BackgroundFirmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both. The recent renewed interest in their nitrogen metabolism has revealed many interesting features, the most striking being their wide variety of dissimilatory nitrate reduction pathways. In the present study, nitrous oxide production from Bacillus licheniformis, a ubiquitous Gram-positive, spore-forming species with many industrial applications, is investigated.ResultsB. licheniformis has long been considered a denitrifier but physiological experiments on three different strains demonstrated that nitrous oxide is not produced from nitrate in stoichiometric amounts, rather ammonium is the most important end-product, produced during fermentation. Significant strain dependency in end-product ratios, attributed to nitrite and ammonium, and medium dependency in nitrous oxide production were also observed. Genome analyses confirmed the lack of a nitrite reductase to nitric oxide, the key enzyme of denitrification. Based on the gene inventory and building on knowledge from other non-denitrifying nitrous oxide emitters, hypothetical pathways for nitrous oxide production, involving NarG, NirB, qNor and Hmp, are proposed. In addition, all publically available genomes of B. licheniformis demonstrated similar gene inventories, with specific duplications of the nar operon, narK and hmp genes as well as NarG phylogeny supporting the evolutionary separation of previously described distinct BALI1 and BALI2 lineages.ConclusionsUsing physiological and genomic data we have demonstrated that the common soil bacterium B. licheniformis does not denitrify but is capable of fermentative dissimilatory nitrate/nitrite reduction to ammonium (DNRA) with concomitant production of N2O. Considering its ubiquitous nature and non-fastidious growth in the lab, B. licheniformis is a suitable candidate for further exploration of the actual mechanism of N2O production in DNRA bacteria and its relevance in situ.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2382-2) contains supplementary material, which is available to authorized users.

Until recently, it has not been generally known that some bacteria can contain the gene inventory for both denitrification and dissimilatory nitrate (NO)/nitrite (NO) reduction to ammonium (NH) (DNRA). Detailed studies of these microorganisms could shed light on the differentiating environmental drivers of both processes without interference of organism-specific variation. Genome analysis of Bacillus azotoformans LMG 9581 shows a remarkable redundancy of dissimilatory nitrogen reduction, with multiple copies of each denitrification gene as well as DNRA genes nrfAH, but a reduced capacity for nitrogen assimilation, with no nas operon nor amtB gene. Here, we explored nitrogen assimilation in detail using growth experiments in media with different organic and inorganic nitrogen sources at different concentrations. Monitoring of growth, NO NO, NH concentration and NO production revealed that B. azotoformans LMG 9581 could not grow with NH as sole nitrogen source and confirmed the hypothesis of reduced nitrogen assimilation pathways. However, NH could be assimilated and contributed up to 50% of biomass if yeast extract was also provided. NH also had a significant but concentration-dependent influence on growth rate. The mechanisms behind these observations remain to be resolved but hypotheses for this deficiency in nitrogen assimilation are discussed. In addition, in all growth conditions tested a denitrification phenotype was observed, with all supplied NO converted to nitrous oxide (NO).

Until now, the exact mechanisms for N2O production in dissimilatory nitrate/nitrite reduction to ammonium (DNRA) remain underexplored. Previously, we investigated this mechanism in Bacillus licheniformis and Bacillus paralicheniformis, ubiquitous gram‐positive bacteria with many industrial applications, and observed significant strain dependency and media dependency in N2O production which was thought to correlate with high residual NO 2 −. Here, we further studied the influence of several physicochemical factors on NO 3 − (or NO 2 −) partitioning and N2O production in DNRA to shed light on the possible mechanisms of N2O production. The effects of NO 3 − concentrations under variable or fixed C/N‐NO 3 − ratios, NO 2 − concentrations under variable or fixed C/N‐NO 2 − ratios, and NH 4 + concentrations under fixed C/N‐NO 3 − ratios were tested during anaerobic incubation of soil bacterium B. paralicheniformis LMG 6934 (previously known as B. licheniformis), a strain with a high nitrite reduction capacity. Monitoring of growth, NO 3 −, NO 2 −, NH 4 + concentration, and N2O production in physiological tests revealed that NO 3 − as well as NO 2 − concentration showed a linear correlation with N2O production. Increased NO 3 − concentration under fixed C/N‐NO 3 − ratios, NO 2 − concentration, and NH 4 + concentration had a significant positive effect on NO 3 − (or NO 2 −) partitioning ([N–NH 4 +]/[N–N2O]) toward N2O, which may be a consequence of the (transient) accumulation and subsequent detoxification of NO 2 −. These findings extend the information on several physiological parameters affecting DNRA and provide a basis for further study on N2O production during this process.

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