Hydrogen production was optimized by varying substrate concentration (10-40 g L-1) in an automated 3m3 pilot-scale continuous dark-fermentative bioreactor with three sequential chambers (# 1-# 3).Hydrogen production rate (HPR) followed the parabolic trend with the substrate concentration (10 to 40 g L-1) where highest HPRof 200.32 mol m-3 d-1 was observed at 30 g L-1. Furthermore, the characteristics of gas/fermentation broth composition showed noticeable changes in the flow direction (# 1 → # 3). From distribution nephogram of velocity in bioreactor, it was observed that high-velocity movement of the corner and the bottom of the baffled plate played conducive effects to the mixing of the nutrients and the functional consortium. Analog computation results were in good correlation to the experiment data. While comparing with photo fermentation, HPRof dark fermentation was remarkably higher when substrate concentration was over 20 g L-1. Our findings may guide to design and develop the bioreactors with high hydrogen yields which can help to support clean energy supply.