Rare bacterium of new genus isolated with prolonged enrichment culture

Dynamic change in microbial flora was monitored with an oxygen electrode. The 1(st) phase microorganisms, which first grew well in LB medium, were followed by the 2(nd) phase microorganisms, which supposedly assimilated microbial cells of the 1st phase and their metabolites. In a similar way, a change in microbial flora was observed from the 1(st) phase to the 4(th) phase in 84 hr. Based on this observation, prolonged enrichment culture was done for as long as two months to increase the ratio of existence of rare microorganisms. From these culture liquids, four slow-growing bacteria (provisionally named Shinshu-ah1, -ah2, -ah3, and -ah4), which formed scarcely visible small colonies, were isolated. Sequence analysis of their 16S rDNA showed that Shinshu-ah1 had 97% homology with Bradyrhizobium japonicum and uncultured alpha proteobacterium clone blaii 16, Shinshu-ah2 91% with Rasbo bacterium, Alpha proteobacterium 34619, Bradyrhizobium genosp. P, Afipia felis and an unidentified bacterium, Shinshu-ah3 99% with Methylobacterium mesophilicum, and Shinshu-ah4 95% with Agromyces ramosus DSM 43045. Phylogenetic study indicated, that Shinshu-ah2 had a possibility to form a new family, Shinshu-ah1 a new genus, and Shinshu-ah4 a new species.