Detection of Methylobacterium spp. Possessing Soluble Methane Monooxygenase (sMMO) Gene Isolated from Rice

강연경 ( Yeongyeong Kang ),( Denver Walitang ),( Poulami Chatterjee ),표채은 ( Chaeeun Pyo ),사동민 ( Tongmin Sa ) 한국환경농학회 2017 한국환경농학회 학술대회집 Vol.2017 No.-

Methane (CH4) is currently receiving great attention due to its severe impact on the environment as a greenhouse gas. The atmospheric concentration of methane is continuously increasing and its average concentration is around 1.87 mg L^-1. Methane is produced in nature when methanogens digest plant material in the absence of oxygen. Methane emission from rice fields is one of the largest individual sources in the global budget of atmospheric CH₄. Methane-oxidizing bacteria (MOB) are phylogenetically diverse groups defined by their ability to use methane as a major carbon and energy source. Aerobic MOB, which are widely present in natural environments, can utilize methane as the sole carbon and energy source through a unique enzyme system of methane monooxygenase (MMO). MMO has a soluble cytoplasmic form (sMMO) and a particulate membrane associated form (pMMO). The objective of this study is to investigate the presence of sMMO in Methylobacterium spp. The selected strains for the current study, Methylobacterium spp. were isolated from rice. MOB are a subset of a physiological group of bacteria known as methylotrophs. The sMMO consists of three components, proteins A, B, and C. Protein A is coded for by the mmoX, mmoY, and mmoZ genes. Protein B is coded for by the mmoB gene. Protein C is the reductase component of the enzyme and is coded for by the mmoC gene. The presence of sMMO in Methylobacterium spp. was proven through polymerase chain reaction (PCR) using mmoC specific primer. This results indicate that there were 18 out of 19 strains of Methylobacterium. spp with sMMO detected through PCR-based amplification of an mmoC gene. PCR products of the correct size (314 bp) were obtained with all of the Methylobacterium spp. used in this study. In the current study confirming the oxidative activity of the strains that contain the sMMO. Future study the growth of these isolates using methane as the sole carbon source and then assess methane oxidation potential of by gas chromatography and their contribution in atmospheric methane oxidation.

Inhibitory effects of sulfur compounds on methane oxidation by a methane-oxidizing consortium

Lee, E.H.,Moon, K.E.,Kim, T.G.,Lee, S.D.,Cho, K.S. Society for Bioscience and Bioengineering, Japan ; 2015 Journal of bioscience and bioengineering Vol.120 No.6

Kinetic and enzymatic inhibition experiments were performed to investigate the effects of methanethiol (MT) and hydrogen sulfide (H<SUB>2</SUB>S) on methane oxidation by a methane-oxidizing consortium. In the coexistence of MT and H<SUB>2</SUB>S, the oxidation of methane was delayed until MT and H<SUB>2</SUB>S were completely degraded. MT and H<SUB>2</SUB>S could be degraded, both with and without methane. The kinetic analysis revealed that the methane-oxidizing consortium showed a maximum methane oxidation rate (V<SUB>max</SUB>) of 3.7 mmol g-dry cell weight (DCW)<SUP>-1</SUP> h<SUP>-1</SUP> and a saturation constant (K<SUB>m</SUB>) of 184.1 μM. MT and H<SUB>2</SUB>S show competitive inhibition on methane oxidation, with inhibition values (K<SUB>i</SUB>) of 1504.8 and 359.8 μM, respectively. MT was primary removed by particulate methane monooxygenases (pMMO) of the consortium, while H<SUB>2</SUB>S was degraded by the other microorganisms or enzymes in the consortium. DNA and mRNA transcript levels of the pmoA gene expressions were decreased to ~10<SUP>6</SUP> and 10<SUP>3</SUP>pmoA gene copy number g-DCW<SUP>-1</SUP> after MT and H<SUB>2</SUB>S degradation, respectively; however, both the amount of the DNA and mRNA transcript recovered their initial levels of ~10<SUP>7</SUP> and 10<SUP>5</SUP>pmoA gene copy number g-DCW<SUP>-1</SUP> after methane oxidation, respectively. The gene expression results indicate that the pmoA gene could be rapidly reproducible after methane oxidation. This study provides comprehensive information of kinetic interactions between methane and sulfur compounds.

Methane Oxidation Potentials of Rice-associated Plant Growth Promoting Methylobacterium Species

강연경,월탱 덴버,Sundaram Seshadri,신완식,사동민 한국환경농학회 2022 한국환경농학회지 Vol.41 No.2

BACKGROUND: Methane is a major greenhouse gas at-tributed to global warming partly contributed by agricul-tural activities from ruminant fermentation and rice pad-dy fields. Methanotrophs are microorganisms that utilize methane. Their unique metabolic lifestyle is enabled by enzymes known as methane monooxygenases (MMOs) catalyzing the oxidation of methane to methanol. Rice ab-sorbs, transports, and releases methane directly from soil water to its stems and the micropores and stomata of the plant epidermis. Methylobacterium species associated with rice are dependent on their host for metabolic sub-strates including methane. METHODS AND RESULTS: Methylobacterium spp. isolated from rice were evaluated for methane oxidation activities and screened for the presence of sMMO mmoCgenes. Qualitatively, the soluble methane mono-oxygenase (sMMO) activities of the selected strains of Methylobacterium spp. were confirmed by the naphthalene oxidation assay. Quantitatively, the sMMO activity ranged from 41.3 to 159.4 nmol min--1 mg of protein-1. PCR-based amplification and sequencing confirmed the presence and identity of 314 bp size fragment of the mmoC gene showing over 97% similarity to the CBMB27 mmoC gene indicating that Methylobacterium strains be-long to a similar group. CONCLUSION(S): Selected Methylobacterium spp. contained the sMMO mmoC gene and possessed methane oxidation activity. As the putative methane oxidizing strains were isolated from rice and have PGP properties, they could be used to simultaneously reduce paddy field methane emission and promote rice growth.

Revie : Biocatalytic Conversion of Methane to Methanol as a Key Step for Development of Methane-Based Biorefineries

( In Yeub Hwang ),( Seung Hwan Lee ),( Yoo Seong Choi ),( Si Jae Park ),( Jeong Geol Na ),( In Seop Chang ),( Choongik Kim ),( Hyun Cheol Kim ),( Yong Hwan Kim ),( Jin Won Lee ),( Eun Yeol Lee ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.12

Methane is considered as a next-generation carbon feedstock owing to the vast reserves of natural and shale gas. Methane can be converted to methanol by various methods, which in turn can be used as a starting chemical for the production of value-added chemicals using existing chemical conversion processes. Methane monooxygenase is the key enzyme that catalyzes the addition of oxygen to methane. Methanotrophic bacteria can transform methane to methanol by inhibiting methanol dehydrogenase. In this paper, we review the recent progress made on the biocatalytic conversion of methane to methanol as a key step for methane-based refinery systems and discuss future prospects for this technology.

Biological conversion of methane to methanol

박동현,이지원 한국화학공학회 2013 Korean Journal of Chemical Engineering Vol.30 No.5

The conversion of methane to methanol is important to economic utilization of natural/shale gas. Methanol is a valuable liquid fuel and raw material for various synthetic hydrocarbon products. Its industrial production is currently based on a two-step process that is energy-intensive and environmentally unfriendly, requiring high pressure and temperature. The biological oxidation of methane to methanol, based on methane monooxygenase activity of methanotrophic bacteria, is desirable because the oxidation is highly selective under mild conditions, but conversion rate and yield and stability of catalytic activity should be improved up to an industrially viable level. Since methanotrophic bacteria produce methanol as only a precursor of formaldehyde that is then used to synthesize various essential metabolites,the direct use of bacteria seems unsuitable for selective production of a large amount of methanol. There are two types of methane monooxygenase: soluble (sMMO) and particulate (pMMO) enzyme. sMMO consisting of three components (reductase, hydroxylase, and regulatory protein) features an (αβγ)2 dimer architecture with a di-iron active site in hydroxlase. pMMO, a trimer (pmoA, pmoB, and pmoC) in an α3β3γ3 polypeptide arrangement is a copper enzyme with a di-copper active site located in the soluble domain of pmoB subunit. Since the membrane transports electrons well and delivers effectively methane with increased solubility in the lipid bilayer, pMMO seems more rationally designed enzyme in nature than sMMO. The engineering/evolution/modification of MMO enzymes using various biological and chemical techniques could lead to an optimal way to reach the ultimate goal of technically and economically feasible and environmentally friendly oxidation of methane. For this, multidisciplinary efforts from chemical engineering,protein engineering, and bioprocess research sectors should be systematically combined.

Biofuel upgrade reactions via phase-transfer catalysis of methanotrophs

박예림,김동호,최규환,김용우,이은열,박범준 한국공업화학회 2021 Journal of Industrial and Engineering Chemistry Vol.95 No.-

Methane, one of the six major greenhouse gases, poses a serious environmental problem with thepotential for global warming 20 times that of CO2. Although a large amount of methane is generatedworldwide, its recovery and utilization are very low. One of the environmentally friendly ways to usemethane is the biological gas-to-liquids (Bio-GTL) process, in which methane is biologically converted touseful products by microorganisms. Methanotrophs are strains that can convert methane to methanol atambient conditions using methane monooxygenase (MMO). Here, we report an efficient phase-transfercatalysis system for methane-to-methanol conversion using methanotrophs. The methanotroph used inthe work is Methylomicrobium alcaliphilum 20Z of which the methanol dehydrogenase (MDH) enzyme isremoved to enhance methanol accumulation. The phase-transfer catalysis system does not require anyseparation processes and facilitates the mass transfer of methane gas, thereby increasing the methanolproductivity and lowering the production cost. The methanol productivity is 0.717 g/L/h, which issuperior to the results reported to date. In addition, the use of a cellulose-membrane reactor systemenables multiple biocatalytic reactions without a significant decrease in productivity.

신규 type I 메탄자화세균 Methylobacterium sp. HG-1을 이용한 메탄올의 생합성

김희곤 ( Hee Gon Kim ),이상귀 ( Sang Gui Lee ),김시욱 ( Si Wouk Kim ) 조선대학교 공학기술연구원 2008 공학기술논문지 Vol.1 No.1

Methanorrophic bacteria are a group of microorganisms possessing the unique ability to utilize methane as the sole source of carbon and energy. In the previous study, an obligate methane-oxidizing bacterium, Methylomicrobium sp. HG-1, which was able to grow on methane and methanol was isolated from the effluent of manure. And a fast and high density cell culture system (compulsory gas circulation system) was developed for the cultivation of methanotrophs. Methylomicrobium sp. HG-1 could oxidize methane to methanol in the presence of a high concentration of Cu(2+). Further oxidation of methanol to formaldehyde was prevented by adding 200 mM NaC1 which acts as a methanol dehydrogenase inhibitor. When cell suspension of Methylomicrobium sp. HG-l was incubated with methane and air mixture an extracellular methanol was accumulated in response to NaC1 concentration. When 0.6 mg dry cell ml(-1) of bacterial cells were incubated with methane/air mixture (1:4, v/v) at 25℃ in a 12.9 mM phosphate buffer (pH 7) containing 20 mM sodium formate and 200 mM NaC1, 12.8 mM methanol was produced for 12 hr.

A novel methanotroph in the genus Methylomonas that contains a distinct clade of soluble methane monooxygenase

Ngoc-Loi Nguyen,유운종,양혜영,김종걸,정만영,박수제,노성운,이성근 한국미생물학회 2017 The journal of microbiology Vol.55 No.10

Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2–97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.

Optimization of Lab Scale Methanol Production by Methylosinus trichosporium OB3b

Kim, Hee-Gon,Han, Gui-Hwan,Kim, Si-Wouk 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.3

Methylosinus trichosporium OB3b is a methanotrophic bacterium containing particulate methane monooxygenase (MMO), which catalyzes the hydroxylation of methane to methanol. The methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH). We developed a novel compulsory circulation diffusion system for cell cultivation. A methane/air mixture (1:1, v/v) was prepared in a tightly sealed gas reservoir and pumped into a nitrate mineral salt culture medium under optimal conditions (5 ${\mu}M$ $CuSO_4$, pH 7.0, $30^{\circ}C$). Cells were harvested, washed, and resuspended (0.6 mg dry cells/mL) in a 500 mL flask in 100 mL of 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA as MDH inhibitors, and 20 mM sodium formate. A single 12 h batch reaction at $25^{\circ}C$ yielded a final concentration of 13.2 mM methanol. The use of a repeated batch mode, in which the accumulated methanol was removed after each of three 8 h cycles over a 24 h period, showed a productivity of 2.17 ${\mu}mol$ methanol/h/mg dry cell wt. Finally, a lab-scale reaction performed using a 3 L cylindrical reactor with a working volume of 1 L produced 13.7 mM methanol after 16 h. Our results identify a simple process for improving the productivity of biologically derived methanol and, therefore the utility of methane as an energy source.

논에서 분리한 메탄산화세균 Methylomonas sp. SM4의 특성과 메탄올 생합성

박성민(Sung Min Park),Lavanya Madhavaraj,김시욱(Si Wouk Kim) 한국생물공학회 2017 KSBB Journal Vol.32 No.2

A methane-oxidizing bacterium was isolated from rice paddy field soil around Jeollanam-do province, Korea, and characterized. The isolate was gram-negative, orange pigmented and short rod (1.1-1.2 × 1.6-1.9 μm). It was catalase and urease-negative but oxidase-positive. The strain utilized methane and methanol as sole carbon and energy sources. It had an ability to grow with an optimum pH 7.0 and an optimum growth temperature 30℃. The strain was resistant to antibiotic polymyxin B but sensitive to streptomycin, kanamycin, ampicillin, chloramphenicol and rifampicin. The isolate required copper for their growth with concentration range of 2-25 μM, with an optimum of 10 μM. Under optimal culture condition, specific cell growth rate and generation time were found to be 0.046 hr<SUP>-1</SUP> and 15.13 hr, respectively. Phylogenetic analysis based on 16S rDNA sequences indicated that the strain formed a tight phylogenetic lineage with Methylomonas koyamae with a value of 99.4% gene sequence homology. So, we named the isolate as Methylomonas sp. SM4. 8.6 mM methanol was accumulated in the reaction mixture containing 70 mM sodium formate and 40 mM MgCl₂ (MDH inhibitor) under atmosphere of methane:air (40:60) mixture for 24 hr at 30℃.