Biological Hydrogen Production from Organic Wastes Fermentation Effluent: Testing Mutant Strains of Rhodopseudomonas palustris

The integration of dark and photo-fermentative processes represents a promising strategy for sustainable hydrogen (H₂) production from organic wastes. However, high ammonium concentrations in dark fermentation (DF) effluents inhibit nitrogenase activity, limiting photo-fermentative yields. This study evaluated the photofermentative hydrogen production potential of Rhodopseudomonas palustris mutant strains compared with the wild-type using DF effluent from sewage and food waste co-fermentation. Four strains were tested: wild-type CGA009, CGA4001 (ΔhupS) with the structural gene of the uptake hydrogenase deleted, CGA4003 (ΔhupS, NifA*), with the transcriptional activator of nif genes constitutively active, CGA4017 (ΔhupS, NifA*, ΔcbbM, ΔcbbLS, ΔcbbP::kmR), lacking RuBisCO and phosphoribulokinase enzymes. Batch experiments were conducted under continuous illumination (150 μmol m⁻² s⁻¹) for 52 days. No H₂ evolution was detected in the wild-type strain, while all mutants exhibited significant production. The CGA4017 strain achieved the highest yield (284 ± 12 NmL H₂), followed by CGA4003 (105 ± 3 NmL) and CGA4001 (27 ± 1 NmL). These results confirm that targeted genetic modifications can overcome ammonium inhibition and substantially improve biohydrogen generation from real waste-derived substrates, supporting the development of integrated, waste-to-energy biotechnologies.