Hemp Cryo-milling and the Impact of Alkaline Pretreatment on Biochemical Conversion

Cannabis sativa (hemp) is a fiber crop that has historically been used for manufacturing textiles. Recently, limitations on cultivating industrial hemp in the United States have been lifted, as hemp has been reclassified as an agricultural commodity with the potential to serve as a bioenergy crop. This work used liquid nitrogen (LN2) to cryo-mill hemp (CMH) before NaOH chemical pretreatment. Pretreatments were conducted at 10% (w/v) solids loading with NaOH loadings ranging from 0 to 0.4 g of NaOH/g of dry biomass. Increasing NaOH chemical loading removed up to 80% percent of the original hemicellulose, but delignification leveled off around 20% even at the highest NaOH loadings. Moderate NaOH pretreatment conditions (0.1 to 0.2 g of NaOH/g of dry biomass) provided optimal conditions for sugar recovery following enzymatic hydrolysis with glucan yields approaching 90% and xylan yields at 80%. Pretreated CMH hydrolysate was fermented by Paenibacillus polymyxa to produce 2,3-butanediol (2,3-BDO). Under anoxic conditions, P. polymyxa generated 19 g/L of 2,3-BDO without conversion into acetoin. Aerobic fermentations produced 2,3-BDO titers near 13 g/L after 24 h, however, the resulting 2,3-BDO was converted to acetoin reaching around 9 g/L after 144 h.