Abstract
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.
Highlights
Firmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both
Dissimilatory nitrate reduction metabolism Three genotypically distinct B. licheniformis strains (Coorevits, A. & De Vos, P., personal communication) from various origins were selected for determination of their dissimilatory nitrate reduction metabolism based on a previous study that demonstrated their capacity to produce N2O [18]
When nitrate was almost depleted, nitrite was reduced (μ2 = 0.16 ± 0.02 h−1), which continued after maximal growth at 8.5 h was achieved and sporulation had started (μ3). This suggested that nitrite reduction did not support growth during the μ2 phase, but rather fermentation was responsible for growth after nitrate depletion
Summary
Firmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both. Denitrification and dissimilatory nitrate/nitrite reduction to ammonium (DNRA) are two key processes, performed by a wide range of Bacteria and Archaea as well as some Eukaryotes [1], responsible for removal of excess nitrate from the environment. Renewed interest in Bacillus has revealed many interesting features like (i) the widespread occurrence of nitrate reduction and denitrification in the genus [18], (ii) the gene inventory for both denitrification and DNRA in one microorganism [19, 20], (iii) a novel type of copper-Adependent, electrogenic nitric oxide reductase (CuANor) [21,22,23,24], or (iv) membrane-bound denitrification [25] with a novel organization for the periplasmic nitrate reductase [19, 26]. Considering the modularity of denitrification and DNRA, a multitude of enzyme combinations for nitrate reduction are imaginable, even within one microorganism
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