The study introduced a proton-pumping channel (Proteorhodopsin PR or Gloeobacter rhodopsin GR) into Rhodobacter sphaeroides HY01 to enhance biomass and hydrogen yield efficiency during periods of resource scarcity. The mutant strains (HY01-PR and HY01-GR) showed a significant increase in cumulative hydrogen yield compared to the original strain when fed with glucose. Notably, HY01-GR displayed tolerance to high acetate concentrations. The proton-pump introduction resulted in reduced carbon flow into the TCA cycle and accumulation of various metabolites. Overexpression of the proton pumps also led to enhanced ATP generation by coupling with ATP synthase. The study highlights the potential of proton pumps for regulating growth metabolism and improving the production of valuable compounds in photosynthetic organisms. Research on biological hydrogen production methods, particularly utilizing photosynthetic bacteria, demonstrates the efficiency and adaptability of these processes. Various approaches, such as blocking competitive pathways and enhancing ATP production, contribute to increased hydrogen yields. Proton-pumping channels like PR and GR offer a promising avenue for sustainable hydrogen production, with implications for optimizing metabolic processes and achieving higher energy conversion efficiencies.