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

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

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

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

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

dNTP Deoxyribonucleotide triphosphate

EDTA Ethylenediaminetetraacetic acid

EPS Extracellular/exo- polymeric substance

pMMO Particulate methane mono-oxygenase

pmoA Gene for alpha subunit of the pMMO

Taq Thermus aquaticus DNA polymerase

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

PREFACE

Since the late 19th century, global warming has been detected with the rise in the average temperature of Earth's atmosphere and oceans During this 21st century, the global surface temperature is predicted to rise a further 1.1 to 6.4 °C (IPPC 2007) Today, climate change shows multi-aspect impacts to the life on earth, e.g sea levels rising, precipitation changing, subtropical desert expansion etc., and subsequently affects to food security of humans due to decreasing crop yield and the loss of habitat from inundation

The primary reason causing the climate change is the increasing emission of greenhouse gases, including water vapor, carbon dioxide, methane, nitrous oxide and

ozone (Karl et al., 2003) Next to carbon dioxide (the most effective greenhouse gas

with fast increasing concentration in the atmosphere, reaching 392.6 ppm in 2012 Trends in Atmospheric Carbon Dioxide, 2012), methane is considered to be the second most important greenhouse gas, the level of which in the atmosphere increased drastically, mainly due to the extensive exploration of anthropogenic sources

-In nature, methane-oxidizing bacteria (MOBs) represent a unique microbial group involving in the methane cycling These microorganisms can utilize methane as

a growth substrate, and thus participate in the control of methane emission as well as many other applications In Vietnam, organic pollution is of great concern, and methane released from organic wastes has not been effectively used From 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

Chapter 1 INTRODUCTION 1.1 Methane and global climate change

Methane is a colorless and odorless gas that makes the major component (97% vol.) of natural gas Methane has a strong absorbance of infrared radiation, which is not able to

escape from the Earth’s atmosphere, leading to the global warming (Lelieveld et al.,

1993) Although carbon dioxide is the single largest greenhouse gas in terms of global warming potential, methane is the most significant contributor to the greenhouse effect after CO2, and on a molar basis it is about 23 times more effective than CO2 (IPCC, 2007)

Global population increases and the consequent rise in consumption of fossil energy as well as the generation of waste have led to a large increase of the anthropogenic methane emission, which now accounts for ~ 70% of 5200 ton CH4

emitted each year to the atmosphere (Breas et al., 2001; IPCC, 1996) The major

natural sources of methane include natural wetlands, paddy fields, ruminants, lakes and oceans Fossil fuel exploitation, livestock feeding operations, landfills and rice cultivation have been counted as the largest anthropogenic sources, that have been rising dramatically since the beginning of the industrial era Consequently, the atmospheric methane concentration has increased from 0.75 – 1.75 ppm in the last 300

years at an enormous rate might reach 4.0 ppm till 2050 (Ramanathan et al., 1985)

Unfortunately, the control of methane emission has not yet received appropriate attention, and in the last decade there were little changes in the atmospheric methane This will be a serious problem for climate change that has become a global concern and more attention needs to be paid to this field of research

1.2 Methanotrophs

Methanotrophs (MOBs) are a unique group of methylotrophic bacteria with the ability

to utilize methane as their sole carbon and energy source (Hanson & Hanson 1996, Murrel 1994) Methylotrophs are phylogenetically widespread, including archaea, eubacteria and yeasts that are capable of utilizing a wide variety of C-1 compounds such as methane, methanol, methylamines, etc 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 oxygen as the terminal electron acceptor (Hanson & Hanson, 1996) Almost all MOBs are obligatory methylotrophic and cannot utilize complex C compounds such as sugars and organic acids (Bowman, 2000) However, facultative methanotrophic strains have been detected recently This could be due to the lack of certain enzymes in the Krebs cycle like α-ketogulatarate dehydrogenase or the lack of specific transporter for

complex C compounds in these organisms (Dedysh et al., 2005)

The first bacterium growing on methane was isolated from pond water and aquatic plants in 1906 by the Dutch microbiologist Nicolaas Sohngen Till 1970s, more than 100 MOB strains were isolated, establishing a new era of research on biochemical properties and pathways of aerobic methane oxidation To date, these organisms have been found in a wide variety of environments including soils

(Whittenbury et al., 1970), sediments (Smith et al., 1997), landfill (Wise et al., 1999), groundwater (Fliermans et al., 1988), seawater (Holmes et al., 1995), peatbog (Dedysh

et al., 2000; Mcdonald et al., 1996) etc., showing their ubiquitous involvement in the

methane cycling

1.2.1 Phylogeny of methanotrophs

Based on morphology and many other physiological properties (table 1.1) such as the arrangement of intra-cytoplasmic membranes, pathways of carbon assimilation,

nitrogen fixation ability, the presence of cysts or spores, colony color and motility, the

methanotrophs were initially classified into five genera Methylosinus, Methylocystis, Methylomonas, Methylobacter and Methylococcus (Whittenbury et al., 1970) Latter,

with the development of molecular tools in microbial taxonomy, these organisms have

been reorganized into two families Methylococcaceae (comprising Type I- and Type X-methanotrophs) and Methylocystaceae (Type II methanotrophs) The type I-

methanotrophs contains nine genera Methylomonas, Methylobacter, Methylomicrobium, Methylosphaera, Methylosarcina, Methylothermus, Methylohalobium, Methylosoma and Methylovulum Two genera Methylocaldum and Methylococcus are grouped in Type X as they are phylogenetically and

morphologically distinct from the other Type I genera Whereas, Type

II-methanotrophs consist of five genera Methylosinus, Methylocystis, Methylocella, Methylocapsa and Methyloferula (Bowman et al., 1993, 1995; Bussmann et al., 2006; Heyer et al., 2005; Rahalkar et al., 2007; Iguchi et al., 2011; Dedysh et al., 2011) The

major distinction between types I and II-methanotrophs is the pathway of incorporating formaldehyde into cell biomass Type I-methanotrophs assimilate formaldehyde via ribulose monophosphate (RuMP) pathway, while type II methanotrophs use serine pathway for the same operation (Fig 1.3, 1.4)

However, it seems that the present classification system as shown in table 1.1

has not yet been covering all MOB species Recently, a filamentous MOB Clorothrix fusca and three extremely acidophilic bacteria of the phylum Verrrucomicrobia have been isolated and characterized as members of the genus Methylacidiphylum, which does not belong to either α or γ-Proteobacteria These bacteria have been found to have striking dissimilarities to other known MOB (Stoecker et al, 2006; Dunfield et

al., 2007; Hou et al., 2008; Vigliotta et al., 2007) (Fig 1.1)

Table 1.1 Characteristics of methanotrophs (Hanson & Hanson, 1996)

Methylobacter Methylomicrobium Methylomonas etc

Methylococcus Methylocaldum Methylosinus Methylocystis

Methylocella Methylocapsa Methyloferula

Cell morphology Short rods, some

coccid or ellipsoids

Coccid, often found as pairs Crescent-shape rods, rods, pear-shape

cells, rosette (sometimes) Resting stages Azotobacter-type

cysts (or none)

Azotobacter-type cysts

Exosprores or lipoidal cysts Membrane arrangement:

Carbon assimilation

Major fatty acid carbon

Figure 1.1.

gene sequences using MEGA4 (Tamura et al., 2007) The tree was constructed using the

neighbor-joining method with 1304 positions of 16S rRNA genes

1.2.2 Physiological diversity of methanotrophs

Methanotrophs are widespread in nature, almost all samples taken from muds, swamps, rivers, rice paddies, oceans, deciduous woods, sewage pludge, sediments… contained MOBs Most cultured methanotrophs are mesophilic and neutrophilic, favoring moderate temperature (~25oC) and pH (pH 6-7) conditions (Hanson & Hanson, 1996) However, investigation of the extreme environments with high and low pH, temperature or salinities have led to the discovery of a variety of

extremophilic and extreme-tolerant methanotrophs (Murell et al., 1998; Trotsenko,

Khmelenina, 2002)

Methylococcus capsulatus, the earliest described heat-tolerant methanotroph,

grows at temperature up to 50 oC (Foster & Davis, 1966; Whittenbury et al., 1970)

Several other moderately thermophilic and thermotolerant species, including

Methylococcus thermophilus, Methylococcus ucrainicus, Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile were subsequently described (Malashenko et al., 1975, 1976; Bodrossy et al., 1995, 1997, 1999; Eshinimaev et al.,

2004) Very recently, the representatives of a novel group of truly thermophilic

methanotrophs (Methylothermus) were isolated from Hungarian and Japanese hot springs (Bodrossy et al., 1999; Tsubota et al., 2011), with temperature limits for

growth were 40 - 72 °C and 37 - 67 °C, respectively

Psychrophilic strains have also been isolated, mostly from Siberian tundra and

antarctic environments (Omelchenko et al., 1993; Bowman et al., 1997; Wartiainen et al., 2006) These methanotrophs can tolerate temperatures as low as 3.5 °C and usually

have optimum growth temperature between 10 °C and 20 °C

A variety of acidophilic, alkaliphilic and halophilic methanotrophs have been

observed (Lidstrom et al., 1988; Fuse et al., 1998; Heyer et al., 2005) Recently,

extreme acidophiles that exhibit optimum activity at pH 2.0– 2.5, even below pH1

have been isolated (Dunfield et al., 2007; Pol et al., 2007; Islam et al., 2008) These bacteria were classified under Verrucomicrobia phylum based on the 16S rDNA

analysis (Figure 1.1) Alkaliphilic methanotrophs have also been isolated with

optimum growth at pH 9.0 and 10.0 (Khmelenina et al., 1997; Sorokin et al., 2000; Kaluzhnaya et al., 2001; Reshetnikov et al., 2005)

Beside the obligatory methanotrophs, facultative methanotrophic strains that can utilize a variety of multi-carbon substrates such as ethanol and acetate for growths

were discovered (Dunfield et al., 2003; Dedysh et al., 2005; Dunfield et al., 2010; Belova et al., 2011; Im et al., 2011; Semrau et al., 2011) A number of characteristics

that are quite unusual for methanotrophs, e.g the absence of intra-cytoplasmic membrane, acidophilicity, and tolerance to low temperature were found in

Methylocella silvestris BL2 (Dunfield et al., 2003) More recently, two newly isolated

methanotrophs in the α-Proteobacteria, Methylocapsa aurea KYG and Methylocystis daltona SB2 were found to maintain viability and growth under the presence of acetate

as the sole carbon source (Dunfield et al., 2010; Im et al., 2011) These strains

exhibited higher specific growth rate and both of them constitutively express pMMO

in the presence of acetate without methane

This great diversity indicates that methanotrophs might be much more diverse

in the natural environments than anticipated from information on isolated laboratory strains The abundant resources of methanotrophs also reveal the great potential for their industrial applications

1.3 Aerobic methane oxidation

In methanotrophs, methane serves as the energy source (electron donor) and the sole or partial carbon source as well In aerobic methane oxidation, the first step is most difficult and takes place in the intra-cytoplasmic membrane system with the help of specialized enzymes known as methane mono-oxygenases (MMOs) (Fig.1.2) These enzymes break the O-O bond in the oxygen molecule by utilizing two reducing equivalents (Hanson & Hanson, 1996) One of the oxygen atoms is converted to water and the other is incorporated into methane to form methanol This reaction requires additional electrons, which are supply by cellular redox carriers such as cytochrome C (for particulate, pMMO) or NADH (for soluble MMO)

CH4 + O2 + H++ NADH = H2O + CH3OH + NAD+Methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH) Formaldehyde is the central metabolite in the anabolic and catabolic pathways

of MOBs In the catabolic pathway, HCHO is further converted to formate and then to CO2 with the help of a multiple enzyme systems: CH3OH → 2H+ + HCHO → 2H+ + HCOO-H+ → CO2 + 2H+

Figure 1.2 Pathways for the oxidation of methane and assimilation of formaldehyde in

MOBs (Hanson & Hanson, 1996) Abbreviation: CytC, cytochrome C (red–reduced, oxidized); FADH, formaldehyde dehydrogenase; FDH, formate dehydrogenase; MDH, methanol dehydrogenase

ox-This process produces most of the reducing power for the metabolism of methane because in these reactions, electrons are donated back to a component of membrane–bound electron transport chain, the cytochrome C (MDH) or NAD (in formaldehyde oxidation system) and FDH Electron flow through the membrane ultimately produces a proton motive force that is converted to ATP by ATPase

complex Oxygen is the terminal electron acceptor (Madigan et al., 2003) HCHO

assimilation takes place via two different pathways in Type I and Type II methanotrophs In Type I methanotrophs, it occurs by the ribulose monophosphate (RuMP) pathway (Fig 1.3), whereas the Type II methanotrophs use the serine pathway (Fig 1.4)

Figure 1.3 RuMP pathway for HCHO assimilation in Type I methanotrophs (Hanson &

Hanson 1996)

Figure 1.4 Serine pathway for the assimilation of formaldehyde in Type II methanotrophs

(Hanson & Hanson, 1996) Abbreviation: Serine hydroxyl-methyltransferase (STHM), hydroxyl pyruvatereductase (HPR), malate thiokinase (MTK), and maleyl coenzyme A lyase (MCL)

Enzyme systems used for assimilating these C1 units and MMOs as well are unique to methanotrophs so that they are used as markers to recognize uncultured species in environmental samples

1.4 Methane mono-oxygenases (MMOs)

1.4.1 The role of MMOs in MOBs

The enzyme methane-monooxygenase (MMO) catalyses the breakdown reaction at the C–H bonds in methane molecule under ambient conditions despite of its high stability (Dalton, 2005) Two forms of MMOs, namely membrane-associated or particulate form (pMMO) and soluble, cytoplasmic form (sMMO) have been found in MOBs Although pMMO and sMMO catalyze the same reaction via putatively similar mechanisms involving metal radicals, they are very different in many aspects, e.g., their distribution among methanotrophic strains, activity, and localization in the cell, structure, and regulation Kinetic studies on purified sMMO have been carried out for many years but the exact mechanism of pMMO, the more prevalent form, is still unknown, largely due to the difficulty in isolation of this enzyme with high activity

Almost all known methanotrophs possess pMMO except members of the genus

Methylocella which are facultative methylotrophs, able to utilize multi-carbon substrate (Dedysh et al., 2005) Whereas, the sMMO is present just in a few MOB species of the genera Methylococcus, Methylomonas, Methylocella, Methylocystis and Methylosinus (Hanson & Hanson, 1996; McDonald et al., 2006) Many methanotrophs, such as Methylomicrobium album BG8 possess only the pMMO, but Methylococcus capsulatus Bath, Methylosinus trichosporium OB3b, and some others

can express either form, depending on the copper concentration in the medium In those methanotrophs, there is a metabolic switch mediated by the availability of copper ions When cells are starved for copper, and the copper-to-biomass ratio is low, sMMO is expressed On the other hand, cells grown under conditions of excess copper

express pMMO and there is no detectable sMMO expression (Murrell et al., 2000) No

evidence of sMMO has been found in acidophilic methanotrophs in the

Verrucomicrobia phylum (Dunfield et al., 2007; Hou et al., 2008; Islam et al., 2008)

Crenothrix polyspora that was detected 135 years ago has been discovered to be a methane oxidizer with an unusual MMO (Stoeker et al, 2006)

Besides the remarkable ability to convert the unreactive methane to methanol, MMOs can also oxidize other organic substrates even though for these organisms, the resultant oxidation products cannot be used as nutrient sources

1.4.2 Soluble methane monooxygenase (sMMO)

sMMO is expressed during growth under low copper–to–biomass conditions and utilizes NADH and H+ as the electron donor This enzyme is a complex of three components (i) non-hemehydroxylase (245 kDa), (ii) a regulatory coupling protein B, and (iii) a reductase (protein C) The hydroxylase is a dimer of three different subunits (α, β and γ, of 60, 45 and 20 kDa, respectively) and has a non-heme di-iron active site

in the α-subunit where methanol is formed from methane and oxygen The reductase enzyme (protein C) transfers electrons to the hydroxylase for the catalysis of methane oxidation and the coupling protein (protein B) links two these enzymes The genes

encoding sMMO have been cloned and sequenced in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b These genes are clustered on the chromosome

in which the α, β and γ subunits of the hydroxylase are coded by mmoXYZ, respectively; mmoB and mmoC code for protein B and protein C, respectively (Coufal

et al., 2000; Cardy et al., 1991) (Fig.1.5.) Merkx et al (2002) detected gene orfY (mmoD) that might play a role in the assembly of the di-iron center of the enzyme in Methylococcus capsulatus Bath.

Figure 1.5 Orientation of soluble mono-oxygenase gene cluster (Murrell et al., 2000)

sMMO has abroad substrate specificity and can co-oxidize a number of other aromatic compounds and hydrocarbons (Hanson & Hanson, 1996) The substrates of sMMO include alkanes, alkenes, alicyclic hydrocarbons, halogenated aliphatics, and aromatic compounds with single oxygenated products generally predominating Some small organic compounds that found to be effective substrates for sMMO are tetrachloromethane, iodomethane, trimethylamine and tetrachloroethene

Figure 1.6 The crystal structure of hydroxylase dimer with cylinders representing helices and

arrows representing β-strands α-chains are colored yellow and pink, β-chains are colored

gray and cyan, and γ-chains are colored green (Elango et al., 1997)

1.4.3 Particulate methane monooxygenase (pMMO)

pMMO is a membrane – bound enzyme containing copper and iron and is expressed only when the copper supply in the medium is high (Prior & Dalton, 1985) Like sMMO, pMMO consists of three subunits including α or pmoA subunit (26 kDa), β or

pmoB subunit (47 kDa) and γ or pmoC subunit (23 kDa), encoded by pmoA, pmoB and pmoC genes, respectively There are two nearly identical copies of pmoCAB cluster in the chromosome of Methylococcus capsulatus Bath, Methylocystis sp strain M and M.trichosporium OB3b (Semrau et al., 1995; Stolyar et al., 1999; Gilbert et al., 2000) Only Methylococcus capsulatus Bath has a third separate copy of pmoC and the pmoC3 sequence was more divergent from the two others (Stolyar et al., 1999)

Figure 1.7 Particulate methane monooxygenase gene cluster of methane-oxidizing bacteria

(Murrell et al., 2000)

Although numerous efforts have been done in the last 20 years, most of the information of this predominant methane monooxygenase had remained unanswered until Lieberman and Rosenzweig (2005) reported the crystal structure of this

membrane protein from M.trichosporium OB3b The crystal structure of pMMO from

this strain was obtained to a resolution of 2.8 Ao The enzyme is now known to be a trimer with α3β3γ3 polypeptide arrangement and has three metal centers (Fig 1.8)

Figure 1.8 Crystal structure of a single promoter of pMMO PmoA, PmoB, and PmoC are shown in magenta, 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 methane, short linear 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)

1.5 Application 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 of this protein for use as sustainable sources in 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 as well the later on a number of animal species such as pigs, mink, fox, dogs, etc fed with bacterial protein meal suggested that the protein derived from natural gas fermentation were useful sources and could be utilized as a future protein for animal

production (Skrede et al., 1998, 2003; Overland et al., 2001, 2005, 2006, 2010; Skrede, Ahlstrom, 2002; Hellwing et al., 2005, 2006, 2007a,b,c; Schoyen et al., 2005, 2007a,b,c; Aas et al., 2006a,b,c)

Commercial BPM using Methylococcus capsulatus Bath was obtained by

continuous aerobic fermentation utilizing 2 m3 of methane per kg of biomass dry

matter, corresponding to approximately 1.7 kg methane per kg crude protein (Bothe et al., 2002) Ralstonia sp., Brevibacillus agri and Aneurinibacillus sp were added in order to enable the growth in continuous system (Bothe et al., 2002) BPM produced

from methane is used in breeding because of its nutrition value that determined by the protein contents and lesser extent lipids The crude protein, fat and ash contents are

approximately 70%, 10% and 7%, respectively (Skrede et al., 1998) Chemical and

amino acids composition of BPM are similar to that of fishmeal and soybean meal as listed in table 1.2

Table 1.2 Chemical and amino acid composition of BPM, fishmeal and soybean meal (SBM) The chemical composition is given as g/100 g and amino acids as g/16 g N (Hellwing

et al., 2005)

Other nutritional values such as digestibility, quantitative energy, protein and nucleic acid metabolism, growth performance and health of different animals fed with BPM have been surveyed and reported in detail The reported data shown that the digestibility of amino acids was reduced by microbial membrane and cell wall

components (Rumsey et al., 1991) This problem can be solved via autolysis and hydrolysis followed by filtration to remove membrane fraction (Schoyen et al., 2005)

Decomposition of dietary RNA and DNA also increases the digestibility of nucleotides

in microbial meal up to 100% when pigs fed with BPM The energy provided by BPM has been utilized as well as that of usual diets based on the data on heat production and

energy retention Also BPM is safe because the production bases on non-pathogenic bacteria and the heat treatment in downstream processing would be an additional step for eliminating contaminations

In Vietnam, the livestock industry has been strongly developed with a growth rate of 6% per year within recent 10 years However, raw materials for animal food industry are mainly imported Of nearly 8.9 tons of imported raw material in 2011, protein – rich materials occupied 3.86 tons (about 43%) The predictive demand of animal food will be 27.4 tons in 2020 (Livestock Newsletter, 2012) Thus, accordant demand in protein sources will increase significantly, leading to an urgent requirement for local production of this source to replace the unstable and expensive imported sources

On the other hand, methane as a main component in biogas produced from anaerobic fermentation of organic wastes has yet not been used effectively in Vietnam Except a small amount that has been used as burning fuel or for generating electricity, most of biogas methane is still let to enter the atmosphere as a component of greenhouse gases Thus, the use of MOB in conversion of methane to valuable, protein rich biomass for animal feeding could be a good solution for both purposes, reducing the methane emission and providing protein source for the livestock industry

1.5.2 Bioconversion of methane to methanol

Methanol or wood alcohol is perhaps one of the most industrially important chemicals Methanol is a major raw material for petrochemical production and now is a convenient liquid fuel because of several unique properties and characteristics such as dispersing easily in the environment, rapid biodegradation, high solubility in water, non carcinogenicity and mutagenicity, easy transportation, high hydrogen–to–carbon ratio, high–energy flame, clean burning, etc (Dave, 2008) In the industry, methanol

is used as starting material for the synthesis of other relevant compounds, including formaldehyde (occupying 32% of total methanol consumed in 2011), dimethyl ether, and acetic acid Besides, methanol is also used in construction, electronic, packaging… In 2011, the global consumption of methanol is over 50 million metric tons and is expected to reach over 90 million metric tons by 2016 (Johnson, 2012) This steadily increasing is mainly due to the expanded formaldehyde production

Methanol can be produced from various feedstock resources It is mainly synthesized from natural gas (mostly methane), which improves the overall yields substantially Methanol can also be produced from coal; biogas as well as renewable resources like forest thinning, agricultural waste and even directly from CO2 captured from power plant and factory emissions Generally, the chemical synthesis of methanol requires high temperature (840 – 900 °C) and high pressure (15 – 30 at) with

a copper catalyst The production process therefore is costly and causes pollution

By using methanotrophs, methanol however can be produced at ambient temperature and pressure (Mehta, 1991; Xin, 2004) These bacteria metabolize methane to cell biomass and CO2 with methanol as an intracellular intermediate Via manipulation of the enzymatic reactions in the metabolic pathway to favor methane monooxygenase (MMO) and/or to inhibit methanol dehydrogenase (MDH), methanol can be obtained extracellularly These two effects can be usually achieved by adding

appropriate chemicals Thus, Corder et al demonstrated that with the obligatory

methanotrophs isolated from digester sludge, an accumulation of methanol up to 1 g/l

in 24 hours after growth started in the reactors (Corder et al., 1986) In order to

prevent complete oxidation of methanol to CO2, the gas phase was switched to 100%

methane instead of the 20:80 methane/air mixture With the same target, Sugimori et

al used the cell suspension treated with cyclopropanol, a selective and irreversible inhibitor for MDH, to produce methanol by Methylosinus trichosproium OB3b The

methanol production yield per methane consumed went up to 71%, reaching methanol

25 mmol/g⋅wet wt⋅cell after 120 hours fermenting (Sugimori et al., 1995)

Dithiothreitol, phenylhydrazine, iodacetate, cyclopropane and EDTA can also act as

MDH inhibitors as well Kim et al (2010) used a 3-liter cylindrical reactor with EDTA

as MDH inhibitor to obtain methanol at the total yield of 13.7 mM after 16 h The 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 change and bioremediation

1.5.3.1 Biological mitigation of methane emission

The total amount of methane released to the atmosphere is about 5200 Ton/year, 90%

of which is oxidized by photochemical processes in the troposphere, and around 10%

is biologically removed from soil or water, mainly by methanotrophs (Reeburgh et al., 1993; Breas et al., 2001) It is estimated that about 70% of the global methane

emission are anthropogenic in which landfill and coalmine gas are the two most important sources

The typical composition of gas produced in a landfill contains 30 – 70% (v/v) methane, 20 – 50% CO2 (Nikiema et al., 2007) By providing optimum conditions for microbial processes and by collecting landfill gas, a number of biological systems have been developed to mitigate the landfill methane emission, e.g long-term biocovers, passively or actively vented biofilter, biowindows, daily–used biotarps

(Huber-Humer et al., 2008) With increasing use of gas collecting systems, biofilter

might be more feasible because of their small footprint and high removal capacity for gases (IPCC 2007)

Figure 1.9 The schematic bench scale plant for treatment of diluted landfill gas in biofilters

1-methane; 2-pressurised air; 3-air humidifying scrubber; 4-biofilters filled with different

biofilter materials; 5-exhaust (Streese et al., 2005).

Biofiltration is an engineered biological treatment process that utilizes the metabolic activity of microorganisms attached onto a variety of packing materials to treat a wide range of organic and inorganic contaminants (Cohen, 2001) In a biofilter, contaminants in the gas phase diffuse into biofilms, a thin layer of a microbial consortium on the packing material, to be consumed by microorganisms therein Biofilters are filled with a variety of packing materials made of natural and synthetic materials e.g soil, compost, wood fibers, peat etc to provide the surface area for establishment of biofilms (Fig.1.10) These tools help to surmount the disadvantage of the use of methanotrophs that demand high concentration of atmosphere methane for

growth Yoon et al designed a model of biotrickling filtration that removes methane at

concentrations greater than 500 ppmv; however, economy is not feasible at current

stage (Yoon et al., 2010)

Figure 1.10 The schematic biofilter (Huang et al., 2010)

Coalmine gas is also a complicated mixture of gases with high concentrations

of methane Emission from coalmines estimated at around 50 – 300 Ton/year (Breas et al., 2001) resulted mainly from desorption of methane during mining, crushing and

inefficient combustion Beside greenhouse effect, coalmine gas can also cause explosions Biological method using methanotrophs has been used to control the coalmine gas, which more economic and efficient than seam gas drainage and air

ventilation The potential of using Methylomonas methanica to remove methane from

coalmine atmosphere was investigated in a bench – scale bioreactor, and 90.4% of the

methane in a 35% methane/air mixture was removed in 24 h (Apel et al., 1991) In another study, Methylomonas fodinarum ACM 3268 was used in a continuous

biofilter Thus, blowing a mixture of 0.25 – 1% methane (v/v) in air, which is common

in coalmine atmosphere, through a biofilter containing M fodinarum resulted in

removal of more than 70% of the methane within 15 min residence time and of 90%

over 20 min (Sly et al., 1993) However, the biological method has not been widely adopted, especially for in situ treatments due to low methane – oxidizing capacity and

operational difficulties

In Vietnam, the average amount of solid waste per year is 25.000 tons, which is

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 with that, other important sources 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 effects to both environment and human health These substances are generally known to resist in conventional biological wastewater treatment processes Aerobic biodegradation of halogenated compounds has been widely studied and seems more effective in comparison to anaerobic degradation that does not complete dechlorination and accumulates more toxic intermediates (such as vinyl chloride in the case of the

reductive dechlorination of tetrachloro-ethylene) (Maymo-Gatell et al., 1999)

Methanotrophs are capable of aerobically degrading halogenated pollutants via oxidation mechanism Due to the omnipresence in various environments, these organisms have been widely applied to decontaminate sites polluted with chlorinated

co-ethenes (Hanson, Hanson, 1996; Semrau et al., 2010)

The treatment processes using methanotrophs can be in situ (in the field) or ex situ (using bioreactors) In situ bioremediation is usually applied in the treatment of

contaminated soil and groundwater systems Indigenous MOBs are stimulated by adding methane and other nutrients and thus enhances the performance of co- metabolic biodegradation of halogenated aliphatics All the primary substrate used for

biostimulation must be safe and cheap (Pfiffner et al., 1997)

Figure 1.11 Horizontal injection extraction of methane, air, and nutrient used in in-situ

bioremediation of TCE Methane, air, and/or nutrients are injected into the lower horizontal well The upper horizontal well extracts air and gases from the vadose zone under vacuum conditions Any alternative gases collected are treated by the catalytic oxidizer and released

into the atmosphere (Hazen et al (1994).

The ex situ bioremediation can be operated in single-stage and multi-stage

bioreactors In single-stage bioreactors, growth on primary substrate (methane) and degradation of contaminants occur in one reactor and methanotrophs are either entrapped in beads or attached to some support media to retain the cells within the bioreactors These include mix tank, expanded-bed reactors, packed-bed reactors,

membrane - attached biofilm reactors, immobilized soil bioreactors… (Karamanev et al., 1998; Ramsay et al., 2001) In multi-stage bioreactors, the growth and the co–

metabolic biodegradation of pollutants take place in different sectors, where the competitive inhibition between the growth substrate and contaminants can be avoided

(Smith et al., 1997)

The degradation of pollutants to various degrees depends on sMMO activity, a highly non–specific enzyme that can insert oxygen into a wide variety of non–growth compounds even in obligate methanotrophs Such oxygenation reactions include hydroxylation of n–alkanes such as ethane, the epoxidation of n–alkenes such as ethylene and dechlorinations of aliphatic and aromatic substances like chloromethanes

(Hou et al., 1979) Of the factors effecting biodegradation by methanotrophs, the form

of MMO expressed decides the rate and range of pollutants degraded expressing cells typically degrade more compounds and with faster initial rates than

sMMO-pMMO-expressing cells (Collby et al., 1977; Burrows et al., 1984) However,

expression of sMMO is usually switched off due to high concentration of copper in contaminated areas In addition, when the concentration of vinyl chloride, trans-dichloroethylene and trichloroethylene above 10 mM, methanotrophs expressing pMMO have higher growth rates but degrades less of these compounds If the concentration was increased to 100 mM, both growth and degradation efficiency of

pMMO-expressing cells increased (Lee et al., 2006) Thus, the determining

appropriate form of MMO should be considered for assessment of relative rates of growth substrate and non-growth pollutant consumption

There exists competition between pollutants and growth substrate, methane, for binding to the MMOs in the bioremediation process by methanotrophs This

competition can be minimized via using facultative methanotroph, Methylocystis strain

SB2 that constitutively express pMMO, to degrade a variety of chlorinated

hydrocarbons when grown on acetate or ethanol (Im & Semrau, 2011; Yoon et al.,

2011) As the result, competition for binding to the MMO of substrate and pollutants can be minimized and it becomes unnecessary to control methane in the cleaning up process

Acidophilic and thermo-acidophilic methanotrophs have a great potential in bioremediation due to their survival in extreme conditions Further, many acidophilic

strains and thermoacidophilic are facultative, making them more applicable for

pollutant degradation (Dunfield et al., 2010; Khadem et al., 2011)

1.6 Objectives of the study

Since aerobic methane 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 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 strains

Chapter 2 MATERIAL AND METHODS 2.1 Sampling

Soil and wastewater samples were collected in three different places in Vietnam A soil sample (PS) was taken from the upper 20 cm in the flooded paddy field in Hungyen province (in May 2011) Two wastewater samples were collected at wastewater treatment systems Hanoi Liquor Company (Yenphong industrial zone – Bacninh province) (W) and at a biogas treatment system (Socson district, Hanoi) (BG)

in December 2011 pH of two wastewater samples was 7, whereas soil sample had pH

6 These samples were stored at 4 oC before use

2.2 Isolation of methanotrophs

Methanotrophs were grown in mineral media supplemented with trace metal and vitamin mix solutions (Table 2.1 and 2.2)

Table 2.1 Fresh water mineral medium

Table 2.2 Metal mix and vitamin mix (Fuse et al., 1998)

Cultural medium contains 10 mg NH4NO3, 1 mg KH2PO4, 0.25 mg Fe-EDTA, 0.25 ml vitamin mix, 0.1 ml metal mix and 99.65 ml fresh water mineral medium pH was adjusted to 6.8 − 7

Enrichment

Freshly collected water and soil samples were used as inoculums for the enrichment of MOBs Three ml of wastewater samples or 3 g of soil were added to 30 ml of cultural medium in 150 ml serum bottles Afterward, the bottles were sealed with rubber stoppers and aluminum caps, injected with methane (40 ml in each bottle) and incubated at 30 0C with moderate shaking at 100 rpm Methane ratio in headspace of the serum bottles was determined with gas chromatography (Agilent 7890A GC) Subculturing was carried out after every 7 days of incubation Size of inoculum was 10% v/v For each sample, three subculturing steps were performed and cultures from

the last transfer were used for isolation of MOBs

Isolation of methanotrophs

Since MOBs grow poorly on solid media, the isolation was carried out on the liquid medium via serial dilution method Thus, 0.1 ml of the 3rd enrichment cultures was carefully mixed with 19.9 ml of liquid mineral medium and diluted in serial dilution with four steps of 1:20 dilution Aliquots (0.3 ml) of the mixture at every dilution step were then filled in wells of 96-well plates (with duplication) Afterward, the plates were placed in a chamber which was then filled with methane/air mixture (ratio 1:2, vol/vol) and incubated at 30 °C in the dark for 1-2 weeks MOB growth was checked via OD600nm measurement using the plate reader Benchmark Plus (BioRad, USA) every

3 days Samples at the highest dilution that showed turbidity were used for the next dilution series After several repetitions of this purification procedure, pure cultures of MOB could be obtained

Purity of the isolates was checked with a phase contrast microscopy (Axiophot, Zeiss) and PCR-DGGE analysis of the 16S rDNA

The isolates were maintained in gas-tight Hungate tubes and stored at 4 °C under a methane/air atmosphere and transferred into fresh medium every 8 weeks

2.3 DNA extraction and PCR amplification

2.3.1 DNA extraction

Total DNA was extracted from enrichment cultures and pure strains using the method

described by Zhou et al (1996) with some modifications Steps were as follow:

- To the mixture of 2 ml enrichment samples and 5.4 ml extraction buffer (100 mM Tris pH 8; 100 mM EDTA pH 8; 120 mM phosphate buffer (Na2HPO4/NaH2PO4); 1,5 M NaCl; 1% CTAB) 40 µl Proteinase K was added, shake horizontally (225 rpm) at 37 0C for 30 min

- Add 600 µl 20% SDS, incubate at 65 °C for 2 h with gently end over end inversion every 25 min

- Add an equal volume of chloroform:isoamylalcohol (24:1, v/v), mix thoroughly and centrifuge at 6000 g for 10 min (room temperature)

- Collect aqueous phase and precipitate DNA with 0.6 volume of isopropanol, incubated at RT for 1 h and harvest the DNA by centrifugation at 12000xg for 25 min at RT

- Decant supernatant and wash pellet with 10 ml cold 70% (v/v) ethanol Centrifuge

at 12000xg for 15 min at RT

- Decant supernatant and dry pellet in air

- Suspend the DNA in 30-50 µl sterilized MQ water and stored at -20 °C

- Check the quality and concentration of the extracted DNA on 1% agarose gel in 1% TAE After electrophoresis, the agarose gel was stained in ethidium bromide

solution (5 mg/ml) for 10 min and observed under UV light with the GelDoc (BioRad, USA)

2.3.2 PCR amplification

Amplification of the 16S rDNA genes

V3 – V5 region (550 bp) of the 16S rDNA genes (Murray et al., 1996) and nearly full

length of 16S rDNA genes (1500 bp) were amplified using primer pairs GM5F

(CCTACGGGAGGCAGCAG) - 907R (CCGTCAATTCCTTTRAGTTT) (Muyzer et al., 1993) and 27f (AGAGTTTGATCMTGGCTCAG) (Edwards et al., 1989) - 1492r (TACGGYTACCTTGTTACGACTT) (Weisburg et al., 1991) To the 5’-end of primer GM5F, a 40-bp GC clamp (CGCCCGCCGCGCGCGGCGGCCGGGGCGGGGGCA-CGGGGGG) was added to stabilize migration of DNA fragments in the denaturing gradient gel

Hot Taq and touchdown PCR (Don et al., 1991) were applied in amplification

reactions to reduce primer-dimer complexes The reaction mixture (50 µl) contained 5

µl of 10 × Taq buffer, 2.5 mM of each deoxynucleotide, 50 pM of each primer, 5 U of Taq DNA polymerase (Promega) and 1 µl (∼ 50 ng) of template DNA

The thermo cycles for touchdown PCR was as follows: initial denaturation at

94 °C for 2 min 30 s and then at 94°C for 40 s, followed by touchdown primer annealing from 65 to 55 °C (the annealing temperature was decreased 0.5 °C for each cycle for the first 17 cycles to 55°C), followed by extension at 72°C for 1 min (for each of the 17 cycles) Then 15 cycles of 94 °C for 40 s, 55 °C for 1 min, and 72 °C for 1 min 30 s were carried out Afterward, 10 more cycles were performed at 94 °C for 40 s, 55 °C for 1 min 15 s, and 72 °C for 2 min The final extension step was at 72

°C for 7 min 30 s

Thermo cycles for the PCR to obtain full-length 16S rDNA gene sequences included an initial denaturation at 94 °C for 3 min, followed by 35 cycles at 94 °C for