Production and consumption of nitrous oxide in nitrate-ammonifying microorganisms

一氧化二氮(N2O；笑气)是一种有效的大气温室气体，导致平流层臭氧消耗。N2O对气候变化的影响使缓解N2O成为一项国际挑战，特别是在世界人口不断增加的背景下。人为排放的N2O主要是由农田土壤微生物造成的，氮肥的施用有利于N2O的排放。许多不同的微生物代谢途径被证明有助于在不同的生境中产生N2O，并且已有多个酶被报道产生N2O。另一方面，只有一种类型的酶(N2O还原酶；NOSZ)被描述为还原N2O，产生N2。在缺氧条件下，硝酸盐通过反硝化或异化硝酸盐还原为铵(DNRA)生成微生物N2O。在反硝化菌中，硝酸盐通过中间产物亚硝酸盐、一氧化氮(NO)和N2O还原为氮气，尽管N2O还原的最后一步可能在反硝化细胞中缺失或受损。在DNRA过程中，N2O似乎是亚硝酸盐的副产物，没有解毒作用。一些执行DNRA的物种含有NOSZ酶，但它们在环境中的作用目前尚未确定。本项目旨在研究两种DNRA模式细菌，即产琥珀酸狼疮杆菌和绿色芽孢杆菌产生和消耗N2O的酶学特性。这两种生物都被证明拥有功能性的一氧化氮合酶基因簇，并将N2O还原与细胞生长联系在一起。在产琥珀酸假单胞菌中，这代表了一种利用N2O作为唯一电子受体的厌氧呼吸。琥珀酸假单胞菌和绿色假单胞菌的细胞具有遗传易感性，并且已在申请人的实验室中成功构建了缺失突变体。本项目旨在利用各种突变体，剖析琥珀酸双胞菌和绿色双胞菌中个体同化和异化酶对N2O产生的贡献。此外，还将探索电池作为N2O汇的潜力。由于N2O是作为NO解毒的产物形成的，因此候选NO还原酶在亚硝化应激防御中的作用也将被讨论。在实验上，该项目将基因工程与微生物生理学和生物化学相结合，并在细菌培养过程中对硝酸盐/亚硝酸盐还原的气体产物进行最先进的检测。可以想象，研究成果将对全球温室气体减排战略产生影响。

Nitrous oxide (N2O; laughing gas) is an effective atmospheric greenhouse gas and contributes to ozone depletion in the stratosphere. The impact of N2O on climate change makes N2O mitigation an international challenge, in particular in the context of the increasing world population. Anthropogenic N2O emissions are mainly caused by microorganisms from agricultural soils and are favoured by the application of nitrogenous fertilizers. Many different pathways of microbial metabolism have been shown to contribute to N2O generation in diverse habitats and multiple enzymes have been reported to produce N2O. On the other hand, only one type of enzyme (N2O reductase; NosZ) has been described that reduces N2O, yielding N2.Microbial N2O generation from nitrate under anoxic conditions results from denitrification or dissimilatory nitrate reduction to ammonium (DNRA). In denitrifiers, nitrate is reduced to N2 via the intermediates nitrite, nitric oxide (NO) and N2O, although the final step of N2O reduction might be absent or impaired in denitrifying cells. In the DNRA process, N2O appears to be a side product of nitrite and NO detoxification. Some DNRA-performing species contain a NosZ enzyme but their environmental role is currently unresolved. This project aims to characterize the enzymology of production and consumption of N2O in two DNRA model bacteria, namely Wolinella succinogenes and Bacillus vireti. Both organisms have been shown to possess functional nos gene clusters and to couple N2O reduction to cell growth. In W. succinogenes this represents a form of anaerobic respiration that uses N2O as sole electron acceptor. Cells of W. succinogenes and B. vireti are genetically tractable and the construction of deletion mutants has been successfully established in the applicant's laboratory.Using various mutants the project aims to dissect the contribution of individual assimilatory and dissimilatory enzymes to N2O production in W. succinogenes and B. vireti. Furthermore, the potential of the cells to act as N2O sinks will be explored. Since N2O is formed as a product of NO detoxification, the role of candidate NO-reducing enzymes in nitrosative stress defence will also be addressed. Experimentally, the project combines genetic engineering with microbial physiology and biochemistry as well as state-of-the-art determination of gaseous products of nitrate/nitrite reduction in growing bacterial cultures.Taken together, the project seeks to broaden our knowledge on the underexplored role of DNRA organisms in release and consumption of atmospheric N2O. Conceivably, research outcomes will impact on global greenhouse gas mitigation strategies.