Integrated Hydrolysis of Mixed Agro-Waste for a Second Generation Biorefinery Using Nepenthes mirabilis Pod Digestive Fluids

To sustainably operate a biorefinery with a low cost input in a commercial setting, the hydrolysis of lignocellulosic biomass must be undertaken in a manner which will impart environmental tolerance while reducing fermenter inhibitors from the delignification process. The challenge lies with the highly recalcitrant lignin structure, which limits the conversion of the holocelluloses to fermentable total reducing sugars (TRS). Due to these challenges, sustainable and innovative methods to pre-treat biomass must be developed for delignocellulolytic operations. Herein, Nepenthes mirabilis digestive fluids shown to have ligninolytic, cellulolytic and xylanolytic activities were used as an enzyme cocktail to hydrolyse mixed agro-waste constituted by Citrus sinensis (orange), Malus domestica (apple) peels, cobs from Zea mays (maize) and Quercus robur (oak) yard waste. The digestive fluids contained carboxylesterases (529.41 ± 30.50 U/L), β-glucosidases (251.94 ± 11.48 U/L) and xylanases (36.09 ± 18.04 U/L), constituting an enzymatic cocktail with significant potential for the reduction in total residual phenolic compounds (TRPCs), while being appropriate for holocellulose hydrolysis. Furthermore, the maximum TRS obtainable was 310 ± 5.19 mg/L within 168 h, while the TRPCs were reduced from 6.25 ± 0.18 to 4.26 ± 0.09 mg/L, which was lower than that observed when conventional methods were used. Overall, N. mirabilis digestive fluids demonstrated an ability to support biocatalytic processes with minimised cellulases hydrolysis interference. Therefore, the digestive enzymes in N. mirabilis pods can be used in an integrated system for feedstock hydrolysis in a second generation biorefinery.