Purple non-sulfur bacteria (PNSB), such as *Rhodobacter sphaeroides*, are a group of photosynthetic microorganisms capable of producing hydrogen through photofermentation. Unlike cyanobacteria, which perform oxygenic photosynthesis, PNSB utilize anoxygenic photosynthesis, making them suitable for hydrogen production under anaerobic conditions. This process is driven by light energy and the breakdown of organic acids, offering a sustainable pathway for biohydrogen generation while simultaneously treating organic waste.

The photofermentative hydrogen production by PNSB relies on nitrogenase, an enzyme complex that catalyzes the reduction of atmospheric nitrogen to ammonia. Under nitrogen-deficient conditions, nitrogenase diverts electrons toward proton reduction, leading to hydrogen evolution. Nitrogenase activity is highly sensitive to oxygen, necessitating strict anaerobic conditions. The enzyme requires ATP and reduced ferredoxin, both supplied by light-dependent reactions. The overall stoichiometry of hydrogen production from organic acids like acetic acid can be summarized as follows:
CH3COOH + 2H2O → 4H2 + 2CO2.

Light intensity plays a critical role in regulating hydrogen production. PNSB operate optimally under moderate light intensities, typically between 100 and 200 W/m². Excessive light can cause photoinhibition, damaging the photosynthetic apparatus, while insufficient light limits ATP synthesis, reducing nitrogenase activity. The wavelength of light also influences efficiency, with near-infrared light (700–900 nm) being most effective due to the absorption spectrum of bacteriochlorophylls.

The choice of organic substrate significantly impacts hydrogen yield. Short-chain organic acids, such as acetate, butyrate, and lactate, are preferred due to their efficient assimilation and high electron content. For instance, acetate can yield up to 4 moles of H2 per mole of substrate, while more reduced substrates like butyrate may yield lower amounts due to thermodynamic constraints. Complex organic wastes, such as those from agricultural or food processing effluents, can also serve as feedstocks, though pretreatment may be required to hydrolyze polymers into fermentable monomers.

Integrating PNSB-based hydrogen production with wastewater treatment offers a dual benefit: energy recovery and pollution reduction. Industrial and domestic wastewaters often contain high levels of organic acids, which PNSB can metabolize while producing hydrogen. This approach reduces the chemical oxygen demand (COD) of wastewater, easing the burden on conventional treatment systems. Pilot-scale studies have demonstrated COD removal efficiencies exceeding 80% alongside hydrogen production rates of 1–2 L/L-culture/day, depending on substrate concentration and operational conditions.

The performance of PNSB systems is influenced by several operational parameters. Temperature must be maintained within a mesophilic range (30–35°C) to sustain microbial activity. pH control is equally important, with neutral to slightly acidic conditions (pH 6–7) being optimal for nitrogenase function. C/N ratio adjustments are necessary to suppress ammonia assimilation, which otherwise inhibits nitrogenase-mediated hydrogen production. Trace metal availability, particularly iron and molybdenum, is critical for nitrogenase cofactor synthesis.

Despite its advantages, photofermentative hydrogen production faces challenges. The process is inherently slower than electrolysis or steam reforming, with typical rates ranging from 10–50 mL H2/g substrate/hour. Light penetration in dense cultures can be limiting, necessitating innovative reactor designs such as flat-panel or tubular photobioreactors with high surface-to-volume ratios. Gas separation and purification are also required to isolate hydrogen from CO2 and trace contaminants.

Research efforts are focused on improving strain performance through genetic engineering. Enhancing nitrogenase expression, reducing pigment content to improve light distribution, and increasing substrate uptake rates are key targets. Immobilization of cells on porous matrices has shown promise in stabilizing long-term production and simplifying biomass recovery. Coupling PNSB systems with dark fermentation stages, where organic wastes are first converted to volatile fatty acids, can further optimize overall hydrogen yields.

The scalability of PNSB-based hydrogen production depends on economic and logistical factors. While the technology is not yet competitive with fossil fuel-derived hydrogen, its ability to valorize waste streams and operate under mild conditions makes it attractive for decentralized applications. Future advancements in reactor design, strain optimization, and process integration could position photofermentation as a viable component of the renewable hydrogen economy.

In summary, purple non-sulfur bacteria offer a unique biological route for hydrogen production, leveraging sunlight and organic waste to drive nitrogenase-catalyzed proton reduction. The process is inherently sustainable, aligning with circular economy principles by converting pollutants into energy. Continued research and development are essential to overcome existing limitations and unlock the full potential of this microbial technology.