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Background  Sealed  Chlamydomonas reinhardtii  cultures evolve significant amounts of hydrogen gas under conditions of sulfur depletion. However, the eukaryotic green alga goes through drastic metabolic changes during this nutritional stress resulting in cell growth inhibition and eventually cell death. This study aimed at isolating  C. reinhardtii  transformants which produce hydrogen under normal growth conditions to allow a continuous hydrogen metabolism without the stressful impact of nutrient deprivation.    Results  To achieve a steady photobiological hydrogen production, a screening protocol was designed to identify  C. reinhardtii  DNA insertional mutagenesis transformants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). The screening protocol entails a new and fast method for mutant strain selection altered in their oxygen production/consumption balance. Out of 9000 transformants, four strains with P/R ratios varying from virtually zero to three were isolated. Strain  apr 1 was found to have a slightly higher respiration rate and a significantly lower photosynthesis rate than the wild type. Sealed cultures of  apr 1 became anaerobic in normal growth medium (TAP) under moderate light conditions and induced [FeFe]-hydrogenase activity, yet without significant hydrogen gas evolution. However, Calvin-Benson cycle inactivation of anaerobically adapted  apr 1 cells in the light led to a 2-3-fold higher  in vivo  hydrogen production than previously reported for the sulfur-deprived  C. reinhardtii  wild type.    Conclusion  Attenuated P/R capacity ratio in microalgal mutants constitutes a platform for achieving steady state photobiological hydrogen production. Using this platform, algal hydrogen metabolism can be analyzed without applying nutritional stress. Furthermore, these strains promise to be useful for biotechnological hydrogen generation, since high  in vivo  hydrogen production rates are achievable under normal growth conditions, when the photosynthesis to respiration capacity ratio is lowered in parallel to down regulated assimilative pathways.

A novel screening protocol for the isolation of hydrogen producing Chlamydomonas reinhardtiistrains