Assessing the potential of mRNA-FISH FACS for isolation of functional soil bacterial populations for quantifying biogeochemical cycle interactions

所有生物的正常生长都依赖于充足的基本元素来源（如碳、氮、硫、磷），在这方面，地球可以被认为是一个封闭的系统，基本元素的供应是有限的。因此，通过环境回收这些元素是避免耗尽的基础，微生物可以被视为驱动地球生物地球化学循环中负责这些元素回收的组成过程的“机舱”。在碳循环中，土壤微生物利用不同的有机和无机形式的碳作为能量和碳源，从而在环境隔间之间转移碳。然而，碳循环并不是自己运作的，而是与其他必需元素的代谢密切相关，要么是通过在能量转导中使用这些元素作为还原剂和氧化剂，要么是通过它们作为多种含有必需元素的生物分子（如蛋白质、DNA）的一部分并入生物质（或从腐烂的死生物质中释放出来）。因此，碳的可用性是决定其他基本元素转化和循环的关键因素，而其他关键元素的可用性控制着微生物消耗和呼吸碳的速度。这种生物地球化学循环的相互作用可以通过土壤微生物的反硝化过程来说明：在低氧条件下，通过硝酸盐的呼吸作用分解有机碳，导致硝酸盐逐步还原为二氮气体（N2），产生一氧化二氮（N2O）作为中间体。微生物生态学的一个中心目标是将生物地球化学过程与环境中特定的微生物类群联系起来，以便微生物群落结构的作用可以在预测模型中得到更好的体现。在过去十年中，为了实现这一目标，在不需要培养和鉴定分离物的情况下开发了一套方法，但这些方法都没有提供以微生物为导向的方式量化生物地球化学循环之间相互作用的机会，例如，关于使用特定碳源作为还原剂来驱动反硝化。因此，在受干扰的碳循环和环境变化情景下，对预测基本元素通量和反馈所需的相互作用的定量理解受到方法的限制。该项目将提供一种新方法的概念验证，以量化细菌对碳的利用，同时转化另一种基本元素。细菌反硝化途径将作为一个案例研究，重点关注细菌利用碳将N2O还原为N2（反硝化的最后一步），因为这一群体在调节大气中N2O浓度中起着至关重要的作用，N2O是一种强效温室气体。新方法包括：(i)使用碳同位素来追踪微生物的碳消耗；（ii）用荧光染料标记活性还原n2o的微生物细胞；（iii）对荧光细胞进行分选，定量测定C同位素含量。概念验证将在简单的实验系统中进行，涉及已知的n2o还原细菌和土壤微生物，在已知的促进反硝化的条件下孵育。作为一个案例研究，我们将测试一个关于n2o还原细菌碳源偏好的理论。该项目汇集了研究人员（使用C同位素、荧光标记和细菌分类、反硝化生物地球化学）和项目合作伙伴（荧光标记在生物地球化学循环中活跃的细菌）的互补专业知识。我们将使用最先进的稳定同位素技术来量化微生物N2O还原，并利用先进的仪器进行细胞分选，从而能够准确检测从土壤中提取的细菌细胞。

The normal growth of all living entities depends on an adequate source of essential elements (e.g. C, N, S, P) and, in this respect, the Earth can be considered a closed system with the supply of essential elements being finite. Therefore, the recycling of these elements through the environment is fundamental to avoid exhaustion and microbes can be viewed as the 'engine room' that drive the component processes responsible for the recycling of these elements in the Earth's biogeochemical cycles. In cycling carbon, soil microbes utilise different organic and inorganic forms of carbon as energy and carbon sources resulting in the transfer carbon between environmental compartments. However, the carbon cycle does not operate on its own but it is closely metabolically linked with that of other essential elements either via the use of these as reductants and oxidants in energy transduction or via their incorporation into biomass (or release from decaying dead biomass) as part of multiple essential element- containing biomolecules (e.g proteins, DNA). Hence, the availability of carbon is a key factor in determining the transformations and cycling of other essential elements whilst the availability of other key elements control the rate at which microbes consume and respire carbon. Such biogeochemical cycle interactions can be illustrated by the soil microbial process of denitrification: the decomposition of organic carbon under low oxygen conditions through the respiration of nitrate resulting in the step-wise reduction of nitrate to dinitrogen gas (N2) with nitrous oxide (N2O) produced as an intermediate. A central goal in microbial ecology is to link biogeochemical processes to specific microbial taxa in the environment so that the role of microbial community structure can be better represented in predictive models. A suite of methods have been developed in the last decade in order achieve this goal without the need for cultivation and characterization of isolates but none of these offer the opportunity to quantify the interactions between biogeochemical cycles in a microbially-oriented way, for example, with respect to the use of a particular carbon source as a reductant to drive denitrification. Gaining the quantitative understanding of the interactions that is required to predict essential element fluxes and feedbacks under perturbed carbon cycle and environmental change scenarios is therefore method- limited.This project will provide proof-of-concept of a new method to quantify use of carbon by bacteria whilst transforming another essential element. The bacterial denitrification pathway will serve as a case study with a focus on the bacteria using carbon to reduce N2O to N2 (the final step in denitrification) due to the crucial role that this group play in regulating the atmospheric concentration of N2O, a potent greenhouse gas. The new method involves: (i) use of C isotopes to trace microbial C consumption; (ii) labelling actively N2O-reducing microbial cells with a fluorescent dye; (iii) sorting the fluorescent cells and quantifying the C isotope content.The proof of concept will be in simple experimental systems involving known N2O-reducing bacteria and soil microcosms incubated under conditions known to promote denitrification. As a case study, we will test a theory concerning the carbon source preference of the N2O-reducing bacteria.The project brings together the complimentary expertise of the investigators (use of C isotopes, fluorescence-labelling and sorting of bacteria, denitrification biogeochemistry) and the project partner (fluorescence labelling of bacteria active in biogeochemical cycling). We will use state-of-the art stable isotope techniques to quantify microbial N2O reduction and exploit advances in instrumentation for cell sorting that enables the accurate detection of bacterial cells extracted from soil.