Is denitrification by ammonia oxidising bacteria a response to nitrite toxicity?

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Production of nitrous oxide (N2O) is of environmental concern due to its involvement in global warming and destruction of stratospheric ozone. As soils account for ~70% of the atmospheric loading of N2O management strategies are urgently required to lower emissions. Such strategies need to be formulated around our understanding of the regulation of microbial N2O-producing processes in soils. This project will determine the conditions under which ammonia oxidising bacteria (AOB) demonstrate flexibility in function to reduce nitrite (NO2-) to nitrous oxide, thereby contributing to global warming and destruction of stratospheric ozone. Specifically this project will examine whether this flexibility is a response to nitrite toxicity during nitrification thus releasing N2O under aerobic conditions. It will test the hypotheses: (i) the ability of ammonia oxidising bacteria to denitrify is primarily a NO2- toxicity response, rather than a facultative response to maintain respiration under reduced Redox conditions (ii) AOB switch to using NO2- as an oxygen source to maintain function when a threshold concentration of NO2- is sensed, reducing it to N2O, and (iii) in the soil environment, the necessity of AOB to reduce NO2- will depend on coupling of AOB with nitrite oxidising bacteria (NOB). The student will undertake culture experiments to test the response of strains of AOB and NOB to NO2- addition rates in culture and ascertain a threshold concentration for nitrite reduction by AOB, the optimum NH4+:NO2- ratio for maximum N2O production by AOB, and the influence of NOB in regulating the NO2- concentration and AOB N2O production. A stable isotope approach (15N-18O-enrichment of NO2- and N2O) will be adopted to ascertain that NO2- is used by AOB as an oxygen source above the threshold NO2- concentration for denitrification. The potential for NOB to regulate NO2- concentrations and therefore reduction of NO2- by AOB will be examined through reduced expression of the AOB nirK gene, in sterilised (gamma-irradiated) soil inoculated with AOB and NOB strains from objective 1, and in a natural soil community adopting a combination of 15N-isotopomer and real time molecular approaches. This project will provide the student with training in a wide range of analytical techniques, including analysis by gas chromatography and mass spectrometry and microbiological techniques, as well as a wide range of soil physical and chemical analyses. The student will obtain training in molecular techniques, sample preparation and analysis from SCRI.