Enhancement of Nitrous Oxide Emissions in Soil Microbial Consortia Via Copper Competition Between Proteobacterial Methanotrophs and Denitrifiers

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized, post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers and thereby enhance NO emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased NO production, with more pronounced responses observed for soils with lower copper content. The NO emission-enhancing effect of the soil's native -expressing methanotrophs on the native denitrifiers was then experimentally verified with a -dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Finally, with microcosms amended with various cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH enrichment, microbiomes with different ratios of methanobactin-producing to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of NO production from denitrification was evident in both -dominant and -dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration, while implying the presence of alternative copper abstraction mechanisms for . These observations support that copper-mediated methanotrophic enhancement of NO production from denitrification is plausible where methanotrophs and denitrifiers cohabit. Proteobacterial methanotrophs-groups of microorganisms that utilize methane as a source of energy and carbon-have been known to employ unique mechanisms to scavenge copper, namely, utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas NO has been suggested in axenic and coculture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments cohabited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allows for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of NO production may account for a significant proportion of NO efflux from agricultural soils.