Abstract The anaerobic digestion of protein-based effluents generally has the risk of ammonia inhibition. While the use of dark-followed by photo-fermentation process, at acidic pH, could be useful for addressing this problem, the activity of photosynthetic bacteria is deteriorated at the low pH values. Hence, in this study, intermittent dark-photo circular baffled reactor (IDP-CBR) was introduced to maintain the pH level (5.5–6.5), where biohydrogen is expected to be efficiently produced from gelatin-based substrate. We designed a four-compartments (i.e., C1 to C4) lab-scale IDP-CBR where C1 and C3 are dark-treated, and C2 and C4 are light-treated. The results revealed that peak hydrogen yield (HY) was achieved at initial gelatin of 2.0 gCOD/L, 24 h-HRT, and initial pH 6.5. The longer HRT provided better substrate conversion efficiency, and the use of higher pH (i.e., 6.5) promoted the photo-fermentation compartments (C2 and C4); further, this relatively acidic pH reduced the availability of free-ammonia. The 16S rRNA gene analysis showed that Clostridiaceae_1 and Rhodospirillaceae were the adapted bacteria that could produce dark- and light-dependent hydrogen, respectively. The comparison between IDP-CBR and the sequential configuration highlighted the superior performance of IDP-CBR in maintaining the medium pH, which promoted the light-dependent biohydrogen production (up to 13%).