Involvement of NO 3 − in Ecophysiological Regulation of Dissimilatory Nitrate/Nitrite Reduction to Ammonium (DNRA) Is Implied by Physiological Characterization of Soil DNRA Bacteria Isolated via a Colorimetric 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 N 2 O reduction. A common prominent physiological feature observed in all DNRA isolates was NO 2 − accumulation observed before NH 4 + production, which was further examined with Citrobacter sp. DNRA3 (possessing nrfA and nirB) and Enterobacter sp. DNRA5 (possessing only nirB ). In both organisms, NO 2 − -to-NH 4 + reduction was inhibited by submillimolar NO 3 − , and nrfA or nirB transcription was down-regulated when NO 3 − was being reduced to NO 2 − . Both batch and chemostat incubations of these isolates with excess organic electron donors produced NH 4 + from reduction of NO 3 − ; however, incubation with excess NO 3 − resulted in NO 2 − buildup but no substantial NH 4 + production, presumably due to NO 3 − presence. This previously overlooked link between NO 3 − repression of NO 2 − -to-NH 4 + reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-vs-DNRA competition in soil. IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO 3 − and NO 2 − . Unlike denitrification leading to nitrogen loss and N 2 O emission, DNRA reduces NO 3 − and NO 2 − to NH 4 + , 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 NO 3 − repression of NO 2 − -to-NH 4 + 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.