Biohydrogen Synthesis from Food Waste: Process Development, Optimization and Intensification

Abstract

This thesis investigates the production of biohydrogen (bioH2) from food waste through an integrated approach combining process optimization, ultrasound-assisted intensification, molecular simulations, and metabolic flux analysis. The research aims to enhance both the hydrolysis of food waste and the subsequent dark fermentation process to achieve higher bioH2 yields and improved process efficiency. Food waste hydrolysis was first optimized using Box–Behnken design, achieving a total reducing sugar (TRS) yield of 263.4 mg/g biomass in 42 hours. Ultrasonic pretreatment (35 kHz, 20% duty cycle) significantly intensified hydrolysis, reducing the required time fourfold and increasing TRS yield by 22% (320 mg/g biomass). Fourier-transform infrared spectroscopy and molecular dynamics simulations revealed that sonication altered the glucoamylase enzyme’s secondary structure by decreasing α-helix and increasing random coil content. These structural changes widened the enzyme’s binding pocket, improving substrate transport and accelerating hydrolysis kinetics.Dark fermentation of the hydrolysate using Clostridium pasteurianum was optimized using response surface methodology (RSM-CCD) and an artificial neural network coupled with a genetic algorithm (ANN-GA). RSM-CCD predicted a bioH2 yield of 1039 mL/L, while ANN-GA identified improved conditions that generated 1108 mL/L (1.73 mol/mol hexose). The modified Gompertz model showed a higher maximum production rate (185.34 mL/L·h) under ANN-GA conditions. Metabolite analysis indicated a metabolic shift toward the acetic acid pathway, reflected by an increase in the acetic-to-butyric acid ratio from 0.9 to 0.94, thereby enhancing hydrogen production.Further intensification through ultrasound-assisted fermentation was evaluated using a metabolic flux analysis model. Sonication increased hexose uptake by ~47% and substantially boosted acetate pathway fluxes, leading to a ~22% improvement in bioH2 yield and a ~37% rise in the acetate-to-butyate ratio. Hypothetical flux simulations demonstrated the potential for further enhancement of hydrogen productivity through complete metabolic redirection or increased sugar uptake. To summarize, the net bioH2 yield from 1 kg of food waste under statistically optimized conditions was 5.1 g per kg of food waste, which further improved to 5.73 g per kg of food waste under sonication. In essence, this thesis has addressed three Sustainability Development Goals of the United Nations, viz., SDG 7 (Affordable and clean energy), SDG 12 (Responsible consumption and production patterns or circular economy) and SDG 13 (Climate action).