Involvement of NO3− 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

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO3− and NO2−. Unlike denitrification, which leads to nitrogen loss and N2O emission, DNRA reduces NO3− and NO2− to NH4+, 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 NO3− repression of NO2−-to-NH4+ 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. ABSTRACT 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 NO3−-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 N2O reduction. A common prominent physiological feature observed in the isolates was NO2− accumulation before NH4+ 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 NO2−-to-NH4+ reduction at submillimolar NO3− concentrations and downregulation of nrfA or nirB transcription when NO3− was being reduced to NO2−. In batch and chemostat experiments, both isolates produced NH4+ from NO3− reduction when incubated with excess organic electron donors, while incubation with excess NO3− resulted in NO2− buildup but no substantial NH4+ production, presumably due to inhibitory NO3− concentrations. 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-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 NO3− and NO2−. Unlike denitrification, which leads to nitrogen loss and N2O emission, DNRA reduces NO3− and NO2− to NH4+, 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 NO3− repression of NO2−-to-NH4+ 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.