Enhancement of Nitrous Oxide Emissions in Soil Microbial Consortia via Copper Competition between Proteobacterial Methanotrophs and Denitrifiers

Abstract

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 in situ and thereby enhance N2O 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 N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soil’s native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Lastly, with microcosms amended with varying cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH4 enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-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 Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit.IMPORTANCE Proteobacterial 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.

Methanotrophs: Discoveries, Environmental Relevance, and a Perspective on Current and Future Applications

“…Similarly, aerobic methanotrophs may also rely on Sphagnum for molecular oxygen, enabling their proliferation in anoxic niches in peatland ecosystems (Raghoebarsing et al, 2005;Ho and Bodelier, 2015); or independently relying on incomplete denitrification (van Grinsven et al, 2020). The interaction of aerobic methanotrophs with their biotic environment, appears to be relevant in modulating community functioning, and may help confer resilience during disturbances prompting the use of alternative strategies and the formation of synergistic/antagonistic interactions (Ho et al, 2014(Ho et al, , 2016(Ho et al, , 2020Veraart et al, 2018;Chang et al, 2020;He et al, 2020;Kaupper et al, 2021). Here, such interactions expand the habitat range of the aerobic methanotrophs to encompass unexpected environments which usually do not meet the physiological requirements for aerobic methane oxidation.…”

Cometabolic Vinyl Chloride Degradation at Acidic pH Catalyzed by Acidophilic Methanotrophs Isolated from Alpine Peat Bogs

“…67,68 Recently, methanobactin-mediated microbial interactions have been suggested, raising the possibility that methanobactin control of Cu availability may be selected for or against certain organismal groups. 42,69,70 Whether these effects on microbial community structure are related to the observed VC cooxidation performance warrants further study. Microbial reductive dechlorination is a cornerstone process for bioremediation at sites impacted with chlorinated ethenes.…”

“…The Cu concentration in the DNMS medium without added Cu did not exceed 0.01 μM. 42 After the bottles were sealed with butyl rubber stoppers (Geo-Microbial Technologies, Inc., Ochelata, OK), 20% of the headspace was replaced with >99.999% CH 4 gas (Deokyang, Ulsan, South Korea), which was provided as the sole electron donor and carbon source. After inoculation, the bottles were incubated on a shaker set to 200 rpm in dark at room temperature (25 °C).…”

“…The serum bottles used for the VC degradation experiments were washed in a 2.2 M HNO 3 bath overnight before use to remove any adsorbed Cu. The Cu concentration in the DNMS medium without added Cu did not exceed 0.01 μM . After the bottles were sealed with butyl rubber stoppers (Geo-Microbial Technologies, Inc., Ochelata, OK), 20% of the headspace was replaced with >99.999% CH 4 gas (Deokyang, Ulsan, South Korea), which was provided as the sole electron donor and carbon source.…”

The methane-driven interaction network in terrestrial methane hotspots

“…Microbial interactions in complex communities, including the methanotroph interactome, have been explored using a co-occurrence network analysis based on specific genes (e.g., 16S rRNA, 18S rRNA genes) amplified from isolated nucleic acids (DNA, RNA) [ 11 – 17 ]. Microbial taxa that are positively correlated in the network analysis can be interpreted as having complementary roles, sharing the same habitat niche, or are driven by cross-feeding [ 1 , 3 , 11 , 13 , 18 ], whereas negative correlations are attributable to competing taxa, predation, or niche partitioning [ 4 , 19 – 21 ]. The aerobic methanotrophs thrive in the presence of other organisms, forming (mutually) beneficial associations (e.g., receiving essential vitamins; [ 22 ]), as well as adverse relationships (e.g., selective predation by protists; [ 23 ]) with their biotic environment.…”