Cultivation of methanogens under low-hydrogen conditions by using the coculture method

Abstract

We previously reported the isolation of novel methanogens by using a new cultivation method, referred to as the coculture method. Here, we extended our coculture method to various anaerobic environmental samples. As a result, we successfully cultivated some uncharacterized methanogens in coculture enrichments and eventually isolated a new methanogen, within the order Methanomicrobiales.

FIG. 1.

Phylogenetic tree showing the placement of 16S rRNA gene sequences/clones obtained in this study. The colored phylotypes were obtained in this study. The difference in color among phylotypes indicates the different substrates used for the enrichment cultures (blue, hydrogen; green, formate; orange, ethanol; pink, butyrate; red, propionate and propionate plus S. fumaroxidans strain MPOB). The name of each phylotype is composed of the sample name, an abbreviation of the substrate for cultivation (H2, hydrogen; For, formate; Eth, ethanol; Buty, butyrate; Pro, propionate; ProM, propionate plus S. fumaroxidans), and the phylotype (for example, SRP-Pro-A is phylotype A recovered from the propionate enrichment culture cultivated from the environmental sample SRP). The number in parentheses indicates the number of identical clones obtained per number of clones analyzed for each phylotype. The accession numbers are also shown after each phylotype name. The phylotypes indicated by the same accession numbers have the same sequences (e.g., SP-For-A and SRP-Buty-C, AR-ProM-A and NR-ProM-A). All of the clonal sequences were greater than 1,000 nucleotides in length, with the exception of Methanospirillum sp. TM20-1 (GenBank acc. no. AB062404; 789 bp). Therefore, the initial tree was constructed with sequences greater than 1,000 nucleotides using the neighbor-joining method. Subsequently, the Methanospirillum sp. TM20-1 sequence was inserted into the tree by using the parsimony insertion tool of the ARB program. The scale bar indicates the estimated number of base changes per nucleotide sequence position. The symbols at the branch nodes indicate bootstrap values.

FIG. 2.

Phylogenetic affiliation of the identified phylotypes based on their cultivation substrates. The panels indicate the results of enrichment cultures with the following substrates: H₂ (A), formate (B), ethanol (C), butyrate (D), propionate (E), and propionate with the addition of the pure culture of S. fumaroxidans (F). The identified phylotypes were classified into their respective genera according to their 16S rRNA gene similarity with previously characterized methanogens. Phylotypes possessing sequence similarity greater than 92% were treated as the same genus. The number of phylotypes for each group is indicated in parentheses.