Effect of Hydrothermal Pretreatment Time on Biochemical Recovery and Hydrogen Production from Lignocellulosic Feedstocks  
[PERSON]  
1 Department of Chemical Engineering and Food Technology, Campus Puerto Real, University of Cadiz, 11510 Puerto Real, Cadiz, Spain; [EMAIL_ADDRESS] (A.R. ); [EMAIL_ADDRESS] (C.J.A.-C.)  
aquasolv, aqueous prehydrolysis, aqueous liquefaction, auto-hydrolysis, autocatalyzed hydrolysis, and water pressure cooking [5,6]. Autoclave-based pretreatment is a reliable and efficient method to overcome the recalcitrance of lignocellulosic biomass by using moist heat under gauge pressure. It operates at temperatures from 120 \({}^{\circ}\)C to 180 \({}^{\circ}\)C and pressures ranging from 0.2 to 1.0 MPa, with residence times from 1 to 5 h. This process breaks lignin-cellulose bonds, making the biomass more accessible for enzymatic or chemical hydrolysis. The even heat distribution achieved through moist air in a sealed chamber enhances delignification and solubilization, optimizing the efficiency of the pretreatment [7,8]. The autoclave method induces significant structural changes in the biomass by degrading hemicellulose and cellulose partially while solubilizing lignin. These modifications improve the accessibility of carbohydrates and release soluble sugars and organic compounds. The sequential degradation of hemicellulose and cellulose increases the yield of soluble dietary fibers, as observed in Brewer spent grain (BSG), a substrate rich in proteins, fatty acids, and phenolic acids, with considerable nutritional benefits [9]. Compared to other pretreatment methods, autoclaving offers distinct advantages. Water bath heating, though simple and cost-effective, lacks the pressure necessary for effective lignin breakdown. Steam explosion, while capable of processing lignocellulosic, may lead to sugar loss and the formation of inhibitory by-products. Autoclaving minimizes such drawbacks through controlled and milder conditions, which enhances process efficiency and reduces the formation of undesirable compounds [10,11]. This method has been applied successfully to a wide range of biomass types, including brewers spent grain (BSG) and soybean curd residue, to enhance phenolic compound content, improve protein functionality, and increase soluble dietary fiber. Its controlled environment also lowers energy requirements compared to more intensive methods, making it a cost-effective and sustainable option for biomass valorization [8,12]. Numerous studies have demonstrated the efficacy of subsequent applications of dark fermentation (DF) following substrate pretreatment [13,14]. The process of DF converts carbohydrates into hydrogen, carbon dioxide, and various organic acids (Figure 1). However, the process also entails the utilization of anaerobic microorganisms, operating within a temperature range that includes mesophilic (25-40 \({}^{\circ}\)C) conditions and thermophilic (50-65 \({}^{\circ}\)C) conditions, with certain processes occurring at extreme thermophilic temperatures that exceed 80 \({}^{\circ}\)C. Thus, the anaerobic fermentation is shared in three steps, where the anaerobic process stops at the second stage of the acidogenesis, and the methanogenesis is suppressed to not convert volatile fatty acids (VFA) and H\({}_{2}\) to CH\({}_{4}\)[15,16]. DF can utilize pure bacterial or mixed microbial cultures [17]. Nevertheless, mixed cultures are frequently advantageous due to their lower economic cost and adaptability to a variety of lignocellulosic biomasses [18,19]. However, during the process of DF, the initial conversion of monomeric sugars into organic acids is followed by the acetogenic bacteria utilization of these organic acids to produce hydrogen and carbon dioxide [20]. Figure 1: Scheme for simplified biochemical pathways of dark fermentation process. The main parameters that influence the efficacy of DF include the substrate type and pretreatment method, the type of inoculum source and enrichment strategy, and the configuration of the bioreactor.