This study investigated the impact of CO2 removal from the headspace of a continuous flow biohydrogen production system on the H-2 yield and microbial community structure. A comparative study was conducted in the Integrated Biohydrogen Reactor Clarifier System (IBRCS) with and without potassium hydroxide in the reactor headspace to sequester CO2 using glucose. Headspace CO2 sequestration increased the H-2 yield by 22% to 2.96 +/- 0.14 mol/molhexose. The impact of headspace CO2 sequestration was not limited to the improvement in H-2 yield and gas quality, as it also influenced the metabolic pathway increasing acetate concentration, and decreasing butyrate and propionate concentrations. Detailed analyses of the microbial community structure in the IBRCS before and after CO2 sequestration revealed that removal of CO2 from the headspace of the bioreactor had a significant impact on the microbial diversity and species distribution which rationalize the observed changes in the metabolic pathways. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.