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

Bacterial strain GP9T (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 μm and stained gram positive. Growth and methane production occurred only with H2-CO2 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 μM 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.