• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.717-724
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• 2016

Methane (CH₄) is the most abundant component in natural gas. To reduce its harmful environmental effect as a greenhouse gas, CH₄ can be utilized as a low-cost feed for the synthesis of methanol by methanotrophs. In this study, several methanotrophs were examined for their ability to produce methanol from CH₄; including Methylocella silvestris, Methylocystis bryophila, Methyloferula stellata, and Methylomonas methanica. Among these methanotrophs, M. bryophila exhibited the highest methanol production. The optimum process parameters aided in significant enhancement of methanol production up to 4.63 mM. Maximum methanol production was observed at pH 6.8, 30℃, 175 rpm, 100 mM phosphate buffer, 50 mM MgCl₂ as a methanol dehydrogenase inhibitor, 50% CH₄ concentration, 24 h of incubation, and 9 mg of dry cell mass ml^-1 inoculum load, respectively. Optimization of the process parameters, screening of methanol dehydrogenase inhibitors, and supplementation with formate resulted in significant improvements in methanol production using M. bryophila. This report suggests, for the first time, the potential of using M. bryophila for industrial methanol production from CH₄.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1234-1241
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• 2016

Methanol is a versatile compound that can be biologically synthesized from methane (CH₄) by methanotrophs using a low energy-consuming and environment-friendly process. Methylocella tundrae is a type II methanotroph that can utilize CH₄ as a carbon and energy source. Methanol is produced in the first step of the metabolic pathway of methanotrophs and is further oxidized into formaldehyde. Several parameters must be optimized to achieve high methanol production. In this study, we optimized the production conditions and process parameters for methanol production. The optimum incubation time, substrate, pH, agitation rate, temperature, phosphate buffer and sodium formate concentration, and cell concentration were determined to be 24 h, 50% CH₄, pH 7, 150 rpm, 30℃, 100 mM and 50 mM, and 18 mg/ml, respectively. The optimization of these parameters significantly improved methanol production from 0.66 to 5.18 mM. The use of alginate-encapsulated cells resulted in enhanced methanol production stability and reusability of cells after five cycles of reuse under batch culture conditions.

• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.753-756
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• 2011

A methane-oxidizing bacterium was isolated from the enriched culture of a landfill cover soil. The closest relative of the isolate, designated M6, is Methylocystis sp. Based on a kinetic analysis, the maximum specific methane oxidation rate and saturation constant were 4.93 mmol gdry cell $weight^{-1}{\cdot}h^{-1}$ and 23${\mu}M$, respectively. This was the first time a kinetic analysis was performed using pure methanotrophic culture. The methane oxidation by M6 was investigated in the presence of aromatic (m- and pxylene and ethylbenzene) or sulfur (hydrogen sulfide, dimethyl sulfide, methanthiol) compounds. The methane oxidation was inhibited by the presence of aromatic or sulfur compounds.

• New & Renewable Energy
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• v.19 no.4
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• pp.46-52
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• 2023

Methane is the second most emitted greenhouse gas after carbon dioxide. Despite lower emissions than those of carbon dioxide, methane receives significant attention owing to its more than 20-fold higher global warming potential. Consequently, the importance of research on methanotrophic bacteria, microorganisms capable of converting methane gas into high-value materials, is increasingly emphasized. In the case of methanotrophic bacteria, knowledge on episomal plasmids that can be used for genetic engineering remains lacking, which poses significant challenges to the engineering process. The replication origin sequences of natural plasmids within methanotrophic bacteria have been predicted through in silico methods. The basic characteristics of the replication origin, such as a high A/T ratio, repetitive sequences, and proximity to proteins related to replication, have been used as criteria for identifying the replication origin. As a result, a region with a sequence of 18 base pairs repeated eight times could be identified. The putative replication origin sequence thus identified generally takes the form of iterons, but it also possesses unique features such as the length of the gap between iterons and the repetition of identical iteron sequences. This information can be valuable for future design of episomal plasmids applicable to methanotrophs.

• Applied Biological Chemistry
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• v.34 no.4
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• pp.386-392
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• 1991

Whole cells of Methylosinus trichosporium OB3b, the obligate methylotroph, were used to produce propylene oxide from propane. This strain has methane monooxygenase, which catalyzes the conversion methane to methanol and can catalyze also the conversion propane to propylene oxide. Optimal condition for the production of propylene oxide was investigated in resting-whole cell system. The optimal pH and temperature was 7.0 and $35^{\circ}C$ respectively. The end product, propylene oxide, didn't inhibit the production of propylene oxide and was not further metabolized in reaction mixture. The addition of methane metabolites (methanol, formaldehde and formic acid) to the reaction mixture stimulated formation of propylene oxide by $3{\sim}4$ times, and methanol was the most effective especially. Under the optimal conditions, the 14.2 mM of propylene oxide was produced after incubation of 60 min. and the conversion ratio of propane to propylene oxide was approximately 8%.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.423-430
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• 2015

Aerobic CH₄ oxidation is an important CH₄ sink in landfills. To investigate the distribution and community diversity of methanotrophs and link with soil characteristics and operational parameters (e.g., concentrations of O₂, CH₄), cover soil samples were collected at different locations and depths from the Mengzi semi-aerobic landfill (SAL) in Yunnan Province of southern China. Specific PCR followed by denaturing gradient gel electrophoresis and realtime PCR were used to examine methanotrophs in the landfill cover soils. The results showed that different locations did harbor distinct methanotroph communities. Methanotrophs were more abundant in areas near the venting pipes because of the higher O₂ concentrations. The depth of 20-25 cm, where the ratio of the CH₄ to O₂ was within the range from 1.3 to 8.6, was more conducive to the growth of CH₄-oxidizing bacteria. Type II methanotrophs dominated in all samples compared with Type I methanotrophs, as evidenced by the high ratio of Type II to Type I methanotrophic copy numbers (from 1.76 to 11.60). The total copy numbers of methanotrophs detected were similar to other ecosystems, although the CH₄ concentration was much higher in SAL cover soil. Methylobacter and Methylocystis were the most abundant Type I and Type II methanotrophs genera, respectively, in the Mengzi SAL. The results suggested that SALs could provide a special environment with both high concentrations of CH₄ and O₂ for methanotrophs, especially around the vertical venting pipes.

• KSBB Journal
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• v.8 no.4
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• pp.341-350
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• 1993

As an effort to develop an alternative transportation fuel, the production of methanol from methane gas was studied using the resting cells of an obligatory methanotroph, Methylosinus trichosporium OB3b. The reaction was carried out in high concentration phosphate buffer solutions with the flask-grown cells containing the exclusively cytoplasmic methane monooxygenase (sMMO) activity. The methanol accumulation rate was observed to be 79nmo1/mg·min during the initial 4.5hr. Phosphate-dependent inhibition was found for both sMMO and methanol dehydrogenase (MDH) activities, and the inhibition constants were 185mM and 42mM, respectively. The inhibition mode was noncompetitive. Methanol was found to be very inhibitory to the sMMO activity and the inhibition constant (noncompetitive) was 21mM when propylene was used as substrate. The sMO activity in the resting cells was declined very fast and the rate became very high during the methanol production. These results indicate that the use of M. trichosporium OB3b as a biocatalyst for the methanol production is heavily dependent on the stable maintenance of the whole-cell SMO activity as well as the effective alleviation of product inhibition.

• Journal of Microbiology and Biotechnology
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• v.32 no.3
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• pp.287-293
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• 2022

The hydroxylation of methane (CH₄) is crucial to the field of environmental microbiology, owing to the heat capacity of methane, which is much higher than that of carbon dioxide (CO₂). Soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily, is essential for the hydroxylation of specific substrates, including hydroxylase (MMOH), regulatory component (MMOB), and reductase (MMOR). The diiron active site positioned in the MMOH α-subunit is reduced through the interaction of MMOR in the catalytic cycle. The electron transfer pathway, however, is not yet fully understood due to the absence of complex structures with reductases. A type II methanotroph, Methylosinus sporium 5, successfully expressed sMMO and hydroxylase, which were purified for the study of the mechanisms. Studies on the MMOH-MMOB interaction have demonstrated that Tyr76 and Trp78 induce hydrophobic interactions through π-π stacking. Structural analysis and sequencing of the ferredoxin domain in MMOR (MMOR-Fd) suggested that Tyr93 and Tyr95 could be key residues for electron transfer. Mutational studies of these residues have shown that the concentrations of flavin adenine dinucleotide (FAD) and iron ions are changed. The measurements of dissociation constants (K_ds) between hydroxylase and mutated reductases confirmed that the binding affinities were not significantly changed, although the specific enzyme activities were significantly reduced by MMOR-Y93A. This result shows that Tyr93 could be a crucial residue for the electron transfer route at the interface between hydroxylase and reductase.

• Journal of Microbiology and Biotechnology
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• v.33 no.7
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• pp.886-894
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• 2023

During the rhizoremediation of diesel-contaminated soil, methane (CH₄), a representative greenhouse gas, is emitted as a result of anaerobic metabolism of diesel. The application of methantrophs is one of solutions for the mitigation CH₄ emissions during the rhizoremediation of diesel-contaminated soil. In this study, CH₄-oxidizing rhizobacteria, Methylocystis sp. JHTF4 and Methyloversatilis sp. JHM8, were isolated from rhizosphere soils of tall fescue and maize, respectively. The maximum CH₄ oxidation rates for the strains JHTF4 and JHM8 were 65.8 and 33.8 mmol·g-DCW^-1·h^-1, respectively. The isolates JHTF4 and JHM8 couldn't degrade diesel. The inoculation of the isolate JHTF4 or JHM8 significantly enhanced diesel removal during rhizoremediation of diesel-contaminated soil planted with maize for 63 days. Diesel removal in the tall fescue-planting soil was enhanced by inoculating the isolates until 50 days, while there was no significant difference in removal efficiency regardless of inoculation at day 63. In both the maize and tall fescue planting soils, the CH₄ oxidation potentials of the inoculated soils were significantly higher than the potentials of the non-inoculated soils. In addition, the gene copy numbers of pmoA, responsible for CH₄ oxidation, in the inoculated soils were significantly higher than those in the non-inoculated soils. The gene copy numbers ratio of pmoA to 16S rDNA (the ratio of methanotrophs to total bacteria) in soil increased during rhizoremediation. These results indicate that the inoculation of Methylocystis sp. JHTF4 and Methyloversatilis sp. JHM8, is a promising strategy to minimize CH₄ emissions during the rhizoremediation of diesel-contaminated soil using maize or tall fescue.

• Microbiology and Biotechnology Letters
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• v.45 no.4
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• pp.322-329
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• 2017

Two pilot-scale biocovers (PBCs) were installed in a landfill, and the methane ($CH_4$) concentrations at their inlets and outlets were monitored for 240 days to evaluate the methane removability. Consequently, the packing materials were sampled from the PBCs, and their potential $CH_4$ oxidizing abilities were evaluated in serum vials. The $CH_4$ concentration at the inlet of the biocovers was observed to be in the range of 23.7-47.9% (average = 41.3%, median = 42.6%). In PBC1, where a mixture of soil, earthworm cast, and compost (7:2:1, v/v) was employed as the packing material, the $CH_4$ removal efficiency was evaluated to be between 60.7-85.5%. In PBC2, which was filled with a mixture of soil, earthworm cast, perlite, and compost (4:2:3:1, v/v), the removal efficiency was evaluated to be between 29.2-78.5%. Although the packing materials had an excellent $CH_4$ oxidizing potential (average oxidation rate for $CH_4=180-199{\mu}g\;CH_4{\cdot}g\;packing\;material^{-1}{\cdot}h^{-1}$), $CH_4$ removal efficiency in PBC1 and PBC2 decreased to the range of 0-30% once the packing materials in the PBCs were clogged and channeled. Furthermore, seasonal effects exhibited no significant differences in the $CH_4$ removal efficiency of the biocovers. The results of this study can be used to design and operate real-scale biocovers in landfills to mitigate $CH_4$ buildup.