The effect of pH regulation on biohydrogen production was studied using suspended and immobilized mixed cultures. Four sets of experiments were conducted using suspended cells under regulated pH (Sus_R) and non-regulated pH conditions (Sus_N) as well as alginate-immobilized cells under pH regulated (Imm_R) and non-pH regulated conditions (Imm_N). Sus_R showed a peak hydrogen fraction of 44% and complete glucose degradation, compared to Sus_N with a peak hydrogen fraction of 36% and a glucose degradation of 37%. Imm_R experiments showed a peak biohydrogen fraction of 35%, while the peak hydrogen fraction observed with Imm_N was 22%. The highest hydrogen fraction was observed using suspended cells under regulated pH conditions. A 100% glucose degradation was observed in both pH regulated and non-regulated processes using immobilized cells. The rate of pH change was slower for immobilized cells compared to suspended cells suggesting a better buffering capacity under non pH regulated conditions. The study showed that biohydrogen production with suspended cells in a non-regulated pH environment resulted in early termination of the process and lower productivity.