Study on methanotrophs and their some potential application aspects nghiên cứu vi khuẩn ôxi hóa metan và tiềm năng ứng dụng sinh học

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     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     TABLE OF CONTENTS Acknowledgements Table of contents List of figures List of tables Abbreviations Abstract Tóm tắt Preface 10 Chapter 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 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     2.7 Chemical analyses 42 Chapter 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     LIST OF FIGURES Figure Title Figure 1.1 Phylogenetic relationships between known methanotrophs based on 16S rRNA gene sequences using MEGA4………………… 15 Pathways for the oxidation of methane and assimilation of formaldehyde in MOBs………………………………………… 18 RuMP pathway for HCHO assimilation in Type I methanotrophs…………………………………………………… 19 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 methaneoxidizingbacteria………………………………………………… 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 1.2 Figure 1.3 Figure 1.4   Figure 1.10   The schematic biofilter ………………………………………… Figure 1.11   Horizontal injection and extraction of methane, air, and nutrient used in in-situ bioremediation of TCE ………………………… Figure 3.1   Page 31 33 Methane consumption in enriched cultures of MOBs after days of cultivation …………………………………………… 43 The increase in culture turbidity through three steps of enrichment of sample PS ……………………………………… 44 Isolation of MOB via liquid dilution series in the wells of 96well 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 Figure 3.2   Figure 3.3       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 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 ……… Figure 3.12   Cultivation of BG3 with methane ……………………………… 52 Figure 3.13   Experimental generation of methane from organic wastes …… Figure 3.14   Control of methane emission from organic wastes in laboratory model using strain BG3 ……………………………………… 55     53 56 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           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           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 68, temperature 25-40 oC, 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 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 m3 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           TĨM TẮT Từ mẫu mơi trường thu thập từ địa điểm khác nhau, ba chủng vi khuẩn oxy hóa metan gồm BG3, PS1 W1 phân lập nhờ phương pháp pha lỗng mơi trường khoáng dịch thể sử dụng metan làm nguồn cacbon lượng Ba chủng nói chứa gen mã hóa cho enzyme methane monooxygenase dạng hạt khơng chứa gen mã hóa cho enzyme dạng hịa tan, chứng tỏ ba chủng khơng có khả sinh trưởng đa dạng loại chất hữu khác Trong ba chủng phân lập được, chủng BG3 có khả sinh trưởng tốt điều kiện có metan chủng lựa chọn sử dụng vi sinh vật mô hình thí nghiệm tiềm ứng dụng Các điều kiện nuôi cấy tối ưu chủng xác định bao gồm: pH 6-8, nhiệt độ 25-40oC, nồng độ muối 1-15g⋅L-1 NaCl Dựa phân tích trình tự đoạn gen 16S rDNA, chủng BG3 xác định thành viên chi Methylomonas (vi khuẩn sử dụng metan tuýp I) với chủng gần gũi Methylomonas methanica (độ tương đồng 95%) Chủng đặt tên Methylomonas sp BG3 trình tự đoạn gen 16S rDNA gửi vào ngân hàng gen mã số KJ081955 Ngoài ra, gen pmoA xác định có mặt chủng với đoạn gen dài 508 bp gửi GenBank với mã số KJ081956 Một số hướng ứng dụng vi khuẩn oxy hóa metan tiến hành nghiên cứu với vi sinh vật mơ hình chủng BG3 Nuôi cấy chủng BG3 với metan tạo sinh khối có trọng lượng khơ tế bào 1,26 g/l, hàm lượng protein thô 69,69g/100 g CDW hiệu suất sử dụng metan 2,85 m3 metan/kg CDW Trong điều kiện thí nghiệm chủng BG3 có khả loại bỏ 77,46 % thể tích metan sinh q trình phân hủy kỵ khí rác hữu Từ khóa: vi khuẩn oxy hóa metan, Methylomonas, pmoA, tạo sinh khối, phát thải metan   10         72 Medigan MT, Martinko JM, Parker J (2003) Brock Biology of 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Methanol:TheClearAlternativeforTransportation URLhttp://www.methanol.org   133 United State National Oceanic and Atmospheric Administration Trends in Atmospheric Carbon Dioxide URLhttp://www.esrl.noaa.gov/gmd/ccgg/trends/global.html Vietnamese   134 Ministry of Agriculture and Rural Development, Department of Livestock Husbandry (2012) Livestock Newsletter URL http://www.cucchannuoi.gov.vn/WebContent/bantinchannuoi/index.aspx?index=detail News&num=15&TabID=1&NewsID=94 135 Ministry of Natural Resources and Environment (2012) National Environment Report 2011   74         APPENDIX I 16S rDNA sequences BG3 - contig (1413bp) CGTAGATTGAACGCTGGCGGTATGCTTAACACTTGCAAGTTTCAACGCTGA AGGGTGCTTGCACCTGGATGAGTGGCGGACGGGTGAGTAATGCATAGGAA TCTGCCTATTAGTGGGGGATAACGTGGGGAAACTCACGCTAATACCGCATA CGCTCTACGGAGGAAAGCCGGGGACCTTCGGGCCTGGCGCTAATAGATGA GCCTATGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACG ATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACG GCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGC AAGCCTGATCCAGCAATACCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAA AGCACTTTCAATGGGAAGGAACACCTATCGGTTAATACCCGGTAGACTGAC ATTACCCATACAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAA TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGTA GGCGGTTTTTTAAGTCAGATGTGAAAGCCCTGGGCTTAACCTGGGAACTGC ATTTGATACTGGGGAACTAGAGTTGAGTAGAGGAGAGTGGAATTTCAGGT GTAGCGGTGAAATGCGTAGAGATCTGAAGGAACACCAGTGGCGAAGGCGG CTCTCTGGACTCAAACTGACGCTGAGGTACGAAAGCGTGGGTAGCAAACA GGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAACCGTTG GGTTCTTAAAGAACTTAGTGGTGGAGCTAACGTATTAAGTTGACCGCCTGG GGAGTACGGCCGCAAGGCTAAAACTCAAATGAATTGACGGGGGCCCGCAC AAGCGGTGGAGCATGGGGGTTTAATTCGATGCAACGCGAAGAACCTTACC TACCCTTGACATCCTCGGAACTTGTCAGAGATGACTTGGTGCCTTCGGGAA CCGAGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGAAGGTT TGGGTTAAGTCCCGTAACGAGCGCAACCCTTATCCTTAGTTGCCAGCGCGT CATGGCGGGAACTCTAGGGAGACTGCCGGTGATAAACCGGAGGAAAGTGG GGACGACGTCAAGTCATCATGGCCCTTATGGGTAGGGCTACACACGTGCTA   75         CAATGGTCGGTACAGAGGGTTGCGAACTCGCGAGAGCCAGCCAATCCCAA AAAGCCGATCCTAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTCG GAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGG CCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTA GG PS1 - contig (1367bp) GCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAACGCCC CGCAAGGGGAGTGGCAGACGGGTGAGTAACACGTGGGGATCTGCCCAATG GTACGGAATAATTCCGGGAAACTGGGACTAATACCGTATGTGCCCGCAAG GGGAAAGATTTATCGCCATTGGATGAACCCGCGTCGGATTAGCTAGTTGGT GAGGTAAAGGCTCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGA TCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA GTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCGCCG ACGAAGATAATGACGGTAGTCGGAGAAGAAGCCCCGGCTAACTTCGTGCC AGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATCACTGGGC GTAAAGCGCACGTAGGCGGACATTTAAGTCAGGGGTGAAAGCCTGGAGCT CAACTCCAGAACTGCCCTTGATACTGGGTGTCTCGAGTCCGGAAGAGGTAA GTGGAACTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCAAGAACACC AGTGGCGAAGGCGGCTTACTGGTCCGGTACTGACGCTGAGGTGCGAAAGC GTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT GGAGGCTAGCCGTTGGTGAGCATGCTCATCAGTGGCGCAGCTAACGCATTA AGCCTCCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTG ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGC GCAGAACCTTACCAGCCTTTGACATGTCCCGGACGGTTACCAGAAGATGGT TTCTTCTCTTCGGAGCCGGGAACACAGGTGCTGCATGGCTGTCGTCAGCTC GTGTCGTGAGATGTTTGGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCC   76         TTAGTTGCCATCATTCAGTTGGGCACTCTTAGGGGGACTGCCGGTGATAAG CCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTG GGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCGAACCCGCGAG GGTAAGCAAATCTCCAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTC GAGTGCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGT GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGG TTTTACCCGAAGGCGCTGCT W1 – contig (1272bp) ATCTGCCCAATGGTACGGAATAATTCCGGGAAACTGGGACTAATACCGTAT GTGCCCGCAAGGGGAAAGATTTATCGCCATTGGATGAACCCGCGTCGGATT AGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAGCTGGTC TGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCATAC GGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGC CATGCCGCCTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCGCCGAC GAAGATAATGACGGTAGTCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAG CAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATCACTGGGCGT AAAGCGCACGTAGGCGGACATTTAAGTCAGGGGTGAAAGCCTGGAGCTCA ACTCCAGAACTGCCCTTGATACTGGGTGTCTCGAGTCCGGAAGAGGTAAGT GGAACTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCAAGAACACCAG TGGCGAAGGCGGCTTACTGGTCCGGTACTGACGCTGAGGTGCGAAAGCGT GGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGG AGGCTAGCCGTTGGTGAGCATGCTCATCAGTGGCGCAGCTAACGCATTAAG CCTCCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGAC GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGC AGAACCTTACCAGCCTTTGACATGTCCCGGACGGTTACCAGAGATGGTTTC TTCTCTTCGGAGCCGGGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTG TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGT   77         TGCCATCATTCAGTTGGGCACTCTAGGGGGACTGCCGGTGATAAGCCGAGA GGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTAC ACACGTGCTACAATGGCGGTGACAGTGGGAAGCGAACCCGCGAGGGTAAG CAAATCTCCAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGC ATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATAC GTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTAC CCGAAGGCGCT pmoA sequences BG3 (415bp) GCGGCTGTTAAAGCTTGCTCGTGGTGGAGATACCGTTTGCCCATCGGCGCA ACCATTTCAGTTGTTGCTCTGATGATCGGTGAGTGGATCAACAGATATTTG AACTTCTGGGGTTGGACATACTTCCCAGTTAACATCTGCTTCCCATCAAACT TGCTGCCAGGCGCTATCGTTCTGGACGTAATCCTGATGTTGGGTAACAGCA TGACCCTGACCGCTATTGTTGGTGGTTTGGCTTATGGTTTGTTGTTCTACCC AGGCAACTGGCCAATCATTGCTCCTCTGCACGTTCCTGTTGAATACGACGG CATGATTATGACTCTGGCTGACTTGCAAGGTTACCACTATGTTCTTACCGGT ACACCTGATTACATCCGTATGGTATAGAAAGGTACATTGAGAACTTTCTGT ATAG PS1 (406bp) TACAAGCTACCCTGCGGTATCGTTTCCGTCTGCCTTTTGGCGCTGTAATTTC TGTTTTAGGTCTACTCTTGGGTGAATGGGTTAACAGATATATGAACTTCTG GGGATGGACATATTTCCCTGTGAACTTCGTATTTCCTTCAAACCTGATGCCA GGTGCTATTGTTCTTGATGTTATCCTGATGTTGTCGAACAGCATGACATTAA CAGCGGTTGTTGGTGGTATGGCATGGGGTCTGTTGTTCTATCCTGGCAACT GGCCAATCATTGCGCCACTGCATATTCCTGTTGAATACAATGGCATGATGT   78   ... GenBank với mã số KJ081956 Một số hướng ứng dụng vi khuẩn oxy hóa metan tiến hành nghiên cứu với vi sinh vật mơ hình chủng BG3 Ni cấy chủng BG3 với metan tạo sinh khối có trọng lượng khô tế bào 1,26... sử dụng metan 2,85 m3 metan/ kg CDW Trong điều kiện thí nghiệm chủng BG3 có khả loại bỏ 77,46 % thể tích metan sinh q trình phân hủy kỵ khí rác hữu Từ khóa: vi khuẩn oxy hóa metan, Methylomonas,... 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