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Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently regained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, the 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 DNRA bacteria were isolated from NO(3)(−)-reducing anoxic enrichment cultures established with rice paddy soils using a newly developed colorimetric screening method. Six of these isolates, each assigned to a different genus, were characterized to improve the understanding of DNRA physiology. All the isolates carried nrfA and/or nirB, and the Bacillus sp. strain possessed a clade II nosZ gene conferring the capacity for N(2)O reduction. A common prominent physiological feature observed in the isolates was NO(2)(−) accumulation before NH(4)(+) production, which was further examined with Citrobacter sp. strain DNRA3 (possessing nrfA and nirB) and Enterobacter sp. strain DNRA5 (possessing only nirB). Both isolates showed inhibition of NO(2)(−)-to-NH(4)(+) reduction at submillimolar NO(3)(−) concentrations and downregulation of nrfA or nirB transcription when NO(3)(−) was being reduced to NO(2)(−). In batch and chemostat experiments, both isolates produced NH(4)(+) from NO(3)(−) reduction when incubated with excess organic electron donors, while incubation with excess NO(3)(−) resulted in NO(2)(−) buildup but no substantial NH(4)(+) production, presumably due to inhibitory NO(3)(−) concentrations. 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-versus-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, which leads to nitrogen loss and N(2)O emission, DNRA reduces NO(3)(−) and NO(2)(−) to NH(4)(+), a reactive nitrogen compound with a higher tendency to be retained in the 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 bacterial ecophysiology. Here, we have developed a new 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 a key to understanding why DNRA activity is rarely observed at substantial levels in nitrogen-rich agricultural soils. |
