “Degradation of polycaprolactone at
50oC by a thermotolerant Aspergillus sp”, Biotechnology Letters, 22, pp 849-853
45 Shah A.A., Hasan F., Hameed A., and Ahmed S (2008), “Biological degradation of
plastics: A comprehensive review”, Biotechnology Advances, 26 (3), pp 246-265
Trang 846 Su-Qin C., Shan C., Liu D.-B., and Xia H.-M (2006), “An extracellular poly
(3-hydroxybutyrate) depolymerase from Penicillium sp.DS9713a-01”, World Journal of
Microbiology and Biotechnology, 22 (7), pp 729-735
47 Suyama T., Tokiwa Y., Ouichanpagdee P., Kanagawa T., and Kamagata Y (1998),
“Phylogenetic affiliation of soil bacteria that degrade aliphatic polyesters available
commercially as biodegradable plastics”, Applied and Environmental Microbiology,
64 (12), pp 5008-5011
48 Tabrez Khan S and Hiraishi A (2001), “Isolation and characterization of a new
poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge”,
FEMS Microbiology Letters, 205 (2), pp 253-257
49 Tansengco M.L and Tokiwa Y (1998), “Thermophilic microbial degradation of
polyethylene succinate”, World Journal of Microbiology and Biotechnology, 14 (1),
pp 133-138
50 Teeraphatpornchai T., Nakajima-Kambe T., Shigeno-Akutsu Y., Nakayama M.,
Nomura N., Nakahara T & Uchiyama H., (2003), “Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics”,
Biotechnology Letters, 25 (1), pp 23-28
51 Tiago I., Teixeira I., Si1va S., Chung P., Veríssimo A., and Manaia C.M (2004),
“Metabolic and genetic diversity of mesophilic and thermophilic bacteria isolated
from composted municipal sludge on poly-ε-caprolactones”, Current Microbiology,
49 (6), pp 407-414
52 Tokiwa Y., Ando T., and T S (1976), “Degradation of polycaprolactone by a
fungus”, Journal of Fermentation Technology, 54, pp 603-608
53 Tokiwa Y and Calabia B (2006), “Biodegradability and biodegradation of
poly(L-lactide)”, Applied Microbiology and Biotechnology, 72 (2), pp 244-251
54 Tokiwa Y and Calabia B (2007), “Biodegradability and biodegradation of
polyesters”, Journal of Polymers and the Environment, 15 (4), pp 259-267
55 Tokiwa Y., Calabia B., Ugwu C., and Aiba S (2009), “Biodegradability of plastics”,
International Journal of Molecular Sciences, 10 (9), pp 3722-3742
56 Tokiwa Y and Jarerat A (2004), “Biodegradation of poly(L-lactide)”, Biotechnology
Letters, 26 (10), pp 771-777
Trang 957 Tokiwa Y and Suzuki T (1977), “Hydrolysis of polyesters by lipases”, Nature, 270
(5632), pp 76-78
58 Tokiwa Y and Suzuki T (1978), “Hydrolysis of polyesters by Rhizopus delemar
lipase”, Agricultural and Biological Chemistry, 42 (5), pp 1071-1072
59 Tomita, K, Kuroki, Y, Nagai, and K (1999), "Isolation of thermophiles degrading
poly(L-lactic acid)", Journal of Bioscience and Bioengineering, 87 (6), pp 752-755
60 Tomita K., Kuroki Y., Hayashi N., and Komukai Y (2000), “Isolation of a
thermophile degrading poly (butylene succinate-co-butylene adipate)”, Journal of
Bioscience and Bioengineering, 90 (3), pp 350-352
61 Tomita K., Nakajima T., Kikuchi Y., and Miwa N (2004), “Degradation of
poly(L-lactic acid) by a newly isolated thermophile”, Polymer Degradation and Stability, 84
(3), pp 433-438
62 Torres A., Li S.M., Roussos S., and Vert M (1996), “Screening of microorganisms
for biodegradation of poly(lactic-acid) and lactic acid-containing polymers”, Applied
and Environmental Microbiology, 62 (7), pp 2393-2397
63 Tseng M., Hoang K.-C., Yang M.-K., Yang S.-F., and Chu W (2007),
“Polyester-degrading thermophilic actinomycetes isolated from different environment in
Taiwan”, Biodegradation, 18 (5), pp 579-583
64 Urayama H., Kanamori T., and Kimura Y (2002), “Properties and biodegradability of
polymer blends of poly(L-lactide) with different optical purity of the lactate units”,
Macromolecular Materials and Engineering, 287 (2), pp 116-121
65 Vink E.T.H., Rábago K.R., Glassner D.A., Springs B., O'Connor R.P., Kolstad J.,
Gruber P.R (2004), “The Sustainability of NatureWorks™ Polylactide Polymers and
Ingeo™ Polylactide Fibers: an Update of the Future”, Macromolecular Bioscience, 4
(6), pp 551-564
66 Vroman I and Tighzert L (2009), “Biodegradable Polymers”, Materials, 2, pp
307-344
Trang 10WEB REFERENCES
67 http://www.wasteonline.org.uk/resources/InformationSheets/Plastics.htm
68 http://www.eia.doe.gov/oiaf/aeo/
69 http://www.copperwiki.org/index.php/Plastic_bags
70 http://www.monre.gov.vn/
71 http://www.cpcb.nic.in/139-144.pdf
72 http:// www.epa.gov/
73 http://en.wikipedia.org/wiki/List_of_solid_waste_treatment_technologies
74 http://khoahoc.com.vn