Agricultural and industrial practices more than doubled the intrinsic rate of terrestrial N fixation over the past century with drastic consequences, including increased atmospheric nitrous oxide (N 2 O) concentrations. N 2 O is a potent greenhouse gas and contributor to ozone layer destruction, and its release from fixed N is almost entirely controlled by microbial activities. Mitigation of N 2 O emissions to the atmosphere has been attributed exclusively to denitrifiers possessing NosZ, the enzyme system catalyzing N 2 O to N 2 reduction. We demonstrate that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitirifers. nos clusters with atypical nosZ occur in Bacteria and Archaea that denitrify (44% of genomes), do not possess other denitrification genes (56%), or perform dissimilatory nitrate reduction to ammonium (DNRA; (31%). Experiments with the DNRA soil bacterium Anaeromyxobacter dehalogenans demonstrated that the atypical NosZ is an effective N 2 O reductase, and PCRbased surveys suggested that atypical nosZ are abundant in terrestrial environments. Bioinformatic analyses revealed that atypical nos clusters possess distinctive regulatory and functional components (e.g., Sec vs. Tat secretion pathway in typical nos), and that previous nosZ-targeted PCR primers do not capture the atypical nosZ diversity. Collectively, our results suggest that nondenitrifying populations with a broad range of metabolisms and habitats are potentially significant contributors to N 2 O consumption. Apparently, a large, previously unrecognized group of environmental nosZ has not been accounted for, and characterizing their contributions to N 2 O consumption will advance understanding of the ecological controls on N 2 O emissions and lead to refined greenhouse gas flux models.nitrogen cycle | climate change

Nitrous oxide (N 2 O) is a potent greenhouse gas with a strong potential to drive climate change (1, 2) and will continue to be the largest contributor to ozone depletion in the stratosphere (2, 3). Anthropogenic activities, predominantly, fertilizer application in agricultural production, have contributed to a steady increase in atmospheric N 2 O concentrations, and a continued upward trend is expected (4-6). Particularly troublesome are the findings of a recent study that concluded that without solutions for the N 2 O problem, carbon dioxide (CO 2 ) emission reductions even greater than those already proposed will be required to avoid climate change (7). Due to its environmental impact, the pathways leading to the generation and consumption of N 2 O have received heightened interest.In the environment, N 2 O is predominantly formed as an intermediate of denitrification and a nitrification by-product (8). Denitrification is the stepwise reduction of NO 3 Ϫ /NO 2 Ϫ to gaseous products (i.e., N 2 O, N 2 ), with each step being mediated by distinct enzyme systems (9). A kinetic imbalance in the rates of reactions producing and consuming N 2 O during denitrification leads to the release of N 2 O to the atmosphere (8, 10). In nitrification, N 2 O is generated by nitrifier denitrification and as a byproduct of ammonia oxidation (8,11,12). A recent report indicated that nitrifiers, rather than denitrifiers, may be the primary source of N 2 O in agricultural soils (12). Other processes contributing to N 2 O formation include respiratory ammonification (also known as dissimilatory nitrate/nitrite reduction to ammonium [DNRA]) and chemodenitrification (i.e., the abiotic reaction of NO 2 Ϫ with ferrous iron) (13,14). In contrast to the diverse pathways of N 2 O generation, the only known major biological pathway for the removal of N 2 O is by reduction to N 2 , catalyzed by the enzyme nitrous oxide reductase (NosZ) (8,(15)(16)(17)

High-performance MS instrumentation coupled with improved protein extraction techniques enables metaproteomics to identify active members of soil and groundwater microbial communities. Metaproteomics workflows were applied to study the initial responses (i.e. 4 days post treatment) of the indigenous aquifer microbiota to biostimulation with emulsified vegetable oil (EVO) at a uranium-contaminated site. Members of the Betaproteobacteria (i.e. Dechloromonas, Ralstonia, Rhodoferax, Polaromonas, Delftia, Chromobacterium) and the Firmicutes dominated the biostimulated aquifer community. Proteome characterization revealed distinct differences between the microbial biomass collected from groundwater influenced by biostimulation and groundwater collected upgradient of the EVO injection points. In particular, proteins involved in ammonium assimilation, EVO degradation, and polyhydroxybutyrate granule formation were prominent following biostimulation. Interestingly, the atypical NosZ of Dechloromonas spp. was highly abundant, suggesting active nitrous oxide (N2 O) respiration. c-Type cytochromes were barely detected, as was citrate synthase, a biomarker for hexavalent uranium reduction activity, suggesting that uranium reduction has not commenced 4 days post EVO amendment. Environmental metaproteomics identified microbial community responses to biostimulation and elucidated active pathways demonstrating the value of this technique as a monitoring tool and for complementing nucleic acid-based approaches.