Methanotrophs, cells that consume methane (CH4) as their sole source of carbon and energy, play key roles in the global carbon cycle, including controlling anthropogenic and natural emissions of CH4, the second-most important greenhouse gas after carbon dioxide. These cells have also been widely used for bioremediation of chlorinated solvents, and help sustain diverse microbial communities as well as higher organisms through the conversion of CH4 to complex organic compounds (e.g. in deep ocean and subterranean environments with substantial CH4 fluxes). It has been well-known for over 30 years that copper (Cu) plays a key role in the physiology and activity of methanotrophs, but it is only recently that we have begun to understand how these cells collect Cu, the role Cu plays in CH4 oxidation by the particulate CH4 monooxygenase, the effect of Cu on the proteome, and how Cu affects the ability of methanotrophs to oxidize different substrates. Here we summarize the current state of knowledge of the phylogeny, environmental distribution, and potential applications of methanotrophs for regional and global issues, as well as the role of Cu in regulating gene expression and proteome in these cells, its effects on enzymatic and whole-cell activity, and the novel Cu uptake system used by methanotrophs.

https://deepblue.lib.umich.edu/bitstream/handle/2027.42/79061/j.1574-6976.2010.00212.x.pdf;sequence=1

Methanotrophs and copper

Methane is an abundant gas used in energy recovery systems, heating, and transport. Methanotrophs are bacteria capable of using methane as their sole carbon source. Although intensively researched, the myriad of potential biotechnological applications of methanotrophic bacteria has not been comprehensively discussed in a single review. Methanotrophs can generate single-cell protein, biopolymers, components for nanotechnology applications (surface layers), soluble metabolites (methanol, formaldehyde, organic acids, and ectoine), lipids (biodiesel and health supplements), growth media, and vitamin B12 using methane as their carbon source. They may be genetically engineered to produce new compounds such as carotenoids or farnesene. Some enzymes (dehydrogenases, oxidase, and catalase) are valuable products with high conversion efficiencies and can generate methanol or sequester CO2 as formic acid ex vivo. Live cultures can be used for bioremediation, chemical transformation (propene to propylene oxide), wastewater denitrification, as components of biosensors, or possibly for directly generating electricity. This review demonstrates the potential for methanotrophs and their consortia to generate value while using methane as a carbon source. While there are notable challenges using a low solubility gas as a carbon source, the massive methane resource, and the potential cost savings while sequestering a greenhouse gas, keeps interest piqued in these unique bacteria.

Methane as a resource:Can the methanotrophs add value?

Methanotrophs: Multifunctional bacteria with promising applications in environmental bioengineering

Environmental deterioration, global climate change, and consequent increases in pollution-related health problems among populations have been attributed to growing consumption of fossil fuels in particular by the transportation sector. Hence, replacing these energy carriers, also known as major contributors of greenhouse gas emissions, with biofuels have been regarded as a solution to mitigate the above-mentioned challenges. On the other hand, efforts have been put into limiting the utilization of edible feedstocks for biofuels production, i.e., first generation biofuels, by promoting higher generations of these eco-friendly alternatives. In light of that, the present review is aimed at comprehensively assessing the role and importance of microorganisms such as bacteria and yeasts as catalysts for sustainable production of liquid biofuels including bioethanol, biomethanol, biobutanol, bio-ammonia, biokerosene, and bioglycerol. Various aspects of these biofuels, i.e., background, chemical synthesis, microbial production (including exploitation of wild and metabolically-engineered species), and product recovery as well as the derivatives produced from these biofuels which are used as fuel additives are thoroughly covered and critically discussed. Furthermore, the industrial features of these green liquid fuels including the industrial practices reported in the literature and the challenges faced as well as possible approaches to enhance these practices are presented.

A review on green liquid fuels for the transportation sector: a prospect of microbial solutions to climate change

A major obstacle preventing the large scale production of polyhydroxyalkanoates (PHAs) has been the lack of a reliable, low cost, large volume feedstock. The abundance and relatively low price of methane therefore marks it as a substrate of interest. This paper presents a techno-economic assessment of the production of poly-3-hydroxybutyrate (PHB) from methane. ASPEN Plus was used for process design and simulation. The design and economic evaluation is presented for production of 100,000 t/a PHB through methanotrophic fermentation and acetone-water solvent extraction. Production costs were estimated at $4.1–$6.8/kg PHA, which compares against a median price of $7.5/kg from other studies. Raw material costs are reduced from 30 to 50% of production for sugar feedstocks, to 22% of production for methane. A feature of the work is the revelation that heat removal from the two-stage bioreactor process contributes 28% of the operating cost. Thermophilic methanotrophs could allow the use of cooling water instead of refrigerant, reducing production costs to $3.2–5.4/kg PHA; it is noted that PHB producing thermophilic methanotrophs are yet to be isolated. Energy consumption for air compression and biomass drying were also identified as significant capital and operating costs and therefore optimisation of bioreactor height and pressure and biomass moisture content should be considered in future research.

https://espace.library.uq.edu.au/view/UQ:410471/UQ410471_OA.pdf

Techno-economic assessment of poly-3-hydroxybutyrate (PHB) production from methane—The case for thermophilic bioprocessing

Slow growth and relatively low cell densities of methanotrophs have limited their uses in industrial applications. In this study, a novel method for rapid cultivation of Methylosinus trichosporium OB3b was studied by adding a water-immiscible organic solvent in the medium. Paraffin oil was the most effective at enhancing cell growth and final cell density. This is at least partially due to the increase of methane gas transfer between gas and medium phases since methane solubility is higher in paraffin than in water/nitrate minimal salt medium. During cultivation with paraffin oil at 5% (v/v) in the medium, M. trichosporium OB3b cells also showed higher concentrations of the intermediary metabolites, such as formic acid and pyruvic acid, and consumed more methane compared with the control. Paraffin as methane vector to improve methanotroph growth was further studied in a 5-L fermentor at three concentrations (i.e., 2.5%, 5%, and 10%). Cell density reached about 14 g dry weight per liter with 5% paraffin, around seven times higher than that of the control (without paraffin). Cells cultivated with paraffin tended to accumulate around the interface between oil droplets and the water phase and could exist in oil phase in the case of 10% (v/v) paraffin. These results indicated that paraffin could enhance methanotroph growth, which is potentially useful in cultivation of methanotrophs in large scale in industry.

Paraffin oil as a “methane vector” for rapid and high cell density cultivation of Methylosinus trichosporium OB3b

Research progresses of methanotrophs and methane monooxygenases

Heterologous expression of particulate methane monooxygenase in different host cells

The invention discloses a method for expressing granular methane monooxygenase and special engineering bacteria thereof. The method obtains zymogen liquid containing granular methane monooxygenase by inducing the engineering bacteria with expression activity of the granular methane monooxygenase. The engineering bacteria inducts a recomposed expression carrier which can experess the granular methane monooxygenase into host bacteria and seens the obtained-recomposed stains that can express the granular methane monooxygenase. The recomposed expression carrier is the carrier that can be expressed in the host bacteria containing monooxygenase promoter and the coding genes of the granular methane monooxygenase connected to the lower reach of the momooxygenase promoter. The engineering bacteria can effectively express the granular methane monooxygenase and can be applied in such catalysis reactions as the transformation of biogas into methanol, the oxidation of propylene into 1, 2-propylene oxide and the like.

Method for expression of granular methane monooxygenase and special engineering strain thereof

Heterologus expression of particulate methane mooxygenase gene in different host bacterial cells for biocatalysis

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