Redefining the metabolism of nitrogen cycling microbes using Dual Stable Isotope Probing

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Nitrification is a key process in the global nitrogen cycle. Ammonia is produced through mineralisation of organic matter. Whilst mineralisation and nitrification are widely regarded as separate processes, many nitrifiers can degrade small organic nitrogen compounds, including urea and cyanate which are ubiquitous in soil and represent actively cycled pools of nitrogen. Furthermore, urea is used as a fertiliser in agriculture. Nitrifiers are considered strict autotrophs, deriving energy from ammonia and fixing carbon via CO2. Growth on urea and cyanate challenges this dogma and indicates that nitrifiers can also use organic substrates for growth. Supporting this notion, organic nitrogen addition stimulates soil nitrification, whereas inorganic ammonia addition does not. This has major consequences for our understanding and management of the nitrogen flux in the environment. The overarching hypothesis is that urea and cyanate represent an important missing link between the nitrogen and carbon cycles in soil.Specific Hypotheses (H):1. Nitrifiers play a key role in the cyanate and urea turnover in soil.2. Nitrifiers derive energy and carbon from urea and cyanate.3. Urea and cyanate give some nitrifiers a competitive advantage in the environment.Research Programme for the PhD student: WP1. Pure cultureUrea experiments will be performed using Nitrosospira multiformis (bacteria) and Nitrosocosmicus franklandus (archaea) (both urease-positive nitrifiers), and cyanate experiments using Nitrososphaera gargensis (archaea), the only cultured cyanase-positive nitrifier2. The student will grow the microbes with 13C- and 15N-labelled urea/cyanate and determine their incorporation into biomass using IR-MS (H2). The student will use microscopy cell counts and colorimetric assays to assess growth yields (H3) and compare the kinetics of urea and ammonia oxidation (H3). Deliverables: Detailed, mechanistic understanding of the substrate affinity, yield, process rates and utilisation of nitrogen and carbon in nitrifiers.WP2: Dual 15N-13C-Stable Isotope Probing with soil microbial communitiesThe PhD student will utilise DNA-SIP to determine the fate of nitrogen and carbon from urea and cyanate in soil. The student will incubate soil microcosms (from Dersingham Bog and Thetford Forest) with either 13C-labelled compound (urea/cyanate), 15N-labelled compound, or control treatments. They will identify of microbes actively metabolising ttest compound by high-throughput DNA amplicon and metagenomic sequencing of the heavy DNA (H1). The PhD student will perform metatranscriptomics. Genomic and transcriptomic data will be used for metabolic reconstruction of microorganisms responsible for soil cyanate and urea turnover.Deliverables: Robust data on key microbial players involved in urea/cyanate turnover in soil, linking the processes in the nitrogen and carbon cycles.All infrastructure is in place and collectively supervisors have outstanding expertise in the techniques involved. The student will gain advanced research training in cutting-edge microbiology techniques and high-quality publications.The PhD student will receive subject-specific and Personal and Professional Development training through the DTP, giving talks at weekly lab meetings and departmental seminars. Students are encouraged to supervise undergraduates, participate in conferences, seminars, workshops, enterprise and engagement. This project will provide the student with a well-rounded research and transferable skillset suitable for a career either within or out of academia.