This project sought to develop a novel technology for the production of ammonia from nitrogen gas, where it was proposed that an enzyme (nitrogenase) could be employed at an electrode surface whereby electrical energy could be supplied to the enzyme to produce ammonia from nitrogen gas. In order to begin the evaluation of such a proposal, nitrogenase had to first be prepared. Nitrogenase is not commercially available; further, this highly complicated enzyme is extremely sensitive to irreversible damage from oxygen present in the atmosphere. It was therefore essential that the researcher could develop an understanding of microbial cultivation and the purification of enzymes from microbes. Within the Minteer research group at the University of Utah, the researcher learned how to cultivate a nitrogenase-producing bacterium, Azotobacter vinelandii, in addition to learning how to purify nitrogenase from the bacterium under an environment free of oxygen. It is anticipated that the researcher will transfer this developed skillset to Donal Leech's research group during the incoming phase of this research project.
Once nitrogenase had been purified to homogeneity, the researcher developed an electrode modification by which the catalytic component of nitrogenase (MoFe protein, where MoFe denotes the presence of iron and molybdenum within the protein) could be electronically-connected. Initial evaluation of this bioelectrochemical system found that the electronically-controlled MoFe protein of nitrogenase was able to produce ammonia following the transfer of electrons to the enzyme, although it could only be produced from azide and nitrite and not from the desired substrate, nitrogen gas. Nevertheless, this electrode architecture later proved useful in gaining an improved understanding of electron transfer within the MoFe protein.
In order to realize the production of ammonia from nitrogen gas, the researcher obtained the electron-supplying protein of the nitrogenase MoFe protein; the Fe protein (where Fe refers to the iron present within the protein). Having the Fe and MoFe proteins of nitrogenase in hand, the researcher was able to construct a novel enzymatic fuel cell that was able to produce ammonia from nitrogen gas. An additional enzyme, hydrogenase, was also employed at a second electrode so that the enzymatic fuel cell could simultaneously produce electrical energy and ammonia when supplied with hydrogen gas and nitrogen gas. In order to preserve the enzymes, the enzymatic fuel cell was operated in the strict absence of oxygen which would otherwise irreversibly deactivate the enzymatic fuel cell.
Finally, the researcher explored the possibility of protecting nitrogenase from irreversible inactivation upon exposure to oxygen, so that ammonia could be produced from air (containing approximately 79% nitrogen gas and 21% oxygen gas) in the place of highly-purified nitrogen gas. This highly ambitious investigation was fueled by the knowledge that the bacterium from which nitrogenase was isolated (A. vinelandii) is able to convert nitrogen gas to ammonia in the presence of ambient oxygen concentrations. Past research had shown that a small oxygen-sensing protein, a ferredoxin protein found within A. vinelandii, was able to offer a degree of protection to nitrogenase under a low concentration of oxygen gas (2%). The researcher produced this protein using an alternative bacterium, Escherichia coli, and it was found that this protein was also able to protect nitrogenase when electronically stimulated. This lead the researcher to construct a novel electrochemical system whereby nitrogenase was able to produce ammonia from air, where the protective ferredoxin was crucial for extended nitrogenase activity.
During the incoming phase of the MSCA, the researcher investigated alternative electron mediators based on osmium-containing complexes. This osmium complex is now the subject on ongoing investigation and it is anticipated that the Leech group and the researcher's own research group will collaborate in this area on future projects.
