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

硝化作用是全球氮循环中的一个关键过程。氨是通过有机物质的矿化作用产生的。虽然矿化作用和硝化作用被广泛认为是两个独立的过程，但许多硝化菌可以降解小的有机氮化合物，包括尿素和氰酸盐，它们普遍存在于土壤中，代表着活跃的循环氮库。此外，尿素在农业中也被用作肥料。硝化菌被认为是严格的自养生物，从氨中获取能量，通过二氧化碳固定碳。在尿素和氰酸盐上的生长挑战了这一教条，并表明硝化菌也可以使用有机基质进行生长。支持这一观点的是，有机氮的添加促进了土壤的硝化作用，而无机氨的添加则没有。这对我们理解和管理环境中的氮素通量具有重大影响。最主要的假设是，尿素和氰酸盐是土壤中氮和碳循环之间的重要缺失环节。具体假设(H)：1.硝化菌在土壤中氰酸盐和尿素的周转中起关键作用。硝化菌从尿素和氰酸盐中获取能量和碳。尿素和氰酸盐使一些硝化菌在环境中具有竞争优势。博士生研究计划：WP1。纯培养将使用多形亚硝酸螺旋体(细菌)和法兰克氏亚硝酸杆菌(古生菌)(两种尿素酶阳性硝化菌)进行尿素实验，并使用氰酸盐实验(古生菌)进行氰酸盐实验，亚硝酸螺旋菌(古生菌)是唯一培养的氰化酶阳性硝化细菌2。学生将用13C和15N标记的尿素/氰酸酯培养微生物，并使用IR-MS(H2)确定它们在生物质中的含量。学生将使用显微镜细胞计数和比色分析来评估生长产量(H3)，并比较尿素和氨氧化的动力学(H3)。交付成果：详细、机械地了解氮肥中氮和碳的底物亲和力、产量、加工速率和利用。WP2：土壤微生物群落的双15N-13C稳定同位素探测博士生将利用DNA-SIP来确定土壤中尿素和氰酸盐中的氮和碳的去向。学生将用13C标记的化合物(尿素/氰酸盐)、15N标记的化合物或对照处理培养土壤微观世界(来自德辛汉姆沼泽和塞特福德森林)。他们将通过高通量DNA扩增和重DNA(H1)的元基因组测序来鉴定活跃代谢Ttest化合物的微生物。这位博士生将进行元翻译手术。基因组和转录组数据将用于负责土壤氰酸盐和尿素周转的微生物的代谢重建。交付成果：关于土壤中尿素/氰酸盐周转的关键微生物的可靠数据，将氮和碳循环中的过程联系起来。所有基础设施都已到位，监管者在涉及的技术方面拥有杰出的专业知识。学生将接受尖端微生物学技术和高质量出版物的高级研究培训。博士生将通过DTP接受特定学科以及个人和专业发展培训，并在每周的实验室会议和部门研讨会上发表演讲。鼓励学生指导本科生，参加会议、研讨会、工作坊、企业和参与。这个项目将为学生提供全面的研究和可移植的技能，适合在学术界内外的职业生涯。

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.