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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SOLENALGAE (IMPROVING PHOTOSYNTHETIC SOLAR ENERGY CONVERSION IN MICROALGAL CULTURES FOR THE PRODUCTION OF BIOFUELS AND HIGH VALUE PRODUCTS)

Teaser

Summary

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 bio-ethanol, bio-diesel and bio-hydrogen. Microalgae exploitation for biofuels production have the considerable advantages of being sustainable and not in competition with food production, since not-arable lands, waste water and industrial gasses can be used for algae cultivation. Considering that only 45% of the sunlight covers the range of wavelengths that can be absorbed and used for photosynthesis, the maximum photosynthetic efficiency achievable in microalgae is 10%. On these bases, a photobioreactor carrying 600 l/m-2 would produce 294 Tons/ha/year of biomass of which 30% to 80%, depending on strain and growth conditions, being oil. However this potential has not been exploited yet, since biomass and biofuels 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 aims to investigate the molecular basis for efficient light energy conversion into chemical energy, in order 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.

Following this concept, the final aim of this research project is to establish a protocol for enhancing light energy conversion efficiency in microalgae by manipulating the thermal dissipation of the excitation energy.

In particular the intermediate objectives of the project are:

I. Understanding the molecular basis for the activity of LHCSR proteins inducing NPQ

II. Investigation of the biomass productivity of C. reinhardtii strains with modulated LHCSR activity

III. Genetic manipulation of selected microalgae species in order to increase the biomass productivity by reducing the heat dissipation of the light absorbed

Work performed

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 (â€œquenchingâ€ activity) of a fraction of excitation energy absorbed by the photosynthetic complexes in microalgae. In order 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.
In particular, a mutational investigation was performed in vitro and in vivo, characterizing the effect of specific mutations on chlorophyll binding sites or protonatable residues, the former being potentially involved in LHCSR excitation quenching activity, the latter in the activation of the quenching mechanism.

Spectroscopic analysis in vitro on LHCSR WT and mutant complexes allowed to identify different quenching mechanisms involved in their functions and the main pigments involved. These findings are now going to be validated by in vivo complementation with lhcsr3 gene mutant variants. Preliminary results in vivo on some chlorophylls and protonatable residues are fully in line with the results obtained in vitro.

In parallel, the function of LHCSR protein have been further investigated focusing on the interaction with other protein subunits. Crosslinking experiments demonstrated the interaction of LHCSR protein with other pigment binding complexes: gene silencing approach allowed to demonstrate in particular the function of three components of the main antenna proteins of photosystem II, LHCBM4-6-8 in the quenching activity of LHCSR subunits, together with LHCBM1 previously identified by other research groups. Additional work, exploiting also the newly developed genome editing methods are currently being applied to identify the role of other components of photosystems as partners of LHCSR complexes.

Objective II: Investigation of the biomass productivity of C. reinhardtii strains with modulated LHCSR activity
The obtainment of C: reinhardtii mutants with altered LHCSR activity allowed for the investigation of the relationship between biomass productivity and LHCSR activity in C. reinhardtii as Non-photochemical quenching (NPQ) induction. Increased biomass production and increased light use efficiency was obtained by introducing in C. reinhardtii a LHCSR3 gene under the control of Heat Shock Protein 70/RUBISCO small chain 2 promoter in a npq4 lhcsr1 background, a mutant strain knockout for all LHCSR genes. This complementation strategy leads to a low expression of LHCSR3, causing a strong reduction of NPQ induction but is still capable of protecting from photodamage at high irradiance, resulting in an improved photosynthetic efficiency and higher biomass accumulation. The evaluation of the performances of the different strains with reduced NPQ allowed to draw an exponential correlation between NPQ reduction and productivity. This strategy, herein reported for the model organism C. reinhardtii, could be transferred to other algal species, such as those with high commercial interest. Further work is currently ongoing to validate this correlation with mutants with reduced LHCSR activity. The investigation of the physiologic features of mutants with altered NPQ phenotype revealed as a key feature for increasing biomass productivity by reducing thermal dissipation of the light absorbed a relative increase of electron acceptors from Photosystem II, as Photosystem I and Cytb6f compared to Photosystem II to ensure a more efficient use of electrons coming from light driven water splitting at the level of Photosystem II. The importance of Photosystem I activity in biomass production and light energy conversion has been further investigated characterizing a C. reinhardtii mutant with impaired NADPH metabolism.

Objective 3: Genetic manipulation of selected microalgae species in order to increase the biomass productivity by reducing the heat dissipation of the

Final results

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
- Demonstration that constitutively low expression of lhcsr gene caused a ~30% increased productivity in C. reinhardtii
- Set up of innovative methods for genome assembly by integration of next generation sequencing and optical mapping. As a first example of application if this method, the genome of C. vulgaris has been assembled and functionally annotated
- Set up of transformation protocols for H. pluvialis
- Determination of the exponential correlation between NPQ reduction and productivity in C. reinhardtii
- Increased biomass and astaxanthin production in H. pluvialis by selecting strains with reduced NPQ induction
- Increased lipid production in N. gaditana by selecting strains with reduced NPQ induction

The expected results until the end of the project are:
- Genome assembly of H. pluvialis and transcriptome analysis
- Consolidation of transformation protocols in C. vulgaris, C. sorokiniana and H. pluvialis
- Set up of genome editing methods for direct genetic analysis in different microalgae species
- Identification of the key genes involved in NPQ and biomass productivity in non-model algae
- Transcriptional profile of high productive strains in C. reinhardtii
- Identification of interaction network of LHCSR subunits in C. reinhardtii