(736f) Rhodopseudomonas Palustris Polyhydroxybutyrate Production from a Lignin Aromatic and Quantification Via Flow Cytometry

Polyhydroxybutyrate (PHB) is a biopolymer that has similar thermomechanical properties as petroleum-based plastics, and is produced by bacteria under certain stress conditions. Unfortunately, the commercial production and widespread adaptation of PHB is limited by higher productions costs that are attributed to the carbon source and extraction method. A renewable and cheaper carbon source, such as lignocellulosic biomass, could help reduce production costs. In this study, PHB was produced and quantified from the metabolically versatile Rhodopseudomonas palustris CGA009 when grown on the lignin breakdown product p-coumarate for the first time. The titer ranged from 0.001 - 0.79 g/L, reaching maximum production at six days. To reduce the bioprocessing burdens of quantifying PHB titer and cell count, a method was optimized that uses either Nile Red or BODIPY 493/503 lipophilic stains followed by flow cytometry analysis. A sample preparation assay revealed that 50% ethanol/PBS solution with 3 µL of either stain resulted in the highest stain to cell count ratio. Linear models for PHB concentration and fluorescence intensity for both Nile Red and BODIPY yielded strong fitness (R² = 0.9384 and R² = 0.9747 respectively). Unlike other studies that show this method to be ill fitted for cell count correlation, the permeability technique created here produced high cell count fitness for both stains (R² = 0.9383 and R² = 0.9955 respectively). Ultimately, this study is novel in that it (I) produces PHB from a lignin carbon source with the metabolically robust R. palustris, (II) innovates a multi-pronged optimization method for quantifying both PHB and cell count via flow cytometry, (III) delivers precise PHB quantification for a wide range of titers that does not require polymer degradation, and (IV) provides flexibility for synthetic biology experiments with cells expressing red and green fluorescent proteins since the stains fluoresce at differing wave lengths.