Nitrogen gas (N2) conversion to ammonium (NH4+) is an essential, but cost- and energy-expensive process, with the industrial Haber-Bosch process constituting the major form of artificial N2 fixation implemented today. Biological nitrogen fixation (BNF) is a more efficient process that has seen significant barriers to commercial implementation. Geobacter sulfurreducens is a free-living N2-fixing soil bacterium that can also respire on electrodes. Recent evidence from our group has indicated that the metabolic rate of G. sulfurreducens while respiring in microbial electrochemical cells (MECs) is correlated to N2 fixation. However, NH4+ is not secreted from cells due to tight regulatory control. The CRISPR-dCas9 system is a robust method for offsetting undesirable regulatory control. In this study, the CRISPR-Cas9/dCas9 system was implemented for the first time in G. sulfurreduencs. As evidence for this new approach, we first targeted acetate utilization in G. sulfurreducens via acetate kinase. Next, we targeted a protein that inhibits the function of nitrogenase in response to high extracellular NH4+ (DraT). In this study, DraT is knocked down with CRISPR-dCas9 to ensure continued function of nitrogenase in the presence of high extracellular NH4+. Lastly, the knockdown mutants were run under N2-fixation conditions in MECs to determine their ability to secrete and accumulate NH4+.

Implementation of CRISPR-Cas9 system in Geobacter sulfurreducens to enable ammonium generation from nitrogen gas

ACS Fall 2020 Virtual Meeting

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