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

使用双稳定同位素探测重新定义氮循环微生物的代谢

• 批准号: 2734239
• 金额: --
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2734239
• 关键词: Redefining; metabolism; nitrogen; cycling; microbes

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.

硝化作用是全球氮循环的关键过程。氨是通过有机物的矿化产生的。虽然矿化和硝化被广泛认为是分开的过程，但许多硝化剂可以降解小的有机氮化合物，包括尿素和氰酸盐，它们在土壤中无处不在，代表着氮的积极循环池。此外，尿素在农业中用作肥料。硝化生物被认为是严格的自养生物，从氨中获取能量，并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，添加有机氮会刺激土壤硝化，而添加无机氨则不会。这对我们对环境中氮通量的理解和管理产生了重大影响。总体假设是尿素和氰酸盐代表了土壤中氮和碳循环之间缺失的重要环节。具体假设(H)：硝化菌在土壤中氰酸盐和尿素的转化中起关键作用。硝化器从尿素和氰酸盐中获得能量和碳。尿素和氰酸盐使一些硝化物在环境中具有竞争优势。博士研究生研究项目：WP1。纯培养实验将使用多种亚硝基螺旋体（细菌）和法兰克亚硝基菌（古细菌）（两者都是脲酶阳性硝化菌）进行，而氰酸盐实验将使用唯一培养的氰化酶阳性硝化菌亚硝基螺旋体（古细菌）进行。学生将用13C-和15n标记的尿素/氰酸盐培养微生物，并使用IR-MS （H2）测定它们与生物质的结合。学生将使用显微镜细胞计数和比色法来评估生长产量（H3），并比较尿素和氨氧化（H3）的动力学。交付成果：对硝化器中底物亲和力、产量、过程速率和氮和碳的利用进行详细的、机械的了解。WP2：双15n - 13c稳定同位素探测土壤微生物群落博士生将利用DNA-SIP来确定土壤中尿素和氰酸盐中氮和碳的命运。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生物（来自Dersingham沼泽和Thetford森林）。他们将通过高通量DNA扩增子和重DNA宏基因组测序（H1）鉴定积极代谢检测化合物的微生物。博士生将执行元转录组学。基因组学和转录组学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。成果：关于土壤中尿素/氰酸盐转化的关键微生物参与者的可靠数据，将氮和碳循环过程联系起来。所有的基础设施都已到位，并且管理人员在所涉及的技术方面具有杰出的专业知识。学生将获得前沿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接受特定学科的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。鼓励学生监督本科生，参加会议、研讨会、讲习班、企业和参与。该项目将为学生提供一个全面的研究和可转移的技能，适合学术界内外的职业生涯。