Selection of Chlamydomonas reinhardtii mutantsfor improved growth in photobioreactor andperspectives for bio-hydrogen production

In the last years, a significant increase in studies concerning different algal species has been realized, with important effects for our knowledge of physiology and genetics, but also for applicative purposes. The possibility to obtain higher yield in term of biomass production, with respect to land plants, makes these organisms of particular interest for biotechnological applications. Indeed, different algal strains are currently investigated for biofuel production, as biodiesel or bio-hydrogen, thanks to their capacity to grow using CO2 as substrate through the photosynthetic process, in a highly efficient way. In particular, among green algae, Chlamydomonas reinhardtii represents the model organism, thanks to the complete genome sequencing and the availability of efficient nuclear and chloroplast transformation technologies. C. reinhardtii possesses an hydrogenase enzyme which allows light-driven hydrogen synthesis, using protons and reducing power generated by the photosynthetic process. However, the exploitation of mass algal cultures in photobioreactor with the final aim of bio-hydrogen production presents some important limiting factors. In fact, algae are equipped with a large light-harvesting antenna system, which constitutes an evolutive advantage in their natural environment, but affecting the light penetration and distribution within the photobioreactor. This causes an energy over absorption in the high light exposed superficial layers, with increased heat dissipation, and at the same time in shading of the inner layers, thus reducing the photosynthetic efficiency. Moreover, hydrogenase activity is strongly inhibited by molecular oxygen, also at very low concentration, which is evolved during the photosynthesis. Finally, some processes affecting the dynamics of electron fluxes inside the cell can compete with the hydrogenase for reducing power, thus diminishing hydrogen production yield. Considering all these elements, in this thesis we decided to adopt an in vivo approach, in order to identify strains with improved characteristics for biomass accumulation at the final aim of bio-hydrogen production. Therefore, we created a Chlamydomonas reinhardtii insertional mutant library, which has been screened by multiple strategies, as described in chapter 2, to select mutants with improved phenotypic characteristics. Moreover, the genetic analysis of these mutants has been initiated in order to identify the genes responsible of the phenotype. Once identified, these genes could offer the possibility to modulate different mechanisms by using inducible expression systems. The screening also allowed to isolate mutants particularly interesting for basic researches, although not suitable for growth in mass culture conditions. This is the case of the gun4 mutant, never characterized so far in Chlamydomonas. The Gun4 protein has been identified in plants and in cyanobacteria as a regulatory factor in the chlorophyll biosynthesis pathway and in the retrograde signaling from chloroplast to nucleus. Therefore, we started a deeper analysis of this mutant, which could provide information about these interesting biological problems, as will be discussed in chapter 3. Finally, chapter 4 presents an in vitro approach for the functional characterization of all Lhca proteins which form the PSI light-harvesting complexes in Chlamydomonas reinhardtii. The different antenna complex subunits are not all functionally equivalent, and it important to elucidate their specific role in the light-harvesting and photo-protection. Their characterization constitutes the basis for proceeding to a selective depletion of the different Lhc proteins in order to improve light-absorption without affecting the photoprotective mechanisms, thus optimising the overall photosynthetic efficiency.