The Isolation and Characterization of Methanomonas Methanooxidans Brown and Strawinski

Abstract

Procedures for the isolation and characterization of Metha7zonzo?zas n~ethatto-oxidans Brown and Strawinslii are described. Isolates from varied sources are alike in cellular morphology, inasmuch a s they form only microcolonies, and in their dependence on methane or methanol as carbon and energy sources for growth. Both organic and inorganic nitrogen sources are used. The organism is a Gram negative non-sporeformillg rod, 1.5 to 3.0 p by 1.0 p in size, and motile by means of a single polar flagellum. In growing cultures the osygen/methane ratio was approsinlately 1.1 and in resting cells 1.7. The R.Q. for methane with resting cells was 0.43. Resting cells were unable t o oxidize organic compounds other than methane, methanol, formaldehyde, and formate. Formic acid was detected in test solutions after cell suspensions had metabolized methane, methanol, and formaldehyde. Using sodium sulphite as trapping agent for formaldehyde, it was found that 60 t o 70y0 of the methane or methanol consumed was converted to formaldehyde. I n the presence of iodoacetate, 70% of the methane consumed was present terminally as methanol. Thus i t was shown that methanol, formaldehyde, and formic acid are sequential intermediates in the osidation of methane by these organisms.In 1905 Kaserer concluded that inethane was oxidized by bacteria but he did not attempt t o isolate the responsible organisms. Shortly thereafter Sohngen (16) described a methane-oxidizing bacterium which he named Bacillus methanicz~s. Since that time, bacteria capable of utilizing inethane have been reported by inany ~vorlters including Munz (13), Giglioli and Masoni (9), Aiyer (I), A4ogilevsltii (12), Boltova, Kusnetsova, and I<usnetsov (3), Nechaeva (14), I-Iutton and ZoBell (lo), Dworltin and Foster (6), and Leadbetter and Foster (11). For a recent review, see Fuhs (8).Strawinslti and Brown (17) and Brown and Strawins1;i (4) reported a new methane-oxidizing bacterium to which they gave the name AJethanomonas methanooxidans sp. n. The present paper gives an account of the procedures ernployed in the isolation and characterization of this species. Aledia Materials and MethodsA mineral salts broth, consisting of I<NO3, 1.0 g; I<~I-IPO.~.3HzO, 0.5 g; ;\4gSO4.7Hz0, 0.2 g ; FeCl3.61-120, 0.05 g/l. distilled water was used for the enrichment and propagation of the methane oxidizer. The same medium, solidified with 2y0 Bacto agar, was used for the isolation of strains of the organism and t o study their colonial characteristics. Nutrient agar and other media enriched with peptone were used t o detect the presence of bacteria capable of utilizing carbon sources other than methane.i\iIethane and other gases employed (A'Iatheson Company, East Rutherford, New Jersey) were of high purity: methane, c.p. grade, 99.0y0 pure;

Enrichment, Isolation and Some Properties of Methane-utilizing Bacteria

“…Subgroup Comparison of isolates with previously described species Some strains were considered to be identical with the previously described methaneutilizing species, Pseudomonas methanica (Leadbetter & Foster, I 958) and Methylococcus capsulatus (Foster & Davis, 1966). None were identified as Methanomonas methanooxidans (Brown et al 1964). The 'Methylosinus' strains may be similar to Pseudomonas methanitrificans (Coty, 1967), as one strain was isolated from a culture originating from Coty.…”

Effect of Leaking Natural Gas on Soil and Vegetation in Urban Areas

“…Methane-oxidizing microorganisms have been described by several authors (Kaserer, 1906;Sohngen, 1906;Harper, 1939;Leadbetter & Foster, 1960;Brown et al, 1964;Nyns & Wiaux, 1969). The organisms mentioned in literature are for example Bacillus methanicus (Sohngen, 1906), Methanonomas methanooxidans (Brown et al, 1964), Methylococcus capsulatus (Foster & Davis, 1966) and Pseudomonas methanica (Dworkin & Foster, 1956;Leadbetter & Foster, 1958).…”

“…The organisms mentioned in literature are for example Bacillus methanicus (Sohngen, 1906), Methanonomas methanooxidans (Brown et al, 1964), Methylococcus capsulatus (Foster & Davis, 1966) and Pseudomonas methanica (Dworkin & Foster, 1956;Leadbetter & Foster, 1958).…”

Particulate malate oxidation in strictly aerobic bacteria

“…On the other hand, Whittenbury (1969) pointed out that cell-yield data obtained in his laboratory and the inhibitor studies by Brown et al (1964) might not be consistent with involvement of an oxygenase. Accordingly he suggested thepossibility ofahydroxylation reaction giving methanol and two electrons for energy transduction: CH4+H20 -+ CH3*OH+2 [H] Direct measurements of the incorporation of 180 into methanol during oxidation of methane by whole cells of M. methanooxidan8 and P. methanica have now been made by using a combined gas chromatograph-mass spectrometer.…”

“…(Brown, Strawinski & McCleskey, 1964) indicate that the route of methane oxidation in this micro-organism is:…”