This study investigated the response of acclimatized anaerobic hydrogen-producing cultures to substrate changes in batches. Two sequential batches were tested using non-acclimatized and acclimatized microbial cultures for biohydrogen production from different sugars. The first set of batches was conducted using non-acclimatized sludge with glucose at a concentration of 10 g/L. The second set of batches was conducted using sludge acclimatized with glucose over a period of 4 days from the first batch and a mixture of pentoses and hexoses (C5 & C6) sugars as the carbon sources. Additionally, two batches were tested: one with glucose, cellobiose, xylose, and arabinose individually with non acclimatized sludge, followed by C5 sugar batches. The acclimatized microbial cultures achieved a better performance compared to the non-acclimatized ones; the average hydrogen yield for glucose with non-acclimatized sludge was 1.71 mol H-2/mol glucose added (225 mL H-2/g COD) compared to 1.9 mol H-2/mol glucose (250 mL H-2/g COD), with glucose-acclimatized cultures. On the other hand, operating integrated biohydrogen reactor clarifier systems (IBRCSs) using equal amounts of glucose, cellobiose, arabinose, and xylose at a concentration of 2.5 g/L each resulted in a hydrogen yield of 1.9 mol H-2/mol sugar added with a hydrogen production rate of 61.7 L H-2/d. (C) 2016 Elsevier B.V. All rights reserved.