Isolation and Characterization of Methanobacterium espanolae sp. nov., a Mesophilic, Moderately Acidiphilic Methanogen

Abstract

Bacterial strain G n T (T = type strain), a nonmotile, nonsporeforming, mesophilic, methanogenic bacterium, was isolated from the primary sludge obtained from the waste treatment facility of a major kraft pulp mill in Canada. Single cells were 6.0 by 0.8 pm and stained gram positive. Growth and methane production occurred only with H,-CO, as the substrate. Acetate, formate, propionate, butyrate, pyruvate, methanol, or trimethylamine could not serve as a sole source of carbon and energy for growth. The optimum pH for growth was between 5.6 and 6.2; consistent growth and methane production were not observed below pH 4.68. The optimum temperature for growth was 35"C, and little or no growth was observed during incubation at 15 and 50°C. Kanamycin and bacitracin were severe inhibitors of growth and methanogenesis, whereas 100 pM bromoethanesulfonic acid caused 30 % inhibition. Supernatant from primary sludge enhanced growth by about 10%. The DNA base composition was 34 mol% guanine plus cytosine. On the basis of physiological characteristics, indirect immunofluorescence typing, and DNA-DNA hybridization studies, the isolate is named Methanobacterium espanolae sp. nov.The optimum pH for growth of most pure cultures of methanogenic bacteria isolated to date lies between 6.0 and 8.0, with the majority of cultures having an optimum close to pH 7.0 (23,25). Methanogenic bacteria with optimum growth pH values greater than 8.0 have been reported recently (27). Although methanogenic activity has been detected in acidic ecosystems, such as bogs and peats (11,24,25), at pH values close to 4.0, methanogenic isolates from these ecosystems have been unable to grow at pH values below 5.3 (25). Moreover, no pure culture of a methanogen that is able to grow at pH values below 5.0 has been reported previously, to our knowledge. The isolation and study of acid-tolerant methanogens was undertaken as part of a program on biomass degradation in anaerobic, acidic environments, with the purpose of extending the potential of anaerobic treatment for disposal of acidic, organic, industrial wastes. In this paper we describe the isolation and characterization of a methanogen that is able to grow and produce methane at pH values below 5.0. MATERIALS AND METHODSMedia. The composition of SA medium, which was used for isolation and stock culture maintenance, was as described previously (5) except for an increase in the cysteineNa,S concentration. This medium contained (per liter) 2,500 mg of CH,COONa -3H@, 480 mg of Na,CO,, 450 mg of mg of vitamin B12. The pH of the mixed ingredients (except cysteine-Na,S and Na,CO,) was adjusted to 4.5 with 5 N HC1. The medium was then reduced with cysteine-Na,S, Na,CO, was added, and 10-ml portions were dispensed into 60-ml serum vials under 80% H2-20% CO, (5). The postautoclaved (121"C, 15 min) pH of the medium was 5.5 k 0.1. Similarily, SA medium with an initial pH of 5.0 and S medium (the same as SA medium but without acetic acid) with pH values of 5.5 and 5.0 were prepared by appropriate pH adjustment before...

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

“…Some methanogens are sensitive to the antibiotics used. Methanotrix soehngenii and the moderately acid-tolerant Methanobacterium espanolae are sensitive to ␤-lactam antibiotics and kanamycin, respectively (25,43). Additional antibiotics that inhibit protein synthesis also inhibit certain methanogens (21).…”

Methanogenic Archaea Isolated from Taiwan's Chelungpu Fault

“…However, the pH tolerance is diverse. Some species are moderately acidic, such as Methanobacterium espanolae, which has a pH range of 4.6 to 7.0 and an optimal pH of 5.6 to 6.2 (43), and some are alkalophiles, such as Methanobacterium alcaliphilum, which has a pH range of 7.0 to 9.9 and an optimal pH of 8.1 to 9.1 (61) ( Table 2). An exception is Methanobacterium subterraneum, the first methanogen isolated from the deep granitic groundwater at depths of 68, 409, and 420 m (27).…”

Methanobacterium lacus sp. nov., isolated from the profundal sediment of a freshwater meromictic lake

“…Growth was stimulated by the addition of acetate (5 mM), vitamins (5 ml l 21 ), yeast extract (0.2 g l 21 ) or rumen fluid (5 ml l 21 ). Strain 17A1 T utilized H 2 /CO 2 and methanol (Ma et al, 2005); 3, Methanobacterium veterum MK4 T (Krivushin et al, 2010); 4, Methanobacterium formicicum MF T (Bryant & Boone, 1987); 5, Methanobacterium palustre F T (Zellner et al, 1988); 6, Methanobacterium espanolae GP9 T (Patel et al, 1990). 2, Negative; +, positive; ND, not determined; E, essential; NR, not required; S, stimulatory.…”