A new genus and a new species of methane-oxidizing bacteria are described. The colonies produced by these bacteria are pink, circular, and convex with entire margins, Cells are gram negative and are normally found singularly with some rosettes. Negative stains indicate polar flagellation. In thin sections, intracytoplasmic membranes, similar to those described as type I1 in other methylotrophs, were present when the cells were grown with methane. No such membranes were apparent when the cells were grown with the other carbon and energy sources tested. The serine pathway for formaldehyde incorporation is the pathway of C, metabolism. The deoxyribonucleic acid base composition is 66 mol% guanine plus cytosine. Methylobacterium is proposed as t,he name for this new genus of rod-shaped, methane-oxidizing bacteria. The specific epithet in the name of the type species, Methylobacterium organophilum sp. nov., denotes the preference of this organism for organic carbon and energy sources more complex than methane. The type strain of M . organophilum is X X (= ATCC 27886). This bacterium differs from all previously described genera and species of methaneoxidizing bacteria in its ability to utilize a variety of organic substrates with carbon-carbon bonds as sources of carbon and energy. The pathways for methane oxidation and the assimilation of one-carbon units are repressed during growth on complex organic substrates.Methylotrophic bacteria are those bacteria that can grow non-autotrophically using carbon compounds containing one or more carbon atoms but containing no carbon-carbon bonds (1). Methylotrophs are considered obligate if they are capable of utilizing only carbon compounds containing no carbon-carbon bonds (methane, methanol, dimethylether, methylamines, etc.) as sole sources of carbon and energy. Facultative methylotrophs are those organisms that can use compounds containing no carbon-carbon bonds as well as organic substrates with carbon-carbon bonds as sole sources of carbon and energy.The previously described methane-utilizing bacteria, isolated in pure culture, all have had an obligate requirement for methane, methanol, or dimethylether (6, 11) as a source of carbon and energy. Patt et al. (8) reported the isolation of facultative methane utilizers. These organisms use methane, methanol, sugars, and organic acids as their carbon and energy sources. In this paper, we define the characteristics of, discuss the taxonomic position of, and propose a name for, a previously described methane-oxidizing bacterium, strain XX (8).MATERIALS AND METHODS lake samples, as described previously (8).Bacterial strain. Strain XX was isolated from Media and growth conditions. The mineral salts medium used contained (per liter of distilled water): KN03, 1 g; MgS04.7H20, 0.2 g; CaCl,, 0.02 g; Na,HP04, 0.23 g; NaH,PO,, 0.07 g; FeS04-7H,0, 1 mg; CuS04.5H20, 5 pg; H3B03, 10 pg; MnSO4.5H,O, 10 pg; ZnS04.7H20, 70 pg; and MOO,, 10 pg. The pH was adjusted to 6.8. A solid medium was prepared by adding purified agar (Difco) to give a...

Bacteria capable of growth on methane and a variety of complex organic substrates as sole sources of carbon and energy have been isolated. Conditions used to rigorously establish the purity of the cultures are described. One facultative methylotroph has been studied in detail. This organism has peripherally arranged pairs of intracytoplasmic membranes characteristic of obligate methylotrophs. This isolate apparently utilizes the serine pathway of formaldehyde fixation. The location of methane oxidizers in a dimictic lake indicates that these organisms prefer less than saturating levels of dissolved oxygen. Laboratory experiments confirmed the preference of these organisms for atmospheres containing less oxygen than air. One of these isolates has been more fully characterized and is described. (The work described in this paper was submitted to the University of Wisconsin by G. C. C. and T. E. P. in partial fulfillment of the requirements for the M.S. degree.) MATERIALS AND METHODS Organisms. Methylosinus sporium 12 and Methylosinus trichosporium PG were used in some experiments for comparison with our isolates (31). These organisms were kindly provided by R. Whittenbury, 955

Methane was oxidized to carbon dioxide in the absence of oxygen by water samples from Lake Mendota, Madison, Wis. The anaerobic oxidation of methane did not result in the assimilation of carbon from methane into material precipitable by cold 10% trichloroacetic acid. Only samples taken at the surface of the sediment of Lake Mendota were capable of catalyzing the anaerobic oxidation of methane. The rate of methane oxidation in the presence of oxygen was highest in samples taken from near the thermocline. Of the radioactive methane oxidized, 30 to 60% was assimilated into material precipitable by cold 10% trichloroacetic acid during aerobic incubation of the samples. These data support the conclusion that two distinct groups of methane-oxidizing organisms occur in stratified lakes. Enrichments with acetate and methane as the sole sources of carbon and energy and sulfate as the electron acceptor resulted in the growth of bacteria that oxidize methane. Sulfate, acetate, and methane were all required for growth of enrichments. Acetate was not oxidized to carbon dioxide but was assimilated by cells. Methane was not assimilated but was oxidized to carbon dioxide in the absence of air.

Intracytoplasmic membranes were present in Methylobacterium organophilum when cells were grown with methane, but not methanol or glucose, as the sole carbon and energy source. Cells grown with methane as the carbon and energy source and low levels of dissolved oxygen had the greatest amount of intracytoplasmic membrane. Cells grown with increased levels of dissolved oxygen had less intracytoplasmic membrane. The amount of total lipid correlated with the amount of membrane material observed in thin sections. The individual phospholipids varied in amount, but the same four were present in M. organophilum grown with different substrates and oxygen levels. Phosphatidyl choline was present as a major component of the phospholipids. Sterols were present, and they differed from those in the type I methylotroph Methylococcus capsulatus. The relative amounts of different sterols and squalene changed with the substrate provided for growth. The greatest amounts of sterols were found in methane-grown cells grown at low levels of dissolved oxygen. None of the unusual or usual membrane components assayed was uniquely present in the intracytoplasmic membranes.

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