Non-Photochemical Quenching (NPQ) and biomass production in microalgae

Photosynthesis is the main solar energy converting system on our planet. In all photosynthetic organisms, photosynthesis takes place in the thylakoid membranes of chloroplasts, which are capable of converting solar energy into chemical energy to fix carbon dioxide into biomass, producing oxygen as a byproduct. Light energy, which intensity and spectral quality change continuously in the environment, can also be harmful since excess irradiances can determine the photosystems over-excitation, with the consequent production of Reactive Oxygen Species (ROS). ROS are toxic molecules that can seriously damage the chloroplast as well as the entire cell. In order to avoid this critical situation, several photoprotective mechanisms evolved. Light harvesting and energy conversion by charge separation occur at the level of membrane protein complexes called Photosystems. In eukaryotic systems, the Light Harvesting Complex (LHCs) multigene family constitutes the pigment binding antenna system of photosystems. LHCs play two fundamental roles in photosynthesis: light harvesting and regulation of excitation energy. The fastest and most important photoprotective inducible strategy is the activation of the thermal dissipation of light energy absorbed in excess, a mechanism called Non-Photochemical-Quenching (NPQ), which is triggered by the generation of a pH gradient across thylakoid membranes. In vascular plants, NPQ depends on the Lhc-like protein PSBS, while a different polypeptide called LhcSR, is required in algae as the model species Chlamydomonas reinhardtii. LhcSR is a chlorophyll and xanthophyll-binding protein, responsive to low pH and it is a strong quencher of Chl excited states, exhibiting a fast fluorescence decay. In microalgae, the NPQ mechanism is really influent: up to 80% of absorbed light energy can be dissipated as heat, with consequent loss of light use efficiency and biomass productivity in favor of photoprotection. This explains the importance of the study of NPQ, in order to find a promising way of domestication to improve overall algal productivity. Algae domestication is an indispensible step for their commercial applications, such as production of biomass for human/animal feeding or extraction of high-value chemicals and biofuels feedstock. For all these reasons, the central point of this thesis is the study of NPQ in microalgae. In the first part of the thesis, the NPQ mechanism was studied in detail in six different green algae, some are planktonic species, others live in aggregation on biofilm structures. The analysis takes into consideration fluorescence measurements, the change in the pigment profiling and the transmembrane DpH formation. From this investigation, the heterogeneity of the NPQ mechanism was demonstrated in green algae: the NPQ behavior was not related to the phylogeny of the algae but rather to the environmental selection pressure. Some green algae present a zeaxanthin-dependent-NPQ, like the vascular plants, while the correlation is absent in other algal species of the same phylogenic group. In a biologic system the productivity and biomass accumulation could be considerably increased by reducing the level of energy dissipation into heat. In order to demonstrate this theoretic hypothesis, the attention was focused on the study of the correlation between biomass production, heat dissipation (NPQ) and the accumulation of LhcSR, which is the crucial protein in triggering NPQ in the model species for algae research Chlamydomonas reinhardtii. Wild type strain and knockout mutants with reduced NPQ induction were grown simultaneously in a small scale photobioreactor, in different conditions of light intensity. The main conclusion of this work is that a mutant in the two genes coding for LhcSR3 subunit, with reduced NPQ induction, showed an improved capacity to convert photons into biomass than the WT. Therefore this is the demonstration that algal productivity can be increased by down-regulating NPQ. On the other hand a second mutant in which NPQ induction was completely abolished by knocking out also the gene coding for LhcSR1, showed a much lower productivity compared to WT: a minimal level of photoprotective quenching is thus necessary. Therefore our results demonstrate that evolution led Chalmydomonas reinhardtii cells to promptly switch upon high irradiance exposure to energy converting states with low efficiency in order to safely dissipate the energy absorbed in excess through heat. Chloroplasts in a highly quenched state are less efficient in photosynthetic biomass production, but they are in a safer photoprotective state with a low risk for Photoinhibition. In Chlamydomonas reinhardtii NPQ is fully dependent on the presence of the LhcSR polypeptide. In addition there are likely other proteins involved among members of the LHCBM family, namely Light Harvesting Complexes subunits predominantly associated with PSII. In Chlamydomonas reinhardtii there are nine highly conserved LHCBM genes: some of them exert a specific role, with a well defined, non-redundant function despite their high homology, while for other genes their functions are still unclear. For example, a role in NPQ was demonstrated for LHCBM1 protein, likely as a partner for the main regulator element LhcSR3, while LHCBM2/7 was reported to be involved in state transitions induction. The specific function of LHCBM4, LHCBM6 and LHCBM8 polypeptide is still unknown, for all these reasons, in this work the biochemical and spectroscopic features of LHCBM4, LHCBM6 and LHCBM8 were first analyzed in the recombinant proteins in vitro and their physiologic function was then studied in vivo by a reverse-genetic approach using amiRNA strains. The results demonstrate the quenching activity of LHCBM4, LHCBM6 and LHCBM8, suggesting their involvement in photoprotective mechanisms. As shown previously, the modulation of the NPQ phenomena, is a key factor for microalgae domestication. The information obtained for the model organism Chlamydomonas reinhardtii was then applied in the case of commercial interesting species. The Eustigmatophyceae oleaginous alga Nannochloropsis gaditana is important for biofuel production: it is characterized by a high oil content (up to 65-70% oil on dry weight) and fast growth in a wide range of conditions. In this work a chemical mutagenesis was done, and time-resolved chlorophyll a fluorescence imaging was used to investigate a large number of colonies and to isolate those with changes in photosynthetic parameters. We analyzed 2100 independent clones, among them we identified three interesting clones for low antenna size of PSII or for a low energy quenching dissipation. Several experiments were done to characterize the phenotype of the selected mutants in order to evaluate their possible increase in light use efficiency. Non-Photochemical-Quenching (NPQ) is a key physiologic mechanism in all photosynthetic cells. It is essential for the correct mode of operation of the photosynthetic process controlling the level of chlorophyll triplets and the ROS production in the chloroplast. The NPQ process is a complex photoprotective mechanism: it can change in the same phylogenetic group of algal species in dependence of the environmental pressure. There are several key factors, which act together, the transmembrane DpH, the xanthophyll pigments, and quencher protein(s). The most important actor involved in NPQ in C. reinhardtii is LhcSR, which can interact with accessory LHC quencher proteins, like LHCBM1 and LHCBM6. In this thesis there is a demonstration that algal productivity can be increased by down-regulating NPQ ensuring to the cells a minimal level of photoprotective quenching. With a better understanding of the NPQ mechanism it is possible to find a way for algae domestication, in order to fill the gap between the theoretic biomass estimation and the real obtainable biomass yield.