PE-064:

Nitrogen fixation depends on the methane oxidation by Methylosinus sp. isolated from paddy rice roots.

Methanotrophs play a key role in CH4 emissions from rice paddies. Previous metagenomic and metaproteomic analyses revealed that in rice roots under a low fertilization (LN) condition, the abundance of typeⅡmethane−oxidizing bacteria (MOB) were significantly increased with highly expressed nif (nitrogen fixation) and pmo (CH4-oxidizing) genes of MOB, suggesting CH4-dependent N2 fixation of MOB in paddy rice roots (Bao et al. 2014). Because acetylene is a strong inhibitor of CH4 monooxygenase enzyme, the acetylene reduction assay has been conducted for the evaluation of N2-fixing activity of MOB cells supplied with methanol as an energy source. Thus, there is no direct evidence for CH4-dependent N2 fixation of MOB so far. The aims of this study are to isolate Metylosinus sp. dominated in LN rice roots, and to examine whether the MOB isolates have the capability of CH4-dependent N2 fixation with Methylosinus trichosporium OB3b as a reference MOB. When MOB strains were cultured in sealed bottles (120 ml) in NMS medium (20 ml) containing KNO3 (1 g/L) or N-free NMS medium under a gas phase of 5% CH4, 10% O2 and 85% N2, CH4 and O2 were consumed along with the cell growth (OD600) and CO2 production even in N-free NMS medium. However, the cell growth was not observed at all in N-free NMS medium when N2 was replaced to Ar (5% CH4, 10% O2 and 85% Ar) in the gas phase. These results indicate that the MOB cells fixed atmosphere N2 as N source in N-free NMS medium. Finally, 15N was apparently incorporated into MOB cells from 15N2 when the MOB cells were cultivated in N-free NMS medium under a gas phase of 5% CH4, 10% O2 and 85% 15N-labelled N2 (0.008 atom% excess). From these results, we concluded that the MOB cells isolated from paddy rice roots are able to fix atmosphere N2 by using CH4 as a sole energy source.

keywords:methane−oxidizing bacteria,Rice paddy field,endophyte,Nitrogen fixation