A new strategy in hydrogen production via biophotolysis developed previously was implemented in mutant strains of Chlamydomonas reinhardtii. Implementing PSI-light successfully demonstrated improved hydrogen production in the wild type strain of C. reinhardtii in a previous study, however, the results also showed rapid attenuation of hydrogen production even under PSI-light due to inhibited hydrogenase activity caused by oxygen, which was simultaneously produced through the water splitting reactions of PSII under light. In order to further decrease oxygen generation under PSI-light during the hydrogen production phase, use of some mutant strains of C. reinhardtii, that are known to show limited oxygen generation, were investigated. Continuous supply of PSI-light after a short anaerobic adaptation under dark conditions achieved significantly prolonged hydrogen production up to approximate to 18 h in a chlorophyll b deficient mutant (cbn 1-48) and a very high light tolerant mutant (VHLR-S4) yielding chlorophyll content based H-2 production of 220 and 176 dm(3) kg(-1) (equivalent to dry cell weight based H-2 production of 4.24 and 8.73 dm(3) kg(-1)), respectively. In addition, by iterating light and dark every 1.5 h with PSI-light, hydrogen production was successfully extended to 27 h yielding chlorophyll content based H-2 production of 366 dm(3) kg(-1) (equivalent to dry cell weight based H-2 production of 8.81 dm(3) kg(-1)) in cbn 1-48. Further, greater energy conversion efficiency from light to the formation of hydrogen molecules was achieved with the combination of PSI-light and some mutant strains compared to alternate methods of biophotolysis. (C) 2013 Elsevier Ltd. All rights reserved.