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.