Enhancement of nitrous oxide emissions in soil microbial consortia via copper competition between proteobacterial methanotrophs and denitrifiers
Authors: Chang Jin , Kim Daehyun Daniel , Semrau Jeremy D. , Lee Juyong , Heo Hokwan , Gu Wenyu , Yoon Sukhwan 
Published: September 2020

AbstractUnique 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 denitrifiersin situand thereby enhance N2O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin fromMethylosinus trichosporiumOB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soil’s nativembnA-expressingMethylocystaceaemethanotrophs on the native denitrifiers was then experimentally verified with aMethylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Lastly, with microcosms amended with varying cell numbers of methanobactin-producingMethylosinus trichosporiumOB3b before CH4enrichment, microbiomes with different ratios of methanobactin-producingMethylocystaceaeto gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in bothMethylocystaceae-dominant andMethylococcaceae-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 forMethylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit.ImportanceProteobacterial methanotrophs, groups of microorganisms that utilize methane as source of energy and carbon, have been known to utilize 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 N2O has been suggested in axenic and co-culture 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 co-habited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allow for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N2O production may account for significant proportion of N2O efflux from agricultural soils.