The Order Methanobacteriales
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The Order Methanobacteriales ADAM S. BONIN AND DAVID R. BOONE
Characteristics of Methanobacteriales Members of the order Methanobacteriales are distinguished from other methanogens by their limited range of catabolic substrates, their morphology, lipid composition, and rDNA sequence. The Methanobacteriales are generally hydrogenotrophic, using H2 to reduce CO2 to CH4. Some members of this order can use formate, CO, or secondary alcohols as electron donors for CO2 reduction. However, members of one genus within this order, Methanosphaera, use H2 to reduce methanol to methane. The predominant cell wall polymer of Methanobacteriales is pseudomurein, which distinguishes this order from the Methanomicrobiales. Lipids composing the cell membranes include caldarchaeol and myo-inositol. Cells usually stain Gram positive and are generally rod-shaped, often forming chains or long filaments up to 40 µm in length. The order Methanobacteriales is divided into two families, the Methanobacteriaceae and Methanothermaceae, on the basis of phylogenetic analysis of 16S rRNA gene sequences as well as phenotypic characteristics (Boone, 2001).
Habitats of Methanobacteriales Similarly to other methanogenic orders, members of the Methanobacteriales are very strict anaerobes inhabiting a variety of anoxic habitats including freshwater and marine sediments, groundwater, rice paddy fields, terrestrial subsurface environments, anaerobic sewage digestors, environments containing accumulated geothermal H2, and the gastrointestinal tracts of animals. Specific habitats known to harbor specific strains are indicated below in the sections describing each species.
Isolation and Characterization Enrichment Media for Growth of Methanobacteriales MS Mineral Medium and MS Medium are appropriate for the growth of Methanobacteriales. The recipes for these media are given below. MS Mineral Medium with H2 added as a catabolic substrate is an effective medium for the enrichment of autotrophic members of this order. MS Medium, which contains yeast extract and trypticase peptones (2 g each per liter), is recommended for the enrichment of species that have organic nutrient requirements. Reducing the concentrations of yeast extract and trypticase peptones to 0.5 g of each per liter (MS Enrichment Medium) can reduce the numbers of heterotrophic bacteria growing in enrichment cultures. The use of the higher concentrations of organic compounds in MS medium has some advantages in the routine cultivation of pure cultures: it is easy to detect the growth of heterotrophic contaminants, when they occur, in this rich medium. The Methanobacteriales generally grow and produce methane by reducing CO2 with H2, although some cells can use formate, CO, secondary alcohols, or methanol as electron donor. Species of Methanospaera grow only by reducing methanol to methane, with H2 as the electron donor. MS Medium (Boone et al., 1989) NaHCO3 Yeast extract Trypticase peptones Mercaptoethanesulfonic acid Na2S · 9H 2O NH4Cl K2HPO4 · 3H 2O
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