Engineering Applications Using The Innate Redox Environment Of Rhodopseudomonas Palustris

Rhodopseudomonas palustris is currently the most metabolically versatile organism known. Because of this, it has become a model organism not only for it's many forms of metabolism, but also for the complex coordination required to regulate them. Under anaerobic conditions, R. palustris has received considerable attention regarding how it handles excess reducing equivalents. Herein, I investigated alternate routes for either pushing or pulling reducing equivalents into the metabolism of R. palustris for applications relating to bioenergy. Of particular interest to my research are the anaerobic metabolic processes of: i. anoxygenic photosynthesis; ii. metabolism of the molecules n-butyrate and p-coumarate; iii. photoautotrophic growth with iron(II) as a sole electron donor; iv. nitrogenase based production of H2; and v. fixation of CO2. In my first study, I focused on the lethal redox imbalance that R. palustris encounters when growing on n-butyrate with no viable electron sinks. Excess reducing equivalents were harnessed to drive the metabolically engineered production of the biofuel nbutanol. Because the reduction of n-butyrate to n-butanol became the only route of maintaining redox balance, this metabolically engineered activity became obligate for growth. In the second study, I co-cultured R. palustris and the model exoelectrogen Geobacter sulfurreducens together in a bioelectrochemical system to investigate metabolite sharing of the lignin monomer pcoumarate by R. palustris. The electrochemical system functioned as a tool for measuring the effect of G. sulfurreducens aiding R. palustris in maintaining redox balance. The third study characterized the ability of R. palustris to take up electrons from a negatively poised electrode through mediated iron cycling as a sole source of electrons for growth. It was observed that volumetric rates of electron uptake were very low, so to improve these rates and demonstrate that this mechanism could be the foundation of a future microbial electrosynthesis technology, improvements in reactor design were made. The electrode was removed from the growth reactor, which allowed optimization of both the photosynthetic bioreactor and the abiotic electrochemical reactor. These two components were then connected through a closed recirculation loop to recycle and regenerate the iron substrate. This resulted in increased volumetric iron consumption 56 times higher in the illuminated reactor compared to previously measured in conventional bioelectrochemical reactors