Physiological characterization of nitrate ammonifying bacteria isolated from rice paddy soil via a newly developed high-throughput screening method

ABSTRACT Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently gained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, ecophysiology of DNRA bacteria inhabiting agricultural soils has remained largely unexplored, due to the difficulty in targeted enrichment and isolation of DNRA microorganisms. In this study, >100 microbial isolates capable of DNRA have been isolated from rice paddy soil with apparent dominance of denitrification using a novel high-throughput screening method. Six of these isolates, each assigned to a disparate genus, was examined to improve understanding of DNRA physiology. All isolates carried nrfA and/or nirB, and an isolate affiliated to Bacillus possessed a clade II nosZ gene and was capable of N2O reduction. A common prominent physiological feature observed in all DNRA isolates was NO2? accumulation observed before NH4+ production, which was further examined with Citrobacter sp. DNRA3 (possessing nrfA and nirB) and Enterobacter sp. DNRA5 (possessing only nirB). In both organisms, NO2?-to-NH4+ reduction was inhibited by submillimolar NO3?, and nrfA or nirB transcription was down-regulated when NO3? was being reduced to NO2?. Both batch and chemostat incubations of these isolates with excess organic electron donors produced NH4+ from reduction of NO3?; however, incubation with excess NO3? resulted in NO2? buildup but no substantial NH4+ production, presumably due to NO3? presence. This previously overlooked link between NO3? repression of NO2?-to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-vs-DNRA competition in soil. IMPORTANCEDissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO3? and NO2?. Unlike denitrification leading to nitrogen loss and N2O emission, DNRA reduces NO3? and NO2? to NH4+, a reactive nitrogen with higher tendency to be retained in soil matrix. Therefore, stimulation of DNRA has often been proposed as a strategy to improve fertilizer efficiency and reduce greenhouse gas emissions. Such attempts have been hampered by lack of insights into soil DNRA ecophysiology. Here, we have developed a novel high-throughput screening method for isolating DNRA-catalyzing organisms from agricultural soils without apparent DNRA activity. Physiological characteristics of six DNRA isolates were closely examined, disclosing a previously overlooked link between NO3? repression of NO2?-to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity, which may be key to understanding why significant DNRA activity is rarely observed in nitrogen-rich agricultural soils.