Application of physicochemical and biological pretreatments to enhance volatile fatty acids production via Dark Fermentation from agri-food waste
In the field known as the Green Economy, one of the most important pillars for developing a future, more sustainable production model is the incorporation of sustainable production processes as substitutes for conventional ones. In this regard, Volatile Fatty Acids (VFAs) constitute the so-called carboxylate platform, which plays a central role in biorefinery processes. The benchmark bioprocess for VFA production is Dark Fermentation (DF), a mode of anaerobic digestion in which the methanogenic phase is inhibited. This approach is framed within the biorefinery concept, where residual biomass is processed comprehensively and sustainably to obtain multiple value-added products, thereby promoting a circular economy model and reducing the environmental impact of agro-industrial waste management. The use of residual biomass as raw material contributes to lowering the production costs of these processes. However, it necessitates the application of costly pretreatments to ensure adequate solubilization of the organic matter present in the biomass. Residual biomass is a cheap carbon source with high volume availability and decentralized geographical distribution, but its use requires the optimization of applicable pretreatments. Waste and byproducts from the agro-food sector -which is immensely important both nationally and in the autonomous community of Andalusia- already represent one of the main sources of renewable residual biomass. They can be valorized through bioprocesses to obtain high value-added compounds. This Doctoral Thesis aimed to collect a significant sample of the principal types of agro-food biomass generated in the Andalusian autonomous community, also considering their diverse physicochemical characteristics. The following substrates were selected for this study: - Rice Husks (RH) - Sugar Beet Pulp (SBP) - Brewers' Spent Grain (BSG) - Orange Peel (OP) The goal is to optimize the valorization of agro-food substrates by applying different pretreatment strategies, including physical (ultrasonic sonication and microwave), biological, and chemical (hydrothermal) pretreatments, using only water as a solvent. These pretreatments will allow for improved solubilization of organic compounds in the biomass and the extraction of bioproducts of interest, such as reducing sugars, proteins, and polyphenols, which have high potential for application in the biotechnology industry. Following pretreatment, the substrates will be subjected to Dark Fermentation, an acidogenic anaerobic digestion process aimed at the production of Volatile Fatty Acids (VFAs) and a biogas with a high hydrogen content. Additionally, although not originally planned in the Doctoral Thesis project, the application of Enzyme Hydrolysis (EH) was also tested to enhance the valorization strategy. So, the general objective can be expressed as: "To develop and optimize physicochemical and biological pretreatments applied to agro-food residual biomass to favor the solubilization of organic matter with the purpose of maximizing the production of volatile fatty acids through a dark fermentation process and to evaluate the different alternatives." And the Specific objectives were: 1. Evaluate and determine the most suitable pretreatment (hydrothermal, microwave, ultrasonic sonication, and biological) for solubilizing and hydrolyzing the organic matter present in four agro-food sector substrates (rice husks, sugar beet pulp, brewers' spent grain, and orange peel) in terms of organic matter solubilization. 2. Evaluate the Volatile Fatty Acid production capacity of a dark fermentation process using the pretreated substrates. 3. Select the most favorable overall alternative for the generation of volatile fatty acids. The development of these partial objectives was carried out sequentially and coordinated with the experimental stages of the Doctoral Thesis work plan. As a result of the work conducted, four scientific articles corresponding to the contents of the Doctoral Thesis have been published in JCR® indexed journals. The DOIs for these publications are listed below: - Article 1: https://doi.org/10.1016/j.jenvman.2023.118332 - Article 2: https://doi.org/10.1021/acs.energyfuels.5c03953 - Article 3: https://doi.org/10.3390/app15158736 - Article 4: https://doi.org/10.1016/j.biombioe.2025.108634 The investigation began with the (Article 1) of a passively aerated biological pretreatment applied to all four residues to evaluate its capability for initial organic matter solubilization. This low-energy approach proved highly effective for biomasses with specific fiber profiles, with OP achieving the highest solubilization yield (sCOD 58.6%) under optimized conditions. These findings confirmed the feasibility of a biological pre-hydrolysis stage to enrich substrate quality and reduce the severity of subsequent processes. The second research axis involved a direct comparison between Microwave-assisted (MW) and Hydrothermal Reactor (HTR) pretreatments (Article 2) to quantify the recovery of Total Reducing Sugars (TRS), proteins (PR), and VFAs. MW pretreatment emerged as the most energy-efficient technique for rapidly solubilizing accessible biomasses such as OP and SBP. Conversely, HTR generated significantly higher extraction yields of TRS and VFAs for more complex substrates (RH, BSG), underscoring that pretreatment effectiveness depends not only on energy input but also on target compounds and biomass structural characteristics. The final phase investigated the integration of Ultrasound (US) and Hydrothermal Pretreatment (HTP) to optimize the sequential production of biohydrogen (H2) and (VFA) within a biorefinery framework, as detailed in Article 3 and Article 4. pretreatment effectively enhanced the solubilization of organic matter, serving as a crucial first-stage intensification method prior to (DF). Specifically, the integrated strategy combining (US) pretreatment, dark fermentation, and enzyme hydrolysis was developed to maximize the output of (H2), (VFA), and Total Reducing Sugars (TRS) from the four feedstocks. Optimized HTP conditions were critically assessed to determine the optimal reaction time necessary to maximize (H2)production from the lignocellulosic feedstocks. This research conclusively demonstrated that combining these tailored physicochemical strategies maximizes the overall biohydrogen and VFA yields, underscoring the necessity of multi-step pretreatment designs for achieving high-efficiency bioconversion. The main conclusions of the work have been: *Effective pretreatment strategies for the valorization of agro-industrial biomasses depend on a careful balance between structural disruption of the biomass, precursor release, and process energy cost. *Pretreatment optimization based on substrate: Method selection must be based on the intrinsic complexity of the biomass. Microwave is preferable for simple substrates (OP, SBP) due to its speed and lower energy consumption (40.1 kJ/g). Hydrothermal pretreatment is necessary to maximize the solubilization of recalcitrant biomasses (BSG, RH) and achieve high VFA yields, although it requires higher energy (70.85 kJ/g). *Decoupling organic matter solubilization and biohydrogen production pretreatment must avoid excessive release of proteins, as this diverts dark fermentation metabolism and limits H2 production. Moderately pretreated SBP (HTP for 20 min) is superior to OP for biohydrogen production. *US pretreatment as the best option for sequential processes: Integration of ultrasound with enzyme hydrolysis of the solid phase of the pretreated effluent is an effective biorefinery strategy. Ultrasound pretreatment reduces recalcitrance (although it uses green solvents with less delignification capacity), allowing subsequent enzyme hydrolysis to significantly increase TRS yield, especially for SBP (4.5-fold increase). *Lignin as the main problem: RH was shown to be consistently resistant to all pretreatments, underlining that its high lignin content (14.0%) and low enzymatic accessibility limit its use as an efficient substrate for dark fermentation without more severe chemical treatments. *The integration of pretreatment, dark fermentation, and enzyme hydrolysis is demonstrated to be an effective strategy for the sustainable conversion of agro-industrial residues into biofuels and value-added biochemicals.
Pretreatment, Agri-food waste, Biomasses, Biological pretreatment, Sugar beet pulp, Brewery bagasse, Rice husk, Orange peel, Solubilization yield, Microwave, Hydrothermal, Bioproduct, Reducing sugar, Total Protein, Volatile fatty acids, Lignocellulosic biomass, Brewers spent grain, hydrothermal pretreatment, hydrogen, total polyphenols, total reducing sugars, biorefinery, Ultrasound pretreatment, Dark fermentation, Enzyme hydrolysis, BSG, RH, TRS, PR, PT, H2, CO2, VFA
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