Comparative optimization of apple pomace pretreatment conditions to maximize sugar generation for dark fermentative biohydrogen production

This study compared autoclave-assisted extraction (AAE), heat-assisted extraction (HAE), and ultrasound-assisted extraction (UAE) techniques for producing fermentable sugars. The Box–Behnken response surface experimental design was employed to optimize the independent variables of AAE (apple pomace particle size-Dp, pH, hydrolysis temperature-T, hydrolysis time-t), HAE (Dp, pH, T), and UAE (Dp, ultrasonication power-P, t) in order to maximize the dependent variables of total sugars (TS) yield and supernatant recovery (SR). The optimum conditions for AAE, HAE, and UAE resulted in TS yields of 0.39 g/g, 0.54 g/g, and 0.53 g/g of dry apple pomace, respectively. Micronutrients essential for the fermentation process, such as boron, manganese, iron, copper, zinc, and molybdenum, were analyzed from extracts produced under optimal conditions for each hydrolysis technique. Potential microbial inhibitors such as carboxylic acids, furfural, ketones, and phenols in the hydrolysate were also analyzed. The HAE optimum conditions (Dp=170.8 μm, pH=3.73, T=99.1 °C), showed significantly higher yields of TS and essential elements, and lower concentrations of potential microbial inhibitors. As a result, it was selected as the best hydrolysis technique and used to produce apple pomace hydrolysate (APH) for the fermentation. Batch fermentation was then conducted with APH and glucose (control) substrates employing Clostridium tyrobutyricum. Biohydrogen, lactic, acetic, propionic, and butyric acids production were monitored daily. The reassimilation of acetic acid for enhanced butyric acid production was observed. The volumetric hydrogen production rates (VHPR) were 433.92 mL/L·day for glucose and 744.51 mL/L·day for APH. The findings highlight apple pomace waste's potential as a feedstock for biohydrogen and organic acid production, supporting waste valorization, carbon neutrality, and the circular economy.

本研究对比了高压辅助萃取（autoclave-assisted extraction, AAE）、热辅助萃取（heat-assisted extraction, HAE）与超声辅助萃取（ultrasound-assisted extraction, UAE）三种技术在可发酵糖制备中的应用效果。本研究采用Box-Behnken响应面实验设计，对三种萃取技术的自变量进行优化，以最大化总糖（total sugars, TS）得率与上清液回收率（supernatant recovery, SR）这两个因变量：其中高压辅助萃取的优化变量为苹果果渣粒径（apple pomace particle size, Dp）、pH值、水解温度（hydrolysis temperature, T）与水解时间（hydrolysis time, t）；热辅助萃取的优化变量为Dp、pH值与T；超声辅助萃取的优化变量为Dp、超声功率（ultrasonication power, P）与t。
三种技术的最优条件下，总糖得率分别达到干苹果果渣的0.39 g/g、0.54 g/g与0.53 g/g。针对各水解技术最优条件下制备的萃取液，本研究分析了发酵过程必需的微量营养素，包括硼、锰、铁、铜、锌及钼；同时还检测了水解液中潜在的微生物抑制剂，如羧酸、糠醛、酮类与酚类物质。
热辅助萃取的最优条件为Dp=170.8 μm、pH=3.73、T=99.1 ℃，在此条件下总糖得率与必需元素含量均显著更高，且潜在微生物抑制剂的浓度更低。因此，热辅助萃取被选为最优水解技术，用于制备发酵用苹果果渣水解液（apple pomace hydrolysate, APH）。
随后以苹果果渣水解液与葡萄糖（对照组）为底物，采用酪丁酸梭菌（Clostridium tyrobutyricum）进行批次发酵，每日监测生物氢、乳酸、乙酸、丙酸及丁酸的生成量。研究观察到乙酸的再同化作用可促进丁酸的生成。葡萄糖组与苹果果渣水解液组的体积产氢速率（volumetric hydrogen production rates, VHPR）分别为433.92 mL/(L·d)与744.51 mL/(L·d)。
本研究结果证实，苹果果渣废弃物可作为生物氢与有机酸制备的原料，为废弃物资源化利用、碳中和及循环经济发展提供支撑。