Involvement of NO 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) 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-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 and/or , and the sp. strain possessed a clade II gene conferring the capacity for NO reduction. A common prominent physiological feature observed in the isolates was NO accumulation before NH production, which was further examined with sp. strain DNRA3 (possessing and ) and sp. strain DNRA5 (possessing only ). Both isolates showed inhibition of NO-to-NH reduction at submillimolar NO concentrations and downregulation of or transcription when NO was being reduced to NO In batch and chemostat experiments, both isolates produced NH from NO reduction when incubated with excess organic electron donors, while incubation with excess NO resulted in NO buildup but no substantial NH production, presumably due to inhibitory NO concentrations. This previously overlooked link between NO repression of NO-to-NH reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-versus-DNRA competition in soil. Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO and NO Unlike denitrification, which leads to nitrogen loss and NO emission, DNRA reduces NO and NO to NH, 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 repression of NO-to-NH 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.