Novel insights into DNRA physiology gained from ammonifying microorganisms isolated from denitrification-dominant agricultural soil with the newly developed high-throughput screening method

Abstract

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently gained attention as a nitrogen dissimilation pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Physiological characteristics of DNRA-catalyzing bacteria inhabiting agricultural soils remains largely unexplored, as DNRA is outcompeted by denitrification in the enrichment process with $NO_3^-$ amendments and thus, targeted enrichment or isolation of DNRA-catalyzing microorganisms was simply not possible. Using a simple high-throughput DNRA screening method, we have successfully screened more than 200 microbial isolates capable of dissimilatory ammonification from denitrification-dominant agricultural soils. Ammonification reactions of six isolates, each affiliated to a different genus, were examined. All isolates carried either nrfA or nirB, and the isolate affiliated to the Bacillus genus possessed clade II nosZ genes and was capable of $NO_2$ reduction. The common prominent physiological feature of isolated DNRA bacteria was $NO_2^-$ accumulation observed before $NH_4^+$ production, which was further examined with Citrobacter sp. (nrfA) and Enterobacter sp. (nirB). In both organisms,$NO_2^-$-to-$NH_4^+$ reduction activity was inhibited by the presence of $NO_3^-$, and nrfA or nirB transcription was down-regulated when $NO_3^-$ was being reduced to $NO_2^-$. Incubations of these two isolates with excess of organic electron donors produced $NH_4^+$ from reduction of $NO_3^-$

however, incubation with excess $NO_3^-$ resulted in buildup of $NO_2^-$ but not $NH_4^+$. These observations, at least in part, explain why DNRA pathway is favored in environments with high C/N ratios and anoxic batch $NO_3^-$ enrichment always results in denitrification-dominant cultures.