Abstract
Synthetic plastic pollution has exacerbated environmental degradation and the depletion of fossil fuel resources. Transitioning to biodegradable, sustainably sourced alternatives such as polyhydroxybutyrate (PHB) bioplastic can help alleviate the crisis. Currently, PHB production relies heavily on expensive and limited food crops as feedstocks, hindering its upscaling and commercial viability. To overcome these challenges, this dissertation examines the potential of lignocellulosic industrial hemp (Cannabis sativa sp.) waste from the cannabidiol (CBD) industry as a sustainable, low-cost feedstock for PHB production and advances bioprocessing methods for its efficient conversion.
Innovative bioprocessing strategies were developed to upcycle two major waste fractions of the CBD industry, fibrous hemp stalks (HS) and leafy and flowery biomass (LF), for enhanced PHB production through fermentation by recombinant bacteria E.coli LSBJ. The HS waste was subjected to a two-stage, low-severity, chemical-free liquid hot water pretreatment (LHW) coupled with sequential disk refining to obtain a high yield of fermentable sugars. Pretreatment at 200℃ for 10 min achieved a high glucose concentration (56 g/L) and 92% cellulosic conversion. Fermentation of the sugar-rich hydrolysate adjusted to 20 g/L total sugar concentration resulted in a PHB titer of 1.8 g/L and 48% accumulation, underscoring the potential of utilizing LHW-pretreated HS biomass for bioplastic production. To advance understanding of pretreatment severity on PHB production, hydrolysates generated from HS residues pretreated at 180, 190, and 200℃ for 10 min were fermented at the same substrate concentration. Hydrolysates from 180 and 190 °C supported higher PHB titers than those from 200 °C, reflecting the advantages of balanced sugar profiles and reduced inhibitor formation.
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These insights guided the fermentation of admixtures formulated by combining the untreated LF hydrolysate with HS hydrolysate, enabling the valorization of the entire industrial hemp waste. The biomass-representative 190HS–LF (7:3 w/w) admixture yielded a PHB titer of 3.04 g/L, demonstrating the effectiveness of co-fermenting HS with LF hydrolysate. This formulation benefited from higher hydrolysis conversion efficiency at 190 °C and improved substrate balance, with LF contributing additional sugars and intrinsic nitrogen that enhanced overall fermentation performance. Finally, the Box–Behnken design elucidated key interaction effects among inoculum size, substrate concentration, C:N ratio, and fermentation time, strengthening understanding of fermentation behavior and identifying optimal conditions that enhanced PHB yields. The optimal cell dry weight, inclusion levels and titers were identified as 5.86 g/L, 38.46% (w/w) and 2.23 g/L respectively.