Methane-oxidizing bacteria (methanotrophs) play an ecological role in methane and nitrogen fluxes because they are capable of nitrogen fixation and methane oxidation, as indicated by genomic and cultivation-dependent studies. However, the chemical relationships between methanotrophy and diazotrophy and aerobic and anaerobic reactions, respectively, in methanotrophs remain unclear. No study has demonstrated the cooccurrence of both bioactivities in a single methanotroph bacterium in its natural environment. Here, we demonstrate that both bioactivities in type II methanotrophs occur at the single-cell level in the root tissues of paddy rice (Oryza sativa L. cv. Nipponbare). We first verified that difluoromethane, an inhibitor of methane monooxygenase, affected methane oxidation in rice roots. The results indicated that methane assimilation in the roots mostly occurred due to oxygen-dependent processes. Moreover, the results indicated that methane oxidation-dependent and methane oxidation-independent nitrogen fixation concurrently occurred in bulk root tissues. Subsequently, we performed fluorescence in situ hybridization and NanoSIMS analyses, which revealed that single cells of type II methanotrophs (involving six amplicon sequence variants) in paddy rice roots simultaneously and logarithmically fixed stable isotope gases ¹⁵N₂ and ¹³CH₄ during incubation periods of 0, 23, and 42 h, providing in vivo functional evidence of nitrogen fixation in methanotrophic cells. Furthermore, ¹⁵N enrichment in type II methanotrophs at 42 h varied among cells with an increase in ¹³C accumulation, suggesting that either the release of fixed nitrogen into root systems or methanotroph metabolic specialization is dependent on different microenvironmental niches in the root. IMPORTANCE Atmospheric methane concentrations have been continually increasing, causing methane to become a considerable environmental concern. Methanotrophy may be the key to regulating methane fluxes. Although research suggests that type II methanotrophs are involved in methane oxidation aerobically and nitrogen fixation anaerobically, direct evidence of simultaneous aerobic and anaerobic bioreactions of methanotrophs in situ is still lacking. In this study, a single-cell isotope analysis was performed to demonstrate these in vivo parallel functions of type II methanotrophs in the root tissues of paddy rice (Oryza sativa L. cv. Nipponbare). The results of this study indicated that methanotrophs might provide fixed nitrogen to root systems or depend on cells present in the spatially localized niche of the root tissue. Furthermore, our results suggested that single type II methanotrophic cells performed simultaneous methane oxidation and nitrogen fixation in vivo. Under natural conditions, however, nitrogen accumulation varied at the single-cell level.

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