In this study the effect of different buffering agents, pH control and N-2 sparging on biohydrogen production in Thermobrachium celere was investigated in batch cultivations. Among the tested buffers, none was able to prevent the medium acidification resulting in a premature interruption of the hydrogen production. Controlling the pH helped to sustain the growth, the complete substrate consumption and the H-2 production. However, in these conditions the increase of H-2 partial pressure induced a partial metabolic shift towards ethanol production resulting in a decreased H-2 yield. Analysis of formate accumulation during growth suggests that this compound might play a relevant role in the anabolic routes in T. celere. When frequent N-2 sparging was applied for H-2 removal, together with pH control, the H-2 yield was remarkably enhanced from 2.26 to 3.53 mol H-2/mol glucose, and the maximum H-2 production rate and specific H-2 production rate reached 41.5 mmol H-2/l/h and 142.3 mmol H-2/h/g, respectively. This result suggests that under proper conditions T. celere is able to produce hydrogen at high yield and production rate. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.