Screening and study on microorganisms degrading biopolymers in vietnam sinh học 60 42 80

UNIVERSITY OF LIEGE *** VIETNAM NATIONAL UNIVERSITY, HANOI INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY *** Trinh Thi Van Anh SCREENING AND STUDY ON MICROORGANISMS DEGRADING BIOPOLYMERS IN VIETNAM MASTER THESIS MAJOR BIOTECHNOLOGY HANOI – 2010 LIEGE UNIVERSITY *** VIETNAM NATIONAL UNIVERSITY, HANOI INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY *** Trinh Thi Van Anh SCREENING AND STUDY ON MICROORGANISMS DEGRADING BIOPOLYMERS IN VIETNAM Speciality: Biotechnology Code: 60 42 80 MASTER THESIS MAJOR BIOTECHNOLOGY SUPERVISOR: Dr NGUYEN QUANG HUY HANOI – 2010 Table of contents INTRODUCTION CHAPTER 1: LITERATURE REVIEW 1.1 PLASTIC WASTE POLLUTION 1.1.1 Plastic waste pollution in the world 1.1.2 Plastic waste pollution in Vietnam 1.1.3 Treatment of plastic waste 1.1.3.1 Landfill 1.1.3.2 Recycling 1.1.3.3 Incineration 1.2 BIODEGRADABLE PLASTICS 1.2.1 Biodegradable plastics 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 Master thesis 2010 Abstract Nowaday, plastic waste is the most attrative issue in the world Biodegradable polymers are seen as a promising solution to this problem because they are environmentally-friendly Because of chemical and physical feature that are suitable for application in many fields and their degradable ability, they were used more and more for reducing the plastic waste pollution Among many biodegradable polymers, poly(L-lactic acid) (PLA), poly(3-hydroxybutyrate) PHB, and poly(ε-caprolactone) (PCL) seem to be of the most attention PHB and lactic acid (raw materials for PLA) can be produced in fermentative biotechnological processes using agricultural products and microorganisms, while PCL was a kind of polymer derived from petroleum, and all of them can be degraded by microorganisms Several aerobic and anaerobic polymer-degrading microorganisms have been isolated from soil, activated and aerobic sludge, seawater and lake water From 24 samples collected from different locations in Hanoi, Vietnam strains degrading PLA, strains degrading PHB and strains degrading PCL were selected by clear zone formed method Among them, strain G5 and strain Cz1 had the highest PLA-degrading activity, strain B2 and strain B1 presented the highest degrading activity on PHB and PCL, respectively Based on morphological, 16S rDNA sequencing, biochemical and physiological characteristics, strain G5 was identified as Streptomyces thermoflavus, strain Cz1 was proposed as Penicillium citrinium, strain B2 was named as Bacillus gelatini, and strain B1 was Brevibacillus agri All strains grew well at pH 6-7, and NaCl concentration from 1% to 3% Among these strains, the optimum temperature of B gelatini B2 and Br agri B1 were around 50oC, S thermoflavus G5 was 37oC, and P citrinium was 30oC To study the polymer degradation of these strains, total organic carbon concentration (TOC) and polymer weight in the medium after culture were determined After 30 days of cultivation in the optimum conditions, strain S thermoflavus G5 degraded 46.8% and strain P citrinum Cz1 degraded 55.5% of initial PLA residual in the medium, while strain B gelatini B2 and Br agri B1 degraded 58.5% and 84% of PHB and PCL initially added into the medium, respectively Study on the degradation of polymers showed that these strains were capable of degradation PLA, PHB and PCL and these strains presented higher activity of degradation PCL than PLA and PHB In the future, we also intend to study further about the enzymes that degrade PLA, PHB and PCL and apply them to biodegradation in the nature environment Key words: biopolymer, poly(L-lactic acid), poly(3-hydroxybytyrate), poly(ε-caprolactone), biodegradable Trinh Thi Van Anh Master thesis 2010 Luận văn thạc sỹ: Screening and study on microorganisms degrading of biopolymers in Vietnam Chuyên ngành: Công nghệ Sinh học Sinh viên: Trịnh Thị Vân Anh Cán hướng dẫn khoa học: TS Nguyễn Quang Huy TÓM TẮT Trong năm gần việc sử dụng cách tràn lan sản phẩm nhựa tổng hợp có nguồn gốc từ hoá dầu polypropylen (PP), polyetylen (PE), polyvinylclorua (PVC)… làm gia tăng ô nhiễm môi trường giới Để giảm thải ô nhiễm rác thải việc sử dụng loại nhựa tổng hợp nhiều quốc gia sử dụng thay thể chúng sản phẩm thân thiện môi trường loại polymer sinh học Trong số polymer sinh học poly(lactic acid) (PLA), poly(3hydroxybutyrat) (PHB) poly(ε-caprolactone) (PCL) polymer ý nhiều Các polymer sinh học khác tính chất lý, hố học chúng có ưu điểm chung phân huỷ thành thành phần CO2 H2O, khơng gây nhiễm mơi trường Các polymer sinh học điều kiện tự nhiên thường phân hủy chậm phải khoảng thời gian dài để trình tự phân hủy bắt đầu diễn Việc phân lập chủng vi sinh vật có khả phân hủy polymer sinh học mạnh tồn tự nhiên việc cần thiết góp phần kiểm sốt, thúc đẩy tốt việc phân huỷ polymer sinh học Từ 24 mẫu đất lấy từ địa điểm khác Hà Nội, phương pháp tạo vòng phân huỷ môi trường bổ sung polymer sinh học phân lập chủng vi sinh vật phân huỷ PLA, chủng vi sinh vật phân huỷ PHB chủng vi sinh vật phân huỷ PCL Trong số chủng G5 Cz1 có hoạt tính phân huỷ PLA mạnh nhất, chủng B2 B1 chủng phân huỷ PHB, PCL mạnh Dựa vào đặc điểm hình thái, sinh lý, sinh hóa trình tự gen 16S rADN, chủng G5 định danh Streptomyces thermoflavus, chủng Cz1 Penicillium citrinium, chủng B2 Bacillus gelatini chủng B1 Brevibacillus agri Các chủng phát triển tốt pH 6-7, nồng độ muối NaCl từ 1-3% Chủng B gelatini B2 Br agri B1 chủng ưa nhiệt, phát triển tối ưu nhiệt Trinh Thi Van Anh Master thesis 2010 độ 50oC, S thermoflavus G5 P citrinium phát triển tốt 37oC 30oC Khả phân hủy polymer chủng tiến hành môi trường chứa polymer nguồn cácbon lượng đánh giá qua hàm lượng bon tổng số (TOC) dịch nuôi cấy sau ly tâm lượng polymer lại Sau 30 ngày, S thermoflavus G5 phân hủy 46.8% P citrinum Cz1 phân hủy 55.5% lượng PLA bổ sung ban đầu, B gelatini B2 Br agri B1 tương ứng phân hủy 58.5% 84% PHB PCL Các chủng vi sinh vật có khả phân huỷ loại polymer sinh học dùng nghiên cứu, hoạt tính phân huỷ PCL cao so với PLA PHB Các enzyme phân hủy polymer sinh học điều kiện phát triển tối ưu chủng vi sinh vật nghiên cứu nhằm mục đích ứng dụng chúng việc xử lý nhiễm mơi trường Từ khóa: biopolymer, poly(L-lactic acid), poly(3-hydroxybytyrate), poly(εcaprolactone), biodegradable Trinh Thi Van Anh ... different locations in Hanoi, Vietnam strains degrading PLA, strains degrading PHB and strains degrading PCL were selected by clear zone formed method Among them, strain G5 and strain Cz1 had the... poly(ε-caprolactone), biodegradable Trinh Thi Van Anh Master thesis 2010 Luận văn thạc sỹ: Screening and study on microorganisms degrading of biopolymers in Vietnam Chuyên ngành: Công nghệ Sinh học Sinh. .. UNIVERSITY *** VIETNAM NATIONAL UNIVERSITY, HANOI INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY *** Trinh Thi Van Anh SCREENING AND STUDY ON MICROORGANISMS DEGRADING BIOPOLYMERS IN VIETNAM Speciality: