luận văn Study on methanotrophs and their some potential application aspects nghiên cứu vi khuẩn ôxi hóa metan và tiềm năng ứng dụngluận văn Study on methanotrophs and their some potential application aspects nghiên cứu vi khuẩn ôxi hóa metan và tiềm năng ứng dụngluận văn Study on methanotrophs and their some potential application aspects nghiên cứu vi khuẩn ôxi hóa metan và tiềm năng ứng dụng
! 1! LIEGE UNIVERSITY *** ! VIETNAM NATIONAL UNIVERSITY, HANOI Institute of Microbiology and Biotechnology *** Nguyen Thi Hieu Thu STUDY ON METHANOTROPHS AND THEIR SOME POTENTIAL APPLICATION ASPECTS Specialty: Biotechnology Code: 60 42 02 01 MASTER THESIS SUPERVISOR: Dr. DINH THUY HANG Hanoi, 2014 ! 2! ACKNOWLEDGEMENTS Foremost, I would like to express my deep gratitude to my advisor Dr. Dinh Thuy Hang for her patience, motivation, enthusiasm, and immense knowledge. Her guidance helped me in all the time of research and writing of this thesis. I am indebted to all the lecturers of Vietnam National University, Hanoi (Vietnam) and University of Liege (Belgium) for sharing their valuable scientific knowledge. I thank my lab mates in Microbial Ecology Department (Institute of Microbiology and Biotechnology) for the stimulating discussions, for providing guidance, and for all the fun we have had. Finally, and most importantly, I would like to thank my family, especial my husband, for unconditional supports that made this thesis possible. Hanoi, December 2013 Nguyen Thi Hieu Thu ! 3! TABLE OF CONTENTS Acknowledgements 1 Table of contents 2 List of figures 4 List of tables 6 Abbreviations 7 Abstract 8 Tóm tắt 9 Preface 10 Chapter 1. Introduction 11 1.1. Methane and global climate change 11 1.2. Methanotrophs 12 1.2.1. Phylogeny of methanotrophs 12 1.2.2. Physical diversity of methanotrophs 15 1.3. Aerobic methane oxidation 17 1.4. Methane monooxygenase 20 1.4.1. The role of MMOs in MOB 20 1.4.2. Soluble methane monooxygenase 21 1.4.3. Particulate methane monooxygenase 23 1.5. Application potential of Methanotrophs 25 1.5.1. Food for animal 25 1.5.2. Bioconversion of methane to methanol 27 1.5.3. Environmental bioengineering 29 1.6. Objectives of this study 35 Chapter 2. Material and Methods 36 2.1. Sampling 36 2.2. Isolation of methanotrophs 36 2.3. DNA extraction and PCR amplification 38 2.4. DGGE 40 2.5. Sequencing and phylogenetic analysis 41 2.6. Morphological and physiological characterization 41 ! 4! 2.7. Chemical analyses 42 Chapter 3. Results and discussion 43 3.1. Enrichment and isolation of MOBs from environmental samples 43 3.1.1. Enrichment of MOBs 43 3.1.2. Isolation of MOBs and preliminary identification 44 3.2. Study the presence of MMO encoding genes in the isolates 46 3.3. Growth of the MOB isolates with methane 48 3.4. Morphology, physiology and phylogeny of strain BG3 49 3.5. Application experiments using Methylomonas sp. BG3 as model organism 52 3.5.1. Study on bacterial meal production 52 3.5.2. Study on reduction of methane emission from organic wastes 55 Conclusion and Prospective works 58 References 59 Appendix 74 ! 5! LIST OF FIGURES Figure Title Page Figure 1.1. Phylogenetic relationships between known methanotrophs based on 16S rRNA gene sequences using MEGA4………………… 15 Figure 1.2. Pathways for the oxidation of methane and assimilation of formaldehyde in MOBs…………………………………………. 18 Figure 1.3. RuMP pathway for HCHO assimilation in Type I methanotrophs…………………………………………………… 19 Figure 1.4.! Serine pathway for the assimilation of formaldehyde in Type II methanotrophs…………………………………………………… 19 Figure 1.5.! Orientation of soluble mono-oxygenase gene cluster…………… 22 Figure 1.6.! The crystal structure of hydroxylase dimer…………………… 22 Figure 1.7.! Particulate methane monooxygenase gene clusters of methane- oxidizingbacteria………………………………………………… 23 Figure 1.8.! Crystal structure of a single promoter of pMMO……………… 24 Figure 1.9.! The schematic bench scale plant for treatment of diluted landfill gas in biofilters………………………………………………… 30 Figure 1.10.! The schematic biofilter. ………………………………………… 31 Figure 1.11.! Horizontal injection and extraction of methane, air, and nutrient used in in-situ bioremediation of TCE. ………………………… 33 Figure 3.1.! Methane consumption in enriched cultures of MOBs after 7 days of cultivation. …………………………………………… 43 Figure 3.2.! The increase in culture turbidity through three steps of enrichment of sample PS. ………………………………………. 44 Figure 3.3.! Isolation of MOB via liquid dilution series in the wells of 96- well plates. ……………………………………………………… 45 Figure 3.4. ! DGGE analysis of PCR-amplified 16S rDNA fragments of the isolates obtained from the MOB-enrichment cultures. …………. 46 Figure 3.5. ! PCR products of pmoA gene fragments (508 bp). ……………… 47 ! 6! Figure 3.6. ! Agarose gel electrophoresis of the mmoX gene PCR products yielded from genome of the isolates (800 bp). …………………. 48 Figure 3.7. ! Growth of the MOB isolates with methane as shown by optical density of the liquid cultures after 4 days cultivation. …………. 49 Figure 3.8. ! Phase – contrast micrographs of the MOB isolates grown in liquid cultures with methane (viewed at 1000× magnifications). 49 Figure 3.9. ! Phylogenetic tree based on the 16S rRNA gene sequences showing the relationship of strains BG3 and other known methanotrophs. …………………………………………………. 50 Figure 3.10. ! Phylogenetic analysis of partial amino acid sequences encoded by the pmoA gene from the three MOB isolates. ………………. 51 Figure 3.11. ! Cultivation condition-dependent growth of strain BG3. ……… 52 Figure 3.12. ! Cultivation of BG3 with methane. ……………………………… 53 Figure 3.13. ! Experimental generation of methane from organic wastes. ……. 55 Figure 3.14. ! Control of methane emission from organic wastes in laboratory model using strain BG3. ……………………………………… 56 ! 7! LIST OF TABLES Table Title Page Table 1.1. Characteristics of methanotrophs. 14 Table 1.2. Chemical and amino acid composition of BPM, fishmeal and soybean meal (SBM). 26 Table 2.1. Fresh water mineral medium. 36 Table 2.2. Metal mix and vitamin mix. 36 Table 3.1. Bacterial strains isolated from MOB-enrichment samples by using liquid serial dilution method. 45 Table 3.2. Crude protein content in biomass of MOB and other bacterial species. 54 ! ! ! ! 8! ABBREVIATIONS 16S rDNA Gene coding for small subunit of ribosomal deoxyribonucleic acid Bp Base pair BSA Bovin serum albumin CI Chloroform-isoamyl alcohol DGGE Denaturing gradient gel electrophoresis DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate EDTA Ethylenediaminetetraacetic acid EPS Extracellular/exo- polymeric substance ICM Intracytoplasmic membrane MOB Methane oxidizing bacteria MQ Mili-Q OD Optical density PCR Polymerase chain reaction pMMO Particulate methane mono-oxygenase pmoA Gene for alpha subunit of the pMMO SDS Sodium dodecyl sulfate sMMO Soluble methane mono-oxygenase TAE Tris-Acetic-EDTA Taq Thermus aquaticus DNA polymerase BPM Bacterial protein meal ! ! ! ! 9! ABSTRACT From environmental samples of different locations, three freshwater strains of methane oxidizing bacteria (MOBs), i.e. BG3, PS1 and W1, were isolated by using serial dilution method in liquid mineral medium with methane as the only carbon and energy sources. These three isolates contained genes encoding for the particulate methane-mono-oxygenase (pMMO) but not the soluble one (sMMO), indicating that they would not be expected to growth on a broad range of organic substrates. Of the three isolates, strain BG3 showed the highest growth with methane and thus was selected and used as model organisms in further experiments on application aspects. Optimal cultivation conditions for this strain were also determined, i.e. pH 6- 8, temperature 25-40 o C, salinity of 1-15 g. L -1 NaCl. Based on phylogenetic analyses of the 16S rDNA partial gene sequences, strain BG3 was identified as a member of the Methylomonas genus (type I methanotroph), the most closely related species was Methylomonas methanica (95% homology). This strain was designated with the name Methylomonas sp. BG3 and its 16S rDNA partial sequence was deposited at the GenBank under accession number of KJ081955. In addition, pmoA gene has also been detected in this strain and a gene sequence fragment (508 bp) was deposited the GenBank under accession number of KJ081956. Studies on the application aspects of MOBs were conducted with the use of strain BG3 as the model organism. It has been shown that methane-fed culture of strain BG3 could yield 1.26 g⋅l − 1 cell dry weight (CDW), accordingly produce 68.69 g crude protein per 100 g CDW and the efficiency of methane consumption in this respect was 2.85 m 3 per kg CDW. In the study on control of methane emission by MOB, strain BG3 showed the capability of reducing 77.46 % of total volume of methane emitted from anaerobically decomposing organic wastes. Key words: methanotroph, Methylomonas, pmoA, biomass production, methane emission ! ! ! ! 10! TÓM TẮT Từ các mẫu môi trường thu thập từ các địa điểm khác nhau, ba chủng vi khuẩn oxy hóa metan gồm BG3, PS1 và W1 đã được phân lập nhờ phương pháp pha loãng trong môi trường khoáng dịch thể sử dụng metan làm nguồn cacbon và năng lượng duy nhất. Ba chủng nói trên chứa gen mã hóa cho enzyme methane monooxygenase ở dạng hạt nhưng không chứa gen mã hóa cho enzyme này ở dạng hòa tan, chứng tỏ ba chủng này không có khả năng sinh trưởng trên đa dạng các loại cơ chất hữu cơ khác nhau. Trong ba chủng phân lập được, chủng BG3 có khả năng sinh trưởng tốt nhất trong điều kiện có metan do đó chủng này được lựa chọn và sử dụng như vi sinh vật mô hình trong các thí nghiệ m tiếp theo về tiềm năng ứng dụng. Các điều kiện nuôi cấy tối ưu của chủng này đã đ ượ c xác định bao gồm: pH 6-8, nhiệt độ 25-40 o C, nồng độ muối 1-15g⋅L -1 NaCl. Dựa trên các phân tích trình tự đoạn gen 16S rDNA, chủng BG3 được xác định là một thành viên của chi Methylomonas (vi khuẩn sử dụng metan tuýp I) với chủng gần gũi nhất là Methylomonas methanica (độ tương đồng 95%). Chủng này được đặt tên là Methylomonas sp. BG3 và trình tự đoạn gen 16S rDNA của nó đã được gửi vào ngân hàng gen dưới mã số KJ081955. Ngoài ra, gen pmoA cũng đã được xác định có mặt ở chủng này với đoạn gen dài 508 bp được gửi tại GenBank với mã số KJ081956. Một số hướng ứng dụng của vi khuẩn oxy hóa metan đã được tiến hành nghiên cứu với vi sinh vật mô hình là chủng BG3. Nuôi cấy chủng BG3 với metan tạo sinh khối có trọng lượng khô tế bào là 1,26 g/l, hàm lượng protein thô là 69,69g/100 g CDW và hiệu suất sử dụng metan là 2,85 m 3 metan/kg CDW. Trong điều kiện thí nghiệm chủng BG3 có khả năng loại bỏ 77,46 % thể tích metan sinh ra trong quá trình phân hủy kỵ khí rác hữu cơ. Từ khóa: vi khuẩn oxy hóa metan, Methylomonas, pmoA, tạo sinh khối, phát thải metan. [...]... oxidation with the involvement of methanotrophs has not been studied in Vietnam, the present work focused on obtaining pure cultures of MOBs from environmental samples and using the isolates as model organisms for some application experiments The working plan would be as following: • Isolate methanotrophs from environmental samples in Vietnam via liquid dilution series using methane as the only energy and. .. scientific viewpoint, methane-oxidizing bacteria have not been looked at and considered as a tool for resolving these problems In the present work, for the first time methanotrophs have been enriched and isolated from environmental samples and primary studies on their application were carried out 11 Chapter 1 INTRODUCTION 1.1 Methane and global climate change Methane is a colorless and odorless... of methane emission in Vietnam are coalmine gases, rice paddies and biogas from anaerobic digesters Thus, studies on the application of methanotrophs for reducing the methane emission could be a good solution for improving the existing pollution situation 1.5.3.2 Biodegradation of difficult to degrade pollutants Halogenated compounds are commonly used in various industrial practices and have serious... yellow, and blue, respectively (Lieberman & Rosenzweig, 2005) To catalyze reactions pMMO uses a higher – potential electron donor than that of sMMO, thus methanotrophs that posses pMMO have higher growth yields on methane and have greater affinity for methane than do methanotrophs that contain sMMO alone (Hanson & Hanson, 1996) However, pMMO has relatively narrow substrate specificity and can oxidize only... capacity and operational difficulties In Vietnam, the average amount of solid waste per year is 25.000 tons, which is 32 increasing at a rate of 10% annually (National Environment Report, 2011) This huge amount of waste however has been treated mainly by the inefficient landfill technology, causing serious pollution problems, one among that is the emission of greenhouse gases CO2 and CH4 Along... hydrocarbon (up to five carbons in length) and trichloroethene (DiSpirito et al., 1992) Comparison of pMMO and ammonia monooxygenase (AMO) gene sequences suggests that these could be evolutionarily related, as conserved residues are found throughout the entire length of pmoA and amoA amino acid sequences, implying their structural similarities (Holmes et al., 1995) 25 1.5 Application potentials... immobilization of cells on polymeric carriers and the addition of stimulating chemicals such as phosphate and formate could increase the methanol production up to above 60 mg/ml (Senko et al., 2007) 1.5.3 Environmental bioengineering The capability of methanotrophs to degrade a wide variety of potential pollutants including methane and halogenated hydrocarbons has been studied for applications in controlling climate... only energy and carbon source • Study physiological characteristics of the isolates and select suitable strains for the use in application experiments • Determine phylogenetic position of the selected strains based on the analyses of 16S rDNA and pmoA gene sequences • Initially investigate application potentials of the MOBs in term of bacterial protein production and methane mitigation by using the selected... potentials of methanotrophs 1.5.1 Food for animal In the 1960s – 1970s, production of microbial protein (bacterial protein meal, BPM) as supply for human and animal nutrition from hydrocarbons such as methane and methanol was considerably researched and industrially developed However, commercial products could not compete with the low priced conventional protein sources Recently, the attention on the potential. .. animal production has been renewed due to the problem in increasing population, environmental pollution and climate change (Overland et al., 2010) With feasible characteristics, i.e carbon containing in a reduced and energy efficient form, a high yield of microbial cell supporting, methane is expected to be an ideal substrate for producing BPM (Hanson & Hanson, 1996) The early studies on young chickens . VIETNAM NATIONAL UNIVERSITY, HANOI Institute of Microbiology and Biotechnology *** Nguyen Thi Hieu Thu STUDY ON METHANOTROPHS AND THEIR SOME POTENTIAL APPLICATION ASPECTS. physiology and phylogeny of strain BG3 49 3.5. Application experiments using Methylomonas sp. BG3 as model organism 52 3.5.1. Study on bacterial meal production 52 3.5.2. Study on reduction of. Whereas known MOBs belong only to the α and γ-subclass of Proteobacteria and are unique as they grow mainly on methane, sometimes also methanol, as their sole carbon and energy source with