Nitrous oxide emission by the non-denitrifying, nitrate ammonifier Bacillus licheniformis

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.

Influence of nitrate and nitrite concentration on N2O production via dissimilatory nitrate/nitrite reduction to ammonium in Bacillus paralicheniformis LMG 6934

“…Percentages of NO 3 − or NO 2 − converted to N 2 O or NH 4 + under different conditions are shown in Figure . Percentage of NO 3 − recovery as N 2 O and growth (∆OD 600 ) under 10 mmol/L NO 3 − condition agreed with previous observations (Sun et al., ).…”

“…After 72‐hr incubation, growth was observed under all NO 2 − concentrations tested, with all NO 2 − converted to NH 4 + or N 2 O, thus confirming its high tolerance to NO 2 − (Table ; Figure ). Indeed, compared with other DNRA strains (Sun et al., ) belonging to Bacillus sp. and Citrobacter sp.…”

“…Our previous study demonstrated that B. paralicheniformis LMG 6934 had a high NO 2 − tolerance of 10 mmol/L and could efficiently perform DNRA by reducing all intermediary NO 2 − to NH 4 + and N 2 O (Sun et al., ), while B. paralicheniformis LMG 7559 showed a NO 2 − tolerance of 6.29 ± 0.39 mmol/L, and both LMG 7559 and B. licheniformis LMG 17339 had residual NO 2 − (2.76 mmol/L ± 0.57 mmol/L, 4.88 mmol/L ± 0.60 mmol/L) after 72‐hr incubation probably due to their lower tolerance to the toxic effect of NO 2 − . Less N 2 O was produced by LMG 6934 than by LMG 7559 and LMG 17339, and less NO 3 − partitioning to N 2 O was observed as well ([N‐NH 4 + ]/[N–N 2 O] of 4.24 ± 0.29 vs 1.49 ± 0.82, 0.71 ± 0.09, respectively) (Sun et al., and unpublished data therein). To uncover factors affecting N 2 O production during DNRA, here, NO 2 − reduction was anaerobically tested in LMG 6934 at concentrations of 1 mmol/L, 5 mmol/L, and 10 mmol/L under variable C/N‐NO 2 − ratios of 180, 36, and 18 and fixed C/N‐NO 2 − ratios of 18.…”

“…In our previous study, we investigated three strains of B. (para)licheniform is (as mentioned above) which were disguised as denitrifiers and proved that they are N 2 O emitters performing DNRA probably by expression of narG , nirB , qNor, and hmp , with up to one‐third of all NO 3 − converted to N 2 O (Sun et al., ). They are therefore suitable model organisms to study the mechanism of N 2 O production during DNRA and to supplement the insights of environmental drivers influencing DNRA.…”

“…For instance, Bacillus vireti LMG 21834 T performs DNRA by NarG, NrfA, and Nor (CbaA), with additional NosZ partially reducing N 2 O to N 2 (Mania, Heylen, Spanning, & Frostegård, ; Mania et al., ). Similarly, Bacillus paralicheniform is LMG 6934, LMG7559 (renamed since 2015 (Dunlap, Kwon, Rooney, & Kim, )), and Bacillus licheniform is LMG17339 possess NarG, NirBD, and Nor, but not NosZ (Sun et al., ). While, the mutants of Salmonella typhimurium Typhimurium lacking Hmp, NorV, and NrfA and of E.coli lacking NirB, NrfA, NorV, and Hmp still can reduce NO, suggesting that there are other mechanisms of NO reduction uncharacterized (Mills, Rowley, Spiro, Hinton, & Richardson, ).…”