Production of Polyhydroxyalkanoates through Soybean Hull and Waste Glycerol Valorization: Subsequent Alkaline Pretreatment and Enzymatic Hydrolysis

Alkaline pretreatment and sequential enzymatic hydrolysis of soybean hull were investigated to obtain fermentable sugars for polyhydroxyalkanoates production along with residual glycerol as low-cost carbon sources. Soybean hull is composed of approximately 32% cellulose, 12% hemicellulose, 6% lignin, and 11% protein. Alkaline pretreatment was carried out with 2% NaOH concentration, 10% (w/v) biomass loading, and 60 min incubation time in an autoclave at 120 degrees C. The response surface methodology (RSM) based on the central composite design (CCD) tool was employed to optimize the enzymatic hydrolysis process, where the variables of biomass loading, enzymes' concentration, and time were considered. The maximum total reducing sugars concentration obtained was 115.9 g center dot L-1 with an enzyme concentration of 11.5 mg protein/g dry substrate for enzyme preparation B1, 2.88 mg protein/g dry substrate for XylA, and 57.6 U/g dry substrate for beta-glucosidase, after 42 h at 45 degrees C, and pH was 4.5. Subsequently, the saccharification step was conducted by increasing the processing scale, using a 1 L tank with stirring with a controlled temperature. Implementing the same enzyme concentrations at pH 4.5, temperature of 45 degrees C, 260 mL working volume, and incubation time of 42 h, under fed-batch operation with substrate feeding after 14 h and 22 h, a hydrolysate with a concentration of 185.7 g center dot L-1 was obtained. Initially, to verify the influence of different carbon sources on Cupriavidus necator DSMz 545 in biomass production, batch fermentations were developed, testing laboratory-grade glucose, soybean hull hydrolysate, and waste glycerol (a by-product of biodiesel processing available in large quantities) as carbon sources in one-factor-at-a-time assays, and the mixture of soybean hull hydrolysate and waste glycerol. Then, the hydrolysate and waste glycerol were consumed by C. necator, producing 12.1 g center dot L-1 of biomass and achieving 39% of polyhydroxyalkanoate (PHB) accumulation. To the best of our knowledge, this is the first time that soybean hull hydrolysate has been used as a carbon source to produce polyhydroxyalkanoates, and the results suggest that this agro-industrial by-product is a viable alternative feedstock to produce value-added components.