Efficient Utilization of Crude Glycerol as Fermentation Substrate in the Synthesis of Poly(3-hydroxybutyrate) Biopolymers
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One refined and two crude glycerol (from biodiesel production) samples were utilized to produce poly(3-hydroxybutyrate) (PHB) by Pseudomonas oleovorans NRRL B-14682. A batch culture fermentation protocol including 1% glycerol and an aeration rate of 3 standard liters per minute proved best for PHB synthesis (av. yield = 1.0 ± 0.2 g/L at 48 h) and efficient glycerol utilization. PHB molecular weights decreased as MeOH concentration increased. Refined glycerol resulted in PHB polymers with number average molecular weights (Mn) of 314,000 g/mol which decreased by 17 and 90% as MeOH media concentrations increased to <0.005 and 0.85%, respectively. Proton (1H) NMR demonstrated the presence of glycerol- and methoxy-based end-capping, which was confirmed by 1H diffusion experiments (DOSY analyses). NMR diffusion analyses of the PHB polymers established their diffusivities, and confirmed that their relative molecular sizes were dependent on the impurities in the glycerol. In addition, DOSY analyses indicated that each end-capped PHB polymer and the glycerol or methoxy groups bound to it had the same diffusion constants, demonstrating that they migrated together as covalent complexes. Non-covalent complexation was eliminated by physically mixing free glycerol with PHB synthesized from oleic acid; their respective diffusivities were notably faster.