(748c) Conversion of Lignin into Bioplastic By Rhodopseudomonas Palustris CGA009

Polyhydroxyalkanoates (PHAs) are biopolymers produced by bacteria under certain stress conditions that have similar properties as petroleum-based plastics. Due to their biodegradation and thermoprocessing properties, PHAs have been used in a wide array of applications from packaging to drug delivery systems. Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) is a PHA that is more desirable due to its fraction of hydroxyvalerate that significantly enhances thermal properties compared to PHB alone. However, PHBV it is inhibited by higher production costs primarily due to the carbon source. While it has been shown that metabolically robust Rhodopseudomonas palustris CGA009 produces PHBV, most studies use hydroxybutyrate or valerate as the carbon source, which is costly and not ideal for real-world processes. Thus, the objective of this study was to engineer R. palustris to produce PHBV for the first time on lignocellulosic biomass. R. palustris was grown anaerobically on the lignin breakdown products p-coumarate (1mM) or coniferyl alcohol (1mM) and on Kraft lignin (3 g/L). Cells were nitrogen starved at mid-exponential growth to promote PHBV production. Samples were harvested after each day of nitrogen starvation, and the PHBV concentration was quantified using methanolysis and gas chromatography-mass spectrometry. For the lignin breakdown products, the titer ranged from 0.16 - 0.79 g/L, reaching maximum production at six days. The quantification of PHBV production on Kraft lignin supplemented with acetate is ongoing. We found that R. palustris does store PHBV without nutrient deprivation, but that nitrogen starvation results in significantly higher accumulations. In a comparison between phosphorous and nitrogen deprivation, nitrogen deprivation resulted in significantly higher PHBV production. To conduct a holistic analysis of the PHBV metabolism for this non-model organism, RNA samples from PHBV-producing cells grown on either p-coumarate, coniferyl alcohol, or acetate were submitted for Total RNA Sequencing. Furthermore, our ongoing efforts using RT-PCR and q-PCR are focused on illuminated the gene expression of the granule associated proteins involved in R. palustris PHBV production. In particular, R. palustris has four phasins that are merely predicted by protein homology that have shown significant variations in gene expression. Lastly, samples were submitted for Transmission Electron Microscopy to assess variations in PHBV granules. Ultimately, this study is novel in that for the first time it (i) produces PHBV from R. palustris on lignin breakdown products and Kraft lignin, and (ii) delivers a holistic analysis of R. palustris’ PHBV metabolism and granule-associated proteins.