Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced in conditions of sulfur deprivation. In this study, we investigated the repeated and enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the optimal concentration of re-added sulfate was 30 mu M for the hydrogen production. By the re-addition of 30 mu M of sulfate and the adjustment of the pH during 4 cycles of repeated production, we obtained the maximum amount of 789 ml H(2) l(-1) culture, which is 3.4 times higher than that of one batch production without adjustment of pH, 236 ml H(2) l(-1) culture. This means that the enhancement of the hydrogen production can be achieved by the careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.