Screening 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 1.2.2.3. 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 3.2. 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 TB- 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. 14. 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 able 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 50 o C by a thermotolerant bacteria which isolated from soil”, Journal of Sciences and Technology, Vietnam National University, 26, pp. 568-574. 35. 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 50 o C 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. 46. 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. 57. 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. WEB 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. . Screening 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;. 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. PCL -DEGRADING ORGANISMS 35 3.2. 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