Nghiên cứu vi khuẩn oxy hóa metan và tiềm năng ứng dụng

Phylogenetic analysis of partial amino acid sequences encoded by the pmoA gene from the three MOB isolates.. Control of methane emission from organic wastes in laboratory model using s

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 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

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

LIST OF FIGURES

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

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

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

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 3.6 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

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

LIST OF TABLES

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

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

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

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 6-

8, 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− 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 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

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 nghiệm tiếp theo về tiềm năng ứng dụng Các điều kiện nuôi cấy tối ưu của chủng này đã được xác định bao gồm: pH 6-8, nhiệt độ 25-40oC, 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 m3 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

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