EAGER: Chasing the elusive syntrophic partners in direct interspecies electron transfer

厌氧消化在全球范围内被广泛用于处理废气并将其转化为沼气，如甲烷。厌氧消化产生的沼气通常含有50%-70%的甲烷，因此需要进行处理以去除包括二氧化碳和水蒸气在内的杂质。为了消除对现有厌氧消化器中沼气的额外处理和提纯的需要，了解和量化参与沼气生产的微生物群体的代谢途径是至关重要的。这个高风险和高回报的渴望项目的首要目标是描述和量化在厌氧消化过程中驱动甲烷产生的代谢途径。为了推进这一目标，该项目的首席研究员(PI)建议开展一项综合实验研究计划，以检验物种间氢转移(IHT)和物种间直接电子转移(DIEW)在导电环境中甲烷生成沼气过程中发挥同等重要作用的假说。这一迫切需要的项目的成功完成可提供新的基本知识，可用于开发和实施新的工程反应堆设计和操作战略，以增加厌氧消化器产生的沼气中的甲烷含量。将通过学生教育和培训，包括对博士生的指导，为社会带来进一步的好处。物种间氢转移(IHT)和物种间直接电子转移(DICE)已被证明有助于在导电环境中通过产甲烷产生沼气。然而，由于缺乏直接测量相关微生物代谢和活性的实验技术，对间歇性羟色胺和饮食对产甲烷的相对贡献的基本了解和量化仍然难以实现。该项目的首要目标是解决这一知识鸿沟。为了推进这一目标，该项目的首席研究员(PI)假设，在导电环境中通过产甲烷产生沼气的过程中，间歇性羟色胺和饮食扮演着同样重要的角色。这一假说是基于PI的初步研究结果，该研究鉴定了一种新的地杆菌物种(Candidatus Gebacter Eutroica)，该种菌在配备导电颗粒活性碳的厌氧反应器中大量存在。PI还发现，假丝酵母富营养化细菌活跃地表达编码细胞外电子传递和氢代谢的蛋白质的基因。为了验证这一新假说，PI建议开展一项围绕两个特定目标构建的综合实验研究计划：1)丰富能够摄食的地杆菌并阐明它们的胞外电子传递机制(特定目标1)和2)丰富能够摄食的甲烷菌并表征它们的胞外电子摄取机制(特定目标2)。为了丰富能够饮食的地质杆菌和产甲烷细菌，PI建议在具有特殊设计的电极的生物电化学系统中使用电化学刺激。通过将循环伏安法与沼气产生、离子色谱、共振拉曼显微镜和组学(元基因组学和元转录组学)测量相结合，PI希望揭开供电子和接受电子的微生物伙伴中负责饮食的代谢途径。该项目的成功完成具有潜在的变革性影响，通过产生新的基础知识来推动新的生物过程的开发，如电甲烷合成，可以将废流转化为沼气，甲烷产率比现有的厌氧消化反应器高得多。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Anaerobic digestion is widely utilized worlwide to treat waste streams and convert them to biogas such as methane. Biogas fom anerobic digestion typically consists of 50-70% methane and thus needs to be treated to remove impurities including carbon dioxide and water vapor. To eliminate the need for addtional treatment and purification of biogas from existing anaerobic digesters, it is critical to understand and quantify the metabolic pathways of the microbial consortial involved in biogas production. The overarching goal of this high-risk and high-reward EAGER project is to characterize and quantify the metabolic pathways that drive methane production during anaerobic digestion. To advance this goal, the Principal Investigator (PI) of this project proposes to carry out an integrated experimental research program to test the hypothesis that interspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) play an equally important role during biogas production by methanogenesis in electrically conductive environments. The successful completion of this EAGER project could provide new fundamental knowledge that could be leveraged to develop and implement new engineering reactor design and operational strategies to increase the methane content of biogas produced by anaerobic digestors. Further benefits to society will be achieved through student education and training including the mentoring of a doctoral student. Interspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) have been shown to contribute to biogas production by methanogenesis in electrically conductive environments. However, a fundamental understanding and quantification of the relative contributions of IHT and DIET to methanogenesis have remained elusive due to the lack of experimental techniques to directly measure the associated microbial metabolisms and activities. The overarching goal of this project is to address this knowledge gap. To advance this goal, the Principal Investigator (PI) of this project hypothesizes that IHT and DIET play an equally important role during biogas production by methanogenesis in electrically conductive environments. This hypothesis is based on the results of preliminary studies by the PI that identified a novel Geobacter species (Candidatus Geobacter eutrophica) that was abundant in anerobic reactors supplied with conductive granular activated carbon. The PI also found that the Candidatus Geobacter eutrophica bacteria actively expressed genes encoding proteins for both extracellular electron transfer and hydrogen metabolism. To test this new hypothesis, the PI proposes to carry out an integrated experimental research program structured around two specific aims: 1) enrich DIET-capable Geobacter bacteria and elucidate their extracellular electron transfer mechanisms (Specific Aim 1) and 2) enrich DIET-capable methanogens and characterize their extracellular electron uptake mechanisms (Specific Aim 2). To enrich the DIET-capable Geobacter and methanogen bacteria, the PI proposes to use electrochemical stimulation in bioelectrochemical systems with specially designed electrodes. By combining cyclic voltammetry with measurements of biogas production, ion chromatography, resonance Raman microscopy and omics (metagenomics and metatranscriptomics), the PI hopes to unravel the metabolic pathways responsible for DIET in both electron-donating and electron-accepting microbial partners. The successful completion of this project has the potential for transformative impact through the generation of new fundamental knowledge to advance the development of new bioprocesses such as electro-methanogenesis that could convert waste streams to biogas with much higher methane yields than existing anerobic digestion reactors.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.