ISOLATION AND STUDY OF PURPLE NON‐SULFUR BACTERIA FROM FUEL SYNTHESIS WASTEWATER CULTURE TO UNDERSTAND AND IMPROVE THEIR POLYHYDROXYALKANOATE PRODUCTION

Abstract

Polyhydroxyalkanoates (PHAs) are biodegradable polymers synthesized by various microorganisms as a means of carbon and energy storage under nutrient-limited conditions. Light-dependent microorganisms such as cyanobacteria and purple non-sulfur bacteria (PNSB) have garnered attention as sustainable platforms for biopolymer and bioenergy production. Recent studies highlight the potential of mixed cultures of purple phototrophic bacteria to produce bioplastics from waste streams. However, the high production cost remains a barrier to large-scale PHA deployment. Enhancing the catalytic efficiency of PHA synthase (PhaC), the key enzyme responsible for polymerizing PHA monomers, offers a promising strategy to reduce costs. This study explores the potential of PNSB for wastewater-based PHA production and investigates how mutations in the phaC gene affect enzyme performance. The research was conducted in four stages. First, PNSB were isolated from non-axenic enrichment cultures using serial dilution and anaerobic plating on Van Niel’s yeast agar under light. DNA was then extracted from selected colonies and the enrichment medium, with PCR amplification of the pufM gene confirming phototrophic identity. 16S rRNA sequencing further classified the isolates as purple non-sulfur bacteria. Next, computational protein modeling and molecular docking were performed on wild-type and mutated phaC enzymes from Rhodovulum sulfidophilum and Rhodoplanes piscinae. The mutated protein-ligand complexes demonstrated improved docking scores (−4.9 and −4.29 kcal/mol), indicating enhanced substrate interaction. Finally, gene expression was evaluated in lab-scale, non-axenic bioreactors. The results showed higher phaC expression in larger bioreactor volumes, while increased phaP expression in 20 L setups suggested a correlation between container size and polymer formation. phaZ expression was inconsistent, likely due to sample variability. These findings offer insights into improving PNSB-based PHA biosynthesis and contribute to scalable, cost-effective biopolymer production strategies.

Date of Award	2025
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences