Photoautotrophic and sustained H2 production by the pgr5 mutant of Chlamydomonas reinhardtii in simulated daily light conditions

Green algae, such as Chlamydomonas reinhardtii, can produce H2 efficiently using their hydrogenases, but these enzymes are O2-sensitive and compete with CO2-fixation for electrons. To overcome these limitations, we pre-viously developed an anaerobic, carbon-limited protocol that keeps the Calvin-Benson cycle inactive, and the evolved O2 is scavenged by an absorbent. The PROTON-GRADIENT REGULATION5 (PGR5)-deficient mutant performs better than the wild type in this system, as it produces at least 2.5-fold more H2 and maintains its photosynthetic apparatus and hydrogenase activity even at the intensity of sunlight. However, as the pgr5 mutant is known to be sensitive to fluctuating light conditions, it is of paramount interest to determine how it reacts to changes in light intensity during H2 production. Therefore, we developed an automated system to monitor H2 production in thin layer cultures under simulated daily light conditions. We found that the pgr5 mutant out-performed the wild type strain by 100 % when light intensity was varied stepwise between 0 and 1000 mu mol photons m - 2s- 1during the day. Photosynthetic subunits, including PsbA, PSBO, CP47, PetB and PsaA, were fully preserved in the pgr5 mutant, and approximately 29 % of its original hydrogenase activity was sustained after 85 h of H2 production in simulated daily light conditions. Hence, the pgr5 mutant is a promising candidate for H2 production under the adverse light conditions that algae may encounter in bioindustry settings.