Dark fermentation is a biological process that produces hydrogen gas through the anaerobic breakdown of organic substrates by microorganisms. Unlike photobiological methods, it does not rely on light energy, making it a versatile and scalable approach for hydrogen generation. The process leverages the metabolic activity of anaerobic bacteria to convert carbohydrates, organic wastes, and other biodegradable materials into hydrogen, carbon dioxide, and volatile fatty acids.

The biochemical pathways in dark fermentation are primarily driven by two key mechanisms: the pyruvate-ferredoxin oxidoreductase (PFOR) pathway and the NADH reoxidation pathway. In the PFOR pathway, pyruvate, a product of glycolysis, is cleaved by pyruvate-ferredoxin oxidoreductase to produce acetyl-CoA, carbon dioxide, and reduced ferredoxin. The reduced ferredoxin is then utilized by hydrogenase enzymes to generate hydrogen gas. The NADH reoxidation pathway involves the oxidation of excess NADH produced during glycolysis, which, under anaerobic conditions, is coupled with proton reduction to form hydrogen.

Key microorganisms responsible for dark fermentation include strict anaerobes such as Clostridium species and facultative anaerobes like Enterobacter. Clostridium species, including C. butyricum and C. acetobutylicum, are highly efficient hydrogen producers due to their high hydrogenase activity and ability to utilize a wide range of substrates. Enterobacter species, such as E. aerogenes, can also produce hydrogen under both anaerobic and microaerophilic conditions, though their yields are generally lower compared to Clostridium. Other notable bacteria include Thermoanaerobacterium, which thrives at elevated temperatures, and mixed microbial consortia derived from anaerobic sludge or compost, which offer robustness in industrial applications.

Anaerobic conditions are critical for dark fermentation because oxygen inhibits the activity of hydrogenase enzymes, which are essential for hydrogen production. The absence of oxygen ensures that metabolic pathways favor fermentation over aerobic respiration, redirecting electron flow toward proton reduction instead of oxidative phosphorylation. Maintaining strict anaerobiosis is typically achieved through nitrogen or argon sparging, reducing agents like cysteine or sodium sulfide, and sealed bioreactors to prevent oxygen ingress.

Substrate selection significantly impacts hydrogen yield and process efficiency. Carbohydrates, particularly glucose and sucrose, are the most commonly used substrates due to their rapid degradation and high hydrogen potential. However, dark fermentation can also utilize complex organic wastes such as lignocellulosic biomass, food waste, and agricultural residues. Pretreatment methods like acid hydrolysis, enzymatic digestion, or thermal processing are often required to break down complex polymers into fermentable sugars. The choice of substrate influences the metabolic end-products; for example, glucose fermentation typically yields hydrogen alongside acetate, butyrate, or ethanol, depending on the microbial strain and environmental conditions.

The metabolic processes in dark fermentation vary based on the type of microorganisms and the substrate. Two primary fermentation types are observed: acetate-type and butyrate-type fermentation. In acetate-type fermentation, glucose is converted into acetate, carbon dioxide, and hydrogen with a theoretical maximum yield of 4 moles of hydrogen per mole of glucose. Butyrate-type fermentation produces butyrate, carbon dioxide, and hydrogen, yielding 2 moles of hydrogen per mole of glucose. The actual yields are often lower due to metabolic bottlenecks, substrate diversion toward biomass growth, and competition from non-hydrogen-producing pathways like propionate or lactate formation.

Dark fermentation differs from other biological hydrogen production methods in several ways. Unlike photobiological processes, which depend on photosynthetic microorganisms like cyanobacteria or green algae to split water using sunlight, dark fermentation operates independently of light, enabling continuous hydrogen production in opaque bioreactors. Additionally, dark fermentation does not require costly photobioreactors or transparent systems, simplifying scale-up. Compared to microbial electrolysis cells (MECs), which use external voltage to drive hydrogen evolution from organic matter, dark fermentation is self-sustaining and does not rely on electrochemical inputs. However, it generally produces lower hydrogen purity due to the coexistence of carbon dioxide and other gases.

The advantages of dark fermentation include its ability to utilize low-cost organic waste streams, rapid reaction rates, and compatibility with existing anaerobic digestion infrastructure. Challenges include low substrate conversion efficiency, accumulation of inhibitory metabolites like volatile fatty acids, and the need for careful pH control (optimal range 5.0–6.5) to prevent metabolic shifts toward solventogenesis or lactate production. Strategies to improve performance include metabolic engineering of bacterial strains, co-culturing with syntrophic partners, and integrating dark fermentation with secondary processes like anaerobic digestion to maximize energy recovery from residual organics.

In summary, dark fermentation represents a promising route for sustainable hydrogen production by leveraging anaerobic microbial metabolism to convert organic substrates into clean energy. Its independence from light and adaptability to diverse feedstocks make it a practical option for decentralized hydrogen generation. Ongoing research focuses on optimizing microbial consortia, refining pretreatment methods, and developing hybrid systems to enhance yield and economic viability. By addressing current limitations, dark fermentation could play a significant role in the transition to a hydrogen-based energy economy.