EAGER Electrically Driving the Microbial Conversion of Nitrogen Gas into Ammonia

氨是农业和工业活动的关键资源。大部分氨是通过一种名为哈伯-博世工艺的工业工艺产生的。这种改变模式的方法是推动农业绿色革命的重要因素，并帮助氨成为世界上产量最大的化学品之一。这种成功是以高昂的成本为代价的，无论是在资金还是对环境的影响上。生产氨需要极高的温度和压力条件，该过程消耗了全球1-2%的能源，每年产生2.5%的二氧化碳排放。因此，迫切需要找到哈伯-博施进程的可持续和低成本替代办法。自然产生的微生物可以将大气中的氮气转化为氨，并可以在环境温度和压力下这样做。阻碍工业规模微生物氨生产的挑战包括氮转换酶对氧气的敏感性、无法推动高氨产率以及从微生物中回收氨。因此，该项目将结合电化学和微生物学领域来表征和优化一种电力驱动的氨生产生物技术。研究小组将使用模型发电细菌来了解它们的氮转化途径对电力驱动力的反应，然后利用这些信息来设计高效的产氨细菌。这一结果将扩大我们对微生物氮转化过程的了解，并为氨生产技术奠定基础，该技术可以从小农场的规模扩大到大型工业规模的过程。该团队还将通过利用他们所在机构已有的研究实习暑期体验(RISE)计划，吸引未被充分代表的本科生和研究生参与研究。这些学生将有一个独特的机会进行横跨电化学、微生物学和工程学的研究。几种新兴的氨生产技术分别利用电化学和微生物方法。它们取得的成功有限，而且面临着固有的可扩展性挑战。这些限制可以通过将电化学过程和生物过程结合起来而不是分开处理来克服。电子微生物学的研究已经证明，微生物的新陈代谢可以在输入不到1伏特的电力的情况下得到电驱动。几种被称为外电的发电细菌也是强有力的固氮微生物。通过开发和优化微生物独特的生理，微生物电化学技术(METS)可能被开发出来，以电驱动氨氮的固定。该项目的总体目标是确定对电力驱动力的固氮调节变化，并利用这些信息来优化产氨菌株。这项工作有三个主要任务：(1)确定MET操作变量对固氮完整性和固氮速率的影响；(2)利用整个转录组RNA测序确定MET操作期间固氮和氨产生途径的调控变化；以及(3)使用簇状规则间隔短回文重复序列(CRISPR)基因组编辑工具设计新的分泌氨的外生菌株。预计这项工作将对与一系列生物技术平台相关的固氮途径产生新的见解。在外生微生物中调节CRISPR也将为从废水处理到生物电合成化学生产的应用提供一种新的工具。这一奖项反映了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。

Ammonia is a critical resource for farming and industrial activities. The majority of ammonia is generated through an industrial process called the Haber-Bosch process. This paradigm changing method was a significant factor driving the agricultural green revolution and helped ammonia become one of the most produced chemicals in the world. This success has come at a high cost in terms of dollars and environmental impact. Requiring extremely high temperature and pressure conditions to produce the ammonia, the process consumes 1-2% of global energy and generates 2.5% of all carbon dioxide emissions annually. There is, therefore, a critical need to find sustainable and low-cost alternatives to the Haber-Bosch process. Naturally occurring microorganisms can convert atmospheric nitrogen gas into ammonia and can do so at ambient temperature and pressure. Challenges preventing industrial-scale microbial ammonia production include the sensitivity of the nitrogen-converting enzyme to oxygen, the inability to drive high ammonia production rates, and ammonia recovery from the microorganisms. Accordingly, this project will combine the fields of electrochemistry and microbiology to characterize and optimize an electrically-driven ammonia production biotechnology. The research team will use model electricity-generating bacteria to understand the response of their nitrogen conversion pathways to an electrical driving force and then use that information to engineer highly efficient ammonia-generating bacteria. The results will expand our knowledge of microbial nitrogen conversion processes and lead to the foundation of an ammonia production technology that can be scaled in size for small farms to large, industrial-scale processes. The team will also engage underrepresented undergraduate and graduate students in the research by leveraging the established Research Internship Summer Experience (RISE) program at their institution. These students will have a unique opportunity to conduct research spanning electrochemistry, microbiology, and engineering.Several emerging ammonia production technologies utilize electrochemical and microbial methods separately. They have had limited success and face inherent scalability challenges. These limitations may be overcome by combining electrochemical and biological processes, rather than treating them separately. Research in electromicrobiology has demonstrated that microbial metabolisms can be electrically driven with inputs of less than one volt of electricity. Several electricity-generating bacteria, known as exoelectrogens, are also vigorous nitrogen-fixing microorganisms. By exploiting and optimizing their unique physiology, microbial electrochemical technologies (METs) may be developed to electrically drive nitrogen fixation into ammonia. The overall objective of this project is to identify nitrogen fixation regulatory changes in response to electrical driving forces and to use that information to optimize ammonia-generating strains. This work has three main tasks: (1) determine the impact of MET operational variables on the completeness and rates of nitrogen fixation; (2) identify regulatory changes in nitrogen fixation and ammonia generation pathways during MET operation using whole transcriptome RNA sequencing; and (3) engineer new exoelectrogenic strains that excrete ammonia using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing tools. It is expected that this work will yield new insight into nitrogen fixation pathways that will be relevant to an array of biotechnological platforms. Modulating CRISPR in exoelectrogenic microorganisms will also provide a new tool for applications ranging from wastewater treatment to bioelectrosynthetic chemical production.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.