Effects of fbcFBC overexpression on ATP content and photo-fermentative hydrogen production performance of Rhodobacter sphaeroides HY01

Photosynthetic fermentation for hydrogen production can convert some organic compounds into hydrogen, thus realizing the dual benefits of waste utilization and energy production. However, the low H₂ production rate is currently a major constraint restricting its further development. ATP synthesis during photophosphorylation could be a key factor on hydrogen production. In this work, the vector pRK415 and three endogenous promoters P_pufQ, P_pucB and P_phaC were used to optimize the expression intensity of the fbcFBC gene, which encodes cytochrome bc₁, a key protein in the photosynthetic electron transport chain. The effect of fbcFBC expression level on the hydrogen production performance of R. sphaeroides HY01 and its hydrogenase gene deletion mutant strain WH04 was studied. The hydrogen yields of pRKfbc/HY01 and pRKfbc/WH04 increased by 15.5% and 5.1% compared with the control. Substitutions of the fbcFBC promoter with promoters P_pufQ, P_pucB and P_phaC, the transcription level of fbcFBC and ATP content were significantly improved. In particular, the transcription level of fbcFBC and ATP content in WHpuc were respectively increased by 734% and 57.0% compared with that in WH04. The results demonstrate that Cyt bc₁ is a limiting factor affecting ATP synthesis in the process of photo-fermentative hydrogen production. The mutants overexpressing fbcFBC are more efficient in hydrogen production than the naturally isolated strains under the medium light intensity of 5000 lux.