The selection of optimal substrates for dark fermentation is a critical factor in maximizing hydrogen yield and process efficiency. Substrates vary widely in composition, availability, and biodegradability, influencing their suitability for dark fermentation. Key criteria for substrate selection include carbohydrate content, biodegradability, nutrient balance, and the presence of inhibitory compounds. Lignocellulosic biomass, food waste, and industrial effluents are among the most studied substrates due to their abundance and potential for sustainable hydrogen production.

Lignocellulosic biomass, such as agricultural residues (rice straw, wheat straw), forestry waste, and energy crops, is rich in cellulose and hemicellulose, which are prime sources of fermentable sugars. However, its complex structure, comprising lignin, cellulose, and hemicellulose, necessitates pretreatment to break down the recalcitrant lignin matrix and expose the polysaccharides for microbial action. Food waste, on the other hand, contains high levels of easily degradable carbohydrates, proteins, and lipids, making it a highly efficient substrate. Industrial effluents, such as those from food processing or dairy industries, often contain organic compounds that can be directly utilized by hydrogen-producing bacteria, though they may require dilution or nutrient supplementation to optimize conditions.

Pretreatment methods are essential to enhance substrate digestibility and improve hydrogen yield. Mechanical pretreatment, such as milling or grinding, reduces particle size, increasing surface area for microbial attack. This method is energy-intensive but avoids the formation of inhibitory byproducts. Chemical pretreatment involves the use of acids, alkalis, or oxidizing agents to break down lignin and hemicellulose. Acid pretreatment, typically using sulfuric or hydrochloric acid, hydrolyzes hemicellulose into monomeric sugars but may generate furfural and hydroxymethylfurfural (HMF), which can inhibit fermentation. Alkaline pretreatment, using sodium hydroxide or ammonia, effectively removes lignin but may require neutralization before fermentation. Enzymatic pretreatment employs cellulases, hemicellulases, and ligninases to degrade the biomass selectively, offering high specificity and mild conditions but at a higher cost due to enzyme production.

Biological pretreatment, such as fungal treatment using white-rot fungi, can degrade lignin through enzymatic action, though it is slower compared to chemical methods. Combined pretreatment strategies, such as thermo-chemical or mechano-enzymatic approaches, often yield better results by synergistically addressing the limitations of individual methods. For instance, steam explosion followed by enzymatic hydrolysis has been shown to significantly improve sugar release from lignocellulosic biomass.

Despite the advantages of pretreatment, challenges arise from the formation of inhibitory compounds that can impede microbial activity and reduce hydrogen production. Furans (furfural and HMF), phenolic compounds, and weak acids (acetic, formic, and levulinic acids) are common inhibitors generated during pretreatment. Furans and phenolics disrupt microbial cell membranes and enzyme functions, while weak acids uncouple proton gradients, reducing energy available for microbial growth. Mitigation strategies include detoxification methods such as overliming (calcium hydroxide treatment), activated charcoal adsorption, and biological detoxification using specific microorganisms capable of degrading inhibitors. Dilution of the substrate or adaptive evolution of microbial consortia to tolerate higher inhibitor concentrations are also effective approaches.

The choice of microbial inoculum is another critical factor in dark fermentation. Mixed cultures, derived from anaerobic sludge or compost, are commonly used due to their robustness and ability to utilize diverse substrates. However, mixed cultures may include hydrogen-consuming bacteria, such as methanogens, which compete for substrates and reduce hydrogen yield. Pretreatment of the inoculum, such as heat shock or acid treatment, can selectively suppress methanogens and enrich for hydrogen-producing bacteria like Clostridium and Enterobacter. Pure cultures offer higher specificity but are less resilient to environmental fluctuations and substrate variability.

Process parameters, including pH, temperature, and hydraulic retention time (HRT), must be optimized to maximize hydrogen production. A pH range of 5.0–6.0 is generally optimal for hydrogen-producing bacteria, as lower pH favors solventogenesis, redirecting metabolic pathways away from hydrogen production. Mesophilic temperatures (30–40°C) are commonly used, though thermophilic conditions (50–60°C) can enhance hydrolysis rates and hydrogen yields. Short HRTs (less than 10 hours) are preferred to wash out slow-growing methanogens and maintain a dominant population of hydrogen producers.

Nutrient supplementation may be necessary to balance the carbon-to-nitrogen (C/N) ratio and provide essential trace elements. A C/N ratio of 20–30 is typically optimal, as excessive nitrogen can lead to ammonia inhibition, while insufficient nitrogen limits microbial growth. Iron, nickel, and magnesium are critical cofactors for hydrogenase enzymes, and their addition can enhance hydrogen production rates.

The scalability of dark fermentation systems depends on the integration of pretreatment, fermentation, and downstream processes. Continuous stirred-tank reactors (CSTRs) and upflow anaerobic sludge blanket (UASB) reactors are widely used for their operational simplicity and efficiency. However, biofilm-based reactors, such as anaerobic packed-bed or fluidized-bed reactors, offer higher biomass retention and improved resistance to inhibitory compounds.

Economic feasibility remains a challenge due to the costs associated with pretreatment, inoculum management, and hydrogen purification. Advances in enzyme production, process integration, and waste valorization (e.g., using fermentation residues for biogas or fertilizer) can improve the overall sustainability and cost-effectiveness of dark fermentation. Policy support and incentives for renewable hydrogen production can further drive adoption and innovation in this field.

In summary, the selection of substrates for dark fermentation requires careful consideration of their composition and pretreatment needs. Lignocellulosic biomass, food waste, and industrial effluents each present unique advantages and challenges, necessitating tailored approaches to maximize hydrogen yield. Pretreatment methods must balance efficiency with the mitigation of inhibitory compounds, while process optimization and microbial management are essential for stable and scalable operation. Addressing these factors can unlock the potential of dark fermentation as a sustainable pathway for hydrogen production.