Nitrogen gas (N2) conversion to ammonium (NH4+) is an essential but cost- and energy-intensive process, with the Haber-Bosch method constituting the major form of artificial N2 fixation implemented today. Biological nitrogen fixation is an alternative process that is more energy efficient, but faces significant barriers to commercial implementation including slow rates of fixed nitrogen production. Geobacter sulfurreducens is a nitrogen-fixing soil bacterium that can respire on anode electrodes. The metabolic rate of G. sulfurreducens while respiring in microbial electrochemical cells (MECs) is connected to nitrogen fixation, wherein higher applied voltages can drive higher N2 fixation rates. The cells do not secrete NH4+ during anode respiration. Tight regulatory control quickly converts NH4+ into molecules such as amino acids. Enabling excretion of NH4+ requires genetic manipulation of relevant regulatory pathways. The CRISPR-dCas9 system is a robust method for offsetting undesirable regulatory control. In this study, the CRISPR-Cas9/dCas9 system was implemented in G. sulfurreducens to encourage nitrogen release. Nitrogenase activity and ammonium uptake were targeted through the generation of various knockdown mutants. These knockdown mutants were evaluated for nitrogen fixation rate, respiration rate and ability to secrete fixed ammonium. The generation of Geobacter sulffureducens mutants capable of ammonium secretion via CRISPR-dCas9 demonstrates a valuable new genetic tool for metabolic engineering and provides a useful example of MEC-driven biochemical production.

Implementation of CRISPR-dCas9 system in Geobacter sulfurreducens to enhance the release of fixed ammonium

ACS Fall 2021

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