Influence of methanotrophs on nitrogen cycling and N2O emissions

Abstract

Methanotrophs synthesize methanobactin, a secondary metabolite that binds copper with an unprecedentedly high affinity. Such a strategy may provide methanotrophs a ‘copper monopoly’ that can inhibit the activity of copper-containing enzymes of other microbes, e.g., copper-dependent N$_2$O reductases. Here, we show that methanobactin from Methylosinus trichosporium OB3b inhibited N$_2$O reduction in denitrifiers. When Pseudomonas stutzeri DCP-Ps1 was incubated in cocultures with M. trichosporium OB3b or with purified methanobactin from M. trichosporium OB3b, stoichiometric N$_2$O production was observed from NO3- reduction, while no significant N$_2$O accumulation was observed in cocultures with a mutant defective in methanobactin production. In addition, copper uptake by P. stutzeri DCP-Ps1 was inhibited by the presence of purified methanobactin, leading to a significant down-regulation of nosZ transcription. Similar findings were observed with three other denitrifier strains. Furthermore, we investigate that multiple copper sequestration strategies of soil methanotrophs may interfere with Cu uptake of soils’ denitrifier and non-denitrifying N$_2$O reducer communities, and result in enhanced N$_2$O emissions from soil denitrification. Denitrification of soil slurry microcosms prepared with rice paddy soils with varying Cu contents exhibited distinctive N$_2$O production trends when amended with purified methanobactin, indicating that methanobactin affected N$_2$O emission from soils’ complex denitrifying communities. The rice paddy soil slurry microcosms enriched with CH$_4$ enhanced N$_2$O production upon transition to anoxic condition, regardless of methanobactin-producing alphaproteobacterial population, suggesting an additional Cu sequestration mechanism in gammaproteobacterial methanotrophs negatively influencing N$_2$O reduction activity. The multimodal influence of methanotrophs on N$_2$O emissions was further investigated with microcosms amended with varying cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH$_4$ enrichment. N$_2$O production was enhanced to a greater extent in the OB3b-dominant enrichments than in the Gammaproteobacteria-dominant enrichments

however, significant N$_2$O production enhancement occurred regardless of methanotroph composition. These observations suggest that involuntary inhibition of N$_2$O reduction is a plausible N$_2$O emission mechanism in subsurface soil environments where methanotrophs and denitrifiers coexist.