OCE-PRF Uncovering the Genetic Basis of Novel Oxidative Extracellular Electron Transfer Mechanisms in an Electrochemically Active Marine Bacterial Consortium

该奖项全部或部分由《2021年美国救援计划法案》（公法117-2）资助。最近，人们发现一些微生物能够从位于细胞外的固相矿物质中呼吸（“呼吸”）或获取能量（“进食”）。这是由细胞外电子转移（EET）促进的：一些微生物将电子从细胞和固体物质之间进出的过程。EET可以在实验室环境中进行评估，在实验室环境中，微生物在电极上生长，并且可以测量生物电流的产生——电子进出电极的流动。在这些实验中，电极被放置在特定的电压（氧化还原电位）下，作为矿物表面的代理。我们对EET的大部分知识都是从少数能够呼吸性矿物还原的模式生物中获得的，在这种模式生物中，电子从细胞转移到氧化的固相物质上，如铁和锰的氧化物，这些物质普遍存在于淡水和海洋沉积物中。然而，人们对氧化EET的机制或生态影响知之甚少，氧化EET是指电子从还原矿物质转移到细胞中作为能量来源（“进食”）的过程。来自微生物群落调查的证据表明，生活在各种栖息地的生物，包括淡水和海洋环境，可能会产生EET。该项目将扩大我们对从海洋沉积物中分离的微生物中氧化EET的遗传基础的理解，这些微生物分别在单独培养和共同培养的财团中培养。此外，这些信息将有助于深入了解EET在全球元素循环中的作用，并可能为使用电化学活性生物的替代清洁能源生产、生物修复和废水处理工作提供信息。该项目将为本科生提供教育和培训机会，他们将被招募来协助研究任务。将组织社区外展活动，特别是在辛辛那提服务不足的地区，为中学生提供以电微生物学和生物能源生产为重点的动手教育机会。细胞外电子转移（EET）的发现对广泛的生物地球化学重要过程具有重要意义。在海洋地下沉积物中，微生物生物量估计占地球总生物量的0.6%，岩石营养代谢难以检测，因为氧化EET的遗传基础在很大程度上仍然未知和未表征，“组学”研究未能识别参与该过程的基因或蛋白质。这一重大的知识差距，加上对“深”和“暗”海洋微生物代谢的直接测量，远远超出了基于有机碳流入系统的预期模型，强烈表明，岩石营养代谢的重要性，以及这些代谢运作的机制，一直被忽视，理解和检测这些过程在自然界是至关重要的。此外，对多微生物群落中EET的生态学知之甚少，但各种数据表明，不同的微生物群落对稳定和进程的环境功能至关重要。因此，该项目的总体目标是深入了解氧化过程中EET的遗传基础，以及这些过程在纯培养和共培养两种遗传上易于处理的电极氧化的岩石营养海洋沉积物细菌中的生态影响。尖端的、有针对性的高通量测序技术将用于鉴定可能参与氧化EET的基因和促进共培养中种间电子转移的基因。将构建基因缺失突变体，并检测突变株的氧化EET能力和种间电子转移能力。这不仅可以更好地理解海洋系统中的微生物群落生态学，而且这项研究还将提供对各种栖息地中氧化EET的深入了解，包括海洋和陆地地下环境以及普遍存在还原电子源的工程环境。从应用和生物技术的角度来看，这项工作的结果将提高评估遗传易感生物中微生物-电极相互作用的能力，并可能为电化学活性生物的工业应用提供信息，如清洁能源发电、废水处理和电合成。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).Recently, it was discovered that some microbes are capable of respiring (‘breathing’) or deriving energy (‘eating’) from solid-phase minerals located outside the cell. This is facilitated by extracellular electron transfer (EET): the process by which some microbes shuttle electrons into and out of their cells from/to solid materials. EET can be evaluated in a laboratory setting, in which microbes are grown on electrodes and the generation of biological current – the flow of electrons to/from the electrode – can be measured. In these experiments, the electrodes are poised at specific voltages (redox potentials) that function as proxies for mineral surfaces. Much of our knowledge of EET has been obtained from just a few model organisms capable of respiratory mineral reduction, in which electrons are transferred out of the cell onto oxidized solid-phase materials, such as iron and manganese oxides, which are prevalent in freshwater and marine sediments. However, little is known about the mechanism or ecological implications of oxidative EET, the process by which electrons are transferred from reduced minerals into the cell as a source of energy (‘eating’). Evidence from microbial community surveys suggests that organisms inhabiting a wide variety of habitats, including both freshwater and marine environments, may be capable of EET. This project will expand upon our understanding of the genetic basis of oxidative EET in microbes isolated from marine sediments grown individually and in a co-culture consortium. Further, this information will provide insight into the role of EET in global elemental cycling and may inform alternative clean energy generation, bioremediation, and wastewater treatment efforts using electrochemically active organisms. This project will provide educational and training opportunities to undergraduate students, who will be recruited to aid with research tasks. Community outreach activities, particularly in underserved areas of Cincinnati, will be organized to provide middle-school aged children with hands-on educational opportunities focused around electromicrobiology and bioenergy production.The discovery of extracellular electron transfer (EET) has important implications for a wide range of biogeochemically important processes. In marine subsurface sediments, where microbial biomass is estimated to account for 0.6% of the total biomass on Earth, lithotrophic metabolisms are difficult to detect since the genetic basis of oxidative EET remains largely unknown and uncharacterized, and ‘omics’ studies fail to identify genes or proteins involved in this process. This significant knowledge gap, coupled with direct measurements of microbial metabolism in the ‘deep’ and ‘dark’ ocean far exceeding the expected models based on the influx of organic carbon to the system, strongly suggest that the importance of lithotrophic metabolisms, and the mechanisms by which these metabolisms operate, has been overlooked and understanding and detection of these processes in nature is crucial. Furthermore, the ecology of EET in polymicrobial communities is poorly understood, but a variety of data imply that diverse microbial communities are important for stable and processive environmental function. Thus, the overarching goal of this project is to gain insight into the genetic basis of EET in oxidative processes, and into the ecological implications of these processes, in two genetically tractable, electrode-oxidizing lithotrophic marine sediment bacteria in pure culture and in co-culture. Cutting-edge, targeted high-throughput sequencing techniques will be used to identify genes putatively involved in oxidative EET and genes that facilitate interspecies electron transfer in co-cultures. Gene deletion mutants will be constructed, and mutant strains will be assayed for their capacity for oxidative EET and interspecies electron transfer. Not only will this allow for a better understanding of microbial community ecology in marine systems, but this study will also provide insight into oxidative EET in a wide array of habitats, including marine and terrestrial subsurface environments and engineered environments where reduced electron sources are prevalent. From an applied and biotechnological standpoint, results from this work will enhance the capacity for evaluating microbe-electrode interactions in genetically tractable organisms, and may inform industrial applications of electrochemically active organisms, such as clean energy generation, wastewater treatment, and electrosynthesis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.