Photo-fermentation is an efficient hydrogen production pathway in which purple non-sulfur bacteria (PNSB) play an active role and produce hydrogen as a part of their metabolism under optimal conditions. These bacteria work under the influence of light to advance their metabolism and use various substrates, such as simple sugars and volatile fatty acids, to produce hydrogen. This article presents a comparative review of several bacterial strains that have been efficiently used to produce hydrogen by photo-fermentation under different optimized conditions, including the substrate, its concentration, type and capacity of the bioreactor, light sources and intensities, and process conditions to achieve the maximum biohydrogen production rate. The analysis showed that the Rhodopseudomonas palustris is the main bacterium used for hydrogen production, with a maximum hydrogen production rate of 3.2 mM/h using 27.8 mM of glucose in a 165 mL serum bottle and 3.23 mM/h using 50 mM of glycerol at pH 7, followed by Rhodobacter sphaeroides, which gave a hydrogen production rate as high as 8.7 mM/h, using 40 mM of lactic acid, pH 7, and 30 °C temperature in a single-walled glass bioreactor. However, it is not preferred over R. palustris due to its versatile metabolism and ability to use an alternative mode if the conditions are not carefully adjusted, which can be a problem in hydrogen production.

Dark fermentation is a promising method for hydrogen (H₂) production utilizing the metabolic pathways of diverse microbial communities. This process can be carried out without the need for light, making it easier and more efficient to operate in different environments and at a lower cost. It also utilizes a wide range of substrates, making it highly adaptable to waste-to-energy applications. Clostridium spp. are particularly favored in this method due to their versatile metabolism, ability to utilize a wide range of substrates, and high H₂ yields. Anaerobes and facultative anaerobes are mostly used in studies due to their efficient hydrogenase enzyme activity and metabolic pathways. A pH range of 5.5–6.5 and a temperature of 30–37 °C for mesophiles and 55–60 °C for thermophiles are usually preferred in addition to the other parameters such as hydraulic retention time and substrate used. The highest H₂ yield of 9.39 mol H₂/mol sucrose consumed was obtained by C. beijerinckii using sucrose as a substrate under batch mode conditions at 37 °C and pH 6–7. The review analyzes different bacterial species and examines the influence of optimized parameters required on H₂ yield in different bioreactor operating modes.

The integration of coupled dark fermentation (DF) and photofermentation (PF) systems represents a promising approach for concurrent H₂ production and organic load reduction in carbohydrate–rich wastewater. However, their behavior under non–optimized, continuous industrial operation remains inadequately characterized, hindering practical implementation. This case study evaluated an integrated DF–PF system treating a synthetic sugar mixture mimicking acidic fruit and dairy processing wastewater. The system presented herein (5-L reactors) serves as an initial prototype to facilitate scaling to both pilot (40-L reactors) and, ultimately, industrial (600-L) scales using real effluents within the framework of a research project. The bioreactors were operated continuously with a 10 h hydraulic retention time and an organic loading rate of 2.5 g COD L⁻¹ d⁻¹. The pH and temperature were monitored but intentionally left uncontrolled. The DF stage facilitated by hydrogen–producing bacteria achieved H₂ concentrations of up to 57% (v/v) and a maximum production rate of 177 mL H₂ L⁻¹ d⁻¹; however, it demonstrated notable process instability due to the absence of controls. In contrast, the PF stage exhibited negligible H₂ production (1.8% v/v) attributable to the displacement of Rhodopseudomonas species by microbial competition, thereby functioning predominantly as a polishing step. Overall, the coupled system achieved an average COD removal of 34%, highlighting the functional differentiation between stages and identifying microbial competition as the primary constraint under suboptimal conditions.