Dilution Methods to Deprive Chlamydomonas reinhardtii Cultures of Sulfur for Subsequent Hydrogen Photoproduction

Sulfur deprivation of Chlamydomonas reinhardtii cultures gradually inactivates photosynthetic O₂ evolution and leads to the establishment of anaerobiosis in the medium. Sulfur-deprived algal cultures kept under anaerobic conditions will then produce H₂ gas for 3-5 days under continuous illumination. Currently, sulfur deprivation is achieved by mechanical centrifugation of cultures grown in sulfur-replete medium, followed by extensive and costly washing. The cells are finally resuspended in sulfur-free medium. The current study investigates two procedures to deprive algal cultures of sulfur that eliminate the centrifugation step. These procedures involve sulfur deprivation by dilution of sulfur-replete cultures into either sulfur-limited medium or sulfur-free medium. We demonstrate that efficient H₂ photoproduction can be achieved on a timely basis using either procedure. However, the dilution of sulfate-replete algal cultures 1:10 v/v into sulfur-free medium is the most appropriate procedure. These observations serve as the basis for developing an algal H₂-production system that is cheaper, less time-consuming, and less amenable to contamination with other microorganisms than systems employing centrifugation for sulfur deprivation. Published by Elsevier Science Ltd. on behalf of the International Association for Hydrogen Energy.