EAGER Electrically Driving the Microbial Conversion of Nitrogen Gas into Ammonia

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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.