We studied the effects of pH and ammonia concentration on the growth of three methanogens. These three halophilic, methylotrophic methanogens, Methanolobus bombayensis, Methanolobus taylorii, and Methanohalophilus zhilinaeae, grew at environmental pH ranges that overlapped with each other and spanned the pH range from 7.0 to 9.5. During growth they had reversed membrane pH gradients (⌬pH) at all pH values tested. The ⌬pH was in the range of ؊0.4 to ؊0.9 pH units, with the cytosol being more acidic than the environmental pH. Methanohalophilus zhilinaeae had the most negative ⌬pH (؊0.9 pH units). These negative pH gradients resulted in the accumulation of ammonium (NH 4 ؉), and when grown at the highest external ammonia concentrations that allowed good growth, cells had cytosolic NH 4 ؉ concentrations as high as 180 mM. The high concentrations of cytosolic NH 4 ؉ were accompanied by greater ⌬pH and lower concentrations of the major cytosolic cation K ؉ (compared with cells grown in medium with only 5 mM ammonia). Methanolobus bombayensis and Methanolobus taylorii were more sensitive to total external ammonia at higher external pH values, but the inhibitory concentration of un-ionized ammonia that resulted in a 50% reduction of the growth rate was about 2 to 5 mM, regardless of the pH. This is consistent with growth inhibition by ammonia in other bacteria. However, Methanohalophilus zhilinaeae was more resistant to un-ionized ammonia than any other known organism. It had a 50% inhibitory concentration for un-ionized ammonia of 13 mM at pH 8.5 and 45 mM at pH 9.5. We examined the effects of pH on three ammonia-assimilating activities (glutamine synthetase, glutamate dehydrogenase, and alanine dehydrogenase) in cell lysates and found that the pH ranges were consistent with the observed ranges of intracellular pH.

Methanolobus bombayensis B-lT (= OCM 43ST) (T = type strain) was isolated from Arabian Sea sediments obtained near Bombay, India. This strain grew on methylamines, methanol, and dimethyl sulfide, but it did not catabolize H,-CO,, acetate, or formate. The cells were nonmotile, irregular coccoids (diameter, 1.0 to 1.5 pm) and occurred singly. Electron micrographs revealed that a cell membrane and a protein cell wall were present. The cells grew fastest at mesophilic temperatures, at a neutral pH, and at salinity levels near the salinity level of the ocean, and they required about 30 mM divalent cations (M$+ and Ca2+). The cells grew in mineral medium, but growth was greatly stimulated by yeast extract and peptones. The guanine-plus-cytosine content of the DNA was 39.2 +-0.1 mol%. A comparison of 16s rRNA sequences showed that strain B-lT was phylogenetically related to Methanolobus vukani, but the sequences of these organisms differed by 2%. Some marine methanogens use either H,-CO, (6-8, 11, 14, 34) or acetate (30), but many marine isolates and nearly all isolates obtained from hypersaline sources are obligately methylotrophic (3, 17, 19, 26, 35, 36). Obligately methylotrophic methanogens grow exclusively on methyl compounds (such as methyl amines and methanol). Some of these obligately methylotrophic organisms can also catabolize dimethyl sulfide and methanethiol (7, 10, 22, 25). Methanosphaera species, which grow by using H, to reduce methanol to methane, are not considered obligate methylotrophs, nor are Methanosarcina species that catabolize acetate or H,-CO, as well as methylotrophic substrates.The family Methanosarcinaceae contains all known halophilic, methylotrophic methanogens. These organisms include extreme halophiles (in the genus Methanohalobium), moderate halophiles (in the genus Methanohalophilus), and slight halophiles (in the genera Methanolobus and Methanococcoides and one Methanosarcina species, Methanosarcina siciliae). Methanosarcina siciliae is a synonym of Methanolobus siciliae (24).In this paper we describe the isolation of a slightly halophilic, methylotrophic methanogen that requires high concentrations of Mg2+, Methanolobus bombayensis sp. nov. strain B-lT (T = type strain). Fe(NH,),(SO,),, 2 g of yeast extract, 2 g of peptones, 1.0 mg of resazurin, and 10 ml of a trace element solution (33). All of the constituents except sulfide were dissolved in water, boiled, and cooled under an 0,-free mixture of N, and CO, (4:l). The medium was distributed to serum bottles under the same gas mixture, and then the bottles were sealed and autoclaved at 121°C for 20 min. Sulfide from a sterile anoxic stock solution was added before inoculation. The pH of the medium was 7.2. For solid medium, 18 g of purified agar per liter was added. MATERIALS AND METHODS SourceFor other experiments we used a modification of MSH medium (22), a bicarbonate-buffered medium containing yeast extract, peptones, and 29.2 g of NaCl per liter. This medium was modified by increasing the concentration of MgC1, * 6H,O from 2.7 ...

Methanolobus vulcani PL-12/MT (T = type strain) grew on methylamines and methanol but not on dimethyl sulfide, formate, acetate, or H,-CO,. The cells grew rapidly at mesophilic temperatures, at neutral pH values (pH 6 to 7.5), and in medium supplemented with 0.1 to 1.2 M NaCl and 13 mM M$+. The cells grew in mineral medium containing biotin and a catabolic substrate, but growth was stimulated by yeast extract and peptones. M. vulcani was physiologically similar to Methanolobus tindarius and had a similar 16s rRNA sequence, although the results of DNA-DNA hybridization experiments indicated that these organisms should be considered separate species.

Sea sediments in tropical regions have been less studied for methanogenesis and methanogens present therein. Three species of methanogens viz. Methanobacterium bryantii, Methanococcus voltae and Methanosarcina barkeri were isolated from Arabian sea sediments collected near the west coast of India. Maximum methane was formed by M. voltae at 3.0% (w/v) NaCl and other two methanogens at 0.06% (w/v) NaCl. M. bryantii and M. barkeri tolerated 2.5 and 3.0% (w/v) NaCl respectively due to which these methanogens must have survived in salt conditions of the sea sediments.