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Okres sprawozdawczy: 2020-09-01 do 2021-08-31

Solar Energy is the most abundant renewable energy source available for our Planet. Light energy conversion into chemical energy by photosynthetic organisms is indeed the main conversion energy step, which originated high energy containing fossil deposits, now being depleted. By the way, plant or algae biomass may still be used to produce biofuels, as bioethanol, biodiesel and bio-hydrogen, bioproducts (antioxidants, biostimulants, fertilizers, pharmaceuticals) or biomass for human nutrition or animal feed. Microalgae exploitations have the considerable advantages of being sustainable and not in competition with food production, since not-arable lands, wastewater and industrial gasses can be used for algae cultivation. However, this potential has not been exploited yet, since biomass yield on industrial scale obtained up to now were relatively low and with high costs of production. The main limitation encountered for sustained biomass production in microalgae by sunlight conversion is low light use efficiency, reduced from the theoretical value of 10% to 1-3%. This low light use efficiency is mainly due to a combined effect of reduced light penetration to deeper layers in highly pigmented cultures, where light available is almost completely absorbed by the outer layers, and an extremely high (up to 80%) thermal dissipation of the light absorbed. This project as aimed to investigate the molecular basis for efficient light energy conversion into chemical energy, to significantly increase the biomass production in microalgae combining a solid investigation of the principles of light energy conversion with biotechnological engineering of algal strains. SOLENALGAE project allowed to understand to molecular details of photoprotection and heat dissipation of the light energy absorbed in microalgae going deep into details about the protein subunits involved, their interactors, and the protein domains and specific residues at the base of the quenching activity observed, also highlighting the energy pathways and the molecular species at the base of the excitation energy quenching. This information was then considered to plan different biotechnological intervention in different microalgae species, which allowed to boost photosynthetic activity and production of specific high value products as astaxanthin, on e of the strongest antioxidant molecule found in nature, proteins for human nutrition of biostimulants to be used in agriculture.
Objective I: Understanding the molecular basis for the activity of LHCSR proteins inducing NPQ
LHCSR are the pigment binding subunits involved in the thermal dissipation (“quenching” activity) of a fraction of excitation energy absorbed by the photosynthetic complexes in microalgae. To investigate the molecular basis of LHCSR photoprotective activity, these subunits have been analyzed in vitro and in vivo in the model organism for green algae C. reinhardtii identifying different quenching mechanisms involved in their functions, the protein domains and the main pigments involved. The results herein obtained were published in 12 peer-reviewed papers and presented in several international congresses.

Objective II: Investigation of the biomass productivity of C. reinhardtii strains with modulated LHCSR activity
The evaluation of the performances of the different strains with reduced NPQ allowed to draw a correlation between NPQ reduction and productivity. However, this correlation is valid only in conditions of CO2 limitation, while at high CO2 concentration the influence of thermal dissipation of the energy absorbed is minimal, likely due to the efficient regeneration of the ADP and NADP+ by the carbon fixation mechanisms. The investigation of the photoprotective properties of C. reinhardtii and the role of specific carotenoids, led to the generation of strains overexpressing the enzyme beta-carotene ketolase (BKT) which were characterized by a high accumulation of the ketocarotenoid astaxanthin, revealing as an efficient system for the industrial production of this important antioxidant, which was the subject of a dedicated ERC PoC ASTEASY. The results for Objective II were disseminated in 5 papers published in international peer reviewed journal.

Objective 3: Genetic manipulation of selected microalgae species in order to increase the biomass productivity by reducing the heat dissipation of the light absorbed.
Advanced genome editing based on CRISR/CAS9 or CRISPR/CAS12 were established in different species. C. vulgaris CCAP211/11P and Haematococcus pluvialis nuclear genome, organelle genomes, and transcriptome were obtained allowing to identify potential targets for the biotechnological manipulation of this organism, for its exploitation for biomass and high value products or for transferring peculiar properties to other species. In the case of Chlorella sorokiniana an high-quality transcriptome has been obtained revealing, a possible C4-like carbon fixation pathway. Strains with increased productivity of biomass/biomolecules were generated in H. pluvialis, C. vulgaris and Nannochloropsis gaditana, in the latter case being at the base of the dedicated ERC PoC ASTAOMEGA project. Several collaborations with international research groups and companies were established, with an ongoing negotiation with the Host Institution and Investors for the launch of our own start-up company for the exploitation of the results herein obtained.
The main progress beyond the state of the art achieved are the following:
- Identification of the quenching mechanisms at the base of the LHCSR activity in C. reinhardtii
- Identification of interaction network of LHCSR subunits in C. reinhardtii
- Demonstration that constitutively low expression of lhcsr gene caused a ~30% increased productivity in C. reinhardtii
- Demonstration of photoprotective events occurring at the level of Photosystem I
- Set-up of encapsulation methods for Photosystem I in organic polymers to improve its stability and use it in extracellular environment for redox based reactions
- Set up of genome editing method in Chlamydomonas reinhardtii and Nannochloropsis oceanica (knock-out and knock-in mutations)
- Increased biomass and astaxanthin production in H. pluvialis by selecting strains with altered NPQ induction
- Generation of an efficient system for astaxanthin production in C. reinhardtii (subject of ERC PoC ASTEASY)
- Set up of innovative methods for genome assembly by integration of next generation sequencing and optical mapping the case of the genomes of C. vulgaris and H. pluvialis, which has been assembled and functionally annotated
- Definition of an innovative strategy to increase photosynthetic efficiency in C. reinhardtii (patent pending)
- Set up of transformation protocols for H. pluvialis
- Increased lipid production in Nannochloropsis gaditana by selection of mutants with altered pigment profile
- Generation of strains of Nannochloropsis gaditana being able to efficiently accumulate in a single growth phase astaxanthin and EPA (subject of ERC PoC ASTAOMEGA, patent pending)
- Increased photosynthetic efficiency and biomass productivity in Chlorella vulgaris by using strains with altered NPQ properties, demonstrating the requirement of a proper balance between the irradiance, CO2 availability and photoprotective properties
- Demonstration of the activity as biostimulant for plant growth of Chlamydomonas reinhardtii and Chlorella sorokiniana biomass