(70g) Conversion of Lignin into Biodegradable Plastic By Rhodopseudomonas Palustris

A sustainable alternative to conventional plastics is polyhydroxyalkonoates (PHAs), biopolymers produced by microorganisms as energy storage granules in certain environmental conditions. PHAs have similar properties as synthetic plastics, are the only 100% truly biodegradable polymer, and can be produced from a variety of substrates. As the most abundant storage compound in bacteria, poly-3-hydroxybutanoate (PHB) is a widely studied short-chain-length PHA. However, PHAs are inhibited by higher production costs due primarily to the selected strain, carbon source, and the need for more efficient extraction methods. Thus, the goal of this study was to engineer the metabolically versatile Rhodopseudomonas palustris CGA009 to produce PHB from lignocellulosic biomass as a renewable carbon source and promote the valorization of PHAs. As part of this study, R. palustris’ native metabolic and regulatory PHB production pathways on lignin breakdown products (LBPs) were evaluated. Results confirmed that R. palustris grows both anaerobically and aerobically on the major LBPs p-coumarate and coniferyl alcohol, as well as on Kraft lignin supplemented with acetate. Furthermore, chemical oxygen demand testing confirmed R. palustris catabolizes these carbon sources. Using aqueous two-phase extraction and gas chromatography-mass spectrometry, we found that R. palustris does generate PHB without nutrient deprivation, but that nitrogen starvation results in significantly higher accumulations. Preliminary results show that R. palustris produces approximately 5g PHB in 13.5mL 1mM LBP after four days of nitrogen starvation, while the optimum timing for maximum production is being investigated. R. palustris’ PHB gene expression was analyzed via qPCR to further characterize its native pathways. Our ongoing genetic engineering efforts utilize these findings to improve PHB production in R. palustris by creating new strains with optimized granule associated proteins. Additionally, our continuing efforts to develop an efficient extraction method specific for this strain using non-chlorinated and cost-effective solvents will further enhance PHB yields. Ultimately, this study is novel in that it combines genetic engineering and efficient fermentation process design to advance PHB production from a non-model and metabolically flexible bacteria using lignin as a renewable carbon substrate through a multifaceted approach: (i) characterizing R. palustris’ native growth and PHB production on LBPs, (ii) genetically engineering R. palustris’ granule associated proteins for the overproduction of PHB, and (iii) designing an effective PHB recovery method specific for this strain. These efforts thwart the two major production costs for PHB, expensive carbon source and extraction techniques, that are preventing PHB production from becoming cost-effective replacements for some petroleum-based plastics.