Regulation, ecophysiology, and kinetic parameters of uncultured N-gas flux associated anaerobic microbial communities in agricultural soils: Towards a microscale spatial resolution

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Denitrifiers (reducing N-oxides to N2O and/ or N2), non-denitrifying N2O-reducers (reduce N2O to N2), and dissimilatory nitrate reducers (DNRA; reduce N-oxides to NH4+) impact the emission of the greenhouse gas N2O as well as retention of nitrogen and thus plant nutrition. N2O-reducers and DNRA compete with denitrifiers. Ecophysiological traits determining their competitiveness and N-gas production capabilities and are essentially unknown for uncultured taxa. Thus, the following hypotheses will further be addressed: (i) Denitrification response to environmental factors is shaped by contrasting microbial communities including hitherto unknown species, and can be explained by their intrinsic ecophysiological properties. (ii) Gene expression and community structure associated with denitrification, N2O reduction and DNRA reflect metabolic states and potentials, thus increasing predictability of N2O and N2 fluxes. Outcomes from phase I were summarized in two publications and three submitted manuscripts or manuscripts in preparation (attached). High-throughput incubations under various conditions (including 15N-tracers) combined with functional gene expression, as well as gene and transcript based next generation sequencing will be applied to identify apparent Michaelis-Menten and physiological parameters. Functional genes of denitrifiers (nirK/S encoding nitrite reductases yielding NO; nosZ I and II encoding N2O reductase yielding N2), and DNRA (nrfA encoding nitrite reductases yielding NH4+), will be primarily analyzed. Phase II focuses on the effect of pH, temperature, plant roots and organic carbon on response patterns of target groups. Active root exudate fueled target organisms will be identified by RNA and DNA based stable isotope probing. Regulation of target groups in response to environmental control factors will be analyzed in mesocosms and field studies performed as central experiments. The spatial distribution of target groups will likewise be addressed. Such data will extend the denitrifier-regulatory-phenotype-concept and give insights into the ecophysiology and competition of target groups under various conditions. A molecular microbial community-based proxy for N2O to N2 ratio potentials based on denitrifiers, DNRA, and clade II N2O reducers will be developed. Various kinetic parameters, response function, and microbial community structure data will be provided for modelling of denitrification and N-gas fluxes.