Microbial Oxidation of Methane and Methanol: Isolation of Methane-Utilizing Bacteria and Characterization of a Facultative Methane-Utilizing Isolate

Abstract

A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanolor succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfatelinked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ionindependent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methanegrown cells contained low levels of enzymes of the tricarboxylic acid cycle, including a-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.Among the methylotrophic organisms are both obligate and facultative methylotrophs. Obligate methylotrophs are capable of utilizing only carbon compounds containing no carboncarbon bonds (methane, methanol, dimethylether, methylamine, etc.) as sole sources of carbon and energy. Facultative methylotrophs can use compounds containing no carbon-carbon bonds as well as complex organic compounds with carbon-carbon bonds as sole sources of carbon and energy (6).Almost all methane-utilizing bacteria isolated in pure culture exhibit obligate growth requirement for methane, methanol, or dimethylether as a source of carbon and energy (5,9,11,19,32,35). Organic compounds were unable to serve as a carbon and energy source. Recently, Patt et al. (25) reported the isolation and characterization of bacteria in pure culture that utilize methane as well as the complex organic compounds as sources of carbon and energy.In this paper we report on the isolation of both obligate and facultative methane-utilizing bacteria and on the characterization of one of the facultative isolates.MATERIALS AND METHODS Bacterial strain. Methylobacterium organophilum strain xx (ATCC 27886) (27) was obtained from the American Type Culture Collection, Rockvile, Md. Media a...

Facultative methanotrophy: false leads, true results, and suggestions for future research

“…Other early studies reported the isolation of facultative methanotrophs from a rice paddy in South China, as well as from soils collected from an oil refinery in the Northeastern United States (Patel et al, 1978;Zhao & Hanson, 1984a, b). These strains were found to have the complete TCA cycle and two of them, strains R6 and 761H, were able to grow solely on glucose, but not with other sugars such as fructose, galactose, or sucrose.…”

Characterization of Two New Facultative Methanotrophs

“…Although whole cells oxidized secondary butanol, the secondary alcohol dehydrogenase activity described by Hou et al (11) was not detected in either isolate. This unusual activity may therefore not be as widespread in methanotrophs as suggested by Hou and co-workers. In terms of presumptive classification, isolates H4-14 and 37W are similar to the other facultative methanotrophs M. organophilum XX (22, 23) and M. organophilum R6 (20) in that they have the ability to use both methane and more complex compounds as sole sources of carbon and energy. Before the work of Patt et al (22), this was not considered to be a feature of methanotrophs (28).…”

“…Since 1974, two facultative methanotrophs have been reported, both of which contained peripheral (type II) internal membrane systems and utilized the serine pathway for formaldehyde fixation (20,22). The isolation of these organisms has opened the way for detailed study of C-1 assimilation processes in methanotrophs (17)(18)(19).…”

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n -Alkanes by Methane-Utilizing Bacteria

“…Cell-free extracts of methylotrophic bacteria and yeasts contained an oxidized nicotinamide adenine dinucleotidelinked secondary alcohol dehydrogenase (9, 22). Oxidation of straight-chain aldehydes in methylotrophic bacteria was catalyzed by an aldehyde dehydrogenase (11,19,20). The ability to oxidize n-alkanes was observed only with cells of the methylotrophic bacteria that had been grown on methane.…”

“…The methane-utilizing bacteria are geneiraly obligately dependent on methane or methanol as sole sources of carbon and energy for growth (1,5,7,12,25,28,32). Recently, there have been reports on the isolation and charac-720 terization of bacteria in pure culture that utilize methane as well as complex organic compounds as sources of carbon and energy (19,24,25).…”