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Bioelectroreduction of nitrogen to ammonia: the incorporation of nitrogenase within enzymatic biological fuel cells for simultaneous production of electrical energy and ammonia.

Periodic Reporting for period 2 - Bioelectroammonia (Bioelectroreduction of nitrogen to ammonia: the incorporation of nitrogenase within enzymatic biological fuel cells for simultaneous production of electrical energy and ammonia.)

Reporting period: 2017-06-01 to 2018-05-31

"The overall objective of this project was to further develop and enhance the researcher's knowledge in and understanding of biochemistry, synthetic chemistry, the electrochemistry of enzymes and proteins, and alternative fuel cell technologies. In order to realize these objectives, the research project (Bioelectroammonia) was carefully designed to enable the researcher to undertake two periods of postdoctoral training within two complementary research groups located on two different continents. A 2-year ""outgoing phase"" was planned to be performed within Shelley Minteer's research group at the University of Utah, followed by a 1-year ""incoming phase"" planned to be performed within Donal Leech's research group at the National University of Ireland Galway. It was also anticipated that the researcher would develop his career within the scientific community through a combination of the publication of scientific reports and the attendance of international conferences, while doing so was also anticipated to develop a network of contacts for the researcher's future career path. During the incoming phase of the MSCA, the researcher transferred the knowledge and skills necessary to perform electrochemistry under strict anoxic or controlled hypoxic conditions. This included assembling and configuring an anoxic glovebox in addition to training a graduate student (under Donal Leech's supervision) on its operation.

From a scientific perspective, the objective of this project was to develop a novel technology for the production of ammonia from nitrogen gas, providing an alternative technology to the Haber Bosch process. It was expected that the researcher would develop practical skills pertaining to the growth of microbes, the purification of enzymes, and the interfacing of enzymes with electrode surfaces. The importance of the proposed scientific project surrounds the economic impact of producing ammonia and the expenses by which it is produced. Currently, between 1-2% of global energy is consumed to produce ~500 million metric tons of ammonia per year, where high pressures and temperatures are required to produce ammonia from nitrogen and hydrogen gas. In doing so, around 3% of global carbon dioxide emissions are attributed to the Haber Bosch process. Thus, a novel technology that could offer an alternative approach to produce ammonia from nitrogen gas has the potential to be economically important on a global scale."
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 Leec
This project has initiated a new stream of enzymatic fuel cells and heterogeneous bioelectrochemical research into nitrogenase and N2 reduction at enzyme-functionalized electrodes. It it highly anticipated that this research project will directly result in an improved understanding of nitrogenase and alternative nitrogenase-based biotechnological by other researchers in the fields of metalloproteins and enzymatic electrochemistry, in addition to the field of inorganic catalysis.

This project has substantially improved the researcher's understanding of biologically relevant reactions (such as nitrogen reduction) and the thermodynamic and kinetic aspects surrounding such reactions. During the incoming phase of this research project, it is anticipated that the researcher will further develop the enzymatic electrochemical platforms outlined herein where alternative fuels to hydrogen gas may be explored.
Hydrogenase/nitrogenase enzymatic fuel cell simultaneously produces ammonia and electrical energy.