Caproate always appears during fermentative H(2) production but its formation was not well explained. It possibly results from the secondary fermentation of ethanol and acetate or butyrate by some special species like Clostridium kluyveri. This study attempts to elucidate caproate formation during the fermentation H(2) production by using C. kluyveri as an example and evaluating several possible pathways of caproate formation. A detailed energetic analysis of the empirical data of an H(2)-producing reactor demonstrated that caproate can be formed from two substrates, either ethanol and acetate or ethanol and butyrate. The analysis showed that at least 5 mol ethanol per mole reaction was essential to support caproate formation under the experimental condition. The analysis also indicated that the secondary fermentation by C. kluyveri might be another pathway to spontaneously produce H(2), butyrate, and acetate in addition to the butyrate-acetate pathway. Co-production of caproate and H(2) from ethanol was thermodynamically feasible and contributed to at least 10-20% of total H(2) production in the reactor studied. It is also clarified that caproate formation is hydrogenogenic rather than hydrogenotrophic. (C) 2010 Elsevier Ltd. All rights reserved.