CH4 oxidation-dependent 15N2 fixation in rice roots in a low-nitrogen paddy field and in Methylosinus sp. strain 3S-1 isolated from the roots

Abstract

Methane (CH4) oxidation and nitrogen (N2) fixation are simultaneously activated in the roots of rice plants grown in paddy fields with low N input (LN). However, the mechanism of CH4 oxidation-dependent N2 fixation remains largely unknown. In the present study, a15N2-feeding experiment was adopted to evaluate methanotrophic N2 fixation in LN rice roots and in a methanotroph isolated from the rice roots. The presence of CH4 significantly enhanced 15N incorporation from 15N2 gas in LN rice roots, indicating methanotrophic N2 fixation. Methylosinus sp. strain 3S-1 was isolated from LN rice roots, and it grew well in N-free liquid medium under different levels of oxygen stress (2%, 10%, and 20% O2). N2 fixation of strain 3S-1 was directly measured using 15N in biomass; when 3S-1 cells were exposed to a gas mixture consisting of 15N2 (35%), CH4 (5%), and O2 (2% or 10%) in argon (Ar) balance, the 15N concentration in the cells rapidly increased at both O2 concentrations. The addition of difluoromethane, a potent inhibitor of methane monooxygenase, immediately stopped CH4 oxidation and reduced the 15N enrichment rate, indicating that N2 fixation depended on CH4 oxidation in pure culture. These results suggest that N2 fixation was stimulated by CH4 oxidation in LN rice roots and that type II methanotrophs in LN rice roots, including Methylosinus, are responsible for CH4 oxidation-dependent N2 fixation.