Microbial consortia and pure cultures represent two distinct approaches to dark fermentation for hydrogen production, each with unique advantages and limitations. The choice between these systems depends on factors such as substrate type, process stability, and desired hydrogen yield. Understanding their differences is critical for optimizing biohydrogen production.

Microbial consortia consist of diverse microbial populations that work synergistically to degrade complex substrates. One major advantage is their robustness in handling variable feedstock compositions. Consortia can process a wide range of organic materials, including lignocellulosic biomass, food waste, and industrial effluents, due to the presence of hydrolytic, acidogenic, and hydrogen-producing bacteria. This substrate versatility reduces pretreatment requirements and enhances process flexibility. Additionally, microbial consortia exhibit greater resilience to environmental fluctuations, such as pH shifts or inhibitory compounds, as the metabolic diversity within the community buffers against system disturbances.

However, consortia face challenges related to microbial competition. Non-hydrogen-producing bacteria, such as methanogens or homoacetogens, can consume hydrogen or intermediates, lowering overall yields. Maintaining selective pressure—through pH control, heat treatment, or operation at short hydraulic retention times—is necessary to suppress these competitors. Another limitation is the difficulty in predicting and controlling metabolic fluxes due to the complex interactions within the consortium.

In contrast, pure cultures involve a single microbial strain with well-defined metabolic pathways, offering precise control over hydrogen production. High-yield strains, such as Clostridium butyricum and Enterobacter aerogenes, are frequently used due to their efficient hydrogen generation. These organisms typically follow the butyrate or acetate fermentation pathways, with theoretical yields of 4 and 2 moles of H2 per mole of glucose, respectively. Pure cultures minimize competition for resources, ensuring that substrate carbon is directed toward hydrogen rather than other byproducts. Process optimization is also more straightforward, as parameters like nutrient supply and growth conditions can be tailored to the specific strain.

The primary drawback of pure cultures is their narrow substrate range. Most high-yield strains require simple sugars or pretreated substrates, increasing process costs. They are also more sensitive to environmental stressors, such as oxygen exposure or pH changes, necessitating strict operational control. Contamination risks are higher in pure culture systems, as invasive microbes can outcompete the desired strain.

Several high-yield strains have been studied for their metabolic efficiency. Clostridium acetobutylicum produces hydrogen via the butyrate pathway, achieving yields of up to 2.5 moles H2 per mole of glucose under optimal conditions. Its ability to form spores enhances survivability in adverse conditions. Enterobacter cloacae employs the formate pathway, converting formate into hydrogen and carbon dioxide, with reported yields of 1.8–2.2 moles H2 per mole of glucose. Thermoanaerobacterium thermosaccharolyticum, a thermophilic bacterium, exhibits high hydrogen productivity at elevated temperatures (50–60°C), reducing contamination risks while achieving yields of 2.6 moles H2 per mole of glucose.

Microbial consortia derived from natural environments, such as anaerobic sludge or compost, demonstrate varied hydrogen production capabilities. Heat-treated sludge often enriches for Clostridium species, yielding 1.5–2.2 moles H2 per mole of glucose. Mixed cultures from soil or manure can utilize complex substrates like cellulose, though yields are generally lower (0.8–1.5 moles H2 per mole of glucose equivalent) due to energy losses in hydrolysis and competing pathways.

The choice between consortia and pure cultures depends on the application. For waste-to-hydrogen systems, consortia are preferable due to their ability to handle heterogeneous feedstocks. In contrast, pure cultures are better suited for high-purity hydrogen production from refined substrates. Hybrid approaches, where consortia are pretreated to eliminate hydrogen consumers or where defined co-cultures are used, offer a middle ground by combining substrate flexibility with improved yield control.

In summary, microbial consortia provide robustness and substrate versatility but require careful management to prevent yield losses. Pure cultures offer higher predictability and yields but are limited by substrate specificity and operational sensitivity. Advances in process design, such as dynamic pH control or continuous systems, can mitigate some limitations in both approaches. The selection of strains or consortia should align with the target feedstock and production goals to maximize hydrogen output efficiently.