Greenhouse gas emissions from plastic pollution and deplorable conditions of plastic waste management have necessitated the conversion of minimally costing lignocellulosic waste to bioplastic. One such prospective feedstock is fibrous stalk waste of industrial hemp (Cannabis sativa sp.) arising from the cannabidiol (CBD) oil industry. Our previous scientific studies demonstrated that chemical-free low-severity hot water pretreatment coupled with sequential disk refining could deconstruct the recalcitrant lignocellulosic matrix of the fibrous stalk, yielding high sugar yields during enzymatic hydrolysis. The hydrolysate was fermented by E.coli LSBJ to produce Poly(3-hydroxybutyrate) (PHB), and the dry cell weight (g/L), PHB titer (g/L) and inclusion (%w of dry cell) were found 3.71,1.77 and 47.73 respectively. The current study focuses on elucidating the effect of optimizing multiple parameters involved in P-3HB fermentation: inoculum size, substrate concentration, C/N ratio, and fermentation time for enhancing PHB titer and inclusion levels. Fibrous hemp stalk waste was subjected to hot water pretreatment at 190℃ for 10 minutes, followed by mechanical disk refining, as per optimum conditions in our previous work. The pretreated biomass was hydrolyzed using commercial cellulase enzymes, and the hydrolysate was fermented to produce PHB. Box–Behnken design was used to determine the optimal conditions, utilizing the levels of the parameters and the outcomes sought, as mentioned above. A verification experiment will then ensure the validation of the optimal criteria. Following the fermentation optimization in the shake flasks, the bioreactor operation strategy will be investigated to achieve high cell density and high productivity of PHB.
