Samples for isolation of MOB were collected from seven compartments (floodwater, soil, surface soil, rhizosphere soil, root, stem and stump) at a Japanese rice field during mid-season drainage and under flooded condition. MOB were isolated directly from the samples without enrichment and from the enrichment cultures by four different methods using nitrate mineral salts (NMS) medium at 30°C for 3-4 weeks. Purity of MOB isolates was confirmed by observing morphology microscopically. In addition, we examined their growth on NMS agar with and without methane addition. DNA was extracted from MOB isolates and subjected to amplification of 16S rDNA by PCR then sequencing.
We obtained twenty-four MOB isolates. 16S rDNA sequence analysis showed that five isolates belonged to Methylomonas or Methylocaldum in Gammaproteobacteria (i.e. type I MOB), thirteen to Methylocystis in Alphaproteobacteria (i.e. type II MOB), four to Flexibacter in Bacteroidetes (CFB group) and two to Acidovorax in Betaproteobacteria.
Members of type II MOB were more abundant in MOB isolates from the rice field ecosystem than type I MOB in this study. Type I MOB have never been isolated from rice field ecosystem so far, though the predominance of them has been shown in the previous studies with molecular approaches. We, however, successfully isolated type I MOB strains from floodwater, rhizosphere soil, root and stump. The Methylomonas- or Methylocaldum-like isolates were aerobic, motile, rod-shaped bacteria with pigmentation (pink, yellow to orange, purple to pink or opaque) during growth on agar medium. Strains FW12E-Y isolated from floodwater and RS11D-Pr obtained from rhizosphere soil were most closely related to Methylomonas methanica and Methylocaldum szegediense, respectively, with 93.9% and 92.4% similarities of 16S rDNA sequence to the type strains of the species, Methylomonas methanica NCIMB 11130T and Methylocaldum szegediense OR2T. This suggests that strains FW12E-Y and RS11D-Pr are novel genera or species within type I MOB. The isolates belonging to type II MOB were aerobic, motile, rod-shaped bacteria that produced pigments (pink, yellow to orange, purple to pink, red or opaque). Strains FW1B-WF isolated from floodwater, SS37A-Re from surface soil, RS6A-Re from rhizosphere soil and Stu20C-Re from stump showed 98.2%-98.7% identities of 16S r DNA sequences with the type strain of the species, Methylocystis parvus OBBPT.
Strains Rt4B-Y and S1A-W belonging to CFB group were most closely related to Flexibacter sancti IFO 16033. The similarity values of 16S rDNA sequences with the type strain, Flexibacter sancti IFO15057T were 96.8%-97.4%. Particulate methane monooxygenase gene (pmoA) was amplified by PCR from the both isolates and partial sequencing of pmoA from strain Rt4B-Y showed 95.9% deduced amino acid identity to uncultured bacterium pmoA. Strain FW5B-W affiliated to the genus Acidovorax showed closest relations to Acidovorax sp. BSB421 and 97.1% similarity of 16S rDNA sequence to the type strain, Acidovorax delafieldii ATCC17505T. Strain FW5B-W had genes of pmoA and methanol dehydrogenase (mxaF), which exhibited 98.2% and 96.2% deduced amino acid identities to pmoA of Methylocystis parvus OBBPT and mxaF of type II methanotroph strain CSC-1, respectively. Although no methanotroph has been known so far in CFB group and Betaproteobacteria, these isolates consumed methane on NMS medium supplemented with 18%(v/v) methane in gas phase as a sole carbon and energy source. These results indicate that CFB group and Betaproteobacteria contain novel methanotrophs. Further investigation is needed to characterize these novel MOB isolates, especially on physiological features including methanotrophy.