Our recent work showed the feasibility of using a 400 L pilot-scale bioreactor for hydrogen production via dark fermentation. However, the H(2) production performance of the pilot system was unsatisfactory when compared with that of lab-scale fermentors under the same conditions. This study applied engineering approaches to enhance the hydrogen production performance of the pilot bioreactor. First, a higher agitation rate was used to promote mass transfer efficiency. Next, the pilot system was operated under different combinations of hydraulic retention time (HRT) and substrate concentration (C(s)) that gives different sets of organic loading rate (OLR) to improve bioH(2) production efficiency. With 25-30 rpm agitation rate and a OLR of 60 g COD/L/d (from combination of 8 h HRT and 20 g COD/L C(s)), the H(2) production rate (HPR) of the pilot system reached 0.55 mol/L/d (13.4 m(3)/ m(3)/d), which is 3.1 fold of that obtained from using a lower agitate rate (10-15 rpm). When operating at HRT = 6 h and C(s) = 30 g COD/L (i.e., OLR = 120 g COD/L/d), the pilot system obtained the highest HPR, hydrogen yield and overall hydrogen production efficiency of 1.18 mol/L/d, 3.84 mol H(2)/mol sucrose and 47.2%, respectively. This performance is similar to that obtained from the lab-scale system and is significantly higher than that from the original pilot tests prior to process optimization. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.