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

LIEGE UNIVERSITY
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VIETNAM NATIONAL UNIVERSITY, HANOI
Institute of Microbiology and Biotechnology
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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


 

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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


 

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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


 

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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


 

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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 7
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.
 


 

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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. ………..
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


 

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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


 

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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


 

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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 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


 

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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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REFERENCES
1.

Aas TS, Grisdale-Helland B, Terjesen BF, Helland SJ (2006a) Improved growth and
nutrient utilization in Atlantic salmon (Salmon salar) fed diets containing a bacterial
protein meal. Aquaculture 259:365-376.

2.

Aas TS, Hatlen B, Grisdale-Helland B, Terjesen BF, Bakke-McKellep AM, Helland
SJ (2006b) Effect of diets containing a bacterial protein meal on growth and feed
itilisation in Rainbow trout (Oncorhynchus mykiss). Aquaculture 261:357-368.

3.

Aas TS, Hatlen B, Grisdale-Helland B, Terjesen BF, Penn M, Bakke-McKellep AM,
Helland SJ (2006c) Feed intake, growth and nutrient utilization in Atlantic halibut
(Hippoglossus hippoglossus) fed diets containing a bacterial protein meal. Aquacult
Res. 38:351-360.

4.

Apel W (1991) Use of methanotrophic bacteria in gas phase bioreactors to abate
methane in coal mine atmospheres. Fuel 70:1001–1003.

5.

Bodrossy L, Holmes EM, Holmes AJ, Kovacs KL, Murrell JC (1997) Analysis of 16S
rRNA and methane monooxygeanse gene sequences reveals a novel group of
thermotolernat and thermophilic methanotroph, Methylocaldum gen. nov. Arch
Microbiol. 168: 493-503.

6.

Bodrossy L, Kovács KL, McDonald IR, Murrell JC (1999) A novel thermophilic
methane-oxidising γ-Proteobacterium. FEMS Microbiol Lett. 170: 335-341.

7.

Bodrossy L, Murrell JC, Dalton H, Kalman M, Puskas LG, Kovacs KL (1995) Heattolerant methanotrophic bacteria from the hot water effluent of a natural gas field.
Appl. Environ. Microbiol. 61:3549–3555.

8.

Boleva SE, Baani M, Suzina NE, Bodelier PLE, Liesack W, Dedysh SN (2011)
Acetate utilization as a survival strategy of peat-inhibiting Methylocystis spp. Environ.
Microbiol. Rep. 3:36-46.


 

60
 



 

 

 

9.

Bothe H, Jensen KM, Mergel A, Larsen J, Jørgensen C, Bothe H, Jørgensen L (2002)
Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath)
in a single cell protein production process. Appl Microbiol Biotechnol. 59:33–39

10. Bowman J (2000) The methanotrophs - the families Methylococcaceae and

Methylocystaceae. The Prokaryotes. Edited by Dworkin M, Springer, New York.
11. Bowman JP, McCammon SA, Skerratt JH (1997) Methylosphaera hansonii gen. nov.,
sp. nov., a psychrophilic, group I methanotroph from antartic marine-salinity,
meromictic lakes. Microbiology. 143: 1451-1459
12. Bowman JP, Sly LI, Nichols PD, Hayward AC (1993) Revised taxonomy of the
methanotrophs: description of Methylobacter gen. nov., emendation of Methylococcus,
validation of Methylosinus and Methylocystis species, and a proposal that the family
Methylococcaceae includes only the group I methanotrophs. Int J Syst Bacteriol. 43:
735.
13. Bowman JP, Sly LI, Stackebrandt E (1995) The phylogenetic position of the family
Methylococcaceae. Int J Syst Bacteriol 45: 182-185.
14. Breas O, Guillou C, Reniero F, Wada E (2001) The global methane cycle: isotopes
and mixing ratios, sources and sinks. Isotopes Environ. Health Stud. 37: 257–379.
15. Burrows KJ, Cornish A, Scott D, Higgins IJ (1984) Substrate specificities of the
soluble and particulate methane mono-oxygenase of Methylosinus trichosporium
OB3B. J. Gen. Microbiol. 130:3327-3333.
16. Bussmann I, Rahalkar M, Schink B (2006) Cultivation of methanotrophic bacteria in
opposing gradients of methane and oxygen. FEMS Microbiol Ecol. 56: 331- 344.
17. Cardy DLN, Laidler V, Salmond GPC, Murrell JC (1991) Molecular analysis of the
methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b.
Mol Microbiol. 5: 335-342.
18. Cohen Y (2001) Biofiltration - the treatment of fluids by microorganisms immobilized
into the filter bedding material: a review. Biores Technol 77:257-274.


 

61
 

 

 

 

19. Colby J, Stirling DI, Dalton H (1977) The soluble methane mono-oxygenase of
Methylococcus capsulatus (Bath) - its ability to oxygenate n-alkanes, n-alkenes,
ethers, and alicyclic aromatic and heterocyclic compounds. Biochem J. 165: 395-402.
20. Corder RE, Johnson ER, Vega JL, Clausen EC, Gaddy JL (1986) Biological
production of methanol from methane. Department of Chemical Engineering,
University of Arkansas.
21. Costello AM, Lidstrom ME (1999) Molecular characterization of functional and
phylogenetic genes from natural populations of methanotrophs in lake sediments.
Appl. Environ. Microbiol. 65:5066–5074
22. Coufal DE, Blazyk JL, Whittington DA, Wu WW, Rosenzweig AC, Lippard SJ (2000)
Sequencing and analysis of the Methylococcus capsulatus (Bath) soluble methane
monooxygenase genes. Eur. J. Biochem. 267:2174–2185.
23. Dalton H (2005) The Leeuwenhoek Lecture 2000 - The natural and unnatural history
of methane-oxidizing bacteria. Philosophical Transactions of The Royal Society B
360: 1207-1222.
24. Dave BC (2008) Prospects for Methanol Production. Bioenergy. Chapter 19:235-245.
25. Dedysh SN (2011) Methyloferula stellate gen.nov., sp. nov., an acidophilic, obligatory
methanotrophic bacterium possessing only a soluble methane monooxygenase. Int. J.
Syst. Evol. Microbiol.
26. Dedysh SN, Knief C, Dunfield PF (2005) Methylocella species are facultatively
methanotrophic. J Bacteriol 187: 4665-4670.
27. Dedysh SN, Liesack W, Khmelenina VN, Trotsenko YA, Semrau JD, Bares AM,

Panikov NS, Tiedje JM (2000) Methylocella palustris gen.nov., sp.nov., a new
methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype
of serine-pathway methanotrophs. Int J Syst Evol Microbiol 50: 955-969.


 

62
 



 

 

 

28. DiSpirito AA, Gulledge J, Murrell JC, Shiemke AK, Lidstrom ME, Krema C (1992)
Trichloroethylene oxidation by the membrane-associated methane monooxygenase in
type I, type II, and type X methanotrophs. Biodegradation 2: 151-164.
29. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991) ’Touchdown’ PCR to
circumvent spurious priming during gene amplification. Nucleic Acids Res. 19:4008
30. Dunfield PF, Belova SE, Vorob'ev AV, Cornish SL, Dedysh SN (2010) Methylocapsa
aurea sp. nov., a facultatively methanotrophic bacterium possessing a particulate
methane monooxygenase. Int J Syst Evol Microbiol.
31. Dunfield PF, Khmelenina VN, Suzina NE, Trotsenko YA, Dedysh SN (2003)
Methylocella silvestris sp. nov., a novel methanotroph isolated from an acidic forest
cambisol. Int J Syst Evol Microbiol 53: 1231-1239.
32. Dunfield PF, Yuryev A, Senin P, Smirnova AV, Stott MB, Hou, S (2007) Methane

oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia.
Nature 450: 879-882.
33. Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct
complete nucleotide determination of entire genes. Characterization of a gene coding
for 16S ribosomal RNA. Nuc Acids Res 17: 7843-7853.
34. Elango NA, Radhakrishnan R, Froland WA, Wallar BJ, Earhart CA, Lipscomb JD,
Ohlendorf DH (1997) Crystal structure of the hydroxylase component of methane
monooxygenase from Methylosinus trichosporium OB3b. Protein Sci 6:556-568.
35. Felsenstein J (1985) Confidence limit on phylogenies: an approach using the boottrap.
Evolution 39: 783 – 791.
36. Fliermans CB, Phelps TJ, Ringelberg D, Mikell AT, White DC (1988) Mineralization
of trichloroethylene by heterotrophic enrichment cultures. Applied and Environmental
Microbiology 54:1709-1714 (1988).


 

63
 



 

 

 

37. Foster JW, Davis RH (1966) A methane-dependent coccus, with notes on
classification and nomenclature of obligate, methane-utilizing bacteria. J Bacteriol 91:

1924-1931.
38. Fuse H, Ohta M, Takimura O, Murakami K, Inoue H, Yamaoka Y, Oclarit JM, Omori
T (1998) Oxidation of trichloroethylene and dimethyl sulfide by a marine
Methylomicrobium strain containing soluble methane monooxygenase. Biosci.
Biotechnol. Biochem. 62:1925–1931.
39. Gilbert B, McDonald IR, Finch R, Stafford GP, Nielsen AK, Murrell JC (2000)
Molecular analysis of the pmo (particulate methane monooxygenase) operons from
two type II methanotrophs. Appl Environ Microbiol 66: 966-975.
40. Hanson RS, Hanson TE (1996) Methanotrophic bacteria. Microbiol Rev 60, 439-471.
41. Hazen TC, Lombard KH, Looney BB, Enzien MV, Dougherty JM, Fliermans CB,
Wear J, Eddy-Dilek CA (1994) Summary of in-situ bioremediation demonstration
(methane biostimulation) via horizontal wells at the Savannah River Site integrated
demonstration project. (pgs. 137-150). IN: In-Situ Remediation: Scientific Basis for
Current and Future Technologies. Battelle Press, Columbus, Ohio.
42. Hellwing ALF, Tauson AH, Ahlstrøm Ø, Skrede A (2005) Nitrogen and energy
balance in growing mink (Mustela vison) fed different levels of bacterial protein meal
produced with natural gas. Archives of Animal Nutrition 59:335-352
43. Hellwing ALF, Tauson AH, Kjos NP, Skrede A (2007a) Bacterial protein meal effects on protein and energy metabolism in pigs. Animal 1:45-54.
44. Hellwing ALF, Tauson AH, Skrede A (2006) Effect of bacterial protein meal on
protein and energy metabolism in growing chickens. Archives of Animal Nutrition
60:365-381
45. Hellwing ALF, Tauson AH, Skrede A (2007c) Excretion of purine base derivatives
after intake of bacterial protein meal in pigs. Livest Sci. 109:70-72.


 

64
 

 

 

 

46. Hellwing ALF, Tauson AH, Skrede A, Kjos NP, Ahlstrøm Ø (2007b) Bacterial protein
meal in diets for pigs and mink: Comparative studies on protein turnover rate and
urinary excretion of purine base derivate. Arch Anim Nutrit 61:425-443.
47. Heyer J, Berger U, Hardt M, Dunfield PF (2005) Methylohalobius crimeensis gen.
nov., sp. nov., a moderately halophilic, methanotrophic bacterium isolated from
hypersaline lakes of Crimea. Int. J. Syst. Evol. Microbiol. 55:1817–1826.
48. Holmes AJ, Owens NJP, Murrell JC (1995) Detection of novel marine methanotrophs
using phylogenetic and functional gene probes after methane enrichment.
Microbiology 141, 1947-1955.
49. Holmes AJ, Owens NJP, Murrell JC (1995) Detection of novel marine methanotrophs
using phylogenetic and functional gene probes after methane enrichment.
Microbiology 141, 1947-1955.
50. Hou CT, Patel R, Laskin AI, Barnabe N (1979) Microbial oxidation of gasnous
hydrocarbons: epoxidation of C2 to C4 n-alkenes by methanotrophic bacteria. Appl
Environm Microbiol 38:127-134.
51. Hou S, Makarova K, Saw J, Senin P, Ly B, Zhou Z (2008) Complete genome
sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum
infernorum, a representative of the bacterial phylum Verrucomicrobia. Biol Direct 3:
26-50.
52. Huang Q, Zhang Q, Cicek N, Mann DD (2010) Biofilters For Mitigating Methane
Emission From Covered Hog Manure Storage. Manitoba Sustainable Agriculture
Practices Program (Msapp) Research & Development Projects 2009-2010 Final

Report.
53. Huber-Humer M, Gebert J, Hilger H (2008) Biotic systems to mitigate landfill
methane emissions, Waste Manag. Res. 26:33–46.


 

65
 



 

 

 

54. Iguchi H, Yurimoto H, Sakai Y (2011) Methylovulum miyakonense gen. nov., sp. nov.,
a type I methanotroph isolated from forest soil. Int. J. Syst. Evol. Microbiol. 61:810–
815.
55. Im J, Lee SW, Yoon S, DiSpirito AA, Semrau JD (2011) Characterization of a novel
facultative Methylocystis species capable of growth on methane, ethanol, and acetate.
Environ. Microbiol. Rep. 3:174-181.
56. Im J, Semrau JD (2011) Pollutant degradation by Methylocystis strain SB2 grown on
ethanol: bioremediation via facultative methanotrophy. FEMS Microbiol. Lett.
318:137-142.
57. Islam T, Jensen S, Reigstad LJ, Larsen Ø, Birkeland NK (2008) Methane oxidation at
55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia
phylum. Proc Nat Acad SciUSA 105: 300-304.

58. Kaluzhnaya M, Khmelenina V, Eshinimaev B, Suzina N, Nikitin D, Solonin A, Lin
JL, McDonald I, Murrell C, Trotsenko Y (2001) Taxonomic characterization of new
alkaliphilic and alkalitolerant methanotrophs from soda lakes of the Southeastern
Transbaikal region and description of Methylomicrobium bury- atense sp. nov. Syst.
Appl. Microbiol. 24:166–176
59. Karamanev D, Samson R (1998) High-rate biodegradation of pentachlorophenol by
biofilm developed in the immobilized soil bioreactor. Environ. Sci. Technol. 32:994–
999.
60. Karl TR, Trenberth KE (2003) “Modern global climate change”. Science302 (5651):
1719–1723.
61. Khadem AF, Pol A, Wieczorek A, Mohammadi S, Francoijs KJ, Stunnenberg HG,
Jetten MSM, Op den Camp HJM (2011) Autotrophic methanotrophy in
Verrucomirobia: Methylacidiphilum fumariolicum SolV uses the Calvin-BensonBassham cycle for carbon dioxide fixation. J. Bacteriol. 193:4438-4446.


 

66
 



 

 

 

62. Khmelenina VN, Kalyuzhnaya MG, Starostina NG, Suzina NE, Trotsenko YA (1997)
Isolation and characterization of halotolerant alkaliphilic methanotrophic bacteria from

Tuva soda lakes. Curr Microbiol 35: 257-261.
63. Kim HG, Han GH, Kim SW (2010) Optimization of Lab Scale Methanol Production
by Methylosinus trichosporium OB3b. Biotechnology and Bioprocess Engineering
15:476-480
64. Lee SW, Keeney DR, Lim DH, Dispirito AA, Semrau JD (2006) Mixed pollutant
degradation by Methylosinus trichosporium OB3b expressing either soluble or
particulate methane monooxygenase: can the tortoise beat the hare? Appl Environ
Microbiol 72: 7503-7509.
65. Lelieveld J, Crutzen PJ, Bruhl C (1993) Climate effects of atmospheric methane.
Chemosphere 26, 739-768.
66. Lelieveld J, Crutzen PJ, Dentener FJ (1998) Changing concentration, lifetime and
climate forcing of atmospheric methane. Tellus 50B:128–150.
67. Lidstrom ME (1988) Isolation and characterization of marine methanotrophs. Antonie
van Leeuwenhoek 54: 189-199.
68. Lieberman RL, Rosenzweig AC (2005) Crystal structure of a membrane-bound
metalloenzyme that catalyzes the biological oxidation of methane. Nature 434, 177182.
69. Malashenko IR, Romanovskaia VA, Bogachenko VN, Shved AD (1975) Thermophilic
and thermotolerant bacteria that assimilate methane. Mikrobiologiia 44: 855-862.
70. Maymo-Gatell X, Anguish T, Zinder SH (1999) Reductive dechlorination of
chlorinated ethenes and 1,2-dichloroethane by "Dehalococcoides ethenogenes" 195.
Appl Environ Microbiol 65:3108-3113.
71. McDonald IR, Miguez CB, Rogge G, Bourque D, Wendlandt KD, Groleau D, Murrell
JC (2006) Diversity of soluble methane monooxygenase-containing methanotrophs
isolated from polluted environments. FEMS Microbiol Lett 255: 225-232.


 

67
 

 

 

 

72. Medigan MT, Martinko JM, Parker J (2003) Brock Biology of microorganisms. Upper
Saddle River, NJ: Pearson Education.
73. Mehta P (1991) Methanol biosynthesis by covalently immobilized cells of
Methylosinus trichosporium: batch and continuous studied. Biotech. Bioeng. 37:551556.
74. Merkx M, Lippard SJ (2002) Why OrfY? Characterization of mmoD, a long
overlooked component of the soluble methane monooxygenase from Methylococcus
capsulatus (Bath). J Biol Chem 277: 5858-5865.
75. Murray AE, Hollibaugh JT, Orrego C (1996) Phylogenetic compositions of
bacterioplankton from two California estuaries compared by denaturing gradient gel
electrophoresis of 16S rDNA fragments. Appl. Envi- ron. Microbiol. 62:2676–2680.
76. Murrell JC, Gilbert B, Mcdonald IR (2000) Molecular Biology And Regulation Of
Methane Monooxygenase. Arch Microbiol 173: 325–332
77. Murrell JC, McDonald IR, Gilbert B (2000) Regulation of the expression of methane
monooxygenases by copper ions. Trends in Microbiology 8, 221-225.
78. Nakamura T, Hoaki T, Hanada S, Maruyama A, Kamagata Y, Fuse H (2007) Soluble
and particulate methane monooxygenase gene clusters in the marine methanotroph
Methylomicrobium sp. strain NI. FEMS Microbiol Lett. 277:157-164.
79. Nikiema J, Brzezinski R, Heitz M (2007) Elimination of methane generated from
landfills by biofiltration: a review, Rev. Environ. Sci. Biotechnol. 6:261–284.
80. Omelchenko MV, Vasilyeva LV, Zavarzin GA (1993) Psychrophilic methanotroph
from tundra soil. Curr Microbiol 27: 255-259.

81. Øverland M, Kjos NP, Olsen E, Skrede A (2005) Changes in fatty acid composition
and improved sensory quality of back fat and meat of pigs fed bacterial protein meal.
Meat Sci.71:719–729.


 

68
 



 

 

 

82. Øverland M, Romarheim OH, Ahlstrøm Ø, Storebakken T, Skrede A (2006) Technical
quality of dog food and salmon feed containing different bacterial protein sources and
processed by different extrusion conditions. Anim Feed Sci Technol. 134:124–139.
83. Øverland M, Skrede A, Matre T (2001) Bacterial protein grown on natural gas as feed
for pigs. Acta Agr. Scand. A-AN. 51:97–106.
84. Øverland M, Tauson AH, Shearer K, Skrede K (2010) Evaluation of methane-utilising
bacteria products as feed ingredients for monogastric animals. Archives of Animal
Nutrition 64(3): 171–189
85. Pfiffner SM, Palumbo AV, Phelps TJ, Hazen TC (1997) Effects of nutrient dosing on
subsurface methanotrophic populations and trichloroethylene degradation, J. Ind.
Microbiol. Biotechnol. 18:204–212.
86. Pol A, Heijmans K, Harhangi HR, Tedesco D, Jetten MSM, Op den Camp HJM

(2007) Methanotrophy below pH 1 by a new Verrucomicrobia species. Nature 450:
874-878.
87. Rahalkar M, Bussmann I, Schink B (2007) Methylosoma difficile gen. nov., sp nov., a
novel methanotroph enriched by gradient cultivation from littoral sediment of Lake
Constance. Int. J. Syst. Evol. Microbiol. 57:1073–1080.
88. Ramanathan V, Cicerone J, Singh HB, Kiehl JT (1985) Trace gas trends and their role
in climate changes. Journal of Geophysical Research 90, 5547-5566.
89. Ramsay B, Karamanev D, Pierre S, Lafontaine C, Sakamoto T, Ramsay J (2001)
Efficient TCE mineralization in a novel methanotrophic bioreactor system.
International In Situ and On-site Bioremediation Symposium, 6th, San Diego, CA,
United States, pp. 171–178.
90. Reeburgh WS, Whalen SC, Alpern MJ (1993) The role of methylotrophy in the global
methane budget, in: J.C. Murrell, D.P. Kelly (Eds.). Microbial Growth on C1
Compounds, Intercept Press, Andover, UK.


 

69
 



 

 

 

91. Rehm

HJ,

Reed

G,

Puhler

A,

Stadler

P

(1993)

Biotechnology.

VCH

Verlagsgesellschaft mbH, Germany.
92. Reshetnikov AS, Mustakhimov II, Khmelenina VN, Trotsenko YA (2005) Cloning,
purification, and characterization of diaminobutyrate acetyltransferase from the
halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. Biochem- Moscow
70: 878-883.
93. Rumsey GL, Hughes SG, Smith RR, Kinsella JE, Shetty KJ (1991) Digestibility and
energy values of intact disrupted and extracts from brewers dried yeast fed to rainbow
trout (Oncorhynchus mykiss). Animal Feed Science and Technology 33: 185–193.
94. Saitou N, Nei M (1987) “The neighbor – joining method: a new method for

reconstructing phylogenetic trees”, Mol Biol Evol, 4, pp.406 – 425.
95. Schøyen HF (2007a) Bacterial protein meal from natural gas as protein source in feed
for monogastric animal. PhD thesis. Norwegian University of Life Sciences, As,
Norway.
96. Schøyen HF, Frøyland JRK, Sahlstrøm S, Knutsen SH, Skrede A (2005) Effects of
autolysis and hydrolysis of bacterial protein meal grown on natural gas on chemical
characterization and amino acid digestibility. Aquaculture 248: 27–33.
97. Schøyen HF, Hetland H, Rouvinen-Watt K (2007b) Growth performance and ileal and
total tract amino acid digestibility in broiler chickens fed diets containing bacterial
protein produced on natural gas. Poult. Sci 86: 87–93.
98. Schøyen HF, Svihus M, Storebakken T, Skrede A (2007c) Bacterial protein meal
produced on natural gas replacing soybean meal and fishmeal in broiler chicken diets.
Archives of Animal Nutrition 61:276-291.
99. Semrau JD, Chistoservdov A, Lebron J (1995) Particulate methane monooxygenase
genes in methanotrophs. J Bacteriol 177, 3071-3079.
100. Semrau JD, DiSpirito AA, Vuileumier S (2011) Facultative methanotrophy: false
leads, true result, and suggestion for future research. FEMS Microbiol Lett. 323:1-12.


 

70
 



 

 

 

101. Semrau JD, DiSpirito AA, Yoon S (2010) Methanotrophs and copper. FEMS
Microbiol Rev. 34:496-531.
102. Senko O, Makhlis T, Bihovsky M, Podmasterev V, Efremenko E, Razumovsky S,
Varfolomeyev S (2007) Methanol production in the flow system with immobiblized
cells Methylosinus sporium. XVth International Workshop on Bioencapsulation,
Vienna, Au. P2-16.
103. Singh A, Abidi AB, Agrawal AK, Darmwal NS (1991) Single cell proteinproduction
by Aspergillus niger and its evaluation. Zentralbl Mikrobiol 146(3):181-184.
104. Şişman T, Gür Ö, Doğan N, Özdal, Algur ÖF, Ergon T (2013) Single-cell protein as an
alternative food for zebrafish, Danio rerio: a toxicological assessment. Toxicol Ind
Health 29:792-799
105. Skrede A, Ahlstrøm Ø (2002) Bacterial protein produced on natural gas: a new
potential feed ingredient for dogs evaluated using the blue fox as a model. J. Nutr
132:1668-1669.
106. Skrede A, Berge GM, Storebakken T, Herstad O, Aarstad KG, Sundstøl F (1998)
Digestibility of bacterial protein grown on natural gas in mink, pigs, chicken and
Atlantic salmon. Anim. Feed Sci. Tech. 76:103– 116.
107. Skrede A, Schøyen HF, Svihus B, Storebakken T (2003) The effect of bacterial protein
grown on natural gas on growth performance and sensory quality of broiler chickens.
Can. J. Anim. Sci. 83:229–237.
108. Sly L, Bryant L, Cox J, Anderson J (1993) Development of a biofilter for the removal
of methane from coalmine ventilation atmospheres. Appl. Microbiol. Biotechnol.
39:400–404.
109. Smith KS, Costello AM, Lidstrom ME (1997) Methane and trichloroethylene
oxidation by an estuarine methanotroph. Methylobacter sp. Strain BB5.1. Appl.
Environ. Microbiol. 63:4617-4620.

 

71
 



 

 

 

110. Smith LH, McCarty PL (1997) Laboratory evaluation of a two-stage treatment system
for TCE cometabolism by a methane-oxidizing mixed culture, Biotechnol. Bioeng.
55:650–659.
111. Sorokin DY, Jones BE, Kuenen JG (2000) An obligate methylotrophic, methaneoxidizing

Methylomicrobium

species

from

a

highly

alkaline

environment.

Extremophiles 4: 145-155.
112. Stoecker K, Bendinger B, Schoning B, Nielsen PH, Nielsen JL, Baranyi C, Toenshoff
ER, Daims H, Wagner M (2006) Cohn’s Crenothrix is a filamentous methane oxidizer
with an unusual methane monooxygenase. Proc Natl Acad Sci USA 103: 2363-2367.
113. Stolyar S, Costello AM, Peeples TL, Lidstrom ME (1999) Role of multiple gene
copies in particulate methane monooxygenase activity in the methane oxidising
bacterium Methylococcus capsulatus (Bath). Microbiology 145: 1235-1244.
114. Streese J, Stegmann R (2005) Potentials And Limitations Of Biofilters For Methane
Oxidation. Proceedings Sardinia 2005, Tenth International Waste Management And
Landfill Symposium S. Margherita Di Pula, Cagliari, Italyiniew
115. Sugimori D, Takeguchi M, Okura I (1995) Biocatalytic methanol production from
methane with Methylosinus trichosporium Ob3B—an approach to improve methanol
accumulation. Bio- technol Lett 17:783–784
116. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary
genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599.
117. Trotsenko YA, Khmelenina VN (2002) Biology of extremophilic and extremotolerant
methanotrophs. Arch Microbiol 177: 123-131.
118. Tsubota J, Eshinimaev B, Khmelenina VN, Trotsenko YA (2005) Methylothermus
thermalis gen. nov., sp. nov., a novel moderately thermophilic obligate methanotroph
from a hot spring in Japan. Int. J. Syst. Evol. Microbiol. 55:1877–1884.


 

72
 

 

 

 

119. Vhile SG, Skrede A, Ahlstrøm Ø, Szymeczko R, Hove K (2005) Ideal and total tract
nutrient digestibility in blue foxes (Alopex lagopus) fed extruded diets containing
different protein sources. Arch Anim Nutr, 59: 61–72.
120. Viet Nam Standard 4328:2001. Animal feeding stuffs – Determination of nitrogen
content and calculation of crude protein content- Kjeldahl method
121. Vigliotta G (2007) Clonothrix fusca Roze 1896, a filamentous, sheathed,
methanotrophic gamma-proteobacterium. Appl. Environ. Microbiol. 73:3556–3565.
122. Wartiainen I, Hestnes AG, McDonald IR, Svenning MM (2006) Methylobacter
tundripaludum sp. nov., a methane-oxidizing bacterium from Arctic wetland soil on
the Svalbard islands, Norway (78° N). Int J Syst Evol Microbiol 56: 109-113.
123. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA
amplification for phylogenetic study. J Bacteriol 173: 697-703.
124. Whittenbury R, Phillips KC, Wilkinson JF (1970) Enrichment, isolation and some
properties of methane-utilizing bacteria. J Gen Microbiol 61, 205-218.
125. Wise MG, McArthur J, Shimkets LJ (2001) Methylosarcina fibrata gen. nov., sp. nov.
andMethylosarcina quisquiliarum sp. nov., novel type I methanotrophs. Int J Syst Evol
Microbiol 51: 611-621.
126. Wise MG, McArthur JV, Shimkets LJ (1999) Methanotroph diversity in landfill soil:
isolation of novel types I and type II methanotrophs whose presence was suggested by
culture-independent 16S ribosomal DNA analysis. Appl Environm Microbiol 65:
4887-4897.
127. Xin JY (2004) Production of methanol from methane by methanotrophic bacteria.
Biocatal. Biotransfor. 22 (3): 225-229.

128. Yoon S (2010). Towards practical application of methanotrophic metabolism in
chlorinated hydrocarbon degradation, greenhouse gas removal, and immobilization of
heavy metals. PhD Thesis. The University of Michigan USA.


 

73
 



 

 

 

129. Yoon S, Im J, Bandow N, DiSpirito AA, Semrau JD (2011) Constitutive expression of
pMMO by Methylocystis strain SB2 when grown on multi-carbon substrates:
implications for biodegradation of chlorinated ethenes. Environ. Microbiol. Rep.
3:182-188.
Webs:
130. Intergovernmental Panel on Climate Change (IPCC) (2007) Climate change 2007: the
physical science basis: contribution of working group I to the fourth assessment report
of the intergovernmental panel on climate change; technical summary. URL
report/ar4/wg1/ar4-wg1-ts.pdf
131. Johnson

D

(2012)

Global

Methanol

Market

Review.

URL

/>0PEMEX%20Petroqu%C3%ADmica/2012/PEMEX_DJohnson.pdf
132. Methanol

Institute.

Methanol:TheClearAlternativeforTransportation.

URL
 
133. United State National Oceanic and Atmospheric Administration. Trends in
Atmospheric

Carbon

Dioxide.

URL />Vietnamese

 
134. Ministry of Agriculture and Rural Development, Department of Livestock Husbandry
(2012)

Livestock

Newsletter.

URL

/>News&num=15&TabID=1&NewsID=94
135. Ministry of Natural Resources and Environment (2012). National Environment Report
2011.


 

74