Production and consumption of nitrous oxide in nitrate-ammonifying microorganisms

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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.