Regulation of denitrification at the cellular level: a clue to the understanding of N2O emissions from soils

Denitrifying prokaryotes use NOx as terminal electron acceptors in response to oxygen depletion. The process emits a mixture of NO, N2O and N2, depending on the relative activity of the enzymes catalysing the stepwise reduction of NO3− to N2O and finally to N2. Cultured denitrifying prokaryotes show characteristic transient accumulation of NO2−, NO and N2O during transition from oxic to anoxic respiration, when tested under standardized conditions, but this character appears unrelated to phylogeny. Thus, although the denitrifying community of soils may differ in their propensity to emit N2O, it may be difficult to predict such characteristics by analysis of the community composition. A common feature of strains tested in our laboratory is that the relative amounts of N2O produced (N2O/(N2+N2O) product ratio) is correlated with acidity, apparently owing to interference with the assembly of the enzyme N2O reductase. The same phenomenon was demonstrated for soils and microbial communities extracted from soils. Liming could be a way to reduce N2O emissions, but needs verification by field experiments. More sophisticated ways to reduce emissions may emerge in the future as we learn more about the regulation of denitrification at the cellular level

Biological sources and sinks of nitrous oxide and strategies to mitigate emissions

Nitrous oxide (N 2 O) is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Global emissions continue to rise. More than two-thirds of these emissions arise from bacterial and fungal denitrification and nitrification processes in soils, largely as a result of the application of nitrogenous fertilizers. This article summarizes the outcomes of an interdisciplinary meeting, ‘Nitrous oxide (N 2 O) the forgotten greenhouse gas’, held at the Kavli Royal Society International Centre, from 23 to 24 May 2011. It provides an introduction and background to the nature of the problem, and summarizes the conclusions reached regarding the biological sources and sinks of N 2 O in oceans, soils and wastewaters, and discusses the genetic regulation and molecular details of the enzymes responsible. Techniques for providing global and local N 2 O budgets are discussed. The findings of the meeting are drawn together in a review of strategies for mitigating N 2 O emissions, under three headings, namely: (i) managing soil chemistry and microbiology, (ii) engineering crop plants to fix nitrogen, and (iii) sustainable agricultural intensification. </jats:p

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB

Recent structural insights into the function of copper nitrite reductases.

Copper nitrite reductases (CuNiR) carry out the first committed step of the denitrification pathway of the global nitrogen cycle, the reduction of nitrite (NO2(-)) to nitric oxide (NO). As such, they are of major agronomic and environmental importance. CuNiRs occur primarily in denitrifying soil bacteria which carry out the overall reduction of nitrate to dinitrogen. In this article, we review the insights gained into copper nitrite reductase (CuNiR) function from three dimensional structures. We particularly focus on developments over the last decade, including insights from serial femtosecond crystallography using X-ray free electron lasers (XFELs) and from the recently discovered 3-domain CuNiRs

The consequences of niche and physiological differentiation of archaeal and bacterial ammonia oxidisers for nitrous oxide emissions

The authors are members of the Nitrous Oxide Research Alliance (NORA), a Marie Skłodowska-Curie ITN and research project under the EU's seventh framework program (FP7). GN is funded by the AXA Research Fund and CGR by a Royal Society University Research Fellowship (UF150571) and a Natural Environment Research Council (NERC) Standard Grant (NE/K016342/1). The authors would like to thank Dr Robin Walker and the SRUC Craibstone Estate (Aberdeen) for access to the agricultural plots, Dr Alex Douglas for statistical advice and Philipp Schleusner for assisting microcosm construction and sampling.Peer reviewedPublisher PD

Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605pRespiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mössbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mössbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ∼ 6 and g ∼ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ∼ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction