Bioplactics Biofertilizers

Environmental Energy Nutrition Food Feed CO 2 sequestration Wastewater treatment

Keywords: Biotechnology, Green microalgae, Photosynthesis, Light, Acclimation vii viii Biotechnologie, Microalgues vertes, Photosynthèse, Lumière, Acclimatation Microalgae culture Extraction Light CO 2 Nutrients Strain selection Genetic engineering Culture system Abiotic factors Microalgae biomass biohydrogen, biodiesel, biomethane, bioethanol

icroalgae are emerging today as part of the answer to the major challenges of this century: climate change, population growth, and depletion of fossil fuel reserves. However, their autophototrophic production is limited by light resource. In addition to the total quantity, the way this light is delivered (intensity, temporality, spectrum) is considered to impact the performance of the culture. Understanding this to manipulate it could lead to increased culture yields. To characterize the effect of light fluctuations, an original experimental device was designed. This device allows ensuring homogeneous lighting of all the culture cells over long periods. In this way, only acclimated biological responses are measured.

First, continuous illumination was studied for intensities from 25 to 800 µmolPhotonPAR/m 2 /s.

The results allowed to delimit a photo-limitation zone from 25 to 200 µmolPhotonPAR/m 2 /s and a photo-saturation zone from 200 to 800 µmolPhotonPAR/m 2 /s. The absence of photo-inhibition suggests that the cells' photoprotection mechanisms are sufficient to protect them and maintain the same performance (µ = 0.088 h -1 on average).

Then, intermittent illumination was investigated (1 Hz, 10 Hz, and 100 Hz, illuminated fraction: 0.5). The results showed no difference from the continuous regime. These experiments highlight the importance of the acclimation state of the cultures. Thus, they invalidate, at long times (those of continuous bioprocesses), the hypothesis that light modulation impacts microalgae performance.

Introduction Introduction

Since the 1950s, the world's population has grown considerably. According to the United Nations Population Division, the world's population is estimated at 7.8 billion in 2020 and will stabilize at 10.9 billion or shortly after the end of the 21st century [1]. This population explosion undeniably increases the total ecological footprint and decreases the available biocapacity per capita. The pressures of human activities on resources such as fossil fuels, water, arable land, and biodiversity are ever-increasing. Despite technological advances, our ecosystem is being severely challenged. A global indicator of this trend is the overshoot day (Figure 1), decreasing every year. In addition to putting pressure on our environment, the modern lifestyle (mainly sedentary and excessive consumption of processed products) induces adverse health consequences: cancers, cardiovascular diseases, diabetes, and, more generally, metabolic syndrome. [START_REF] Pozzobon | Effet de la photopériode sur la croissance des microalgues -Journée microalgues[END_REF]. Data from Global Footprint Network [START_REF]Supply and Demand[END_REF].

Among the various existing alternatives, microalgae are emerging as good candidates to tackle part of these current challenges. They are a promising source of quality animal feed (proteins, micronutrients) as well as bio-sourced molecules with numerous applications in cosmetics and pharmaceuticals (pigments, texturizers, antioxidants). In addition, their cultivation is accompanied by environmental benefits: sequestration of carbon dioxide and NOx, water purification (nitrates, and phosphates), possible recovery of the remains 2

Table of contents

of the extraction process into biofuels. Finally, they can be grown on non-arable land, avoiding competition with food crops.

Several microalgal-based products are already available on the market. However, the production of microalgae is not yet viable on a large scale on various aspects (energy, economic). To fully realize its promise, three major challenges must be met or circumvented: improving the efficiency of production processes, reducing the energy costs associated with harvesting, and efficiently extracting molecules in an environmentally friendly manner. Among these challenges, the first is undoubtedly the most important.

Light is a predominant parameter to consider because it provides all the energy necessary for the metabolism of any photosynthetic organism. Its availability within a photobioreactor is one of the main problems to be solved to increase the efficiency of the production processes. Indeed, the light reaching the culture surface is rapidly absorbed along the light path. This results in a light gradient across the depth of the photobioreactor. More precisely, cells far from the surface will receive insufficient light and will therefore restrict the subsequent accumulation of biomass. In contrast, cells close to the illuminated surface may be exposed to excessive light intensity inducing photo-inhibition. Furthermore, although the main functions of culture mixing are to maintain the cells in suspension and allow a homogeneous distribution of nutrients, it also plays a role in the light intensity and photoperiod perceived by the microalgae [4]. Indeed, the microalgae move more or less rapidly between the illuminated zones located near the external surfaces, and the dark zones located in the heart of the culture. Thus, the cells are subjected to light cycles whose duration and intensity depend on the photobioreactor's geometry and the agitation. Even tough these light variations seem insignificant at first sight, microalgae have been shown to be sensitive to these cycles. This finding paves the way for manipulating light input to improve the economic viability of bioprocesses using microalgae. However, even if this biological phenomenon is known in the scientific community, its exploitation in biotechnology is limited due to significant disparities reported on the biological responses that prevent the results' appropriation. The ambition of this thesis work is to produce quality biological data allowing an accurate extrapolation to the industrial scale.

In this context, an original device ensuring homogeneous lighting of all the cells was designed and characterized. This device allows the measurement of the biological responses acclimatized according to the light patterns. The experimental campaigns were conducted in two steps. First, the cultures were exposed to continuous illumination of varying intensities from 25 to 800 µmolPhotonsPAR/m /s. These results serve as a reference for comparison. Second, intermittent illumination was investigated (1 Hz, 10 Hz and 100 Hz, illuminated fraction: 0.5). The first two frequencies explored correspond to the fluctuating light frequently encountered in PBR cultures. The last frequency corresponds to a flashing light and coincides with the characteristic times of photosynthesis. At the end of each run, the cultures were analyzed. Three parameters were monitored. The first parameter is the growth rate. This is an indicator widely used in biotechnology to describe both the general well-being of a culture and the process productivity. The pigment profile in chlorophylls and carotenoids was also evaluated. Finally, to move closer to photosynthesis, the state of the photosynthetic apparatus was quantified. Although these last two types

Introduction

This chapter aims at providing the context for understanding the following work. Considering the complexity of the cellular machinery that is photosynthesis, and the disagreements of scientists on certain mechanisms, it modestly attempts to provide an up-to-date state of the art.

The first part presents microalgae: their classification and applications. A focus on the model of the study is proposed (morphology, biochemical composition and mode of cultivation). The objective is to provide a general overview to familiarize the reader with microalgae and their possible applications in biotechnology.

In a second part, the mechanism of photosynthesis is presented in relative depth with a focus on green microalgae. For this, all the actors involved are first introduced. Then, the different steps and reactions are described and schematized to help the reader.

The third and last part outlines the different damage and protection mechanisms that microalgae can put in place in response to their light environment. As before, they are described and illustrated to ease the reading.

Microalgae

Definition

The term algae has no official taxonomic status. It is, however, commonly used to refer to a polyphyletic assemblage including macroalgae and microalgae. The former, commonly known as seaweed, are macroscopic and multicellular organisms while the latter are microscopic organisms. This second category is considered to be one of the oldest forms of life on Earth. It encompasses unicellular or simple multicellular photosynthetic microorganisms with the size ranging from a few micrometers to a few hundreds of micrometers. As a result of billions of years of evolution, microalgae have been able to evolve and acquire new phenotypic and genotypic characteristics allowing them to survive in different environments. While most microalgae are preferentially found in aquatic environments (marine and freshwater), they also populate other spaces including deserts, volcanic waters, highly acidic and frozen soils [START_REF] David | Microalgae selection and improvement as oil crops: GM vs non-GM strain engineering[END_REF]. Although their size may seem insignificant, these microorganisms are essential to the survival of the Earth. On the one hand, they form the primary component of food chains in the world's ecosystems. On the other hand, they play an important role in the retrocontrol of the global climate. As such, they are the main producers of oxygen and contribute to about 40% of global photosynthesis [START_REF] Andersen | Diversity of eukaryotic algae[END_REF][START_REF] Sharma | Biodiversity and biogeography of microalgae: Progress and pitfalls[END_REF][START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF].

Microalgae are a phylogenetically diverse group of microorganisms with an estimated total number of species between 30,000 and 1 million [START_REF] Michael | How many species of algae are there?[END_REF]11]. These microorganisms can have extremely diverse morphologies: most often as spherical shape, they can also be found as spindle-shaped, filament form, spiral-shaped, or even caltrop-shaped with colors ranging from blue to red, green and brown (Figure 1.1). In addition, microalgae include organisms with two different types of cell structure. On the one hand, prokaryotic oxygenic photosynthetic bacteria, better known as cyanobacteria or blue-green algae, are small, simple unicellular organisms lacking certain cell structures. They are considered to be the progenitors of chloroplasts. On the other hand, eukaryotic organisms representing the vast majority of microalgae. They have well-defined membrane structures that they would allegedly have acquired from evolution through endosymbiosis [START_REF] Pulz | Valuable products from biotechnology of microalgae[END_REF][START_REF] Stark | An Introduction to Photosynthetic Microalgae[END_REF][START_REF] Rockwell | Primary endosymbiosis and the evolution of light and oxygen sensing in photosynthetic eukaryotes[END_REF].

This great versatility is also found in the differentiation of their metabolic pathways. As relative plants, microalgae primarily grow photoautotrophically. In particular, they derive their energy from light for use in cellular functions and are capable of using inorganic compounds as a source of carbon (mainly carbon dioxide (CO 2 ), carbonic acid (H 2 CO 3 ) and bicarbonate (HCO - 3 )). Nevertheless, some of them are unable to synthesize their own organic carbon-based compounds from inorganic sources. Hence, they survive by degrading organic substances produced by, or available in, other organisms. They are then referred to as heterotrophs. Although the pigment profile of microalgae makes photosynthesis the main metabolic model, some microalgae can also grow under mixotrophic conditions. In this case, microalgae use a mix of different sources of energy (light and chemical) and carbon (organic and inorganic) [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF].

Microalgae classification

Traditionally, microalgae have been classified according to their photosynthetic pigments. Yet the current systems of classification take account of others criteria, among them cytological and morphological characters, cell wall constituents, and chemical nature of storage products. Based on these features, some methods are commonly employed to identify and classify algal species including morphological observations under a microscope, molecular-based classification using specific short gene sequences [START_REF] Groendahl | The best of both worlds: A combined approach for analyzing microalgal diversity via metabarcoding and morphology-based methods[END_REF] or more recently semi-automated or fully automated classification using a flow cytometer combined with computational methods [START_REF] Sosik | Automated taxonomic classification of phytoplankton sampled with imaging-in-flow cytometry[END_REF][START_REF] Drews | Microalgae classification using semi-supervised and active learning based on Gaussian mixture models[END_REF][START_REF] Correa | Deep Learning for Microalgae Classification[END_REF]. Regardless of the approaches used to identify algal species, the classification system has changed many times over the years. Currently, there is no consensus among the taxonomists around the world to use one classification over another [START_REF] Heimann | Chapter 3 -Microalgal Classification: Major Classes and Genera of Commercial Microalgal Species[END_REF][START_REF] Malcata | Marine Macro-and Microalgae : An Overview[END_REF]. One of the latest classification models of organisms is given by Ruggiero et al. [START_REF] Michael | A Higher Level Classification of All Living Organisms[END_REF]. These authors proposed a classification based on two main domains, Prokaryota and Eukaryota gathering seven kingdoms: Archaebacteria, Eubacteria, Protozoa, Chromista, Fungi, Plantae and Animalia.

Study model: Chlorella vulgaris

As mentioned above, Chlorophyta is a phylum with a high biotechnological potential. To date, more than 6,714 species have been reported belonging to this phylum [START_REF] Michael | How many species of algae are there?[END_REF]11]. Among them, Chlorella vulgaris is a species widely used as study model in the biological community along with Chlamydomonas reinhardtii. For these reasons, Chlorella vulgaris was chosen as the model organism for this work.

Taxonomy, morphology and ultrastructure

Chlorella is a genus of microalgae frequently encountered in the biological community. As such, more than 100 different species belonging to this genus have been reported in literature [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF]. It is considered as a model organism to study and understand the mechanism of photosynthesis [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF]. It was first described by the Dutch microbiologist Martinus Willem Beijerinck in 1890 [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF]. Taxonomically, the genus Chlorella is divided into two classes: Chlorophyceae and Trebouxiophyceae [START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF]. The species Chlorella vulgaris studied in this thesis belongs to the second class.

Chlorella vulgaris is a non-mobile eukaryotic green microalgae that can be found in both aquatic and terrestrial habitats [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF][START_REF] Richmond | Handbook of Microalgal Culture: Biotechnology and Applied Phycology[END_REF][START_REF] Barka | Microalgae as a potential source of singlecell proteins. A review[END_REF]. Mainly unicellular, the cells can also be found grouped in colonies. Morphologically, they are small, spherical or ellipsoidal cells whose diameter varies from 2 to 10 µm [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF][START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF][START_REF] Barka | Microalgae as a potential source of singlecell proteins. A review[END_REF][START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF].

By focusing on its ultrastructure, Chlorella vulgaris contains organelles similar to higher plants [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. From the outside to the inside, the cells are composed of a cell wall enclosing a cell membrane containing the cytoplasm shown in Figure 1. 4. The primary function of the cell wall is to provide structural support and protection against predators and hostile environmental conditions [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. Its structure can vary according to growth phases, environmental conditions and from one species to another [START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF][START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. As a result, discrepancies in the cell wall structure of the genus Chlorella have been reported in literature. In most cases, in the early stages of growth, Chlorella has a microfibrillar monolayer of about 2 nm thick. As growth progresses, the wall thickens to finally reach an average thickness of 21 nm [START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF][START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. Its structure also evolves. At the end of the growth, the cell wall is formed of different layers: a thick outer layer surrounding a thinner inner layer. These two layers are separated by a translucent intercellular space [START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF]. The cell wall covers the cell membrane that encloses the cytoplasm. This latter is a gel-like substance composed of water, soluble proteins and minerals. It contains various organelles including nucleus, mitochondria, endoplasmic reticulum (ER), Golgi body, vacuoles, ribosomes and chloroplast [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. Chlorella vulgaris has a single, cup-shaped chloroplast located at the periphery of the cytoplasm. The latter occupies about half of the cell volume [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF]. It is delimited by a double phospholipid membrane: the outer membrane is permeable to ions and metabolites while the inner membrane has a more specific function dedicated to protein transport [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. Inside the chloroplast are thylakoids containing photosynthetic pigments. A pyrenoid, generally present in most Chlorella species, is located in the center of the chloroplast [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF]. It contains high levels of the enzyme RuBisCO for carbon dioxide fixation [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. Finally, starch and lipid granules may form in the chloroplast, especially under unfavourable conditions. The reproduction of Chlorella vulgaris is asexual by autosporulation [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF][START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF]. In most cases, 4 daughter cells are produced in reproduction. They are formed inside the mother cell and, after rupture of the cell wall, are released into the extracellular medium. Generation time can vary from approximately 6 hours to more than a day depending on surrounding conditions.

Biochemical composition

The elemental composition of the Chlorella vulgaris biomass consists mainly of carbon, up to 50% of the cells [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF]. The second most abundant element is oxygen, which represents about 33% of the chemical composition. Then, nitrogen is the third element which accounts for about 9% of the biomass. Finally, hydrogen is the last major chemical element with 7% of the composition [START_REF] Babich | Catalytic pyrolysis of microalgae to high-quality liquid bio-fuels[END_REF].

Moving to a biochemical point of view, the biomass composition is likely to vary between species, and is affected by culture conditions (abiotic and nutritional factors). Keeping this concept in mind, it is estimated that for favourable growth conditions (no nutrient or light limitation, no stress applied), Chlorella vulgaris would be made up of about 60% protein, 10-15% carbohydrates, 12-15% lipids [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. In addition to these macronutrients, Chlorella vulgaris biomass is also rich in fibers, vitamins, minerals and pigments [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF][START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. Among the pigments expressed by this strain, the main ones are chlorophylls a and b, giving the cells its characteristic green colour. In addition to chlorophylls, carotenoids are also expressed (β-carotene, zeaxanthin, violaxanthin, neoxanthin, antheraxanthin, astaxanthin and canthaxanthin), the most abundant being lutein (up to 0.45% dry weight (DW)) [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF][START_REF] Gouveia | Evolution of pigment composition in Chlorella vulgaris[END_REF].

Cultivation mode

Historically, Chlorella is one of the oldest cultivated genera of microalgae. Its commercial exploitation began in the 1960s in Japan for human consumption [START_REF] Barka | Microalgae as a potential source of singlecell proteins. A review[END_REF]. Nowadays, it is the second most produced genus worldwide after Arthrospira with 5,000 tons per year [START_REF] Nethravathy | Recent Advances in Microalgal Bioactives for Food, Feed, and Healthcare Products: Commercial Potential, Market Space, and Sustainability[END_REF][START_REF] Wang | Microalgae as a Mainstream Food Ingredient: Demand and Supply Perspective[END_REF].

At present, there are 3 modes of cultivation for Chlorella vulgaris: autotrophy, heterotrophy and mixotrophy [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF][START_REF] Richmond | Handbook of Microalgal Culture: Biotechnology and Applied Phycology[END_REF][START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF]. Although heterotrophy and mixotrophy have undeniable advantages in terms of biomass production, they are relatively expensive production methods to implement because of organic carbon source cost and risk of contamination. In the context of this thesis, the production mode used is autotrophy. The latter can be achieved by two different cultivation approaches: open ponds and closed photobioreactors. The first offers a cost-effective solution on a large scale, at the cost of poor control of growing conditions and a very high risk of contamination. The second allows very strict control of growing conditions, but is very expensive. However, with the rise of the biorefinery concept, closed photobioreactors represent the future of the industry dealing with the production of medium to high added value molecules.

A photoautotrophic culture requires the contribution of two key elements: light and an inorganic carbon source. While light is provided by the sun or artificially, carbon is usually brought in the form of carbon dioxide (and more rarely as carbonic acid or bicarbonate). However, as the atmospheric air does not contain carbon dioxide in large quantities (415 ppm in January 2021 [START_REF]Earth's CO2 Home Page[END_REF]), most cultures are enriched with 1 to 5% of CO 2 [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF]. At the heart of photoautotrophy lies photosynthesis, a sophisticated mechanism detailled hereinafter.

Photosynthesis in green microalgae

Photosynthesis is a process converting light energy into chemical energy useful to support cellular metabolism and provide the basic raw materials from which cell structures are made. It is a light-driven reaction that involves carbon dioxide and water in which they are converted into carbohydrates. As a side-product, molecular oxygen is released. Oxygenic photosynthesis can be expressed as follows:

6CO 2 + 6H 2 O → 6O 2 + C 6 H 12 O 6 (1.1)
This process can be broken down into two stages: light reactions and carbon reactions or Calvin-Benson cycle. During the light reactions, light is taken as an input together with water resulting in the production of a biochemical reductant agent, NADPH, a high-energy compound, ATP, and the release of oxygen. Then, in the carbon reactions, the ATP and NADPH previously produced are used in the Calvin-Benson cycle along with carbon dioxide to synthesize carbohydrates, among others. These different mechanisms are detailed deeper hereinafter.

The nature of light

During photosynthesis, energy is provided by light, whether natural or artificial. In the broadest sense, light is an electromagnetic wave that travels at a velocity around 2.99792 × 10 8 m/s in vacuum. It has dual nature: wave and particle. It is part of a larger ensemble called electromagnetic radiation which extends from short wavelengths referred to as γ rays (from 10 -11 m) to long wavelengths, radio waves (from 10 -1 m) (Figure 1.5). Within this ensemble, the human eye is able to perceive only a small portion of this radiation. This band ranging from 390 to 780 nm is defined as visible light [START_REF] Lehmuskero | Light and photosynthetic microalgae: A review of cellular-and molecular-scale optical processes[END_REF]. The energetic particles at the origin of electromagnetic waves are called quanta or photons. The duality between wave and particle is related by the Planck's equation which allows the energy to be expressed as a function of frequency :

E photons = h × ν = h × c λ (1.2)
Where E is the particule energy (J), h is the Planck's constant (6.626 × 10 34 J/s), c is the speed of light (m/s) and λ the wavelength (m). Thus, the energy of a quantum is proportional to its frequency ν (= c λ ).

Light energy can also be expressed per mole of photon by multiplying the value of photon energy by the Avogadro number according to the following relationship:

E permole = h × c λ × N a (1.3)
From a chemist's perspective, another way to quantify light energy is to express it in electron Volt (eV). As a matter of fact, photons are capable of causing electronic excitations within molecules. These excited electrons acquire a potential energy corresponding to the capacity to produce chemical work (changes in the bonding or chemistry of the molecule). This is called electrical potential. Thus, the electron Volt corresponds to the energy acquired by an excited electron when its electrical potential is modified by one Volt (1 eV= 1.60 × 10 -19 J).

Table 1.1 summarizes the energy content and the electrochemical potential difference of photons at different wavelengths. According to Planck's equation, the shorter the wavelength, the higher its energy. Thus, the energy content of a violet light photon is about twice as high as that of a red light photon (299 kJ/mol photons and 171 kJ/mol photons, respectively). In the visible light spectrum, photons are sufficiently energetic to trigger certain biochemical reactions and, in particular photosynthesis. Infrared light contains less energy per photon and therefore does not have the energy to trigger the metabolic pathway threshold. On the other hand, ultraviolet radiation is known for its harmful and potentially mutagenic power [START_REF] Barsanti | Algae: anatomy, biochemistry, and biotechnology[END_REF]. Thus, the wavelengths of visible light correspond approximatively to the radiations used by photosynthetic organisms [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. The latter is defined as Photosynthetically Active Radiation (PAR) and covers the wavelengths between 400 to 700 nm [START_REF] Lehmuskero | Light and photosynthetic microalgae: A review of cellular-and molecular-scale optical processes[END_REF]. It was recently extended to 750 nm due to the discovery of red shift chlorophyll [START_REF] Barsanti | Algae: anatomy, biochemistry, and biotechnology[END_REF][START_REF] Nürnberg | Photochemistry beyond the red limit in chlorophyll f-containing photosystems[END_REF].

In the biology field, a unit commonly used to quantify the amount of photosynthetically active photons hitting a surface per unit area per unit time is the Photon Flux Density (PFD) expressed in µmolphotons/m 2 /s [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF].

Photosynthetic pigments

Photosynthetic pigments play a major role in the process of assimilating light energy into chemical energy. As such, they are responsible for the absorption of photon energy and its transfer to reaction centers where it is used for photochemistry.

All photosynthetic organisms have pigments capable of collecting light energy. These pigments are classified into three main groups according to their color: chlorophylls (green pigments), carotenoids (yellow to red pigments) and phycobiliproteins (red and blue pigments). The first two are classes of pigments associated in protein-pigment complexes and widespread in many microalgal phyla. The last class gathers hydrophilic pigments mainly found in Cyanobacteria and red algae and, therefore, will not be further developed in this section.

Chlorophylls

Chlorophylls are amphiphilic green pigments that play a central role in the transfer of light energy during photosynthesis. In most cases, they are able to collect light at a wavelength lower than 480 nm and between 550 and 700 nm [START_REF] Heldt | Plant biochemistry[END_REF]. These pigments are found in oxygenated photosynthetic organisms such as higher plants, macroalgae and microalgae including cyanobacteria [START_REF] Da | Impact of culture conditions on the chlorophyll content of microalgae for biotechnological applications[END_REF]. The cell's chlorophyll content varies according to the environmental conditions (light, nutrients, temperature) and the strain [START_REF] Da | Impact of culture conditions on the chlorophyll content of microalgae for biotechnological applications[END_REF] from about 0.5 to 4% of dry weight [START_REF] Spolaore | Commercial applications of microalgae[END_REF][START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF]. Generally speaking, the basic chemical structure of a chlorophyll molecule is composed of an aromatic macrocycle (either chlorine or porphyrin, depending on the chlorophyll type) connected to a hydrocarbon tail, called phytol. The macrocycle contains four pyrrole subunits, called tetrapyrrole, which are linked to a central magnesium atom (Figure 1.6) [START_REF] Edward | Phycology[END_REF]. All chlorophylls have at least two main absorption bands: one in the blue light region (400-475 nm) and one in the red light region (630-700 nm). Although the molecular structures of all chlorophylls are similar, they differ in some peripheral groups of the macrocycle. These structural differences affect their absorption spectrum, giving each type of chlorophyll unique absorption peak [START_REF] Chen | Chlorophyll modifications and their spectral extension in oxygenic photosynthesis[END_REF] (Table 1.2). Currently, five types of chlorophyll are known: a, b, c (c1 and c2 ), d, and f. Among them, only the first two types are found in green microalgae. Owing to its central role in photosynthesis, chlorophyll a is the most abundant pigment present in all photosynthetic microalgae. It represents on average between 0.3 and 3% of the dry weight of the cells [START_REF] Edward | Phycology[END_REF]. Considering only the chromophore, the chlorophyll a has two absorption bands: one in the blue light region at 430 nm and the other in the red region at 663 nm (Figure 1.6b). Thus, chlorophyll a is able to cover only a small part of the visible spectrum. In its natural state, chlorophyll is non-covalently bound to proteins in the thylakoid membrane, forming a pigment-protein complex. Complex formation modifies the maximum absorption of the chlorophyll molecule. While chlorophyll a is a universal pigment, other chlorophylls have a more limited distribution within photosynthetic microalgae (Table 1.2) [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. The latter are rather known to function as accessory pigments, i.e. they collect and transport light to chlorophyll a, thus extending the absorption spectrum for light capture [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Edward | Phycology[END_REF].

Chlorophyll b is the second most common pigment. Its chemical structure differs from chlorophyll a in the presence of a formyl group instead of a methyl group (Figure1.6a). This change has an impact on the absorption spectrum. The chlorophyll b molecule has absorption maxima close to that of chlorophyll a : one at 465 nm and the other at 645 nm (Figure 1.6b). In green microalgae, it is common to speak of a chlorophyll a/chlorophyll b ratio to characterize the effect of light on the photosynthetic apparatus. In general, this ratio varies from 2:1 to 3:1 [START_REF] Edward | Phycology[END_REF].

Carotenoids

Carotenoids are lipophilic pigments with a color ranging from yellow to red and an absorption range of 400 to 550 nm (Figure 1.7) [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. They are also called accessory pigments because of their ability to broaden the absorption spectrum without directly converting light energy into electrochemical energy. Usually, these pigments are located in the thylakoid membranes of the chloroplast. However, in some cases they may be found in the stroma or cytoplasm, incorporated into lipid vesicles [START_REF] Catarina Guedes | Microalgae as Sources of Carotenoids[END_REF]. On average, carotenoids represent 0.1 to 0.2% DW [START_REF] Spolaore | Commercial applications of microalgae[END_REF]. However, under unfavourable conditions, these pigments can be expressed up to 12% in the phylum Chlorophyta [START_REF] Edward | Phycology[END_REF].

With more than 600 representatives, carotenoids form the most diverse and widespread class of pigments [START_REF] Gong | Carotenoids from microalgae: A review of recent developments[END_REF][START_REF] Ejovwokoghene | Potential biotechnological application of microalgae: a critical review[END_REF]. Most of them share a common chemical structure composed of a 18-carbon conjugated double bond chain with two hexacarbon rings at each end (Figure 1.7) [START_REF] Ejovwokoghene | Potential biotechnological application of microalgae: a critical review[END_REF]. Within this large class, carotenoids are divided into two groups: carotenes and xanthophylls. The former are oxygen-free hydrocarbons such as α-carotene and β-carotene, while the latter are oxygenated derivatives of carotenes (lutein, violaxanthin, zeaxanthin, fucoxanthin, astaxanthin, among others) [START_REF] Edward | Phycology[END_REF]. To illustrate this, the Figure 1.7 details the chemical structure of the main carotenoids found in green microalgae.

In general, the synthesis of xanthophylls begins with the synthesis of carotenes, notably with α-carotene and β-carotene. As a matter of fact, lutein is synthesized from α-carotene while β-carotene is the precursor of zeaxanthin, canthaxanthin or astaxanthin [START_REF] Gong | Carotenoids from microalgae: A review of recent developments[END_REF]. During the synthesis of xanthophylls, the oxygen present can be brought into the chemical structure via different chemical groups: hydroxyl groups (lutein and zeaxanthin), ketone groups (canthaxanthin) or a combination of different chemical groups as for astaxanthin (Figure 1.7) [START_REF] Catarina Guedes | Microalgae as Sources of Carotenoids[END_REF].

Despite this apparent high diversity, less than 30 carotenoids play an important role in photosynthesis [START_REF] Gong | Carotenoids from microalgae: A review of recent developments[END_REF]. According to their function, the latter are classified into two groups: * Primary pigments: These are carotenoid pigments whose main function is to absorb light in the green area of the light spectrum, inaccessible to chlorophylls. They are then able to transfer the absorbed energy to the chlorophylls, thus broadening the absorption light spectrum [START_REF] Catarina Guedes | Microalgae as Sources of Carotenoids[END_REF][START_REF] Gong | Carotenoids from microalgae: A review of recent developments[END_REF][START_REF] Ejovwokoghene | Potential biotechnological application of microalgae: a critical review[END_REF]. These pigments are therefore structural and functional components of the photosynthetic apparatus. Chemical structures and absorption spectra of the most common carotenoids found in microalgae * Secondary pigments: These are pigments whose major role is the photoprotection of the photosynthetic apparatus [START_REF] Gong | Carotenoids from microalgae: A review of recent developments[END_REF][START_REF] Ejovwokoghene | Potential biotechnological application of microalgae: a critical review[END_REF][START_REF] Demmig | The role of xanthophyll cycle carotenoids in the protection of photosynthesis[END_REF]. Unlike primary carotenoids, secondary carotenoids are punctually produced in high amounts and encapsulated in lipid droplets in the cytoplasm. When cells are exposed to specific unfavourable environmental stimuli (nutrient deficiencies, extreme temperatures, high light intensity, high salinity, oxydative stress, etc.), they modify their carotenoid metabolism. As a result, they overproduce secondary pigments as an adaptive response to environmental stresses [START_REF] Catarina Guedes | Microalgae as Sources of Carotenoids[END_REF][START_REF] Gong | Carotenoids from microalgae: A review of recent developments[END_REF][START_REF] Gouveia | Microalgae -source of natural bioactive molecules as functional ingredients[END_REF].

In a nutshell, there is a wide variety of pigments, each of which has a role in the photosynthetic mechanism. Depending on their functions, they are located at different places in the cell. Chlorophyll a is the key pigment in photosynthesis. Consequently, it is found in the reaction center (RC) where light energy is converted into chemical energy. This chlorophyll is also found along with chlorophyll b and carotenoids in structures called antennae or Light-Harvesting Complexes (LHCs). The latter, located on the periphery, collect and transfer light energy to the reaction center. These accessory pigments have a dual role. On the one hand, they broaden the absorption spectrum of chlorophyll a and transport the absorbed energy to the reaction center where the chemical conversion of energy takes place. On the other hand, under certain conditions, they can also be used to protect the cell from the harmful effects of light through photoprotection and photoacclimation mechanisms [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. These mechanisms will be discussed in the Section 1.3.

Cell chloroplast structure

Photosynthesis reactions take place within specialized ellipsoid-shaped cellular organelles called chloroplasts. Like mitochondria, chloroplasts are semi-autonomous organelles that have their own genetic material. Owing to the presence of photosynthetic pigments in their structure, chloroplasts are the central cellular components of photosynthesis. Within them, light is captured and all stages of photosynthesis are carried out.

The cellular structure of a chloroplast is generally delimited by a double membrane separated by a space called intermembrane space (Figure 1.8)1 . The outer membrane is a semi-porous lipid bilayer very permeable to ions and metabolites. The inner membrane is a lipid bilayer composed of unsaturated fatty acids that ensure the fluidity of the membrane, and pigments (chlorophyll and carotenoids) often associated with proteins. The latter forms a selective permeability barrier whose main function is to regulate the transport of metabolites within and outside the chloroplast. For this purpose, on the surface of the inner membrane, there are specific membrane proteins called porins that prevent the passage of substances with a molecular weight greater than 10 kDa [START_REF] Heldt | Plant biochemistry[END_REF]. In addition, this membrane delimits the inner part of the chloroplast containing an aqueous matrix rich in protein and enzyme called stroma.

Inside the stroma float various cellular elements, such as ribosomes, pyrenoid, eyespot, starch grains and thylakoids. The latter is a highly organized membrane system arranged in flattened round bags containing photosynthetic pigments. Its origin comes from the invagination of the internal membrane of the chloroplast [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Heldt | Plant biochemistry[END_REF]. Each thylakoid contains a space called intra-thylakoid space or lumen. Its main function is to partition the protons released during photosynthesis to form a gradient between thylakoid lumen and chloroplast stroma [START_REF] Heldt | Plant biochemistry[END_REF]. The thylakoid membranes are very tightly joined together and form piles called granal thylakoids or grana (Figure 1.8). They are connected to each other by tubular structures which are unstacked thylakoids (stromal thylakoids) forming a continuous compartment.

Thylakoids, and in particular thylakoid membranes, play a major role in the process of photosynthesis. Their composition as well as their function are detailed hereinafter.

Photosynthetic membranes of thylakoids

During photosynthesis, light reactions take place in thylakoid membranes while carbon dioxide is fixed in the chloroplast stroma. To carry out these photochemical reactions, protein complexes inserted within the thylakoid membrane play a key role in the harvest of light [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. In particular, thylakoid membranes contain five major protein complexes: LHCs, Photosystem I (PSI), Photosystem II (PSII), cytochrome b 6 /f complex and ATP synthase or ATPase. Together, these membrane protein complexes use the energy of light to drive photophosphorylation, resulting in the production of NADPH and ATP [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. Some of these complexes are protein-protein complexes (cytochrome b 6 /f complex and ATP synthase) while PSI, PSII and LCHs are proteins associated with pigments forming protein-pigment complexes. Depending on the functions of these complexes, their distribution within the thylakoid membrane is different. As such, Figure 1.9 illustrates a schematic representation of the spatial location of the different actors involved in the light reactions of photosynthesis within the membranes of thylakoids. Succinctly, PSI and PSII are found buried in the thickness of the thylakoid membrane in different regions. In general, chloroplasts contain more PSII than PSI and the stochiometric ratio PSII/PSI is often about 1.5. However, this ratio varies depending on environmental conditions [START_REF] Melis | Spectroscopic methods in photosynthesis: photosystem stoichiometry and chlorophyll antenna size[END_REF]. PSII, as well as its light harvesting antenna, are mainly directed towards the inside of the grana membranes and thus not exposed to the stroma. Contrarily, PSI and ATP synthase are located in the periphery of the membranes facing the chloroplast stroma. The cytochrome b 6 /f complex is distributed evenly throughout thylakoid membranes. Owing to the PSI and PSII layout in the thylakoid membrane, electron transfer agents are required to shuttle electrons between them. These carriers are pheophytin, plastoquinone and plastocyanin.

In order to better understand the process of photosynthesis, the structure and composition of each actor is first described. This description is essential to make the link between the spatial organization of the different actors in the membrane and their role in photosynthesis, which is detailed more extensively afterwards.

Photosystem II

PSII is the first complex to intervene in light reactions of photosynthesis. Briefly, it allows the absorption of photons whose energy excites electrons. The latter are transferred within it through a set of reactions supported by co-factors up to a plastoquinol molecule. In green microalgae, about 70-80% of PSII are located towards the inside of the grana membranes of thylakoids. The remaining part of PSII is exposed towards the stroma [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF].

By taking a closer look at its structure, PSII is a large protein complex, comprising about 20 subunits. It is formed of a heterodimer whose composition of each monomer contains three protein domains: the reaction center (RC), the core antenna and the Oxygen Evolving Complex (OEC) [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. A simplified representation of the structure of PSII with its LHCIIs is shown in Figure 1.10. Today, it is possible to obtain very fine resolutions of these structures down to 3.37 Å. They show the quaternary structure, i.e. how each component of the complex is spatially arranged in relation to the others. For the interested reader, they are available in

Reaction center (RC)

At the center of the PSII structure is the reaction center. This domain is the heart of the photosynthetic process. It is structurally symmetrical and consists of a heterodimer of two homologous proteins, D1 and D2, and the α and β subunits of cytochrome b 559 [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Minagawa | Structure, function and assembly of Photosystem II and its light-harvesting proteins[END_REF][START_REF] Raymond | Structural determination of the large photosystem II-light-harvesting complex II supercomplex of Chlamydomonas reinhardtii using nonionic amphipol[END_REF]. In its center, the RC contains a special chlorophyll a pair. These two molecules are different from the others because they are the focus point towards which the collected light energy converges. Their maximum absorption wavelength is 680 nm. This property is used to designate the name of the reaction center of a PS. Hence the PSII reaction center is called P 680 . Proteins D1 and D2 also contain the electron transport intermediates essential to the photosynthesis process, namely, tyrosine Z, iron-sulfur (Fe-S) centers, pheophytin and primary and secondary quinone acceptors called plastoquinone Q A and Q B , which are contained by protein D2 and D1, respectively [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF][START_REF] Minagawa | Structure, function and assembly of Photosystem II and its light-harvesting proteins[END_REF].

Core antenna

On either side of the RC are the proteins CP43 and CP47 responsible for transferring light energy from the peripheral antennas to the reaction center. Together with other low molecular weight subunits, they form the core antenna. The combination of RC and core antenna gives rise to the core complex, containing on average 37 molecules of chlorophyll a [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF][START_REF] Melis | Excitation Energy Transfer: Functional and Dynamic Aspects of Lhc (cab) Proteins[END_REF].

Oxygen evolving complex (OEC)

The complex, bound to proteins D1 and D2, is located on the lumenal side of the thylakoid membrane. It consists of different proteins. It contains a total of 4 manganese atoms, as well as calcium and chlorine which are co-factors involved in water splitting [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF].

Photosystem I

In the electron transport chain, PSI comes after the cytochrome b 6 /f complex on which it depends to recover electrons from the plastocyanin. It is also capable of absorbing photons, whose energy is converted into electrochemical energy within it, just like PSII. All this process leads to the formation of NADPH.

Similar to PSII, the PSI of green algae is composed of a complex core on which an auxiliary antenna (LHCI) is grafted [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF][START_REF] Melis | Excitation Energy Transfer: Functional and Dynamic Aspects of Lhc (cab) Proteins[END_REF][START_REF] Su | Antenna arrangement and energy transfer pathways of a green algal photosystem-I-LHCI supercomplex[END_REF][START_REF] Suga | Structural variations of photosystem I-antenna supercomplex in response to adaptations to different light environments[END_REF]. The PSI core is an intermembrane complex with multiple subunits including 14 protein subunits noted PsaA to PsaL and PsaN to PsaO [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF][START_REF] Su | Antenna arrangement and energy transfer pathways of a green algal photosystem-I-LHCI supercomplex[END_REF][START_REF] Suga | Structural variations of photosystem I-antenna supercomplex in response to adaptations to different light environments[END_REF][START_REF] Qin | Structure of a green algal photosystem I in complex with a large number of light-harvesting complex I subunits[END_REF]. Among these subunits, the two largest, PsaA and PsaB, are located in the center of the complex. These major intrinsic subunits are surrounded by 12 minor polypeptides (PsaC-L and PsaN-O) which are shown in Figure 1.11. All together, they represent the reaction center to which the light energy is directed. The PsaA and PsaB set contains about 100 molecules of chlorophyll a and about 20 molecules of β-carotene [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF]. At its center, there is a special pair of chlorophyll a with a peak wavelength of 700 nm. This complex also carries the co-factors necessary for electron transport in the photosynthetic chain. In particular, two electron acceptors, A0 (chlorophyll a molecule) and A1 (phylloquinone), as well as a primary group 4Fe-4S (Fx) are found [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF].

Light harvesting antennae

The light harvesting complex (LCH), also known as antenna complexe, is an important network of protein molecules and photosynthetic pigments embedded in the thylakoid membrane of microalgae. Each photosystem has its own LHC located within its periphery. This complex is responsible for absorbing photons and transferring their energy by Förster resonance to the reaction centers of each photosystem to concentrate the energy.

Typically, per complex, 60 to 750 chlorophylls a and b are present depending on the organism and light conditions [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF][START_REF] Melis | Excitation Energy Transfer: Functional and Dynamic Aspects of Lhc (cab) Proteins[END_REF][START_REF] Sarada | Development of the lightharvesting chlorophyll antenna in the green alga Chlamydomonas reinhardtii is regulated by the novel Tla1 gene[END_REF]. Thus, the majority of chlorophylls are found in the antennae and not in the reaction centers. In addition to chlorophylls, LHCs also contain additional accessory pigments, including primary carotenoids such as β-carotene, lutein, 9'cis-neoxanthin or violaxanthin [START_REF] Raymond | Structural determination of the large photosystem II-light-harvesting complex II supercomplex of Chlamydomonas reinhardtii using nonionic amphipol[END_REF]. The function of these pigments is to collect photons in the green window located between the abortion maxima of the chlorophylls. Another important function is also to act as antioxidants to prevent the photooxidative damage of chlorophyll molecules forming harmful triplet state.

However, the composition of each antenna differs according to the supercomplex to which it belongs. As a result, their names are defined depending on whether they are associated with PSII (LHCII) or with PSI (LCHI). However, their function remains the same. These complexes absorb the light with very high effectiveness and allow the extension of antennae area for collecting photons and funnelling energy to PS. Both of them contain the necessary pigments for this purpose, namely chlorophylls and carotenoids.

LHCII associated with PSII

In green microalgae, the structure of the LHC has mainly been studied in Chlamydomonas reinhardtii. In these organisms, the LHCII is formed by three major domains as follows (Figure 1.12a) [START_REF] Pessarakli | Handbook of photosynthesis[END_REF].

Chapter 1. Background

The first is the core antenna or internal antenna directly associated with the PSII reaction centers. It is made up by the CP43 and CP47 proteins. The latter are capable of binding 13-14 and 14-17 chlorophyll a molecules, respectively [START_REF] Sarada | Development of the lightharvesting chlorophyll antenna in the green alga Chlamydomonas reinhardtii is regulated by the novel Tla1 gene[END_REF]. In addition, 3 β-caroten are assigned to CP43 and 2 to CP47.

Next is the minor antenna formed from the CP29 and CP26 monomeric proteins. The latter are able to bind respectively 9 and 13 chlorophylls and 2 or 3 xanthophylls (lutein, neoxanthin and violaxanthin) [START_REF] Pessarakli | Handbook of photosynthesis[END_REF]. This complex is located at the interface between the core antenna and the major antenna. Its main mission is to carry the energy from the major antenna to the reaction center.

The furthest out of the PSII is the major antenna consisting of LHCII major proteins. There are nine major LHCII proteins called Lchb1 to 9 organized in trimers [START_REF] Janik | Light-induced formation of dimeric LHCII[END_REF][START_REF] Kawakami | Four distinct trimeric forms of light-harvesting complex II isolated from the green alga Chlamydomonas reinhardtii[END_REF]. Each monomer is composed of a polypeptide of about 232 amino acid associated with photosynthetic pigments. It is capable of binding a total of 18 pigments: 14 chlorophylls (8 molecules of chlorophyll a and 6 molecules of chlorophyll b), and 4 xanthophyll molecules (1 violaxanthin, 1 neoxanthin and 2 luteins) [START_REF] Pessarakli | Handbook of photosynthesis[END_REF][START_REF] Janik | Light-induced formation of dimeric LHCII[END_REF]. Depending on the composition of these trimers, the affinity of their association with the PSII core is different. Three types of degree of trimers' association have been reported: strong (S), moderate (M), and loose (L) [START_REF] Pessarakli | Handbook of photosynthesis[END_REF]. The major antenna complex is responsible for collecting photons from light and transferring the excitation energy to the minor antennae. The set of major and minor antennae is usually denoted as LHCII complex or outer antenna. Spatial reconstructions revealed the presence of 3 trimer LHCII major proteins and 2 minor antenna on each side of the PSII core in green microalgae [START_REF] Raymond | Structural determination of the large photosystem II-light-harvesting complex II supercomplex of Chlamydomonas reinhardtii using nonionic amphipol[END_REF][START_REF] Pessarakli | Handbook of photosynthesis[END_REF]. Due to different affinities, these mobile complexes are capable of lateral migration in thylakoid membranes, depending on their phosphorylation state [START_REF] Pessarakli | Handbook of photosynthesis[END_REF]. In addition to the trimers directly linked to the PSII, it is possible to find additional trimers whose purpose is to increase the light-capture capacity of the photosystem. These are dispersed on the thylakoid membrane at the periphery of the PSII-LHCII supercomplexes and are capable of transferring the collected light energy directly to the LHCII associated with the PSII. 1.2. Photosynthesis in green microalgae 25 Consequently, microalgae are able to modulate the size of their LHCs according to the irradiance to which they are exposed. This change is dependent on the species, but also on growth conditions and other environmental stresses [START_REF] Melis | Excitation Energy Transfer: Functional and Dynamic Aspects of Lhc (cab) Proteins[END_REF].

LHCI associated with PSI

The LHC complex group that serves PSI is known to be larger than that of higher plants [START_REF] Suga | Structural variations of photosystem I-antenna supercomplex in response to adaptations to different light environments[END_REF][START_REF] Kubota-Kawai | Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii[END_REF]. This complex is different from the LHCII in composition and spatial arrangement [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF].

Unlike the LHCII, the LHCI associated with PSI of green microalgae contains only one type of highly homologous major protein (Lhca). Yet the exact number as well as the localization of these proteins remains controversial in literature ranging from 4 to 18 different proteins per PSI core complex according to studies [START_REF] Suga | Structural variations of photosystem I-antenna supercomplex in response to adaptations to different light environments[END_REF][START_REF] Pessarakli | Handbook of photosynthesis[END_REF][START_REF] Kubota-Kawai | Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii[END_REF]. This structural variability can be explained by a high flexibility of LHCI (modulated by environmental conditions). Still the vast majority of models reported in the literature propose a spatial organization of proteins on only one side of the PSI core complex. These proteins would be agglomerated in the shape of a half-moon on two layers called the LHCI belt. Each layer would be composed of 4 to 5 Lhca proteins [START_REF] Suga | Structural variations of photosystem I-antenna supercomplex in response to adaptations to different light environments[END_REF][START_REF] Pessarakli | Handbook of photosynthesis[END_REF][START_REF] Kubota-Kawai | Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii[END_REF][START_REF] Takahashi | Comparison of the Subunit Compositions of the PSI-LHCI Supercomplex and the LHCI in the Green Alga Chlamydomonas reinhardtii[END_REF]. The quaternary structure of the PSI-LCHI supercomplex is available in Figure A.2 in the Appendix A.

Very recently, Kubota-Kawai et al. [START_REF] Kubota-Kawai | Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii[END_REF] proposed a new pattern. The latter would include the existence of 9 different types of major proteins, like LHCII. These major proteins, noted Lhca1-9, are present as monomers. They would each be able to bind with 14 to 17 chlorophyll molecules [START_REF] Su | Antenna arrangement and energy transfer pathways of a green algal photosystem-I-LHCI supercomplex[END_REF] as well as with 2 to 3 carotenoids (lutein, violaxanthin, β-carotene) [START_REF] Pessarakli | Handbook of photosynthesis[END_REF]. In contrast to the LHCII pigment composition, LHCI contains more chlorophyll a molecules than chlorophyll b with a ratio chla/chlb of about 3.5 [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF]. Regarding the assembly of these proteins, the model depicts a PSI-LCHI supercomplex comprising a total of 10 major Lhca proteins. Eight of them form a half-ring structure located near the PsaF and PsaJ polypeptides on one side of the PSI core. The LHCI belt is also organized in two parallel layers, each comprising 4 Lhca proteins. On this belt, the authors showed the presence of two copies of the same protein, Lcha1, each on a different layer. Finally, 2 extra Lhca proteins were also found between the PsaG and PsaH polypeptides of the PSI core complex (Figure 1.12b). Other scholars have also agreed on this LHCI composition and spatial arrangement using the same study model microalga Chlamydomonas reinhardtii [START_REF] Su | Antenna arrangement and energy transfer pathways of a green algal photosystem-I-LHCI supercomplex[END_REF]. However, this study also revealed a higher occurrence of LHCI with 8 subunits instead of 10. The presence of the 8-subunits LHCI would be mainly due to a disintegration phenomenon during the purification process while the 10-subunits LHCI would be the predominant form [START_REF] Suga | Structural variations of photosystem I-antenna supercomplex in response to adaptations to different light environments[END_REF].

Cytochrome b 6 /f complex

PSII and PSI are linked via the cytochrome b 6 /f complex. The purpose of this complex is to mediate the tranfer of electrons between the two reaction centers while pumping protons across the thylakoid membrane. Electrons from PSII are carried by plastoquinol to the cytochrome b 6 /f complex where they are removed and transferred to plastocyanin [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Edward | Phycology[END_REF][START_REF] Stroebel | An atypical haem in the cytochrome b 6 f complex[END_REF]. Commonly called cytochrome b 6 /f complex, it takes its name from its composition. It consists of two symmetrical copies of four main subunits: cytochrome b 6 , cytochrome f, Rieske iron-sulphur protein as well as subunit IV (Figure 1.13) [START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Stroebel | An atypical haem in the cytochrome b 6 f complex[END_REF]. In addition to these components, four small peptides (PetG and PetL-M) are present, as well as one chlorophyll and one β-caroten molecules whose function is still unknown [START_REF] Edward | Phycology[END_REF][START_REF] Stroebel | An atypical haem in the cytochrome b 6 f complex[END_REF].

CP43 CP47

LUMEN

STROMA

Subunit IV

Rieske protein Cytochrome f

Cytochrome b6

Heme-b

Heme-b

Heme-c

Figure 1.13: Schematic representation of the structure of the cytochrome b 6 /f complex inspired from [START_REF] Heldt | Plant biochemistry[END_REF]. For the sake of simplicity, only one copy of each subunits is presented.

Cytochromes are proteins on which heme structures can bind. Hemes are tetrepyrroles with an iron central atom [START_REF] Heldt | Plant biochemistry[END_REF]. Thanks to the ability of this atom to change between the oxidized states Fe 3+ and Fe 2+ , cytochromes can function as one-electron carriers. Cytochrome b 6 is linked to a total of 3 hemes: 2 hemes of type b, and one heme of c. The function of the latter has not yet been elucidated. Thus, it does not appear on the Figure 1.13. On this cytochrome, two binding sites for PQH 2 /PQ are present: one in the stromal region, and one in the lumen region. Close to the cytochrome b 6 is the Rieske protein that crosses the membrane and protrudes into the lumen. This protein contains a 2Fe-2S group within it. Adjacent to it, the cytochrome f is found with a structure similar to that of the Rieske protein. This cytochrome has a single heme c-type group, as well as a binding group allowing the reduction of PC 2+ to PC. Finally, the subunit IV lacks a prosthetic group but it is necessary for the full catalytic activity of the complex.

ATP synthase

The ATP synthase is the last link in the chain. Through subunit conformational changes, the enzyme is capable of catalyzing the regeneration of ATP from ADP. It is a complex composed of two domains: a highly hydrophobic mobile part (F 0 ) located in the thylakoid membrane, linked to a fixed hydrophilic part (F 1 ) which protrudes on the stromal side of the thylakoid membrane [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Heldt | Plant biochemistry[END_REF] (Figure 1.14). These two areas are connected together by a lateral and central connection made by means of two stems.

The F 0 domain consists of 4 main components: subunit a, dimer b and subunit c. The latter is itself composed of 12 to 14 copies forming a cylinder which crosses the membrane. The F 1 domain consists of 3 α subunits, 3 β subunits, 1 γ subunit, 1 ε subunit and 1 δ subunit. The α and β subunits have an alternating arrangement, forming 3 α-β subunits.

It is on these subunits, particularly the β subunit, that the adenosine nucleoside binds to form ATP [START_REF] Heldt | Plant biochemistry[END_REF].

LUMEN

Light driven reactions

During photosynthesis, light reactions take place on the thylakoid membranes of the chloroplast. All this process starts with the capture of photons by a photosynthetic pigment. These photons then pass through a chain of redox reactions, called the electron transport chain, to finally produce the biochemical reductant, NADPH, and the chemical energy, ATP, necessary for the assimilation of inorganic carbon [START_REF] Melis | Excitation Energy Transfer: Functional and Dynamic Aspects of Lhc (cab) Proteins[END_REF]. This set of reactions, also called photophosphorylation, can be expressed as a chemical equation (Eq. 1.4) [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]:

2H 2 O + 2N ADP + + 3ADP + 3P i → 2N ADP H + 2H + + 3AT P + O 2 (1.4)
The main role of light reactions of photosynthesis is to provide sufficient chemical energy for the assimilation and conversion of inorganic carbon. Oxygen is released as a by-product of the reaction [START_REF] Lunka | Effects of flashing light-emitting diodes on algal biomass productivity[END_REF]. This highly endergonic process requires the input of light as energy source.

Another representation widely used by the community to represent the set of reactions taking place in the presence of light is the Z-scheme. It gets its name because of its "Z" shape (Figure 1.15). The advantage of this representation is that it highlights both the qualitative energy requirements (redox potentials) of each stage in the production of NADPH and ATP and the actors involved. It is thus understandable that the two photosystems, placed in tandem, work simultaneously. The electron transport between Chapter 1. Background these two photosystems is carried out by the cytochrome b 6 /f complex and assisted by mobile carriers [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. All these steps, represented by boxes in the diagram, are detailed below.

Electron

Trapping of light: funneling of excitation from the antenna system towards the reaction center

When a photon strikes an electron of a pigment molecule with an energy corresponding to the electron transistion energy of the pigment, light energy is absorbed. Physically, this corresponds to the excitation of the electron, whose captured energy allows it to move to a higher energy state.

Chlorophyll, the flagship pigment of photosynthesis, is able to absorb light energy in two regions of the visible spectrum: the blue region and the red region. When a photon is absorbed, the chlorophyll molecule goes into an excited state. To return to its ground state, the pigment uses different pathways depending on the degree of energy of the absorbed photon. If the pigment receives a short wavelength blue photon, the absorbed energy is capable of creating an excited state of greater energy than the absorption of light in the red region. This state is called the second excited state. In this case, the excited molecule is unstable and very quickly gives up the extra energy in the form of heat through rotation and vibration. The molecule then falls into a lower excited state similar to that of a molecule that absorbs red light, of lower energy. This state is called the first excitation state or singlet [START_REF] Heldt | Plant biochemistry[END_REF]. This electronic transition phenomenon is described in Figure 1. [START_REF] Sosik | Automated taxonomic classification of phytoplankton sampled with imaging-in-flow cytometry[END_REF]. The return of the molecule to its low-energy ground state requires the release of the remaining energy. To do this, the excited electon can transfer the excess energy to another 1.2. Photosynthesis in green microalgae 29 pigment molecule sufficiently close, by electromagnetic interractions. This is called Förster Resonance Energy Transfer (FRET). The success of this transfer depends mainly on the distance between the donor molecule and the acceptor molecule. The architecture of the LHC is precisely organized to allow such a transfer from one molecule to another. It is performed randomly in the direction of increasing wavelengths (decreasing energy) until reaching the RC where the energy will be stored in electrochemical form (Figure 1.17). To ensure proper energy efficiency, each energy transfer step is performed very rapidly (a few femtoseconds) since the electronic energy can only be stored fleetingly by the molecules in the excited state (a few nanoseconds) [START_REF] Fleming | Design principles of photosynthetic light-harvesting[END_REF][START_REF] Mirkovic | Light Absorption and Energy Transfer in the Antenna Complexes of Photosynthetic Organisms[END_REF]. Thus, the energy transport chain from the captured photon to the reaction center takes place on a picosecond time scale [START_REF] Heldt | Plant biochemistry[END_REF].

Energy

Second excitation state

Frist excitation state

Triplet

Heat

Heat Phosphorescence Fluorescence

Chemical work

Transfer (FRET)

Red photon absorption Blue photon absorption

Ground state

ROS

Figure 1.16: Schematic representation of excitation sates of chlorophyll a and their return to ground state inspired from [START_REF] Heldt | Plant biochemistry[END_REF]. The different levels of rotational and vibration energy of electrons are represented by thin lines and the ground states by solid lines

Transfer of electrons through PSII: photo-induced charge separation

According to the mechanism detailed above, the light energy is finally channeled at the reaction center P 680 . More precisely, this energy is transferred to the electrons of the chlorin head of the special pairs of chlorophyll a. As a consequence, each of the chlorophyll molecules rises to the singlet energy state. The return of the pigments to their ground state can be achieved in different ways as follows (Figure 1.16) [START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF]. The excited state of the special pair of chlorophylls is of lower energy than all other upstream photocollecting pigments. Unlike all other pigments, they trap the energy transferred by other molecules and deactivate themselves by yielding an electron to pheophytin which is the primary electron acceptor in the electron transport chain of photosynthesis. This mechanism is called photo-induced charge separation. It represents the beginning of the flow of electrons in photosynthesis in which light energy is converted into a chemical product. This conversion occurs at the picosecond time scale. * Heat: Another possible way of returning to the ground state is heat dissipation. It usually occurs when there is an overload of light. In this case, the excess energy is completely converted into heat by passing through different energy levels. This gradual conversion is achieved through rotation and vibration of chemical bonds within the pigment. * Fluorescence: The return of a chlorophyll molecule to the ground state can also be achieved in the form of fluorescence. Like heat dissipation, emission of fluorescent light occurs when light exceeds the energy requirements of photosynthesis. This emission is usually accompanied by a loss of energy as heat.

long half-life and therefore has harmful consequences on the cellular constituents. Chlorophyll in the triplet state is capable of reacting with oxygen putting it into a singlet state. This formed molecule is highly reactive (Reactive Oxygen Species -ROS).

Transfer of electrons through PSII: splitting of water

Continuing the photochemical route, once the electron is released to the pheophytin by each special pair, P 680 is in a highly oxidized state denoted P + 680 . To replace the donated electrons by the chlorophyll molecules, P 680 has the ability to split a water molecule thanks to its high oxidizing power. Water thus plays the role of electron donor to make up the deficit. In this regard, the splitting of a water molecule releases oxygen, protons as well as electrons [START_REF] Raymond | Structural determination of the large photosystem II-light-harvesting complex II supercomplex of Chlamydomonas reinhardtii using nonionic amphipol[END_REF]. A minimal need of 2 water molecules and 4 photons are necessary for the RC to recover the 4 electrons donated (Equation 1.5) [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF]. This requirement was notably highlighted by the pioneering work of Robert Emerson [START_REF] Emerson | THE PHOTOCHEMICAL REACTION IN PHOTOSYNTHESIS[END_REF].

2H 2 O -→ O 2 + 4H + + 4e - (1.5)
The water splitting takes place within the OEC closely associated with the PSII. The recovery of electrons by P 680 is done indirectly through intermediaries (Figure 1.18). This process is quite complex. In a very simplified manner, for each reduction of a chlorophyll molecule, P 680 withdraws an electron stored by the four manganese atoms via tyrosine. The latter then successively go through four oxidation states (S1 to S4), releasing one electron each time. In their last oxidized state (S4), the manganese atoms acquire the capacity to oxidize two molecules of water allowing them to recover the four electrons previously released. They thus return to their S0 state by releasing the four protons and the oxygen molecule coming from the breaking of the water molecules in the thylakoid lumen [START_REF] Minagawa | Structure, function and assembly of Photosystem II and its light-harvesting proteins[END_REF]. Hence, electrons from the OEC are successively yielded to each of the chlorophylls of P 680 [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF]. The replacement of the electron missing from the chlorophyll allows it to return to its ground state. Once recovered, the molecule can be excited again by the energy coming from another photon.

Although the oxygen released as a co-product plays no role in further photosynthesis, the protons released play an essential role in the rest of the light reactions. Their function is described in more detail shortly afterwards.

Transfer of electrons through PSII: flow of electrons

Still following the photochemical pathway, the electron transferred to the pheophytin by chlorophyll a is then transferred to the primary acceptor, plastoquinone Q A (Figure 1.18). The transfer continues on to the secondary acceptor, plastoquinone Q B [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF]. This plastoquinone has the capacity to accept 2 electrons and 2 protons, successively, generating a plastoquinol (or hydroplastoquinol) molecule (QH 2 ) which is the latest product generated by the PSII. The latter is released into the lipid bilayer of the thylakoid membrane and diffuses towards the Q pool which acts as both energy and substrat reserve [START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Minagawa | Structure, function and assembly of Photosystem II and its light-harvesting proteins[END_REF]. This succession of reactions within the PSII runs on the millisecond time scale due to the limitation imposed by the water splitting mechanism which takes place over a period of 1 to 2 millseconds [START_REF] Fleming | Design principles of photosynthetic light-harvesting[END_REF][START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF]. Moving the quinol (QH 2 ) to the quinone pool leaves the Q B site empty. This site left empty is quickly replaced by an oxidized quinone from the pool [START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF]. The QH 2 released in the pool will then undergo a series of oxidation and reduction reactions between the quinol and quinone forms, called the Q cycle, resulting in a movement of protons through the lipid bilayer. These reactions are made possible by the support of the cytochrome b 6 /f complex which allows the passage of electron flow between PSII and PSI. This cycle is carried out in two successive steps depicted in the Figure 1.19 [START_REF] Heldt | Plant biochemistry[END_REF].

In the first step of the Q cycle, two molecules of QH 2 enter the cycle at the first lumenal binding site. They are oxidized by the iron-sulphur center of the Rieske protein. Due to the high positive redox power, it allows the removal of one electron from each QH 2 , leaving the molecule in an unstable state called semiquinone (QH • ). To regain their stability, the molecules will yield an additional electron to the b-heme of cytochrome b 6 . This step allows, for the 2 QH 2 molecules entering the cycle, to release 4 electrons and to regenerate two Q molecules which return to the Q pool. These reactions are accompanied by the release of 4 protons in the lumen. Of these four electrons, two are transferred to cytochrome f via the Rieske protein where they reduce two plastocyanin (PC) molecules to continue the linear electron transfer chain to P 700 . The other two electrons migrate through the two heme groups of cytochrome b 6 to the second stromal binding site.

The second step is the complete reduction of a Q molecule to QH 2 at the second stromal binding site. This reduction is achieved thanks to the electrons recovered from the first stage of the cycle and the uptake of 2 protons from the stroma. The quinol is In total, the Q cycle allows the uptake of 2 protons from the stroma while releasing 4 protons in the lumen. This proton pump allows the oxidation of two molecules of QH 2 into Q and the reduction of one molecule of Q into QH 2 returning to the Q pool as well as the reduction of 2 molecules of plastocyanine. Moreover, this cycle is conditioned by the reaction speed of the oxidation of the quinol. Indeed, this step takes place between 3 to 5 milliseconds, also constituting the limiting step of the linear electron transfer chain [START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF].

Q B CP 43 CP 47 2 PC 2+ 2 PC + 2 H + LUMEN STROMA 2 Q QH 2 2 QH 2 4 H + Q pool Q 2 e -

Transfer of electrons through PSI: making of NADPH

The PSI is capable of collecting light energy via its photosynthetic antennas within a few femtoseconds and routing it to its reaction center by the same mechanism as the PSII (Figure 1.20). The excitation energy is trapped in the PSI at the P700 reaction center causing a charge separation [START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF][START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF]. The special pair of chlorophylls a yields an electron to the first electron acceptor, another chlorophyll a molecule (A0), resulting in an oxidized state. It is then reduced by the plastocyanin from the cytochrome b 6 /f complex which has diffused through the thylakoid lumen to bind to a positively charged binding site located on the F subunit of PSI. Once its electron yielded to the special pair of chlorophyll a, the oxidized form of the resulting plastocyanin returns to the cytochrome b 6 /f complex to be further reduced. Thus, during photosynthesis, the electron transfer reactions involve the cooperation of the two PS, operating in series, with electrons from the PSII serving to reduce the P700 of the PSI.

In the meantime, chlorophyll A0 transfers an electron to another electron acceptor, phylloquinone (A1). The latter has a similar role as Q A in PSII. It transfers its electron to a 4Fe-4S iron-sulfur center (Fx), which in turn transfers it to two other 4Fe-4S iron-sulfur [START_REF] Heldt | Plant biochemistry[END_REF] centers called F A and F B . The transfer of electrons from A0 to F B takes only a few picoseconds [START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF]. These centers reduce the ferredoxin which is the final electron acceptor of the chain. The latter is a protein with a 2Fe-2S center located at a positively charged binding site on the C subunit of PSI on the stromal side of the thylakoid membrane. It allows the reduction of NADP + to NADPH, a reaction catalyzed by ferredoxin-NADP reductase (FNR). The NADPH thus generated represents the final product of the electron transport chain of photosynthesis.

The rate of electron transport within the PSI is mainly controlled by the flow of electrons from the PSII through the cytochrome b 6 /f complex. Indeed, the transport of plastocyanine to PSI takes place on a scale of several microseconds [START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF].

ATP generation from chemiosmosis

Over the electron transport chain, successive redox reactions release protons into the thylakoid lumen. The build-up of hydrogen ions on the lumenal side creates an electrochemical gradient called the proton driving force. This is formed due to the difference in concentration (chemical potential) and the difference in charge (electrical potential) that the protons create across the thylakoid membrane. This proton imbalance provides the energy necessary to conduct the regeneration of ATP from ADP and inorganic phosphate via ATP synthase. This phenomenon is called chemiosmosis.

Each of the ATP synthase subunits passes successively through three states characterized by different affinity constants of the nucleosides binding site [START_REF] Heldt | Plant biochemistry[END_REF]. The loose state (L) for which the catalytic site has a low affinity with the nucleoside, the tight state (T) which corresponds to a closed conformation in which the catalytic site has a 1.2. Photosynthesis in green microalgae 35 high affinity, and the open state (O) which corresponds to the release of ATP (Figure 1.21). Consequently, at each moment, the 3 α-β subunits are in a different state. The L configuration allows the ADP to be loosely bound with a phosphate. The α-β subunit then switches to the T configuration which catalyzes the formation of ATP. Once the ATP is tightly bound, it is released by switching the subunit to an O-configuration. It is the flow of protons through the complex, driven by the proton motive force, that allows the rotation of the c subunit and the γ stem [START_REF] Heldt | Plant biochemistry[END_REF]. These movements lead to a concerted modification of the conformation of the three binding sites, which is responsible for the release of an ATP molecule. The complex is capable of approximately 130 revolutions per second. A complete rotation of ATP synthase requires about 14 protons to release 3 molecules of ATP [START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF]. The protons can then pass through the thylakoid membrane using this proton pump.

Summary of the light reactions

During this first part of photosynthesis, the photosynthetic pigments absorb the light energy and funnel it at the P 680 of the PSII thanks to the LHCs. The collected energy then passes through the linear electron transport chain. These successive oxidation-reduction reactions cause hydrogen ions to be pumped into the thylakoid lumen, forming an electrochemical gradient. Thanks to the proton driving force, the hydrogen ions cross the ATP synthase to reach the stroma in a process called chemiosmosis. This proton passage allows the release of ATP molecules. At the same time, PSI also absorbs photons, which leads to the regeneration of NADPH molecules through the cooperation of the two PS operating in series. The two types of molecules regenerated during the light reactions, ATP and NADPH, are then used in the second stage of photosynthesis for Calvin-Benson cycle reactions. All these light reactions as well as the characteristic times of the limiting steps involved are summarized in the Figure 1.22. main purposes: the synthesis of structural components in cell walls and the synthesis of energy storage components for metabolic processes. This conversion is achieved through the Calvin-Benson cycle. The latter is entirely enzymatic and unfolds in the order of ten to hundred microseconds [START_REF] Richmond | Handbook of Microalgal Culture: Biotechnology and Applied Phycology[END_REF][START_REF] Daniel | Efficiency of photosynthesis in continuous and pulsed light emitting diode irradiation[END_REF]. These reactions take place in the stroma of the chloroplasts. They can be broken down into 3 main steps schematically shown in Figure 1. 23 [42, 44, 74, 80].

Carbon reactions

Carbon fixation

The first step of the Calvin-Benson cycle consists of the incorporation of a CO 2 molecule into a 5-carbon molecule, ribulose 1,5-biphosphate (RuBP). This combination gives rise to a highly unstable 6-carbon intermediate compound. The latter splits rapidly to release two molecules of a 3-carbon compound: 3-phosphoglyceric acid (3-PGA). This reaction is catalyzed by an enzyme called RuBP carboxylase or RuBisCO. Thus, for each molecule of CO 2 that enters the cycle, 2 molecules of 3-PGA are produced.

Reduction of 3-PGA

In the second stage of the Calvin-Benson cycle, ATP and NADPH are used to convert 3-PGA molecules into glyceraldehyde-3-phosphate (G3P) molecules. This step can be divided into two parts involving two different reactions. First, each 3-PGA molecule is phosphorylated and receives a phosphate group from ATP to form a double-phosphorus molecule called 1,3-bisphosphoglycerate (BPG). This reaction is catalyzed with the enzyme phosphoglycerolkinase (PGK), leaving ADP as a by-product. In the second reaction, the BPG molecules are reduced due to the gain of electrons from NADPH. Thus, each BPG molecule receives 2 electrons from NADPH and loses one of its phosphate to give a 3-carbon compound called glyceraldehyde 3-phosphate (G3P). This reaction is catalyzed by the enzyme glyceraldehyde phosphodehydrogenase (GAPDH) and releases NADP + and inorganic phosphate (Pi) as a by-product.

Regeneration of ribulose 1,5-biphosphate (RuBP)

A small fraction of previously formed G3P is used for the production of carbohydrates. This fraction can either be directly converted into starch within the chloroplast, or it can be exported to the cell cytoplasm by specific transport. However, most of the G3P produced is returned to the cycle to regenerate RuBP. This is step is necessary for the incorporation of new CO 2 molecules. This regeneration process requires ATP and involves a complex set of reactions involving sugars with different carbon numbers (from 3 to 7 carbons).

In summary, to generate a G3P molecule that goes to carbohydrate synthesis, three CO 2 molecules must be incorporated into the Calvin-Benson cycle. When these 3 molecules enter the cycle, a total of 6 molecules of G3P are formed thanks to the support of 9 molecules of ATP and 6 molecules of NADPH [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Heldt | Plant biochemistry[END_REF]. Only one of the G3P molecules leaves the cycle for carbohydrate synthesis while the other five are used to regenerate 3 molecules of RuBP. Although Calvin-Benson cycle is entirely enzymatic, some of its central enzymes require light for regulation [START_REF] Buchanan | The carbon (formerly dark) reactions of photosynthesis[END_REF]. In addition, carbohydrate synthesis depends on ATP and NADPH produced in light reactions. As a result, the cycle is more likely to operate in the presence of light.

Photorespiration

In the carbon reactions of photosynthesis, CO 2 is fixed by carboxylation to RuBP to give rise to two 3-PGA molecules. These molecules are then reduced to G3P by NADPH and ATP provided through the light reactions of photosynthesis. This carboxylation reaction is catalyzed by RuBisCO which has a strong affinity for CO 2 . However, in its natural state, this enzyme also has a lower but considerable affinity for oxygen [START_REF] Reumann | Plant peroxisomes respire in the light: Some gaps of the photorespiratory C2 cycle have become filled-Others remain[END_REF]. This double affinity comes from the configuration of the active site of the enzyme. Oxygen and carbon dioxide are similar molecules in shape and chemical properties, although CO 2 is larger in size [START_REF] Berman | The Protein Data Bank[END_REF]. Consequently, an oxygen molecule can bind to the catalytic site instead of carbon dioxide. This is all the more true since in microalgae, Rubisco's selectivity for CO 2 is described as intermediate [START_REF] Michiel Van Lun | CO2 and O2 Distribution in Rubisco Suggests the Small Subunit Functions as a CO2 Reservoir[END_REF]. As a result, it is estimated that about one molecule in two, to one in four, is oxygenated rather than carboxylated for a standard air composition [START_REF] Reumann | Plant peroxisomes respire in the light: Some gaps of the photorespiratory C2 cycle have become filled-Others remain[END_REF][START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF].

The oxygenase activity of RuBisCO causes the oxidation of a RuBP molecule through the incorporation of an O 2 molecule to form a 3-carbon molecule, 3-phosphoglycerate (3-PGA) and a 2-carbon molecule, 2-phosphoglycolate (2-PG) [START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF][START_REF] Timm | The regulatory interplay between photorespiration and photosynthesis[END_REF]. The 3-PGA molecule returns directly to the Calvin-Benson cycle. By contrast, the 2-carbon molecule (2-PG) can neither enter the Calvin-Benson cycle, nor can it directly be converted into carbohydrates [START_REF] Reumann | Plant peroxisomes respire in the light: Some gaps of the photorespiratory C2 cycle have become filled-Others remain[END_REF]. To minimize the loss of carbon fixing, the 2-PG molecule will undergo a series of complex reactions known as photorespiration, to be recycled. This set of reactions involves the cooperation of three different cellular compartments: chloroplast, peroxisome and mitochondrion (Figure 1.24). Briefly, in the chloroplast, 2-PG is dephosphorylated to form a glycolate. This molecule is then transported to the perixosome where it is changed into a glyoxylate. This reaction is accompanied by the production of hydrogen peroxide which is rapidly detoxified in O 2 and 2 H 2 O by the enzyme catalase, already present in the perixosome. The glyoxylate is then transformed into glycine and reaches the mitochondrion. In this cell compartment, the conversion of glycine to serine releases one molecule of CO 2 and ammonia. The serine then returns to the perixosome to give glycerate which joins the chloroplast. Through the use of ATP, the latter compound allows the regeneration of a 3-PGA molecule [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF][START_REF] Timm | The regulatory interplay between photorespiration and photosynthesis[END_REF].

Using the photorespiratory pathway is an energy-costly metabolic process that reduces photosynthetic efficiency [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF]. On the one hand, it reduces the production rate of 3-PGA compared to the carboxylase activity of RuBisCO. Instead, it generates a two-carbon alternative product, 2-PG, of which only three-quarters is recycled in 3-PGA. Indeed, two molecules of 2-PG (2C) are needed to regenerate one molecule of 3-PGA (3C) while one molecule of CO 2 is released constituting a 25% loss of fixed carbon [START_REF] Reumann | Plant peroxisomes respire in the light: Some gaps of the photorespiratory C2 cycle have become filled-Others remain[END_REF][START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF]. On the other hand, photorespiration does not allow the regeneration of RuBP. Moreover, this process consumes 3.25 mol ATP and 2 mol NADPH per one oxygenated mole of RuBP which cannot be used in the Calvin-Benson cycle [START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF].

The diversion from photosynthesis to photorespiration depends mainly on the relative concentrations of oxygen and carbon dioxyde [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. A high O 2 /CO 2 ratio increases the chances of oxygen fixation, and therefore photorespiration. Conversely, a low O 2 /CO 2 ratio promotes carbon dioxide fixation stimulating the Calvin-Benson cycle [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Michiel Van Lun | CO2 and O2 Distribution in Rubisco Suggests the Small Subunit Functions as a CO2 Reservoir[END_REF]. The affinity of the enzyme for its substrate is also species-dependent [START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF]. Furthermore, it appears that photorespiration is favoured with increasing temperature and water shortage [START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF]. Although photorespiration is prima facie harmful and in direct competition with Calvin-Benson cycle, numerous studies have shown that compromising photorespiration does not result in better photosynthetic efficiency. Rather, photorespiration plays the role of an efficient metabolic repair system -75% of the inorganic carbon is salvaged and used to synthesize RuBP -to repair the consequences of RuBP oxygenation due to substrate competition from RuBisCO [START_REF] Hagemann | Photorespiration and the potential to improve photosynthesis[END_REF][START_REF] Timm | The regulatory interplay between photorespiration and photosynthesis[END_REF]. In addition, it would have other positive effects on the cell. Among others, it would participate in the nitrate assimilation by the C3 plants while supplying glycine and serine to the cell. It would also help maintain the redox balance of cells and would be able to influence multiple signalling pathways controlling growth, environmental and defense responses as well as programmed cell death [START_REF] Peterhansel | Photorespiration. The Arabidopsis Book / American Society of Plant Biologists[END_REF].

Aerobic cellular respiration

As aforementioned, the output of photosynthesis, and more specifically of the Calvin-Benson cycle, is G3P. This intermediate is converted mainly into carbohydrates whose primary purpose is to serve as an energy substrate for cell metabolic processes. In particular, the energy recovered from these molecules is used to support the growth and maintenance of the cell [START_REF] Raven | Dark Respiration and Organic Carbon Loss[END_REF]. To this end, cell respiration, formerly called dark respiration, corresponds to the set of metabolic routes allowing the conversion of chemical energy stored in carbohydrates into ATP. This pathway consists of the interaction of glycolysis and Krebs cycle also known as Tricarboxylic Acid Cycle (TCA) [START_REF] Heldt | Plant biochemistry[END_REF][START_REF]Chapter 8 -Energetics of Dark Respiration and Mechanism of Cytochrome Oxidase Functioning[END_REF][START_REF] Raven | Carbohydrate Metabolism and Respiration in Algae[END_REF]. Through the use of oxygen, these processes enable the oxidation of NADH and FADH 2 in mitochondrial membranes for the production of ATP [START_REF]Chapter 8 -Energetics of Dark Respiration and Mechanism of Cytochrome Oxidase Functioning[END_REF]. In a very simplified way, the cellular respiration of carbohydrates starts with the glycolytic pathways, whose purpose is to catabolize glucose into pyruvate. In most microalgae, the glycolytic pathway is summed up as glycolysis reactions. Glycolysis can take place both in the chloroplast stroma and in the cytoplasm of the cell [START_REF] Raven | Dark Respiration and Organic Carbon Loss[END_REF]. Pyruvate is then transported to the mitochondrion where it enters the TCA cycle as acetyl-Co-A [START_REF] Raven | Dark Respiration and Organic Carbon Loss[END_REF]. These metabolic pathways generate numerous molecules of NADH and FADH 2 which are then oxidized by the electron transfer chain of the mitochondrion, with oxygen as the terminal electron acceptor. Similar to the electron transport chain for the light reactions of photosynthesis in chloroplasts, this transport chain allows the generation of a proton driving force which is used to synthesize ATP by chemiosmosis [START_REF] Raven | Dark Respiration and Organic Carbon Loss[END_REF].

Light damages and protective mechanisms

During cultivation, microalgae can sometimes be subjected to high irradiation that can be harmful. In these conditions, the photosynthetic apparatus experiences excessive excitation resulting in the generation of reactive oxygen species (ROS) and, consequently, photooxidative damages leading to photoinhibition [START_REF] Goss | Biodiversity of NPQ[END_REF]. As a result, microalgae have developed response mechanisms on different time scales to cope with energy overage and alleviate its detrimental effects [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF].

Photodamages

Although the photosynthetic machinery is efficient and carefully regulated, it is still susceptible to a stochastic process of denaturation. In this respect, it is estimated that approximately every 10 to 100 million photons intercepted, one PSII is damaged [START_REF] Krause | Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms[END_REF][START_REF] Raven | The cost of photoinhibition[END_REF]. The occurrence of this process, commonly referred to as photodamage, is an unavoidable phenomenon that occurs at a fixed rate regardless of the incident light intensity. Thus, as light intensity increases, photodamages occurence increases proportionally [START_REF] Wah | The role of inactive photosystem-II-mediated quenching in a lastditch community defence against high light stress in vivo[END_REF]. In addition, the probability of damaging a PSII is wavelength-dependent. It is generally accepted that UV light and visible light in the blue region of the spectrum cause more photodamages than other wavelengths of visible light [START_REF] Raven | The cost of photoinhibition[END_REF].

Photodamage mainly leads to the proteolytic elimination of protein D1 from the reaction center of PSII, and to a lesser extent that of protein D2 [START_REF] Raven | The cost of photoinhibition[END_REF]. However, the mode of action of photodamage is still a matter of debate in the community [START_REF] Keren | Photoinhibition: molecular mechanisms and physiological significance[END_REF]. To date, three main mechanisms occurring simultaneously are described (Figure 1.25). Two of them are associated with the electron transport chain at the level of the PSII reaction center, where one distinguishes the donor side, located upstream of the RC, and the electron acceptor side downstream [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF][START_REF] Han | Effect of photoinhibition on algal photosynthesis: a dynamic model[END_REF][START_REF] Nishiyama | A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II[END_REF]. The remaining mechanism is the direct result of light absorption by the chlorophyll. * Acceptor side: This mechanism corresponds to the limitation of the acceptor side while the donor side is still active [START_REF] Keren | Photoinhibition: molecular mechanisms and physiological significance[END_REF]. Excessive excitation of the PSII leads to an accumulation of plastohydroquinone as well as an inordinate reduction of the photosynthetic electron transport components (Q A and Q B ) due to the slow process of cytochrome b 6 /f complex (3-5 ms) compared to water splitting reaction (around 1-2 ms) [START_REF] Heldt | Plant biochemistry[END_REF]. This imbalance causes a bottleneck in the electron transport chain which, in the long term, favours the appearance of ROS at the reaction center [START_REF] Han | Effect of photoinhibition on algal photosynthesis: a dynamic model[END_REF]. straightforwardly absorbed by the manganese at the OEC, in particular blue light and UV, alters the OEC as well as tyrosine [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF]. This is followed by the release of manganese ions which directly degrade the D1 protein [START_REF] Nishiyama | A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II[END_REF][START_REF] Li | Mechanisms of Photodamage and Protein Turnover in Photoinhibition[END_REF]. These are called primary photodamages. Once the OEC is damaged, the flow of electrons from the water to the P + 680 is blocked. Thus, the OEC is unable to keep up with the speed at which electrons are transferred from the P 680 reaction center to the acceptor side [START_REF] Han | Effect of photoinhibition on algal photosynthesis: a dynamic model[END_REF]. The disturbed charge separation forces the special pair to remain in an excited state, P + 680 , which is a powerful oxidant capable of damaging protein D1 (secondary photodamages) [START_REF] Nishiyama | A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II[END_REF]. This oxidant can also recombine with oxygen. This reaction results in the return of P 680 to its ground state by producing singlet oxygen ( 1 O 2 ), an aggressive ROS [START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Straka | Light-dependent kinetic model for microalgae experiencing photoacclimation, photodamage, and photodamage repair[END_REF]. It has long been believed that ROS directly damages the PSII by targeting protein D1. However, it has recently become apparent that ROS mainly acts as an inhibitor of PSII repair of de novo protein synthesis, particularly protein D1 [START_REF] Nishiyama | A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II[END_REF][START_REF] Torzillo | Environmental Stress Physiology with Reference to Mass Cultures[END_REF]. * Triplet state chlorophyll: This mechanism involves the chlorophyll molecules present in LHCs. Basically, a chlorophyll molecule is excited by the absorbed light generating the singlet state ( 1 Chl + ). The excitation energy is rapidly transferred to the other chlorophyll molecules in the LHC. During overexcitation, the chlorophyll molecules can reach a triplet state ( 3 Chl + ) which is a highly reactive species with a lifetime longer than that of the singlet state. Like the special pair, chlorophyll in the triplet state can return to its ground state by releasing energy to an oxygen molecule Chapter 1. Background in ground state from the LHC complex [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. Similarly, this reaction leads to the formation of singlet oxygen [START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Straka | Light-dependent kinetic model for microalgae experiencing photoacclimation, photodamage, and photodamage repair[END_REF].

To a lesser extent, PSI may also be affected by photodamages [START_REF] Li | Mechanisms of Photodamage and Protein Turnover in Photoinhibition[END_REF][START_REF] Straka | Light-dependent kinetic model for microalgae experiencing photoacclimation, photodamage, and photodamage repair[END_REF]. Under low light, PSI is somehow protected by the action of PSII. Indeed, the latter, which is primarily affected by photodamages, sets up mechanisms to try to regulate the electron transport chain and thus avoid an imbalance that could disturb the downstream PSI. In constrast, under high light, when the flow of electrons coming from PSII exceeds the capacity of the electron acceptors of PSI, photodamage occurs at the PSI. These can be accompanied by the formation of ROS. Indeed, the reduction of oxygen on the acceptor side of PSI leads to the formation of the superoxide radical (O •- 2 ), which can be transformed into hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (OH • ) [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Raven | The cost of photoinhibition[END_REF][START_REF] Nishiyama | A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II[END_REF]. Once again, the electron transport chain is disrupted at PSI. An underlying consequence of a damaged PSI is to cause significant photodamage to PSII due to a lack of electron acceptors [START_REF] Straka | Light-dependent kinetic model for microalgae experiencing photoacclimation, photodamage, and photodamage repair[END_REF].

To cope with the appearance of photodamages, photosynthetic organisms have developed short and long term regulation mechanisms (protection and repair) to avoid the energy overload of the photosynthetic appartus [START_REF] Krause | Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms[END_REF]. Short-term mechanisms are considered to be the first lines of defence in response to light shock. In constrast, the longer-term response leads to an acclimation of the organism to the light environment. It should be noted that the latter can be implemented both in limiting or excess light while the former only occurs when the cells are facing light shots. Because of the confusion regarding the denomination of these mechanisms, in this manuscript we will refer to photoprotection for short-term mechanisms and photoacclimation for long-term ones.

Photoprotection

Photoprotection is the set of mechanisms that aim to balance excitation with the cell's ability to use light energy for photosynthesis during luminous overloading. It takes place on a short time scale ranging from a few seconds to a few minutes [START_REF] Torzillo | Environmental Stress Physiology with Reference to Mass Cultures[END_REF][START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF]. The associated mechanisms can be grouped into two categories. The first category gathers mechanisms whose aim is to limit the entry of excess energy into the photosynthetic chain (upstream of the PSII reaction center). These include physical avoidance as well as dissipation by non-photochemical mechanisms [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF][START_REF] Raven | The cost of photoinhibition[END_REF]. When it is impossible to avoid light overload, the microalgae sets up a second category of mechanism to regulate the excess of energy in the electron transport chain (downstream of the PSII) as best as possible [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF][START_REF] Krause | Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms[END_REF][START_REF] Raven | The cost of photoinhibition[END_REF].

Avoidance by movement and positionning

One of the first rapid response mechanisms to excessive incident light is physical avoidance in order to prevent the photosynthetic organelles from absorbing more light. The latter is made possible either by phototactic movements of the whole cell (phototaxis) for mobile microalgae [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Raven | The cost of photoinhibition[END_REF] and/or by changes in the position and orientation of organelles within the cell [START_REF] Heldt | Plant biochemistry[END_REF].

Phototaxis

The phototactic response is a mechanism in which the orientation of cell movement is influenced by the direction and intensity of incident light, but also by temperature. In chlorophytes, this movement can be completed in two different modes. Although mobile microalgae can move by mucilage secretion, the most commonly encountered movement is achieved by the beating action of the flagellum [START_REF] Edward | Phycology[END_REF]. Commonly, flagellated cells move forward following a helical trajectory due to the beating of the flagellum [START_REF] Arrieta | Phototaxis beyond turning: persistent accumulation and response acclimation of the microalga Chlamydomonas reinhardtii[END_REF]. This rotation induces a periodic modulation of the light signal received by photoreceptors. The latter consist of a chromophore, the 11-cis-retinal, linked to a protein and forming a complex called rhodopsin [START_REF] Edward | Phycology[END_REF]. They are located above the eyespot, in the cytoplasm, and are able to detect the quantity and quality of photon flux by comparing them at two time intervals [START_REF] Edward | Phycology[END_REF][START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. They are mediators for the transduction of the signal from the eyespot to the flagellum. The rapidly relayed signal (ms) allows the control of the opening or closing of the calcium channels as well as the concentration of intraflagellar calcium responsible for the movement of the flagellum [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Arrieta | Phototaxis beyond turning: persistent accumulation and response acclimation of the microalga Chlamydomonas reinhardtii[END_REF]. Depending on the action potential, the response of the flagellum beat can cause the cells to move towards the light (positive phototaxis) or away from it (negative phototaxis) [START_REF] Edward | Phycology[END_REF][START_REF] Arrieta | Phototaxis beyond turning: persistent accumulation and response acclimation of the microalga Chlamydomonas reinhardtii[END_REF].

Screening or positionning

Another mechanism for avoiding high light is the movement of the organelles within the cell. This mechanism occurs at different cellular scales. At the microscopic level, the chloroplast is able to move according to the perceived light intensity [START_REF] Heldt | Plant biochemistry[END_REF]. Under high light conditions, the chloroplast moves from the cell surface to the side walls and positions itself to align itself parallel to the incident light in order to limit excessive light exposure. On the contrary, in low light conditions, the chloroplasts are rather arranged perpendicular to the incident light in order to maximise light capture. By zooming in on the structure of the chloroplast, the thylakoids also react dynamically to light. In particular, they are capable of stacking to form radiation-attenuating structures [START_REF] Raven | The cost of photoinhibition[END_REF]. This phenomenon, known as the package effect, limits the excitation energy reaching the reaction centers of the PSII by decreasing absorption of pigments [START_REF] Raven | The cost of photoinhibition[END_REF]. The package effect is accompanied by changes in the membrane and pigment profile [START_REF] Raven | The cost of photoinhibition[END_REF]. At the smallest scale, a reorganisation of the light harvesting complexes can be carried out. This reorganisation can be structural and/or spatial. From a structural point of view, it has recently been shown that a reorganisation of the LHCII from the trimeric form to dimeric and monomeric forms that are less efficient for the transfer of excitation energy would make it possible to dissipate excess energy [START_REF] Janik | Light-induced formation of dimeric LHCII[END_REF]. Spatially, movements of the LHCII called "state transitions" can also occur [START_REF] Raven | The cost of photoinhibition[END_REF]. This mechanism is discussed in more detail in the dedicated subection 1.3.2.2.

Non Photochemical Quenching (NPQ)

In addition to physical avoidance, cells set up rapid complex cellular responses to dissipate excess energy before it reaches the reaction centre of the photosystems, and in particular PSII. As soon as the energy absorbed by the LHCs exceeds the need for photochemical activity, the processes that lead to the dissipation of excessive excitation energy as heat are activated. These are collectively referred to as Non-Photochemical Quenching (NPQ) [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Goss | Biodiversity of NPQ[END_REF]. They are grouped into three different components, each characterised by a particular kinetic behaviour: energy-dependent quenching (qE), state transition quenching (qT) and photoinhibition (qI) component [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Zhao | Non-photochemical Quenching Plays a Key Role in Light Acclimation of Rice Plants Differing in Leaf Color[END_REF]. The first two are very fast settling components (induced in seconds for qE and minutes for qT), while the last is much slower (hours to days) and will be discussed in the sub-section 1.3.3.2 [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF].

High-energy-state quenching (qE)

The high-energy-state quenching (qE) is the fastest and also the most studied component of the NPQ mechanisms. Studies have highlighted the importance of this component for the survival of organisms to rapid fluctuations in light to avoid light shocks [START_REF] Szabó | Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation[END_REF]. Indeed, when photosynthetic organisms are exposed to high levels of light, the collection of light and its routing become saturated, causing downstream bottlenecks in the electron transport chain. These phenomena lead to a drastic drop in the photosynthetic yield. The establishment of qE makes it possible to act upstream of this reaction cascade in order to release the reaction center of the excitation pressure while protecting the photosynthetic apparatus [START_REF] Murchie | Dynamic non-photochemical quenching in plants: from molecular mechanism to productivity[END_REF].

The qE component of the NPQ can be induced in just a few seconds and is rapidly reversible, relaxing in seconds to minutes. It is triggered by the formation of a proton gradient across the thylakoid membrane responsible for the xanthophyll cycle activation as well as antenna aggregation [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Zhao | Non-photochemical Quenching Plays a Key Role in Light Acclimation of Rice Plants Differing in Leaf Color[END_REF]. While the physiological importance of qE has been recognized for many years, its mechanism remains elusive and under discussion at present [START_REF] Holt | Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants[END_REF][START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. * Formation of a proton gradient Under excessive lighting, the lumen of the thylakoids becomes acidified due to an accumulation of protons generated by the electron transport chain [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Murchie | Dynamic non-photochemical quenching in plants: from molecular mechanism to productivity[END_REF]. In addition, because the carbon reactions are slower, ATP is not consumed fast enough relative to the outflow, which limits the flow of protons through ATPase. As a result, the pH difference (∆pH) fills up between the stroma and the lumen. This sharp decrease in the lumenal pH is a signal of excess light that triggers the feedback regulation of light harvesting by qE [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Raven | The cost of photoinhibition[END_REF][START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF]. On the one hand, it serves to activate the xanthophyll cycle by deepoxidation of violaxanthin to zeaxanthin. On the other hand, it induces the protonation of particular proteins (LHCSR3 in Chlamydomonas reinhardtii) associated with PSII [START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF][START_REF] Ruban | Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage[END_REF]. Together, the presence of protons and xanthophyll pigments causes a conformational change that switches a harvesting PSII-LHCII unit to a quenched state [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Szabó | Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation[END_REF]. * Zeaxanthin-dependent quenching (qZ) Green microalgae exhibit biphasic qE induction kinetics when cells are exposed to intense light [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. The initial rapid increase in qE is attributed to the massive acidification of the lumen of the thylakoids and the presence of lutein. The second phase leading to a slower increase in extinction corresponds to the accumulation of zeaxanthin [START_REF] Demmig | The role of xanthophyll cycle carotenoids in the protection of photosynthesis[END_REF][START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. In practice, the acidification of the lumenal thylakoid pH is accompanied by the interconversion of specific xanthophyll pigments linked to LHC proteins. This interconversion, commonly referred to as the xanthophyll cycle, or VAZ cycle in green microalgae because of the pigments involved, occurs on a time scale of a few minutes [START_REF] Demmig | The role of xanthophyll cycle carotenoids in the protection of photosynthesis[END_REF][START_REF] Jahns | Mechanism and regulation of the violaxanthin cycle: the role of antenna proteins and membrane lipids[END_REF][START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF].

Violaxanthin (two epoxy groups), a xanthophyll pigment naturally present in the thylakoid membrane, is gradually converted into antheraxanthin (one epoxy group) and then into zeaxanthin (without epoxy group) by deepoxidation (Figure 1.26) [START_REF] Zhao | Non-photochemical Quenching Plays a Key Role in Light Acclimation of Rice Plants Differing in Leaf Color[END_REF][START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. This reaction is catalysed by an enzyme, violaxanthine de-epoxidase (VDE), which is activated at lumenal pH values around 5 in response to exposure to excessive light. It is assisted by a co-substrate, ascorbate, to successively reduce the epoxy groups of violaxanthin and antheraxanthin. To complete the cycle, zeaxanthin can be reconverted into violaxanthin by epoxidation [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF]. This feedback occurs at a pH of about 8 and is provided by another enzyme, zeaxanthin epoxidase (ZE) [START_REF] Goss | Evidence for a rebinding of antheraxanthin to the light-harvesting complex during the epoxidation reaction of the violaxanthin cycle[END_REF]. The latter is located on the stromal side of the thylakoid membrane and, like VDE, requires co-substrates to reintroduce the epoxy groups, in particular oxygen and NADPH [START_REF] Goss | Biodiversity of NPQ[END_REF]. However, ZE has a conversion rate about 10 times lower than VDE, allowing zeaxanthin to accumulate during periods of high illumination [START_REF] Goss | Biodiversity of NPQ[END_REF]. Therefore, the pigment level of the VAZ cycle is determined by the activities of the VDE and ZE enzymes, whose activation is conditioned by pH.

The presence of these xanthophyll pigments within the LHCs play an essential role in the thermal dissipation of excessive energy. Their interconversion induces conformational changes that cause the antenna to change from a light harvesting state under low light to a dissipative state under high light correlated with NPQ induction [START_REF] Zhao | Non-photochemical Quenching Plays a Key Role in Light Acclimation of Rice Plants Differing in Leaf Color[END_REF][START_REF] Jahns | Mechanism and regulation of the violaxanthin cycle: the role of antenna proteins and membrane lipids[END_REF][START_REF] Goss | Evidence for a rebinding of antheraxanthin to the light-harvesting complex during the epoxidation reaction of the violaxanthin cycle[END_REF]. In the deepoxidised form, zeaxanthin has a relative energy level close to that of the singlet state chlorophyll a, allowing it to take up the excitation energy of chlorophyll a [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Raven | The cost of photoinhibition[END_REF]. Conversely, epoxidised xanthophyll, violaxanthin, can act as energy donors [START_REF] Raven | The cost of photoinhibition[END_REF]. Nevertheless, some studies have demonstrated the presence of an incomplete VAZ cycle in some green microalgae [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF][START_REF] Goss | Evidence for a rebinding of antheraxanthin to the light-harvesting complex during the epoxidation reaction of the violaxanthin cycle[END_REF]. These microalgae are only able to express violaxanthin and antheraxanthin. This implies that the role of zeaxanthin in qE can be replaced by antheraxanthin [START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF]. In these organisms, antheraxanthin would have a similar extinction efficiency as zeaxanthin [START_REF] Goss | Evidence for a rebinding of antheraxanthin to the light-harvesting complex during the epoxidation reaction of the violaxanthin cycle[END_REF]. It would thus be able to act as a direct extinguisher of singlet state chlorophyll a, or to form a heterodimer with a chlorophyll a molecule. The latter, after excitation, carries out a charge separation and dissipation of the excitation energy in the form of heat [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Goss | Evidence for a rebinding of antheraxanthin to the light-harvesting complex during the epoxidation reaction of the violaxanthin cycle[END_REF]. * LHCII aggregation

The drop in the pH of the thylakoid lumen not only activates the deepoxidation of violaxanthin into zeaxanthin, but it is also necessary to initiate a conformational change in the thylakoid membrane [START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF][START_REF] Ruban | Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage[END_REF][START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF]. These structural changes are reversible and occur on a time scale of a few tens of seconds to a few minutes [START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. They occur on several scales, from the atom to the macroscopic rearrangement of the locations of the pigment-protein complexes within the membrane [START_REF] Jomo Walla | Electronic Carotenoid-Chlorophyll Interactions Regulating Photosynthetic Light Harvesting of Higher Plants and Green Algae[END_REF]. This restructuring modifies the dynamics of energy transfer by creating new interactions and charge and energy transfer pathways between the chlorophyll and carotenoid molecules involved. As a result, LHCIIs move from a state of light harvesting to a state of efficient extinction of excess energy [START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF][START_REF] Jomo Walla | Electronic Carotenoid-Chlorophyll Interactions Regulating Photosynthetic Light Harvesting of Higher Plants and Green Algae[END_REF]. In particular, the change in conformation has been shown to directly influence the NPQ response. The more LHCII is in an aggregated state, the greater the expression of NPQ [START_REF] Goss | Biodiversity of NPQ[END_REF].

The LHCII-PSII supercomplex of green microalgae contains special proteins called Light Harvesting Complex Stress Related (LHCSR) that are accumulated in high light [START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF]. These chlorophyll and xanthophyll-binding proteins are capable of detecting lumenal acidification of thylakoid membranes caused by excess light. This results in protonation of their amino acid residues [START_REF] Goss | Biodiversity of NPQ[END_REF]. It is suggested that this protonation would allow the rapid and reversible triggering of LHCII aggregation [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Murchie | Dynamic non-photochemical quenching in plants: from molecular mechanism to productivity[END_REF][START_REF] Ruban | Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage[END_REF], although at present the triggering mechanism remains under debate [START_REF] Murchie | Dynamic non-photochemical quenching in plants: from molecular mechanism to productivity[END_REF]. In literature, different LHCII conformation models for NPQ induction have been proposed. Among them, two models are more frequently used.

The first presents 4 different extinction states that can occur within a LHCII by mixing the dependence of the proton gradient and the VAZ cycle [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Horton | Molecular design of the photosystem II lightharvesting antenna: photosynthesis and photoprotection[END_REF]. The first state (state 1) is obtained under limiting light conditions, when the LHCII is in a light harvesting state. Violaxanthin is the predominant xanthophyll in the membrane and qE is not induced. This state is characterised by an efficient transfer of excitation energy to the reaction center. By increasing the light, one switches to states 2 and 3 corresponding to intermediate states with a lower NPQ than state 4. State 3 is characterized by moderately aggregated and protonated LHCs in which violaxanthin is still present. State 2 is obtained after a transition from high illumination to limiting light. This state describes unprotonated LHCs which still contain zeaxanthin. Finally, state 4 is obtained when the cells are illuminated with high light intensities. In this state, the LHCs aggregation is triggered by the protonation of LCHSR proteins, in particular LHCSR3, as well as by the transformation of violaxanthin into zeaxanthin within the thylakoid membrane. In this way, a large part of the excitation energy is avoided.

The second model involves two distinct and independent extinction sites within thylakoid membranes [START_REF] Goss | Biodiversity of NPQ[END_REF]. The first extinction site is at the level of the major LHCII. During intense lighting, these are detached from the core complex of the LHCII to form aggregates. These assemblies are characterised by a strong dissipation of excitation energy in the form of heat. Their formation is made possible by the protonation of LHCSRs, in particular LHCSR3, which regulates the detachment, migration and aggregation/disaggregation of LHCII [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. The second extinction site, consisting of the minor LHCII, remains attached to the PSII core complex. It would correspond to the conversion of xanthophyll pigments by the VAZ cycle. Thus, it would present a slower formation kinetics than the first extinction site, spread over a time scale of several minutes [START_REF] Goss | Biodiversity of NPQ[END_REF].

All of the above mechanisms are designed to effectively extinguish the excitation of chlorophyll to prevent energy from reaching a reaction center that could be damaged [START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. In other words, the antennae acquire photophysical mechanisms that make it possible to short-circuit the photochemical pathway. To be effective, these have a shorter relaxation time from the excited state to the ground state compared to fluorescence or the photochemical route [START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. This condition is fulfilled thanks to the reorganisation of the antennae combined with the protonation of LCHSR3 and the presence of certain xanthophyll pigments. Together, they allow the energy connectivity of the pigments to be modified by the reorganisation of their orientation, their spatial distance from other pigments and their local protein environment, so that they become extinction sites [START_REF] Horton | Molecular design of the photosystem II lightharvesting antenna: photosynthesis and photoprotection[END_REF].

Over the years, several hypotheses have been put forward in an attempt to explain the mechanism leading to the deactivation of the excited chlorophyll. Most of them involve pigment-pigment interactions such as chlorophyll-chlorophyll and chlorophyll-carotenoid [START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. Although there is a clear consensus that these pigment-pigment interactions play a major role, the detailed mechanism is still debated as to the precise nature of these interactions, how they are triggered, the pigments actually involved, the location of the dissipation site and how the confluence of these factors leads to the thermal dissipation of excess excitation energy [START_REF] Holt | Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants[END_REF][START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF][START_REF] Jomo Walla | Electronic Carotenoid-Chlorophyll Interactions Regulating Photosynthetic Light Harvesting of Higher Plants and Green Algae[END_REF].

Several reaction pathways between the different pigments have been proposed and are shown in Figure 1.27. Under non-excessive light conditions, the chlorophylls interact with each other to carry energy towards the reaction center. Under high light, this pathway is obstructed. To return to their ground state, the chlorophylls are able to emit fluorescence. Once NPQ is induced, new, faster and more efficient pigment interactions are set up to reduce overloading at the reaction centers. Carotenoids, in particular zeaxanthin, antheraxanthin and lutein [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Holt | Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants[END_REF][START_REF] Wormit | Excitation energy transfer and carotenoid radical cation formation in light harvesting complexes-A theoretical perspective[END_REF], are heavily involved and are believed to contribute in two distinct modes of action called indirect and direct quenching mechanisms [START_REF] Holt | Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants[END_REF][START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF].

On the one hand, they would be able to act indirectly as allosteric regulators of LHCs [START_REF] Holt | Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants[END_REF][START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF]. Basically, once expressed, zeaxanthin induces conformational changes in the pigment protein complexes that lead to the formation of interactions between chlorophyll molecules. These new pathways facilitate the de-excitation of chlorophyll either by internal conversion of the chlorophyll to the ground state by releasing heat [START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF] or by excitonic interactions between neighbouring chlorophyll molecules [START_REF] Holt | Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants[END_REF].

On the other hand, xanthophyll pigments can directly de-excite chlorophyll due to their singlet state excitation which is lower and shorter than that of excited chlorophyll [START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF]. In this case, two different mechanisms have been proposed [START_REF] Wormit | Excitation energy transfer and carotenoid radical cation formation in light harvesting complexes-A theoretical perspective[END_REF]. The first mechanism is the transfer of energy from a chlorophyll excited in the sigulet state to a carotenoid excited in the singlet state (S1) of lower energy. These carotenoids are capable of quenching chlorophyll excitation energy by energy transfer. Then they relax by releasing heat [START_REF] Wormit | Excitation energy transfer and carotenoid radical cation formation in light harvesting complexes-A theoretical perspective[END_REF]. Direct quenching by carotenoids can also be done by electron transfer, which is the second transfer mechanism [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Holt | Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants[END_REF][START_REF] Jomo Walla | Electronic Carotenoid-Chlorophyll Interactions Regulating Photosynthetic Light Harvesting of Higher Plants and Green Algae[END_REF][START_REF] Wormit | Excitation energy transfer and carotenoid radical cation formation in light harvesting complexes-A theoretical perspective[END_REF]. The latter would preferably take place at the level 1 -Electronic interaction between an excited chlorophyll and the PSII reaction center for photochemistry. In light overload, this path is jamed ; 2 -Dissipation by fluorescence of the excited chlorophylls in the singlet state to return to its ground state ; 3 -Electronic interaction between neighbouring excited chlorophyll molecules ; 4 -Formation of a carotenoid radical cation leading to dissipation by charge transfer ; 5 -Electronic interaction between chlorophyll and carotenoid molecules in the singlet state of the minor antennas of the PSII [START_REF] Goss | Biodiversity of NPQ[END_REF]. Unlike energy transfer, charge transfer requires the formation of a chlorophyll-carotenoid heterodimer. The excited chlorophyll (or excited carotenoid) transfers its excess energy to the heterodimer generating a radical carotenoid cation and a chlorophyll anion. This complex then performs a charge separation.

State transition quenching (qT)

In nominal conditions, PSII and PSI work in series. In this configuration, the excitation rate of each photosystem is constantly balanced, thus maintaining efficient linear electron transport [START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF]. However, in non-optimal light conditions, this excitation balance is broken to the disadvantage of the PSII. Subject to a strong excitation stress, the latter is particularly sensitive to inactivation due to the generation of a powerful oxidant and a relatively slow extinction of the excited chlorophyll compared to PSI [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. In order to reduce the excessive excitation pressure exerted on PSII, microalgae have developed a mechanism for moving the LHCII antenna from PSII to PSI by reversible phosphorylation of LHCII proteins [START_REF] Szabó | Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation[END_REF][START_REF] Kono | Long-term and short-term responses of the photosynthetic electron transport to fluctuating light[END_REF]. This mechanism, called state transition quenching (qT), allows a change in the relative absorption capacity of the two photosystems by redistribution of the excitation (Figure 1.28) [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF]. In literature, two transition states have been described in the model microalga Chlamydomonas reinhardtii [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF][START_REF] Kono | Long-term and short-term responses of the photosynthetic electron transport to fluctuating light[END_REF]. In State 1, the excitation is either balanced between the photosystems or in favour of PSI. Consequently, the LHCII antennae are associated with PSII which has a high quantum efficiency. By modifying the light conditions to favour PSII, a change in the electron transport chain occurs, particularly at the PQ pool level. The reduced plastoquinone/plastoquinol imbalance causes activation of a kinase. The latter phosphorylates the LCHII proteins resulting in dissociation at PSII level and subsequent association with PSI, known as State 2 [START_REF] Kono | Long-term and short-term responses of the photosynthetic electron transport to fluctuating light[END_REF]. In this state, cells generally exhibit lower PSII quantum efficiency and high NPQ [START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF]. This process is reversible under Chapter 1. Background the action of a phosphatase [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. However, although it has been reported that up to 80% of LHCII antennae can dissociate from PSII [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Kono | Long-term and short-term responses of the photosynthetic electron transport to fluctuating light[END_REF], their association with PSI is still debated [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. In this context, non-invasive spectroscopic analyses have notably highlighted a population of LHCII antennae which are not associated with any photosystem. The proposed mechanism is that a small fraction of the LHCIIs actually migrate to PSI while the rest form extinction antenna aggregates in the thylakoid membrane. This would explain a decrease in antenna size for LHCIIs without a corresponding increase in the size of PSI antennae in State 2.

Change in electron transport

When the previous mechanisms (NPQ and physical avoidance) are not sufficient to prevent a high energy influx from reaching the electron transport chain, other mechanisms complement them. These provide sinks for excess excitation energy to restore balance in the linear electron transport chain. These alternative pathways for the electrons are the cyclic transport, the water-water cycle, and chlororespiration [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. Finally, while these pathways allow to rebalance the electron transport chain, a last pathway exists to regulate the production/consumption ratio of energy intermediates (ATP, NADPH) of the light reactions of photosynthesis. This is mitochondrial respiration.

Cyclic electron flow

In addition to the linear transport, the electrons can follow a cyclic path which involves only the PSI, the cytochrome b 6 /f complex as well as the FNR (Figure 1.29) [START_REF] Li | Mechanisms of Photodamage and Protein Turnover in Photoinhibition[END_REF]. Contrary to the former, cyclic electron transport allows the production of ATP, without inducing the production of NADPH. This electronic path would have a dual role. On the one hand, it would be considered essential to balance the ATP/NADPH production ratio to meet the changing needs for CO 2 fixation under nominal conditions [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. On the other hand, it would protect both PS from damage caused by over-reduction under stressful conditions, especially when exposed to intense light [START_REF] Li | Mechanisms of Photodamage and Protein Turnover in Photoinhibition[END_REF][START_REF] Iwai | Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis[END_REF]. In particular, it has been shown that the electron flow switches between linear and cyclic transport as a function of the excitation balance of the two PS in response to energy demand [START_REF] Li | Mechanisms of Photodamage and Protein Turnover in Photoinhibition[END_REF]. Thus, in State 2 of the qT, the cyclic transport would be predominant while in State 1 the linear transport would take over [START_REF] Iwai | Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis[END_REF].

In the microalga Chlamydomonas reinhardtii, two possible cyclic electron transfer pathways have been proposed in the literature [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Li | Mechanisms of Photodamage and Protein Turnover in Photoinhibition[END_REF][START_REF] Iwai | Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis[END_REF]. There is a ferredoxindependent pathway and a NADPH dehydrogenase dependent pathway, called PGR5 and NDH pathways respectively [START_REF] Johnson | Physiology of PSI cyclic electron transport in higher plants[END_REF]. However, both of these pathways would involve ferredoxin as a reducing agent. Regardless the pathway used, the electrons are directly transmitted to the PQ pool to return to the linear chain of carriers leading to the PSI [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. This path allows additional protons to be accumulated without reducing NADP + (increased driving force) and leads to the generation of a pH gradient across the thylakoid membrane. The latter can, in turn, lead to the synthesis of ATP but also serve to regulate light harvesting by inducing qE [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Johnson | Physiology of PSI cyclic electron transport in higher plants[END_REF].

The water-water cycle

Another alternative electron sink is the water-water cycle. It corresponds to the photoreduction of oxygen to water in PSI from electrons generated by PSII (Figure 1 conditions of excessive light, the influx of electrons into PSI can exceed the capacity of its electron acceptors inducing the photoreduction of oxygen to superoxide radical (O - 2 ). This reaction is commonly referred to as the Mehler reaction [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Miyake | Alternative Electron Flows (Water-Water Cycle and Cyclic Electron Flow Around PSI) in Photosynthesis: Molecular Mechanisms and Physiological Functions[END_REF]. The superoxide radical is then rapidly converted into H 2 O 2 by the enzyme superoxide dismutase (SOD). H 2 O 2 , a harmful oxidant, is efficiently scavenged into water by ascorbate peroxidase (APX) [START_REF] Miyake | Alternative Electron Flows (Water-Water Cycle and Cyclic Electron Flow Around PSI) in Photosynthesis: Molecular Mechanisms and Physiological Functions[END_REF]. This reaction is made possible by the ascorbate which functions as an electron donor for APX, generating a monodehydroascorbate (MDA) radical. Ascorbate is then regenerated from MDA by either ferredoxin or MDA reductase (MDAR). This second pathway uses NADPH as an electron donor.

The water-water cycle would have different physiological functions [START_REF] Miyake | Alternative Electron Flows (Water-Water Cycle and Cyclic Electron Flow Around PSI) in Photosynthesis: Molecular Mechanisms and Physiological Functions[END_REF]. It would serve both as a way to reduce the lifetime of photoproduced ROS, to consume the surrounding oxygen, to rebalance the NADPH/ATP ratio, as an electron sink to suppress the accumulation of electrons in the photosynthetic electron transport, and consequently as a way to dissipate excess energy in photosynthesis.

Chlororespiration

Chlororespiration is an alternative electron transfer pathway that leads to the nonphotochemical oxidation of the PQ pool [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. In this reaction, plastoquinone acts as a central electron transporter. To function, this pathway involves two enzymes, NADPHdehydrogenase (NHD complex) and plastoquinol terminal oxidase (PTOX) located on the stromal side of the thylakoid membrane (Figure 1.29) [START_REF] Rumeau | Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response[END_REF]. While the first enzyme performs a reduction reaction of the PQ pool (Eq. 1.6) under limiting light conditions, the second enzyme oxidizes the PQ pool under saturating light conditions (Eq.1.7). Thus, chlororespiration, and more specifically PTOX, would play the role of a safety valve to avoid excessive reduction of the PQ pool of PSII acceptors in the presence of excess light by stimulating its reoxidation [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Rumeau | Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response[END_REF][START_REF] Peltier | Chlororespiration[END_REF].

2N ADP H + 2Q -→ 2N ADP + + 2QH 2 (1.6) O 2 + 2QH 2 -→ 2H 2 O + 2Q (1.7)

Mitochondrial respiration

In basal functioning, photosynthesis and the mitochondrial respiratory chain share metabolites (NAD(P)H and ATP). The interaction of these two pathways ensures several functions, including the sharing of energy resources between photosynthesis and respiration and the decrease of ROS levels [START_REF] Curien | The Water to Water Cycles in Microalgae[END_REF]. In addition, mitochondrial respiration may represent an important internal source of CO 2 for photosynthetic carbon fixation. Thus, under sub-optimal or over-optimal light conditions, the redox and/or ATP exchanges between the chloroplast and the mitochondria are modified in favor of one or the other [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Curien | The Water to Water Cycles in Microalgae[END_REF].

Measurements

While most of the mechanisms presented above have been identified and quantified in specific studies, the measurements of the simplest ones have been streamlined to be made available to biotechnology laboratories. This is notably the case for monitoring the activity of photosystems, and in particular PSII. The latter are studied by the fluorescence of chlorophyll which is detected rapidly and non-invasively [START_REF] Raven | The cost of photoinhibition[END_REF][START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF].

In principle, chlorophyll is at the heart of photosynthesis. It is the pigment that channels the absorbed light energy to the reaction centers of PSII and PSI where the photochemical conversion of the excitation energy takes place. There are three de-excitation pathways, which are photochemical, heat dissipation and fluorescence emission. Therefore, the fluorescence measurement can be used as an indicator of PS activity because of its complementarity with the other two alternative de-excitation pathways [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. An increased fluorescence yield is a sign of decreased photochemical and heat dissipation yields, and conversely. Nevertheless, the variable part of chlorophyll fluorescence comes mainly from PSII. Thus, the observed modifications mainly reflect changes in the photochemical efficiency and heat dissipation of PSII.

With this in mind, the researchers developed fluorimetry associated with the method of saturation pulses called PAM (Pulse-Amplitude-Modulation). This is a tool commonly used to measure changes in the performance of chlorophyll fluorescence as organisms acclimatize to changing light conditions [START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF]. A typical PAM fluorometer measurement consists of a succession of different steps (Figure 1.30). After adaptation to darkness, the sample to be analyzed is exposed to non-actinic low light intensity to determine its minimum fluorescence value (Fo) when all reaction centers are open (non-photochemical quenching is minimal). The sample is then exposed to a pulse of saturated light so that all reaction centers are in the closed state [START_REF] Ruban | Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage[END_REF]. This pulse allows to obtain the maximum amount of fluorescence (Fm) of the sample. The difference between these two extreme values (Fm-Fo) is the variable fluorescence (Fv). From these values, the ratio Fv/Fm can be deduced. The latter serves as a proxy for the maximum photochemical efficiency of the PSII [START_REF] Ruban | Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage[END_REF][START_REF] Pilát | Optical trapping of microalgae at 735-1064nm: Photodamage assessment[END_REF]. In healthy, non-stressed microalgae, this measure takes a value around 0.8 [START_REF] Pilát | Optical trapping of microalgae at 735-1064nm: Photodamage assessment[END_REF]. A decrease in this ratio would indicate the onset of photoinhibition. In conjunction with the application of a saturation pulse, the momentary fluorescence efficiency, F, can be measured briefly before the saturation pulse is triggered. Actinic lighting is then applied for about 5 minutes during which pulses of saturated light are emitted. This protocol allows the maximum fluorescence output (Fm') to be achieved. This value is normally lowered with respect to Fm by non-photochemical quenching and stabilizes at the end of the illumination period. From this simple measurement process, one can calculate the effective yield of PSII photochemical energy conversion (Y(II)) under illumination according to the Equation 1.8. This parameter provides information on the relative amount of absorbed photons that are converted into chemically fixed energy by the photochemical charge separation at PSII reaction centers. The part of the photons that is not converted into energy is either dissipated into heat or fluorescence. In principle, this quantum efficiency can vary between 0 and 1.

Y (II) = (F m -F ) F m (1.8)
In addition to the detection of photochemical extinction, PAM fluorometer analyses also allow the observation of the induction of non-photochemical processes [START_REF] Zaks | Models and measurements of energy-dependent quenching[END_REF][START_REF] Pilát | Optical trapping of microalgae at 735-1064nm: Photodamage assessment[END_REF]. From the collected fluorescence parameters described above, two other parameters, called regulated energy dissipation Y(NPQ) and unregulated dissipation Y(NO) at PSII centers, Chapter 1. Background can be calculated. The latter are a measurement of the non-photochemical fluorescence quenching, reflecting the downregulation of PSII as a protection mechanism against excess light intensity. A high value of Y(NPQ) indicates that the photon flux density is excessive but also that the sample has retained the physiological means to protect itself by dissipating excessive excitation energy into harmless heat. On the contrary, a high Y(NO) value indicates that both the photochemical energy conversion and the protective regulation mechanisms are ineffective. It is therefore a proxy for the occurrence of photodamage. In summary, when photons are absorbed at a rate higher than that at which they are processed by the transport chain of electrons, photoprotection mechanisms take over at different levels. At the cellular level, the first action consists, as much as possible, in physically avoiding excess light. At the same time, microalgae set up molecular mechanisms to prevent excess energy from reaching the PSII reaction centers. In this respect, the funneling of light-induced chlorophyll excitation to the PSII core is prevented by the LHC state transition. In parallel, the activation of the xanthophyll cycle discharges excess energy harvested through safe dissipation mechanisms. The coupling of these two mechanisms limits the excitation pressure at the reaction centers as well as the resulting over-reduction of the rest of the electron transport system. In addition to this barrier, other downstream metabolic pathways can be triggered. These alternative electrons pathways fulfill two main functions. First, cylic electron transport at PSI and chlororespiration serve as electrons sinks to rebalance the electron transport chain. Second, the water-water cycle and/or mitochonrial respiration pathways can be used to control the damage. For instance, they are used to reduce the levels of ROS formed during light reactions or as homeostatic systems regulating the NADPH/ATP ratio. All of these mechanisms allow microalgae to fight against the harmful effects of exposure to intense light.

Photoacclimation

Previously, it has been shown that photoprotection is a set of rapid kinetic mechanisms that only occurs when photosynthetic organisms are exposed to high light in order to limit the harmful damage as much as possible. On longer time scales, i.e. from a few hours to a few days, further cellular adjustments may occur leading to the physiological acclimation of the cell to the light conditions commonly called photoacclimation. Unlike photoprotection, photoacclimation is a cellular response that can be initiated at any intensity, whether low or high. Consequently, a distinction must be made between photoacclimation to low light intensity and photoacclimation to high light intensity. In the first case, the main mechanism involved is changes in pigment content. In the second case, the mechanisms involved are more diverse. They include PSII repair mechanisms to maintain photosynthetic capacity as well as phenotypic modifications, in particular changes in protein and pigment activity and expression, and an accumulation of antioxidants [START_REF] Krause | Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms[END_REF][START_REF] Torzillo | Environmental Stress Physiology with Reference to Mass Cultures[END_REF][START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF]. All these changes are to be differentiated from photoadaptation, which involves changes in genetic structure.

Change in LCH and PS size and pigmentation

One of the strategies for the long-term acclimatizing photosynthetic organisms to the surrounding light conditions is to adjust the proportion of energy sent in the photosynthetic chain. This can be achieved in two ways: either by changes in the number of reaction centers of the photosystems or by modulation of their light harvesting capacity [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Minagawa | Structure, function and assembly of Photosystem II and its light-harvesting proteins[END_REF]. However, as mentioned above, it is important to differentiate between adaptation to limiting light and saturating light, which do not involve the same strategies.

Low light intensities

Here, two situations can be distinguished: microalgae grown in light-limiting conditions and those grown in the dark after a period in the light.

In sub-optimal light conditions, the organism tends to increase its light harvesting capacity by increasing its absorption cross-section. To do this, it may have recourse to increasing the number of photosynthetic units as well as increasing the size of light collecting antennas [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. Furthermore, its pigment content is modified in favour of an overexpression of chlorophyll a as well as other light harvesting pigments [START_REF] Torzillo | Environmental Stress Physiology with Reference to Mass Cultures[END_REF][START_REF] Hu | Environmental Effects on Cell Composition[END_REF].

In the event that they no longer perceive any light (dark), the photosynthetic organisms adopt a biphasic acclimation strategy different from the one in low light. First, after the light is extinguished, they will try to capture more light by producing more photocollecting pigments, in particular, chlorophyll a. However, after a certain amount of time( days to weeks), although still genetically capable of producing pigments, they will end up downregulating their production [START_REF] Bunt | DATA ON THE COMPOSITION AN DARK SURVIVAL OF FOUR SEA-ICE MICROALGAE 1[END_REF][START_REF] Hu | Heterotrophic cultivation of microalgae for pigment production: A review[END_REF].

High light intensities

When photosynthetic organisms are exposed to saturating light intensities over a long period of time, they tend to minimize the energy reaching the photosynthetic chain. This acclimation strategy is deployed on two scales. At the ultrastructural level, the reduction of the light harvesting capacity is achieved by decreasing the absorption cross section and the number of photosynthetic units. These modifications of the ultrastructure are accompanied by changes in pigment content [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF]. Cells acclimatized to high light intensity have a pigment profile less rich in chlorophyll a compared to low light intensities. This phenomenon has been investigated, among others, by Myers et al. [START_REF] Myers | CULTURE CONDITIONS AND THE DEVELOPMENT OF THE PHOTOSYNTHETIC MECHANISM[END_REF]. In their pioneering study, the authors highlighted the concomitant decrease in chlorophyll content and cell number with increasing light intensity. This decrease is all the more rapid as it occurs by dilution during cell division and does not result from the active destruction of the pigment [START_REF] Torzillo | Environmental Stress Physiology with Reference to Mass Cultures[END_REF]. These results were confirmed by other researchers and published in reviews [START_REF] Da | Impact of culture conditions on the chlorophyll content of microalgae for biotechnological applications[END_REF][START_REF] Chen | Chlorophyll modifications and their spectral extension in oxygenic photosynthesis[END_REF][START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. While the pigment profile is depleted in chlorophyll, secondary photoprotective carotenoids such as β-carotene, lutein and astaxanthin accumulate [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Torzillo | Environmental Stress Physiology with Reference to Mass Cultures[END_REF][START_REF] Hu | Environmental Effects on Cell Composition[END_REF]. These carotenoids have two fundamental roles. First, and already discussed above, they dissipate the excess of energy at the reaction centers as heat. Second, the accumulation of carotenoids in LHCs act as a screen to prevent excess light from reaching the photosynthetic machinery. Finally, the xanthophyll cycle pigments, especially zeaxanthin and antheraxanthin, are expressed under intense light. These can reduce the excitation of the reaction centers of PSII by dissipating excess excitation energy through NPQ. The time required to achieve pigmentation changes varies from one species to another, from a few hours to several days [START_REF] Torzillo | Environmental Stress Physiology with Reference to Mass Cultures[END_REF].

Protein D1 repair cycle

The last component of NPQ that relaxes very slowly is the photo-inhibitory quenching (qI). As the name implies, this component is commonly associated with damage to the D1 protein that leads to photoinhibition and lower photosynthetic capacity. For some researchers, this component can be considered as a photoadaptive response to excess light.

As detailed above, the reaction center of PSII is particularly sensitive to light-induced oxidative damage. Long exposure to intense light causes unavoidable damage to the reaction centers and more specifically to the D1 protein, which, if not replaced, leads to their inactivation. PSII has a selective and efficient multi-step repair process allowing the replacement of protein D1 that takes place simultaneously with the degradation [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. Overall, the cycle begins with a damaged PSII that undergoes phosphorylation. This phosphorylation allows its disassociation from the LHCII-PSII supercomplex as well as its monomerization. The damaged PSII monomer then migrates laterally from stacked grana thylakoids to stromal thylakoids where the repair process takes place [START_REF] Theis | Revisiting the photosystem II repair cycle[END_REF]. At this point, the PSII is partially disassembled to provide access for the repair machinery, i.e. CP43 and OEC are released leaving the core complex with protein D2, CP47 and cytochrome b 559 only [START_REF] Minagawa | Structure, function and assembly of Photosystem II and its light-harvesting proteins[END_REF][START_REF] Theis | Revisiting the photosystem II repair cycle[END_REF]. The damaged D1 protein is dephosphorylated and then degraded by proteolysis. After degradation, a new copy of D1 protein is inserted into the CP43 and OEC free complex. Then, CP43 and OEC are reassembled to form a fully operational reaction center. Finally, the newly repaired PSII monomer returns to the grana thylakoids to further assembly into supercomplexes.

Light damages and protective mechanisms 57

The inactivation of PSII can be seen as an acclimation process to reduce the number of PSII units [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. At saturating intensities, moderate reduction in the number of active PSII makes it possible to limit the influx of excessive energy without necessarily reducing overall electron transfer rates [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF]. However, the rate of PSII repair takes place over relatively long periods of time ranging from a few tens of minutes to more than an hour [START_REF] Raven | The cost of photoinhibition[END_REF]. Moreover, it is controlled by environmental factors and the energy state of the chloroplast, and slows down in case of excess light [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. Therefore, one can easily understand that prolonged exposure at high intensity causes an imbalance between degradation/repair leading to a chronic decrease in photosynthetic capacity.

Accumulation of antioxydants

Oxidative photodamage induced by exposure to high light intensity leads to the accidental production of ROS (superoxide radical (O •- 2 ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (OH • )) of which the most harmful to photosynthetic organisms is singlet oxygen ( 1 O 2 ). Photosynthetic organisms have a long-term line of defense that helps mitigate the damage caused by increased intracellular ROS levels. This defense strategy involves both enzymatic and non-enzymatic antioxidants [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF].

The main enzymatic antioxidants found in chloroplast include superoyde dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Das | Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants[END_REF]. Each of these enzymes is responsible for an enzymatic reaction that extinguishes either the superoxide radical, hydrogen peroxide or hydroxyl radical [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. SOD catalyzes the removal of O Non-enzymatic agents involved in the removal of ROS in chloroplasts include ascorbic acid, reduced glutathione (GSH), α-tocopherol, the amino acid proline and carotenoids [START_REF] Das | Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants[END_REF]. While the first two participate indirectly as co-substrates for the enzymes mentioned above, the others are direct agents that can fight according to two modes of action: chemical or physical pathways [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF]. The chemical route corresponds to oxidative quenching. It involves α-tocopherol and proline, both of which are effective ROS scavengers. Although the locations are different, their action is similar since they are both effective eliminators of OH • and 1 O 2 [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Das | Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants[END_REF]. Regarding physical quenching, it occurs by energy transfer. This is the path taken by carotenoids [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF]. These molecules exert their antioxidant activity in different ways. They can directly trap 1 O 2 and generate heat as a by-product in the RC of PSII, prevent the formation of 1 O 2 by reaction with 3 Chl + and excited chlorophyll ( 1 Chl + ) in LHCs and finally dissipate excess excitation energy via the xanthophyll cycle [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Das | Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants[END_REF].

In summary, photosynthetic organisms have a variety of mechanisms to protect and adapt themselves to the light fluctuations that can occur in their environment. By modulating the light harvesting capacity and the use of energy, they can limit damage to the photosynthetic apparatus without interrupting the photosynthetic process. In addition to these means of dissipation and trapping of reactive oxygen species, some damage is repaired by specific mechanisms creating a precarious balance between the rate of photodamage and the rate of repair. As long as the rate of repair corresponds to the rate of damage, no net loss of photosynthetic activity is observed [START_REF] Krause | Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms[END_REF]. When photosynthetic organisms are confronted with saturating light intensities that exceed the organism's energy demand for a prolonged period of time, two phenomena occur. In addition to direct damage to the photosynthetic apparatus, excess light is also responsible for the inhibition of PSII repair by the production of ROS [START_REF] Nishiyama | A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II[END_REF]. Under these conditions, the photodamage rate exceeds the repair rate of PSII [START_REF] Li | Mechanisms of Photodamage and Protein Turnover in Photoinhibition[END_REF][START_REF] Straka | Light-dependent kinetic model for microalgae experiencing photoacclimation, photodamage, and photodamage repair[END_REF]. This imbalance leads to a decrease in photosynthetic activity manifested by a decrease in maximal electron yield and in the photosynthetic CO 2 fixation capacity [START_REF] Melis | Dynamics of photosynthetic membrane composition and function[END_REF][START_REF] Krause | Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms[END_REF][START_REF] Han | Effect of photoinhibition on algal photosynthesis: a dynamic model[END_REF][START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF]. This phenomenon is commonly referred to as photoinhibition. Its extent depends both on the physiological state of the organism (effectiveness of protection and repair mechanisms) and on environmental conditions (intensity, duration of exposure, etc.) [START_REF] Krause | Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms[END_REF][START_REF] Murchie | Dynamic non-photochemical quenching in plants: from molecular mechanism to productivity[END_REF]. Two types of photoinhibition are distinguished in the literature. On the one hand, partial and reversible photoinhibition corresponds to a moderate excess energy characterized by a decrease in the quantum efficiency of PSII only, and for which the protection mechanisms are effective. On the other hand, chronic irreversible photoinhibition corresponds to a simultaneous decrease in the maximum rate of photosynthesis and in the quantum efficiency of the PSII and for which the excessive excitation cannot be compensated by the photoprotection mechanisms. The photoinhinition can be accompanied by an autophagy mechanism. The latter allows the degradation of damaged or toxic cellular materials that may be generated as a result of the accumulation of ROS [START_REF] María | Reactive Oxygen Species and Autophagy in Plants and Algae[END_REF]. However, depending on the severity of photoinhibition, autophagy can be selective and target only certain cellular components to increase viability under conditions of abiotic stress [START_REF] Solovchenko | Ultrastructural patterns of photoacclimation and photodamage to photosynthetic algae cell under environmental stress[END_REF], or lead to cell death [START_REF] Ishida | Roles of autophagy in chloroplast recycling[END_REF].

Conclusion

In this chapter, the mechanism of photosynthesis has been presented in its entirety. It is a complex light-driven process, finely regulated, allowing the storage of chemical energy in the cells from light and inorganic carbon fixation. It is commonly divided into two stages involving many actors at different locations in the cell. While the light reactions take place at the thylakoid membranes and generate essential energetic intermediates (NADPH and ATP), the carbon reactions synthesize carbohydrates from the previously formed ATP and NADPH by incorporating carbon dioxide. These latter exclusively enzymatic reactions take place in the stroma of the chloroplast.

In addition, photosynthetic organisms have various mechanisms to protect and adapt themselves to light fluctuations that may occur in their environment. In particular, they have developed short and long term regulation mechanisms to avoid the energy overload of the photosynthetic apparatus and thus limit the appearance of photodamage. Short-term mechanisms, called photoprotection, are considered as the first lines of defense in response to a light shock due to their rapid kinetics of triggering. They include physical avoidance, NPQ, and changes in the electron transport chain. The long-term response, on the other hand, leads to acclimatization of the organism to the light environment commonly referred to as photoacclimation. Unlike photoprotection, photoacclimation is a cellular response that can be triggered at any intensity. It uses different strategies including PSII repair mechanisms as well as phenotypic modifications, particularly changes in protein and pigment activity and expression, and an accumulation of antioxidants. Thus, by modulating light harvesting capacity and energy utilization, photosynthetic organisms can limit damage to the photosynthetic system without interrupting the photosynthetic 1.3. Light damages and protective mechanisms 59 process and thus avoid the occurrence of photoinhibition as much as possible.

From a process point of view, photosynthesis is attractive since it is a bioenergetic mechanism by which organic molecules are created from light energy and an inorganic carbon. This biological process, coupled with the phylogenic variety of microalgae, allows the expression of a large number of high-potential energy molecules. Therefore, it is all the more natural that biotechnologists have investigated microalgae as a solution to current environmental and energy issues. The next chapter is dedicated to how biotechnologists have attempted to manipulate light and photosynthesis through light and culture devices.

Introduction

With the goal of exploiting photosynthesis to make the most of light for the growth of microalgae, it is essential to understand how cells adjust in response to changes in light intensity. This is all the more important since at present, optimization of light supply to cultures remains a crucial issue in microalgal biotechnology due to the low quantum efficiency of photosynthesis. With this in mind, a large number of studies have been carried out involving two main strategies. While some have focused on the features of light, others have been devoted to photobioreactor design. The following chapter presents these two strategies, each with a dedicated section.

The first part makes the reader aware of the light pattern experienced by microalgae when cultured in photobioreactors and its consequences on their growth. This intermittent light has been reported as having considerable effects on microalgae growth. Positive influence reports have sparkled the idea that efficient L/D cycle management could promote culture growth, hence alleviate part of the burden hindering microalgae mass production. Still, in this context, it is necessary to understand the relationship between the periodic light modulations and the resulting variation in photosynthetic efficiency and growth as well as its effect on the production of desired molecules. As a consequence, this part is categorized into three sub-parts corresponding to the three light parameters that can affect growth, i.e. light intensity, light intermittency and spectrum. Here again, given the number of publications in the field, as well as the diversity of microalgae and by extension the variability of the response to light, particular attention is paid to studies carried out on green microalgae.

The second part is devoted to the different culture devices that have emerged to cultivate microalgae. First, it presents the conventional designs currently used for the industrial production of microalgae. Then, special attention is paid to the reduced optical path designs of photobioreactors. These have a double challenge. On the one hand, they make it possible to reduce the light limitation in the cultures. On the other hand, they play on the light pattern experienced by the microalgae to exploit it in the best possible way in order to increase the performance of the microalgae. Finally, this section also briefly discusses the different existing photobioreactor culture modes of operation in order to give a brief overview of the possible methods of culture.

Light and time patterns

Like any living organism, the growth of microalgae is affected by a number of abiotic factors including temperature, salinity, nutrients, gas concentrations (CO 2 , O 2 ) and pH. However, because they are photosynthetic organisms, light is often the main factor limiting growth. This is furthered by the fact that light in itself cannot be stored by cells. Consequently, it has to be continuously supplied to the culture [START_REF] Molina Grima | A study on simultaneous photolimitation and photoinhibition in dense microalgal cultures taking into account incident and averaged irradiances[END_REF]. In addition, the interaction between light and microalgae is complex and changing over the course of a batch culture. At the beginning of the culture, cell suspension is translucent and light penetrates the whole culture vessel supplying energy to all the cells. Then, as the culture grows, cells absorb more and more light resulting in the formation of a light gradient. Thus cellular self-shading starts prevailing, shortening the depth of light penetration inside of the culture vessel. This results in the appearance of two zones: the photic zones near the lit surface of the vessel, and the aphotic zones corresponding to almost complete darkness in the center of the PBR. In addition, as they are cultured under constant agitation, cells are shuttled through the light gradient (Figure 2.1). Hence, microalgae experience light fluctuations on a timescale a few milliseconds to several seconds depending on both the PBR geometry and the mixing rate [START_REF] Grobbelaar | Turbulence in mass algal cultures and the role of light/dark fluctuations[END_REF][START_REF] Perner-Nochta | Simulations of light intensity variation in photobioreactors[END_REF][START_REF] Sforza | Adjusted Light and Dark Cycles Can Optimize Photosynthetic Efficiency in Algae Growing in Photobioreactors[END_REF][START_REF] Janssen | Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/dark cycles[END_REF][START_REF] Bonnanfant | Photosynthetic electron transport transients in Chlorella vulgaris under fluctuating light[END_REF]. This heterogeneous light field can significantly affect microalgae growth. In this context, it is essential to understand the interaction between light and microalgae.

Constant light intensity

To address this issue, the first level of study is the description of the relationship between light intensity and photosynthetic activity. It is commonly completed by exposing a culture to continuous actinic light intensity that gradually increases [START_REF] Masojidek | Photosynthesis in Microalgae[END_REF][START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF]. For each applied light intensity (I), the rate of photosynthesis (P) is measured. The latter corresponds to the rate of carbon fixation (CO 2 or O 2 as a proxy for CO 2 ) per unit of time. This concentration is normalized by a specific biomass unit which can be either the number of chlorophyll a, the number of cells or the intracellular carbon [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF]. Then, the results are plotted resulting in a curve called photosynthesis-irradiance (PI or PE) curve or Light Response Curve (Figure 2.2). Its shape and magnitude reflect the underlying biophysical, biochemical and metabolic processes that regulate photosynthesis when light is the only limiting factor [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF].

The PI curve is widely used in the scientific community. It makes it possible to evaluate the efficiency of photon use by photosynthetic organisms [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF][START_REF] William | Measurement and Interpretation of Photosynthetic Light-Response Curves in Algae in the Context of Photoinhibition and Diel Changes[END_REF]. In practice, this tool is all the more important as it allows the identification of a range of light intensity in which microalgae perform photosynthesis at a maximum rate [START_REF] Janssen | Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/dark cycles[END_REF]. However, it is important to note that the PI curve depends on the light history experienced by the microalgae before analysis [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF]. On this curve, different key areas and points can be identified [START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF][START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF][START_REF] William | Measurement and Interpretation of Photosynthetic Light-Response Curves in Algae in the Context of Photoinhibition and Diel Changes[END_REF][START_REF] Grobbelaar | Mass Production of Microalgae at Optimal Photosynthetic Rates[END_REF]. When no light is applied, the rate of net photosynthesis is represented by a negative number. In darkness, maintenance and cellular respiration phenomena are predominant. By increasing the light intensity, photosynthetic activity is stimulated. The intersection of this activity with the abscissa axis gives rise to the compensation point (I C ). This point represents the amount of light required by the culture for photosynthesis to compensate for respiration-related losses. In other words, at this intensity, the photosynthetic activity equals cell maintenance. At low light intensities, the rate of photosynthesis depends linearly on the light intensity. This is the photolimitation zone. In this zone, all photons captured at the cell level are fully converted into carbohydrates. The slope of the line, α, represents the maximum quantum efficiency of photosynthesis [START_REF] Grobbelaar | Mass Production of Microalgae at Optimal Photosynthetic Rates[END_REF]. However, as light intensity increases, a transition from light-limited to light-saturated photosynthesis occurs. This transition results in a loss of linearity between the absorbed light and the rate of photosynthesis. At saturation, the rate of photosynthesis reaches a maximum called P max . The intersection between the theoretical linear relationship and P max is called I K . This point corresponds to the transition between the two regimes of photolimitation and photosaturation. In the photosaturation zone, the enzymatic processes of photosynthesis become limiting. As a result, the cell is unable to integrate more light energy. The excess energy is dissipated as heat by the NPQ mechanism. This phase is characterized by reaching a plateau where the rate of photosynthesis is constant and independent of light intensity. By further increasing the light intensity, a depression of photosynthesis occurs at an intensity called I i . This phenomenon, called photoinhibiton, results in a drop in photosynthetic activity partly due to the denaturation of protein D1 at high intensities. Commonly, a culture is called photo-inhibited when exposed to a light intensity about 10 times higher than the culture light conditions [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF].

As discussed in the previous chapter, microalgae have developed mechanisms to cope with changes in light quality and intensity [START_REF] Demmig | The role of xanthophyll cycle carotenoids in the protection of photosynthesis[END_REF][START_REF] Grobbelaar | Mass Production of Microalgae at Optimal Photosynthetic Rates[END_REF]. They can, therefore, both modulate their pigment composition and the number and size of antennae to acclimate [START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF][START_REF] Kono | Long-term and short-term responses of the photosynthetic electron transport to fluctuating light[END_REF]. This photoacclimation affects the PI curve although the general trend of the curve is maintained [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF][START_REF] William | Measurement and Interpretation of Photosynthetic Light-Response Curves in Algae in the Context of Photoinhibition and Diel Changes[END_REF][START_REF] Iluz | The Enhancement of Photosynthesis by Fluctuating Light[END_REF][START_REF] Bernd | The effect of dynamic light regimes on Chlorella[END_REF]. Figure 2.2 illustrates the effects of light acclimation on the PI curve. The open arrows indicate the direction of the light response for a culture acclimated to high intensities while the solid arrows indicate the direction of the light response for a culture acclimated to low intensities. One can see that, under nutrient-replete conditions, cultures acclimated to low light intensities exhibit a significant α [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF]. The onset of saturation and photoinhibition occurs at lower light intensities than microalgae acclimated to high light intensities (lower P max and I i ) [START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF][START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF]. This is mainly due to the low initial concentration of photoprotective pigments acting as a screen to protect the reaction centres from excessive excitation. Conversely, cultures grown at high light intensities can tolerate higher light intensities, delaying the onset of photoinhibition. These cultures show a decrease in chlorophyll a content accompanied by a significant increase in photoprotective pigment content [START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF][START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF]. This accumulation of xanthophyll cycle pigments allows the excess excitation energy to be dissipated via the NPQ mechanism [START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF]. However, since these pigments absorb light without a concomitant increase in energy transfer to the reaction centre, these cultures often have lower maximum quantum yields at low light intensities (lower α) [START_REF] Vonshak | Environmental stress physiology. Handbook of microalgal culture: biotechnology and applied phycology[END_REF].

Intermittent light

To delve a little deeper into understanding the feedback of microalgae in response to changes in light, scholars were interested in the light pattern to which microalgae are subjected within a photobioreactor and its effect on cellular response.

Light patterns in photobioreactors

Lighting a photobioreactor optimally is not an easy task. One must take into account the existence of two interacting systems with different characteristic times. The first is the biological system. It corresponds to the biological response in terms of light integration by cells. This system has fixed characteristic times (τ bio ) corresponding to inflexible process time of biological mechanisms. The second system is the light load time (τ c ) that will vary according to agitation, PBR design and cell density. Contrary to the first system, the biotechnologist can play on the second to improve the performance of the microalgae.

According to the ratio of the characteristic times of these two systems, three different light integration responses of microalgae to light loads times have been described (Figure 2.3). • Full light integration response (τ c < τ bio ): For light application times shorter than the biological response, it is considered that the cells do not respond dynamically to light fluctuations. Microalgae perceive light as a continuous supply of average intensity due to the high frequency light load time (Figure 2.3a). Generally, this phenomenon appears when the light load time is less than one second. One of the mechanisms proposed to explain this phenomenon is a light flash time too short to induce a significant oxidation or reduction of the energy intermediates of the photosynthetic chain [START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF]. As a result, photosynthesis functions without interruption as under continuous lighting.

• No light integration response (τ c > τ bio ): Unlike the full light integration response, this type of response assumes that the light load is much longer than the biological response time, generally an order of magnitude of hours. In this case, since the biological response has a characteristic time much smaller than that of light intermittency, it can follow the light stimulus relatively closely (Figure 2.3c). Thus, during the illuminated phase, the photosynthetic conversion of light is relatively faithful to the light pattern, the transition periods being much shorter compared to the stabilized phase. On the other hand, the periods of darkness are long enough to reduce the pool of energy intermediates entirely and do not allow any integration of light during this time [START_REF] Graham | A penalty on photosynthetic growth in fluctuating light[END_REF]. Therefore, the resulting growth rate is the weighted average of the rates obtained in both the light and dark phases.

• Partial light integration response (τ c τ bio ): This type of response can be obtained when both systems, i.e. the biological and light systems, are on characteristic times of the same order of magnitude (between a few seconds and a few minutes).

Here, the duration of the dark phase is sufficient to induce partial depletion of the intermediate products accumulated during the light phase, without observing their total exhaustion. In addition, these latter would make partial reoxidation of the intermediate metabolites possible increasing the capacity to convert light energy into chemical energy during the light [START_REF] Janssen | Photosynthetic efficiency of Dunaliella tertiolecta under short light/dark cycles[END_REF]. As a result, a partial response of the biological system to light load is observed, the growth rate of which is the result of the average amount of light energy received by the cells (Figure 2.3b).

Based on these light time patterns, three light regime have been defined for the operation of a culture in a PBR: photoperiod, fluctuating light and flashing light [START_REF] Grobbelaar | Do light/dark cycles of medium frequency enhance phytoplankton productivity[END_REF]. Photoperiod term refers to long L/D cycles of the order of several hours. It can be natural, such as circadian cycles imposed by the sun, or artificially induced either for growth or culture synchronization purposes. Fluctuating light is defined as medium-frequency light loads ranging from 10 Hz to 0.1 Hz. This light regime is most frequently encountered for cultures grown in PBR. It is dependent on both the mixing rate and the PBR design. Flashing light corresponds to the delivery of light impulsions at frequencies higher than 10 Hz. Of these three regimes, only the last two are explored in this manuscript. While fluctuating light patterns occur naturally in PBR, flashing light was first born out of the will to understand photosynthesis [START_REF] Kok | On the Efficiency of Chlorella Growth[END_REF][START_REF] Phillips | Growth Rate of Chlorella in Flashing Light. 1[END_REF]. Later, it was used with the objective of manipulating photosynthesis to promote cell growth.

Over the years, many studies have been carried out on the impact of the application of intermittent light on microalgae response. This phenomenon, commonly referred to as Light/Dark (L/D) cycles were first investigated by the pioneering works of Emerson [START_REF] Emerson | THE PHOTOCHEMICAL REACTION IN PHOTOSYNTHESIS[END_REF], Kok [START_REF] Kok | On the Efficiency of Chlorella Growth[END_REF] and Myers [START_REF] Phillips | Growth Rate of Chlorella in Flashing Light. 1[END_REF] who established the concepts of photosynthesis and whose outcomes are to be acknowledged. However, since then, conflicting effects have been reported [START_REF] Sforza | Adjusted Light and Dark Cycles Can Optimize Photosynthetic Efficiency in Algae Growing in Photobioreactors[END_REF][START_REF] Janssen | Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/dark cycles[END_REF][START_REF] Grobbelaar | Do light/dark cycles of medium frequency enhance phytoplankton productivity[END_REF] for both fluctuating and flashing light regimes. While some authors have reported stimulating effects on productivity, others have shown no effect or even deleterious effects. The wide variety of microalgal responses to different light loads can be attributed to two main factors. On the one hand, the studies were conducted on microalgal strains belonging to different phyla. However, depending on the strain used, the structure of the photosynthetic apparatus and the pigments involved differ and may call upon different response mechanisms [START_REF] Nickelsen | Photosystem II Assembly: From Cyanobacteria to Plants[END_REF]. On the other hand, the implementation of the experimental design differs from one study to another which greatly affects the light field to which the microalgae are subjected (see Section 2.2) and thus can bias the results.

Facing the large number of publications on the subject as well as the discrepancies in the results, there is a real need of reviewing the studies conducted so far. This synthesis is the subject of the next two sub-sections. From this perspective and keeping in mind the large number of studies as well as the great diversity of experimental protocols, a selection of relevant papers was carried out. In order to compare likes to likes, constraints were applied to studies presenting a precise control over parameters of interest. Therefore, results of the studies conducted on low density cultures in an optically thin bioreactor were focused. In this setup, all microalgae cells are exposed to the same light pattern in the culture vessel which is imposed by the lighting device without being disturbed by the addition of light cycles induced by a heterogeneous light field coupled with agitation. In addition, from a biological point of view, a restriction to studies on green microalgae, specifically on the Chlorophyta phylum as the response may differ from one phylum to another. The final criterion was the availability of a continuous light reference. Beyond the experimental device and the strain used, special attention is also paid to the parameter used to monitor cell growth since it can also influence the response. In the context of studies evaluating the effects of a light treatment, two monitoring strategies have been identified. The first in an instantaneous measurement of the cell response by measuring the rate of photosynthesis (P) (refered to as P O 2 hereinafter). This latter is achieved by the quantification of the rate of carbon fixation per unit of time. The second monitoring strategy is based on parameters that allow for the assessment of long-term effects of light regime on a culture. In this case, the parameter monitored over time may be optical density, dry weight or cell count (refered to as GR hereinafter). Afterwards, they are used to compute the culture growth rate and sometimes also to evaluate productivity.

Once the constraints have been chosen, it is important to define the parameters to be studied carefully. Not only the experimental device, the microalgal strain used and the culture performance monitoring (GR or P O 2 ) were paid attention to. In addition to those important information, other key features have been carefully analysed and reported covering L/D cycle parameters and, when available, statistical assessment. Generally speaking, a L/D cycle is defined by different parameters including the time during which the culture is exposed to incident light (τ L ) of a given intensity (I 0 ), the time during which the culture is left in the dark (τ D ). From those, the total duration of the cycle -for fluctuating light (τ c ) -or the frequency (f = 1 τc ) for flashing light -is obtained as follows: τ c = τ L + τ D . In addition, from these basic parameters, one can deduce the duty cycle (ε) -ratio of the light phase over the cycle duration -, expressed as ε = τ L τc , as well as the average light intensity perceived by the cells during a cycle:

I avg = I 0 × ε.
Over the course of the literature survey, a total of 118 measurements points have been reported for fluctuating light regime and 643 for flashing light regime. For all the reported measurements points, the relative variation of the growth rate (or photosynthesis rate) with respect to its continuous illumination counterpart have been computed (η, Eq. 2.1). A positive value of η represents an improvement over continuous lighting, a negative one a detrimental configuration, and a null one a configuration yielding equal growth. This mathematical treatment aimed at reducing the impact of the specific devices used in each study (PBR type, light source, illumination strategy, ...). Furthermore, in order to compare likes to likes, a weight (study dependent, most of the case ε as incident illumination is often the same between continuous and intermittent light) has been applied so that the amount of light energy supplied is the same between the two quantities µ Intermitent light, Iavg and µ Continuous light, Iavg . While this correction for the supplied light amount has been used by other authors [START_REF] Peter | Flashing light does not improve photosynthetic performance and growth of green microalgae[END_REF], it is not optimal in the sense that it does not account for growth non-linear dependence upon incident light [START_REF] Terry | Photosynthesis in modulated light: Quantitative dependence of photosynthetic enhancement on flashing rate[END_REF]. Still, applying this more elaborate correction procedure would require to have had access to a PI curve for each and every reported study, which was sadly not the case.

η = µ Intermitent light, Iavg -µ Continuous light, Iavg µ Continuous light, Iavg (2.1)
From all these parameters, only the average light intensity (I avg ), the duty cycle (ε), the total cycle time (or frequency) are reported in summary tables for readability reasons. The other parameters can be recalculated from the equations given above. These tables are categorized according to the light regime (fluctuating or flashing light) and the parameter used to monitor cell growth (growth rate or dissolved gases). For the sake of readability of this manuscript, they are presented in Appendices B and C.

Fluctuating light

Based on the Tables B.1 and B.2, at first glance, both positive and negative variations can be seen regardless of the monitoring parameter used. This first observation is in line with what is described in the literature. Given the number of parameters involved, drawing conclusions from these tables appears complicated. For ease of reading, once the values of η have been calculated for each data points in each study, scatter plots were drawn to get a general sens of the dataset. The relative variations are plotted as a function of the different parameters of the L/D cycles mentioned above, i.e. average light intensity (I avg ), total cycle time (τ c ) and duty cycle (ε). The results are presented in Figure 2.4. In addition, studies using the growth rate and gas monitored photosynthesis rate are differentiated according to the color used. From these graphs, no clear relationship between the different variables can be identified with the bare eye due to the wide dispersion of the results. Still, a trend seems to emerge from this crude analysis: higher duty cycle values would promote growth in fluctuating light regime.

In order to dive further into the data and get a better understanding of the data, η was plotted and analysed using descriptive statistics tools. Boxplots agglomerating all the results for fluctuating light regime is displayed in Figure 2.5. In addition to this plot, two other boxes are drawn: one only accounting for GR based measurements and the other only for P O 2 ones. At first glance, the data are widely dispersed ranging from -108 % to +91 %. This calls for a first comment as values lower than -100 % can be observed. They are alleged to originate from the combination of two factors: highly unfavourable conditions promoting respiration (e.g. long dark phases) and measurement uncertainty. With a focus on the dispersion of data between the first (Q1) and third (Q3) quartiles, the fluctuating results seem rather negative (from -74 % to 0 % with a mean of -30 %). Still, diving a bit further and considering specifically at the culture monitoring strategies, one can see that they give quite different results. On the one hand, P O 2 monitoring reports globally more negative (Q1-Q3: from -92 % to 0 %; mean: -53 %) than GR monitoring (Q1-Q3: from -27 % to +4 %; mean: -8 %). In addition, GR monitoring does not reports η values below -100 %.

Based on those results, and despite the number of publications in the field, it is still not clear how medium-duration L/D cycles could affect photobioreactor productivity. Indeed, GR and P O 2 monitorings report conflicting findings. To take the analysis of the results further, inferential statistics are used to assess the statistical significance of the observed trends regarding monitoring strategies. This analysis is proposed in a paper recently accepted [START_REF] Levasseur | Green microalgae in intermittent light: a meta-analysis assisted by machine learning[END_REF]. Although there is no consensus on the results, a mechanism for cell adaptation to the fluctuating light regime appears to be repeatable. In this regard, many authors have reported that a significant increase in the level of chlorophylls a and b following exposure to intermittent light cycles [START_REF] Janssen | Photosynthetic efficiency of Dunaliella tertiolecta under short light/dark cycles[END_REF][START_REF] Quéguiner | Phytoplankton photosynthetic adaptation to high frequency light fluctuations simulating those induced by sea surface waves[END_REF][START_REF] Janssen | Specific growth rate of Chlamydomonas reinhardtii and Chlorella sorokiniana under medium duration light/dark cycles: 13-87 s[END_REF][START_REF] Takache | Investigation of light/dark cycles effects on the photosynthetic growth of Chlamydomonas reinhardtii in conditions representative of photobioreactor cultivation[END_REF]. This finding leads us to believe that there is still a need for research to clarify the effect of fluctuating light in PBR on growth and photosynthetic efficiency.

Flashing light

The same procedure was undergone for flashing light regime. As for studies in fluctuating light, the scatter plot does not provide clear information (Figure 2.6). Regarding the boxplots, results are also widely spread for this regime, from -43 % to +26 % (Figure 2.5). Still, the main fraction of the result (Q3-Q1) are encompassed within -17 % and +2 %, with a mean of -12 %. Here, much fewer values below -100 % are reported and thus do not appear in the boxplot. This is explained by the fact that by definition flashing light regime does not allow for long dark phases promoting respiration. Finally, as for the fluctuating regime, the culture monitoring strategy gives different results. While P O 2 seems to be rather negative results (Q1-Q3: from -17 % to +1 %; mean: -13 %), GR monitoring reports more positive effect (Q1-Q3: from 0 % to +13 %; mean: +14 %).

As with fluctuating light, further analysis of the results is available in the same article [START_REF] Levasseur | Green microalgae in intermittent light: a meta-analysis assisted by machine learning[END_REF]. Still, from descriptive statistics, it becomes obvious that no clear conclusion can be drawn and that further research is required.

Light spectrum

In addition to light intensity and frequency, the spectral quality of light also affects the growth of microalgae. Generally speaking, light can be provided from natural (sunlight) or artificial sources on a microalgal culture. Sunlight covers a wide spectral range of which only the visible portion of the spectrum is used by microalgae. Although this source is the most cost-effective, in the majority of cases, cultures are exposed to artificial lighting. Unlike sunlight, artificial light allows for continuous production while controling the spectrum, photon flux density, and photoperiod [START_REF] Peter | Light emitting diodes (LEDs) applied to microalgal production[END_REF]. There are a wide variety of artificial light sources, each with a specific spectral composition (Figure 2.7) [START_REF] Lehmuskero | Light and photosynthetic microalgae: A review of cellular-and molecular-scale optical processes[END_REF]. They include incandescent lamps (tungsten lamp, halogen lamp, carbon arc lamp), discharge lamps (fluorescent lamp) and more recently, light-emitting diode (LED) lamps. Today, only fluorescent lamps and LEDs are commonly used in routine applications due to the poor efficiency of incandescent lamps in visible light. The former have a radiation comprising visible light as well as a small amount of UV radiation in the UV-A range, and even shorter wavelengths in the UV-C range. Unlike fluorescent lamps, LEDs are capable of emitting monochromatic or polychromatic radiation. Their spectrum can therefore be customized. In addition, compared to other conventional light sources, LEDs have a high electrical conversion efficiency, a long service life and low heat generation. They are also fast-responding which allows them to be used as a high-frequency flashing source [START_REF] Lunka | Effects of flashing light-emitting diodes on algal biomass productivity[END_REF][START_REF] Peter | Light emitting diodes (LEDs) applied to microalgal production[END_REF][START_REF] Park | Optimization of algal photobioreactors using flashing lights[END_REF][START_REF] Fatemeh Mohsenpour | Spectral conversion of light for enhanced microalgae growth rates and photosynthetic pigment production[END_REF]. However, the use of artificial light has a cost. Therefore, the choice of a light source depends mainly on the desired spectrum but also on the energy consumption generated by its use [START_REF] Peter | Flashing LEDs for Microalgal Production[END_REF].

Various studies have looked at the spectral components of light for the cultivation of microalgae. These studies can be divided into two parts depending on the aspect studied. On the one hand, there are studies on the depth of light penetration in a culture system. On the other hand are studies on the metabolic response of microalgae. These two points are briefly described below as they are not the subject of this thesis.

The depth of light penetration in the Photosynthetically Active Radiation (PAR) range is defined as the depth to which the irradiation in the culture is reduced to 10% of the incident irradiation. Depending on the wavelength of the light, it differs. In particular, it has been shown for dense cultures that green light penetrates deeper than blue and red light, the latter being absorbed primarily by the cells [START_REF] Gitelson | Photic Volume in Photobioreactors Supporting Ultrahigh Population Densities of the Photoautotroph Spirulina platensis[END_REF]161]. However, the length of light penetration is also a function of culture concentration [START_REF] Gitelson | Photic Volume in Photobioreactors Supporting Ultrahigh Population Densities of the Photoautotroph Spirulina platensis[END_REF]161]. As a matter of fact, a decrease in penetration depth from 10 to 8 mm in the blue and red ranges of the spectrum is observed when the cell concentration is increased from 1 to 10 mg/L chlorophyll. By further increasing the cell concentration (300 mg/L chlorophyll), the depth of light penetration decreased significantly. In the blue and red regions of the spectrum, it was between 0.03 and 0.05 mm, respectively. This result supports the fact that light is the limiting factor for growth since it is totally absorbed at the surface of the culture device and does not penetrate further into the culture. Contrarily, when green light is applied, it penetrates deeper. For the same cell concentration (300 mg/L chlorophyll), the penetration depth was close to 1 mm, i.e. a twenty-fold increase in comparison with the blue and red lights. Consequently, green light is considered as an additional and essential energy source available for photosynthesis in high cell density cultures.

In addition to the penetration of light in a culture, the light spectrum also plays a role in the metabolism of microalgae. As microalgae respond dynamically to light, they are able to regulate their photosynthetic apparatus and consequently their metabolic response to the color of light. By using customizable light spectra, it is possible to manipulate the biochemical composition of microalgae targeting the accumulation of certain molecules of interest [START_REF] Abu-Ghosh | Flashing light in microalgae biotechnology[END_REF]. In this respect, review works have been carried out [START_REF] Lehmuskero | Light and photosynthetic microalgae: A review of cellular-and molecular-scale optical processes[END_REF][START_REF] Peter | Light emitting diodes (LEDs) applied to microalgal production[END_REF]. First of all, it appears that the induced response is dependent on the color of light applied but also on the initial pigment composition and thus on the phylum to which the strain belongs. By taking a closer look at green microalgae, the application of a blue or red color spectrum would allow the achievement of higher growth rates and biomass accumulation than under green light [START_REF] Lehmuskero | Light and photosynthetic microalgae: A review of cellular-and molecular-scale optical processes[END_REF]. As an example, in Chlorella vulgaris cultures, the greatest biomass accumulation was obtained under violet-blue light (400-450 nm) while the maximum specific growth rate was obtained under orange light (585-620 nm) [START_REF] Fatemeh Mohsenpour | Spectral conversion of light for enhanced microalgae growth rates and photosynthetic pigment production[END_REF]. Furthermore, it was reported that the use of a blue spectrum could influence gene expression, enzyme activation and certain metabolic pathways [START_REF] Lehmuskero | Light and photosynthetic microalgae: A review of cellular-and molecular-scale optical processes[END_REF][START_REF] Peter | Light emitting diodes (LEDs) applied to microalgal production[END_REF]. In particular, blue light would promote the synthesis of RuBisCO, which would directly affect the rate of CO 2 fixation in microalgae [START_REF] Lehmuskero | Light and photosynthetic microalgae: A review of cellular-and molecular-scale optical processes[END_REF]. In addition, the blue range of the light spectrum would allow for increased lipid and pigment production [START_REF] Lehmuskero | Light and photosynthetic microalgae: A review of cellular-and molecular-scale optical processes[END_REF][START_REF] Peter | Light emitting diodes (LEDs) applied to microalgal production[END_REF]. However, the energy of blue photons is higher than that required for photosynthesis. Therefore, blue light can be harmful and may lead to the generation of ROS [START_REF] Peter | Light emitting diodes (LEDs) applied to microalgal production[END_REF][START_REF] Pickett | Monochromatic Light Saturation Curves for Photosynthesis in Chlorella[END_REF]. Finally, the green and red spectra have been reported to induce pigmentary modification [START_REF] Peter | Light emitting diodes (LEDs) applied to microalgal production[END_REF][START_REF] Fatemeh Mohsenpour | Spectral conversion of light for enhanced microalgae growth rates and photosynthetic pigment production[END_REF]. A study showed that under red light, Chlorella vulgaris expressed more chlorophyll a and chlorophyll c. As for exposure to green light, it would promote the chlorophyll pigmentation [START_REF] Fatemeh Mohsenpour | Spectral conversion of light for enhanced microalgae growth rates and photosynthetic pigment production[END_REF].

PBR designs and light investigation

At the laboratory, pilot and commercial scale, the design and operation of culture systems is a key step in the study and production of microalgae. Basically, a microalgae culture device is a three-phase system comprising the culture medium as the liquid phase, the cells as the solid phase, the air as the gas phase, all of them being illuminated by light.

With this in mind, two main types of different culture systems have been developed: open ponds and closed photobioreactors [START_REF] Pulz | Photobioreactors: production systems for phototrophic microorganisms[END_REF][START_REF] Jorquera | Comparative energy life-cycle analyses of microalgal biomass production in open ponds and photobioreactors[END_REF]. In the former, the culture is directly exposed to the environment. They are mainly used for unexpensive large-scale production. In the second type of systems, the culture is completely enclosed in the vessel [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF]. In contrast to open systems, closed systems are divided between small volume designs exclusively used at laboratory scale (flasks, microplates, etc.) and larger volume designs.

The differentiation of these culture systems depends mainly on the cost, the nature of the strain and the desired product, the nutrient source and the CO 2 capture [START_REF] Siqueira | Introductory Chapter: Microalgae Biotechnology -A Brief Introduction[END_REF]. Over time, a wide range of designs have been reported within these two broad categories of culture systems to maximize productivity and competitiveness.

Conventional designs 2.2.1.1 Open ponds

The vast majority of current commercial microalgal culture systems are open ponds. They represent the oldest and simplest culture system in which microalgae are grown directly exposed to the external environment. They can be natural or artificial [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF]. In the course of time, different forms of open ponds have appeared. Currently, the main configurations of open systems used in research and industry include raceways, circular ponds and unstirred ponds [START_REF] Siqueira | Introductory Chapter: Microalgae Biotechnology -A Brief Introduction[END_REF][START_REF] Shen | Microalgae Mass Production Methods[END_REF]. Their schematic representation is shown in Figure 2.8.

Raceways

Raceways are the most commonly used artificial microalgal production system on a commercial scale due to their relatively low construction and maintenance costs [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Shen | Microalgae Mass Production Methods[END_REF]. Their principle is to circulate the microalgae over a small width and depth but over a large distance. To achieve this, they consist of long recirculation channels with semi-circular ends. These can be assembled individually or in groups forming a closed loop. They are generally constructed of concrete or compacted earth covered with plastic [START_REF] Kumar Duran | A review on microalgae strains, cultivation, harvesting, biodiesel conversion and engine implementation[END_REF]. Their depth can vary between 15 and 50 cm although most of the time it is rather between 15 and 20 cm [START_REF] Pulz | Photobioreactors: production systems for phototrophic microorganisms[END_REF][START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF][START_REF] Shen | Microalgae Mass Production Methods[END_REF][START_REF] Chisti | Biodiesel from microalgae[END_REF]. The circulation and mixing of the culture is carried out by means of a paddle wheel. Its purpose is to continuously drive the medium around the circuit. The flow can be guided by the presence of deflectors minimising the formation of dead zones. There are other culture mixing systems such as pumps and elevators but they are less popular than paddle wheels [START_REF] Shen | Microalgae Mass Production Methods[END_REF].

The use of such culture systems generally allows to reach a cell concentration lower than 1 g/L and productivities in the range of 10 to 25 g/m /d [START_REF] Pulz | Photobioreactors: production systems for phototrophic microorganisms[END_REF][START_REF] Shen | Microalgae Mass Production Methods[END_REF]. These relatively small amounts of biomass are explained by the fact that raceways are not able to meet the main requirements of microalgae, i.e. efficient use of light and CO 2 [4,[START_REF] Chisti | Biodiesel from microalgae[END_REF]. The light intensity is dependent on the incoming sunlight. Therefore, the efficiency of open ponds depends on local daylight variations. In addition, although the channels are shallow, light penetration is rapidly limited, providing a heterogeneous light field affecting growth [START_REF] Chisti | Biodiesel from microalgae[END_REF]. In addition, in such systems, CO 2 comes directly from the air in contact with the culture. Submerged aerators can be installed to supplement the culture with CO 2 . Despite this, the mixture does not allow sufficient gas/liquid mass transfer to meet the needs of the culture [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF]. Besides, a poor vertical mixing does not allow in-deepth cells to be regularly exposed to light.

Circular ponds

Circular ponds are among the oldest large-scale microalgal culture systems [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF]. They are currently widespread in Southeast Asia for the production of molecules for human nutrition [START_REF] Shen | Microalgae Mass Production Methods[END_REF]. The main advantage of this type of system is their simplicity, which translates into relatively low production and operating costs [START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF].

Their design is similar to raceways made of concrete. These circular-shaped ponds have diameters of up to 45 m and depths of 30-70 cm [START_REF] Shen | Microalgae Mass Production Methods[END_REF]. The mixing of the culture is ensured by a rotating arm mounted in the centre of the pond. Such a stirring device is at the origin of the limitation in size of the circular ponds because of the low efficiency of the mixing when the rotating arm is too long (> 50 m) [START_REF] Shen | Microalgae Mass Production Methods[END_REF]. Moreover, its operation requires a high energy input [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Shen | Microalgae Mass Production Methods[END_REF].

In circular ponds, productivities of about 15 g/m 2 /d have been reported [START_REF] Shen | Microalgae Mass Production Methods[END_REF].

Unstirred ponds

Unstirred ponds are the most economical and least technical method of commercial microalgae culture [START_REF] Shen | Microalgae Mass Production Methods[END_REF]. These types of open ponds can be natural such as lakes and lagoons, or artificially constructed from natural water ponds. Generally, their depth does not exceed 50 cm. The prerequisite for this type of system is that they must autonomously combine the climatic and nutritional conditions necessary for the development of microalgae [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF]. However, such unmixed systems have a relatively low microalgal density (< 1 g/L) [START_REF] Borowitzka | Large-scale algal culture systems: the next generation[END_REF].

Regardless of the design, open ponds offer ease of use and low energy costs [START_REF] Siqueira | Introductory Chapter: Microalgae Biotechnology -A Brief Introduction[END_REF][START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF][START_REF] Michael | Commercial production of microalgae: ponds, tanks, and fermenters[END_REF]. In addition, the costs of construction, operation and maintenance of these culture ponds are low, making them competitive [START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF][START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF]. However, such simplicity of implementation has its limits. In particular, they are not very technical. These open culture systems allow only limited control of growing conditions [START_REF] Siqueira | Introductory Chapter: Microalgae Biotechnology -A Brief Introduction[END_REF]. Most of the time, the cultures growing there are rapidly limited by growth parameters, especially light and carbon dioxide [START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF][START_REF] Shen | Microalgae Mass Production Methods[END_REF]. As a matter of fact, light can only penetrate water to a limited extent. However, in such systems, the depth of the culture is the result of a compromise between the need to provide adequate light to the cells and the need to maintain sufficient water depth for mixing [START_REF] Michael | Commercial production of microalgae: ponds, tanks, and fermenters[END_REF]. Consequently, in the majority of cases, the depth remains too deep to ensure sufficient light exposure to the microalgae. In addition, the mixing system is relatively inefficient and gas transfer rates are low, leading to a CO 2 deficiency in the culture.

In addition, the direct exposure of open ponds to the surrounding environment creates other issues. More precisely, open ponds are dependent on the local climate in terms of temperature and luminosity [START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF]. Depending on the climatic conditions, cultures can undergo temperature fluctuations as well as changes in the ionic composition of the culture medium due to evaporation. As a result, it is difficult to maintain the performance of a system on an annual basis due to its sensitivity to daily and seasonal variations in light and temperature. In addition to these fluctuations, open ponds are also subject to contamination. This susceptibility limits their exploitation for the production of low or medium added value molecules [START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF]. To reduce the risk of contamination, these culture methods are limited to a restricted number of strains of microalgae that can develop under severe growth conditions (high salinity, high alkalinity, etc.) [START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF][START_REF] Michael | Commercial production of microalgae: ponds, tanks, and fermenters[END_REF]. Another solution to overcome these limitations is the cultivation under greenhouse [START_REF] Alberto | Chapter 1 -An Open Pond System for Microalgal Cultivation[END_REF][START_REF] Luis | Semi-continuous culture of a microalgal consortium in open ponds under greenhouse conditions using piggery wastewater effluent[END_REF]. This method allows to protect the culture from adverse environmental conditions (rainfall, temperature, and luminosity) and reduce the risk of contamination.

All these obstacles lead to cultures with low cell density (mainly between 0.1 and 0.5 g/L) and low productivities: areal (15 to 30 g/m 2 /d for a 30 cm deep open pond) and volume (0.05 to 0.1 g/L/d) [START_REF] Pulz | Photobioreactors: production systems for phototrophic microorganisms[END_REF][START_REF] Michael | Commercial production of microalgae: ponds, tanks, and fermenters[END_REF]. As a result, one of the main bottleneck in this type of culture system is the cost of harvesting due to the achievement of rather low cell concentrations [START_REF] Shen | Microalgae Mass Production Methods[END_REF][START_REF] René | Potential of sponges and microalgae for marine biotechnology[END_REF]. To overcome the limitations associated with open culture systems, culture in closed photobioreactors has emerged as an alternative. With the rise of the biorefinery concept, they are thought to represent the future of the industry driven by nutraceuticals and pharmaceuticals.

Closed PBRs

A closed PBR refers to a transparent culture device for which no direct exchange between the external environment and the culture system is possible. These culture systems offer a closed environment that protects the culture from direct contamination and limits evaporation [START_REF] Kumar Duran | A review on microalgae strains, cultivation, harvesting, biodiesel conversion and engine implementation[END_REF]. They can be located indoors or outdoors and lit by sunlight or by artificial lighting.

Initially designed to overcome the limitations of open ponds, closed systems are known to have a higher illuminated surface to volume ratio, better mixing efficiency and better control of culture parameters such as temperature, carbon dioxide and oxygen contents, and pH [START_REF] Torzillo | Production of Spirulina biomass in closed photobioreactors[END_REF][START_REF] Rawat | Dual role of microalgae: Phycoremediation of domestic wastewater and biomass production for sustainable biofuels production[END_REF]. All these aspects allow closed systems to reach productivities about 8 times higher (about 0.8 -1.3 g/L/d) and cell concentrations about 16 times higher (about 2 -8 g/L) than open ponds [START_REF] Safi | Morphology, composition, production, processing and applications of Chlorella vulgaris: A review[END_REF][START_REF] Pulz | Photobioreactors: production systems for phototrophic microorganisms[END_REF][START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Kumar Duran | A review on microalgae strains, cultivation, harvesting, biodiesel conversion and engine implementation[END_REF][START_REF] Mata | Microalgae for biodiesel production and other applications: A review[END_REF][START_REF] Venugopal | Marine polysaccharides: Food applications[END_REF]. Unlike open ponds, these performances are easily reproducible. In addition, their design allows them to grow single-species in an axenic manner, making these systems particularly suitable for the production of high value-added molecules for the pharmaceutical and cosmetics industries [START_REF] Stark | An Introduction to Photosynthetic Microalgae[END_REF][START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Tredici | From open ponds to vertical alveolar panels: the Italian experience in the development of reactors for the mass cultivation of phototrophic microorganisms[END_REF][START_REF] Huang | Biodiesel production by microalgal biotechnology[END_REF]. However, such systems also suffer from several drawbacks. Their main limitations are overheating, biofouling, oxygen accumulation or damage to the cells by shear stress. They also involve high construction and operating costs and are difficult to expand [START_REF] Siqueira | Introductory Chapter: Microalgae Biotechnology -A Brief Introduction[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF].

Over the last decades, a wide variety of different closed PBRs have been developed to meet the needs of industrial production. These include column, tubular, flat plate, stirred tank, dome-shaped, helical-type, conical and torus reactors, or even hybrid culture systems [START_REF] Singh | Development of suitable photobioreactor for algae production -A review[END_REF]. However, despite this wide variety of designs, only a few of them can be used for mass culture. Currently, there are four geometries of closed PBRs recommended for large-scale operations: column reactors, tubular reactors, plastic bag reactors and flat panel reactors [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF].

Vertical column reactors

Vertical column photobioreactors are cylindrical vessels made of transparent material, usually glass or acrylic [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF]. Their size is relatively limited. For reasons of light availability and penetration into the columns, their diameter generally does not exceed 20 cm. The height of the cylinder is restricted to about 4 meters due to the limits of gas transfer (CO 2 and O 2 ) and the resistance of the transparent materials used [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF][START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. As a result, these systems are characterised by a high surface/volume ratio. In addition, the constant supply of gas in the form of bubbles and without any physical system allows a continuous and gentle agitation of the culture with little shear stress. Thus, these types of PBRs are often associated with a high volumetric gas transfer coefficient and little cell damage [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. Different column reactor geometries exist as simple bubble columns, annular columns and airlift columns [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF]. They are shown in Figure 2.9. The bubble column is a cylinder whose height is more than twice the diameter [START_REF] Singh | Development of suitable photobioreactor for algae production -A review[END_REF]. It is externally illuminated and includes a sparger at its base allowing gas injection. The latter causes bubbles to form and move turbulently responsible for gas transfer and mixing of the culture [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF].

The annular photobioreactors are composed of two concentric cylinders creating an empty space between their centers. These systems can thus be illuminated in their center and/or from the outside. This is the only difference with bubble column reactors.

Airlift photobioreactors are gas/liquid systems composed of two distinct but interconnected sections: the riser and the downcomer. Mixing and mass transfer is achieved by injecting air through a sparger into the riser compartment, which then allows the fluid to flow into the downcomer compartment, which does not receive any gas. Depending on the circulation mode, airlifts reactors can be classified in two categories: internal loop airlift reactor and external loop airlift reactor [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. They are shown in Figure 2.10. The substantial advantage of airlifts is the better agitation they offer compared to conventional bubble columns [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. This efficiency is associated with lower energy consumption, lower shear stress, the absence of different gradients (temperature, nutrients, gas) and a relatively better gas-liquid mass transfer [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF].

To summarize, the column reactors have the advantage of creating a flow circulation allowing better mixing and high mass transfer with low shear stress [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF]. This simple and compact design has low energy consumption and is relatively easy to sterilize [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF][START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. In addition, they can reach higher final biomass density than open ponds (up to 4.4 g/L for airlifts and 3.1 g/L for bubble columns) [START_REF] Kumar | Growth characteristics of Chlorella sorokiniana in airlift and bubble column photobioreactors[END_REF]. However, these systems involve higher capital, construction and operating costs. Finally, because of their relatively large diameter, the light is quickly attenuated on the column depth. This low light availability and the cleaning problems are the main obstacles to their large-scale implementation [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF].

Tubular reactors

Tubular systems represent one of the most suitable configurations for mass culture [START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF]. They generally consist of an array of transparent tubes made of glass, polypropylene-acrylic or polyvinyl chloride [START_REF] Kumar Duran | A review on microalgae strains, cultivation, harvesting, biodiesel conversion and engine implementation[END_REF]. Their internal diameter is generally small (between 10 and 60 mm on average) to increase light penetration compared to column reactors. They can reach significant lengths of several hundred meters [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF]. Inside, the culture is circulated by a pump or an airlift system allowing an exchange between gas and liquid. The nutrient supply is generally carried out by a tank attached to the tubes in the case of semi-continuous or continuous mode cultivation. These systems can be artificially lit or simply exposed to sunlight. They can be arranged in different patterns (straight, bent or spiral) and orientations (horizontal, vertical or inclined) to maximize light capture [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF] (Figure 2.11). While a vertical arrangement of the tubes allows for greater mass transfer and lower energy consumption, the horizontal configuration is more flexible. In this respect, it is possible to obtain a very small diameter since the mechanical stress of the materials is bypassed with this orientation. As a result, horizontal tube systems generally provide a higher surface/volume ratio. In addition, it is also possible to modulate the angle of incident light with respect to bioreactors, which allows for better light harvesting. An inclination of 45°has been shown to be the best compromise between improved light harvesting and gas transfer and no increase in costs associated with structural supports [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. However, this orientation requires a larger area of land and temperature control systems that can be costly [START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF][START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF].

In general, these systems are relatively cheap and easier to scale up than most other culture systems [START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF]. In addition, they have a large light harvesting area associated with sizable final biomass concentrations (from 2 g/L to 7 g/L) [START_REF] Watanabe | Photosynthetic performance of a helical tubular photobioreactor incorporating the cyanobacterium spirulina platensis[END_REF][START_REF] Frumento | Cultivation of Chlorella vulgaris in tubular photobioreactors: A lipid source for biodiesel production[END_REF]. However, like other systems, they suffer from operating difficulties. One of the major challenges in scaling up these tubular reactors is the accumulation of oxygen conditioned by the length of the tubes, which could cause an inhibition of photosynthesis [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF][START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. They can also cause problems with temperature control and fouling [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF]. In addition, they occupy (a) Vertical [START_REF] Daniel | Beneficial Reuse of Industrial CO2 Emissions Using a Microalgae Photobioreactor: Waste Heat Utilization Assessment[END_REF] (b) Horizontal [START_REF] Alaswad | Technologies and developments of third generation biofuel production[END_REF] (c) Helical [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF] Figure 2.11: Schematics of several configurations of tubular photobioreactors a large area of land and cleaning problems are not easy to overcome [START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF]. Finally, depending on their shape and pumping speed, a high shear stress can occur [START_REF] Michiel | Cultivation of shear stress sensitive and tolerant microalgal species in a tubular photobioreactor equipped with a centrifugal pump[END_REF].

Plastic bag photobioreactors

Plastic bags photobioreactors are attractive, and in recent years they have received increased attention for the commercial production of microalgae due to their low cost [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF][START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. They are transparent and cheap plastic sheaths made of polyvinyl chloride (PVC) or polyethylene (PE) that can be exposed indoors or outdoors. These plastic bags can be of different shapes and arranged in different models according to their respective volume. For small volumes (> 5 L), they can be hung directly. Beyond that, they are usually hung at each end from a metal structure as shown in Figure 2.12a [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF]. For large volumes, there is also a floating configuration of these systems. In this case, the bags are immersed in pools to reduce costs and control the temperature in summer (Figure 2.12b) [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF][START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. These bags can also be installed with an aeration system to improve yields [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. Although these systems are very simple and inexpensive, it must be kept in mind that they have limited durability. In addition, gas mixing and exchange is very limited [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF][START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. These systems have a low surface/volume ratio and suffer from biofouling and photolimitation problems. Unlike other closed systems, it is not possible to control the growing conditions. These disadvantages lead to low cell densities and productivity [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF][START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. Finally, the disposal of large quantities of plastic bags is a major challenge for large-scale operations [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF][START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF].

As pointed out by the study of Huang et al. [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF], designing an effective PBR remains a difficult task. Several parameters such as light, mixing, mass transfer, temperature, pH and operating costs must be considered as well as the interaction of these individual factors. With a view to lifting the current levers of light distribution, mass transfer and construction cost, new designs are being explored [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. The challenge today is to find a new, compact cultivation system that guarantees control of growing conditions, with high performances, i.e. high biomass productivity, and low investment and operating costs. Despite numerous developments of photobioreactors, few have been developed on an industrial scale. Although offering many advantages over open systems such as reduced contamination, controllable culture conditions and less CO 2 loss and water evaporation, closed culture systems still have a low light-to-biomass conversion [START_REF] Liao | A novel photobioreactor generating the light/dark cycle to improve microalgae cultivation[END_REF]. One of the reasons for this is the very frequent photolimitation in these cultures due to the relatively large cultivation depth [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF]. Among the proposed strategies to improve light absorption and conversion in photobioreactors, shortening the light path appears to be the most promising.

Reduced optical path designs

The most important factor in the growth of photosynthetic microorganisms is the availability of light. This is all the more true since it cannot be stored by the cells. However, as detailed in the previous sections, the light energy received by photoautotrophic microorganisms at the PBR scale is very often limited due to an exponential attenuation of the irradiation over the depth of a photobioreactor. This phenomenon is the result of a combination of different factors including the density of the photon flux reaching the surface of the culture, the wavelengths involved, the depth of the culture and the cell density [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF][START_REF] Acién | 1 -Photobioreactors for the production of microalgae[END_REF]. It can be expressed mathematically by Beer-Lambert's law (Eq. 2.2), where I l is the available light at the depth l in the PBR, I 0 is the incident intensity, α is the extinction coefficient which depends on the wavelength, and c the species concentration. This relation basically relates the attenuation of light to the properties of the medium through which the light is traveling. However, the law is no longer valid at high concentrations, especially if the medium is highly scattering and/or participating.

I l = I 0 × e -αlc (2.2)
Light penetration, in most cases, is the decisive and engaging step for the success of algae cultures [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. With this in mind and from the Figure 2.1, one can easily understand that one of the levers to increase the length of light penetration within a culture and thus its availability is the shortening of the light path (< 5 cm) [START_REF] Richmond | Handbook of Microalgal Culture: Biotechnology and Applied Phycology[END_REF]. The narrowing of the optical path has a twofold purpose. On the one hand, it provides a more homogeneous light field by increasing the availability of light for the cells [4]. On the other hand, a short optical path makes it possible to best adapt the L/D cycles undergone by the microalgae to the characteristic times of photosynthesis through ligthing conditions manipulation [START_REF] Richmond | Handbook of Microalgal Culture: Biotechnology and Applied Phycology[END_REF]. In this context, it has been demonstrated for years that the productivity of microalgae in narrow optical path photobioreactors has improved [START_REF] Richmond | Handbook of Microalgal Culture: Biotechnology and Applied Phycology[END_REF][START_REF] Gifuni | New ultra-flat photobioreactor for intensive microalgal production: The effect of light irradiance[END_REF]. All this has been made possible by the design of new generations of PBRs called flat plate PBRs (FP-PBR) and thin-layer cascade (TLC) reactors.

Flat panel reactors

The simplest design of a FP-PBR consists of a thin rectangular transparent box made from two panels arranged in parallel. These can be made of polymers such as polyvinyl chloride (PVC), polycarbonate (PC), polymethylmethacrylate (PMMA) or polyethylene (PE), or glass [START_REF] Acién | 1 -Photobioreactors for the production of microalgae[END_REF]. These plates are usually separated by a few centimetres, inside which a thin layer of microalgae suspension flows (Figure 2.13a). This thickness is the central element of the PBR since it will determine the length of the optical path and the surface/volume ratio [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. The panels can be arranged vertically or horizontally with a certain inclination to maximize the incident light intensity. They can be illuminated on one or both sides. To control temperature, a transparent cooling jacket with water flowing through it can be added on one side. In general, the mixing of the culture is achieved by the bubbles rising due to the injection of a gas flow into the bottom of the reactor [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. Thus, it is a bubble column-like PBR but with a particular geometry. Many different types of FP-PBR have been developed since then. They are categorised mainly by their mixing mode. On some FP-PBR desings, mixing can be done via a pump, while on others it is achieved by an arilift system [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. In addition, from the very simplistic basic design, modifications can be made to improve the performance of the PBR. For example, a V-shape and the introduction of horizontal baffles in the PBRs have improved mixing, minimized dead volume and induced faster L/D cycles [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF][START_REF] Acién | 1 -Photobioreactors for the production of microalgae[END_REF][START_REF] Iqbal | A flat-sided photobioreactor for culturing microalgae[END_REF]. Among this wide variety of designs, one is particularly noteworthy as it is a large-scale patented system on the market. These are airlift-type reactors in which the flat plates are divided into a series of internal rectangular channels (Figure 2.13b). These reactors have been proposed in part for their greater structural rigidity, more efficient culture flow, and optimal light distribution within the PBR by guiding the flow induced by the static mixers. As a result, all microalgal cells are transported back and forth between photic and aphotic areas within a range of about 1 Hz compared to about 0.2 s in conventional FP-PBR [START_REF] Huang | Improving performance of flat-plate photobioreactors by installation of novel internal mixers optimized with computational fluid dynamics[END_REF].

Generally speaking, FP-PBR offer a design that guarantees a considerably high surface/volume ratio and thus an increase in the surface area for harvesting the light necessary for the growth of microalgae [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. This feature combined with a narrow optical path allows cultures with high densities ranging from 10 to about 50 g/L and a high volumetric biomass productivity of 0.25 to 3.64 g/L/d to be achieved [START_REF] Janssen | Photosynthetic efficiency of Dunaliella tertiolecta under short light/dark cycles[END_REF][START_REF] René | Potential of sponges and microalgae for marine biotechnology[END_REF]. In this respect, a recent study conducted on a Chlorella sorokiniana culture offered a new design of FP-PBR with the aim of maximizing biomass concentration and productivity. The particularity of the latter is a very thin optical path of 3 mm thickness, which allowed a maximum cell density of 24 g/L and a productivity of 32.2 g/m 2 /d to be obtained [START_REF] Gifuni | New ultra-flat photobioreactor for intensive microalgal production: The effect of light irradiance[END_REF]. In addition, the simple design of the FP-PBR allows the light path to be modulated as desired [START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF]. Further advantages of such a design are to be highlighted. First, they have low energy consumption [START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF]. Second, they generally provide good mixing and a high gas-liquid mass transfer rate, with relatively low mechanical force exerted on the cells [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF]. The open area at the top of the PBR serves as the degassing zone reducing the need for a dedicated degassing unit [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. However, with regard to the oxygen build-up, the literature reports non-consensual results. Some authors advocate a limitation of oxygen accumulation [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF]. While others raise the problem of oxygen accumulation as a major disadvantage of this design due to a high photosynthetic activity coupled with a small thickness of the reactors [START_REF] Acién | 1 -Photobioreactors for the production of microalgae[END_REF]. Finally, the temperature can be easily controlled thanks to the presence of a cooling jacket [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF].

However, FT-PBR also has its limitations. One of the most notable is the risk of photoinhibition of the culture [START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF][START_REF] Acién | 1 -Photobioreactors for the production of microalgae[END_REF]. Another major disadvantage reported with FT-PBRs is the formation of wall biofilm due to the very dense cultures [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF]. In addition, some FT-PBR designs do not have a cooling jacket. Under these conditions, cultures are susceptible to strong temperature variations affecting their growth [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Wang | Closed photobioreactors for production of microalgal biomasses[END_REF]. Finally, these reactors present difficulties in moving to industrial scale due to their limited scalability and their relatively high space and light energy requirements [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF]. In addition, they are relatively expensive to manufacture [START_REF] Brennan | Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF][START_REF] Acién | 1 -Photobioreactors for the production of microalgae[END_REF]. To further reduce the cost of equipment and improve economic feasibility, a new design of FP-PBR consisting of a transparent plastic bag placed on a rigid frame has been proposed [START_REF] Hamed | The Evolution and Versatility of Microalgal Biotechnology: A Review[END_REF][START_REF] Yen | Chapter 10 -Design of photobioreactors for algal cultivation[END_REF].

Thin-layer cascade reactors

The thin-layer cascade reactors are based on the combination of an inclined and illuminated platform system on which the flow of microalgae circulates and a transparent tube in which the suspension is pulled upstream to close the loop (Figure 2.14). These reactors are characterized by a shallow culture depth (< 50 mm) and a fast (0.4 -0.5 m/s) culture flow [START_REF] Pruvost | Development of a thin-film solar photobioreactor with high biomass volumetric productivity (AlgoFilm©) based on process intensification principles[END_REF][START_REF] Grivalský | Development of thin-layer cascades for microalgae cultivation: milestones (review)[END_REF]. In addition, they offer a high surface/volume (> 100 m -1 ) [START_REF] Grivalský | Development of thin-layer cascades for microalgae cultivation: milestones (review)[END_REF]. These systems have the advantages of open systems such as direct solar irradiation, easy heat dissipation, simple cleaning and maintenance, and efficient degassing. Moreover, the originality of these thin-layer cascade reactors lies in the fact that they allow to obtain dense cultures (> 10 gDW/L, up to 40 -50 gDW/L [START_REF] Doucha | Outdoor open thin-layer microalgal photobioreactor: potential productivity[END_REF] with high productivity (> 30 g/m 2 /d) although they are open systems [START_REF] Grivalský | Development of thin-layer cascades for microalgae cultivation: milestones (review)[END_REF][START_REF] Doucha | Outdoor open thin-layer microalgal photobioreactor: potential productivity[END_REF]. However, like any system, it has its own limitations, the main one being the high probability of contamination induced by direct exposure of the culture to the environment.

All the different bioreactor designs described above are used on an industrial or semi- industrial scale. There are laboratory scale equivalents to these bioreactors for research purposes. However, these commercial solutions offer designs whose optical path is still too thick (about 2-3 cm) [START_REF] Straka | Effect of culture density on biomass production and light utilization efficiency of Synechocystis sp[END_REF]. As a result, research teams find themselves in most cases having to develop their own reactor to meet specific needs [START_REF] Gifuni | New ultra-flat photobioreactor for intensive microalgal production: The effect of light irradiance[END_REF].

Operation mode

The complete adequacy of the photobioreactor set-up to metabolic and kinetic conditions is a very important aspect in the development of technologies for the production of microalgae. In addition to the choice of a PBR design, the operation mode must be carefully selected. Cultures of microorganisms can be carried out in different ways, depending on the strain involved, nutrient requirements, desired product, etc. In open or closed systems, microalgae can be cultivated by batch (discontinuous), fed-batch, semi-continuous and continuous processes [START_REF] Koller | Design of Closed Photobioreactors for Algal Cultivation[END_REF][START_REF] João | Microalgae Bioreactors[END_REF].

In batch processes, culture medium and cells are added at the beginning of the culture. No nutrients are added after the initial loading, and the product is only removed at the end of the process. This process is the safest way to maintain aseptic production conditions, to control the genetic stability of the strain as well as the traceability of all related materials [START_REF] João | Microalgae Bioreactors[END_REF]. However, the major disadvantage is the addition of high initial concentrations of substrate which can be inhibitory. Similarly, undesirable products may be formed through the direct metabolic pathway of the organism [START_REF] João | Microalgae Bioreactors[END_REF]. In addition, as the culture grows, the cells are exposed to the mutual shading effect reducing the availability of light to the cells, and therefore negatively affecting the culture.

The fed-batch mode corresponds to a cultivation mode in which one or more nutrients are added to the reactor throughout the culture, while the cells and products remain until the end of the operation. Unlike the batch mode, this mode of operation prevents nutrients Chapter 2. Light use in Biotechnology from becoming limiting factor. In addition, the regulated addition of certain nutrients controls both the deviation of cell metabolism for synthesis of the desired product and the specific growth rate [START_REF] João | Microalgae Bioreactors[END_REF]. However, the major disadvantage of such a system is the risk of overfeeding or underfeeding which can be detrimental to cell growth and/or product formation.

In the semi-continuous process, the culture follows a batch development. Once the maximum cell concentration has been reached and stabilized, part of the culture medium is removed. The remaining part is used as inoculum to start a new culture by adding fresh culture medium to replace the volume removed. This process mainly saves time. The cells are already acclimatized to the culture conditions in the PBR.

Finally, the continuous process is achieved by continuously feeding the reactor with fresh medium at a specific flow rate and by continuously withdrawing the spent medium at the same flow rate to maintain a constant volume and aim at an equilibrium state. The use of this culture mode has several advantages over discontinuous modes. On the one hand, they offer a higher degree of control of the culture. Growth rates are regulated and maintained for extended periods of time and the biomass concentration can be controlled by varying the dilution rate [START_REF] Mata | Microalgae for biodiesel production and other applications: A review[END_REF]. On the other hand, the addition of constant medium limits the phenomenon of cell mutual shading and accumulation of inhibiting agents. This mode of operation is highly sought-after when the objective is to study the effect of a parameter without interference from other factors that vary during growth in traditional batch cultures [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF]. However, such an operation exposes the cells to a higher risk of contamination and genetic deviation of the population over the long term. In addition, another major weakness of such a mode of operation is the impossibility of traceability of related materials which limits the applications.

Conclusion

This chapter aimed to present the problems and stakes of the cultures lighting in microalgal biotechnology. Currently, two systems of culture have been developed: open systems, where the culture is exposed to the atmosphere, and closed systems, where the cultures have no direct contact with the atmosphere. The choice of the production system depends on the degree of control needed to produce the desired product and its value. At the industrial level, microalgae are often cultivated in ponds, but the production of high value-added molecules requires a closed system. Regardless of the chosen culture system, light availability is very often limited. This observation has paved the way to manipulate the light supply in order to improve the economic viability of bioprocesses using microalgae. However, to date, there are a large number of studies on the interaction between light and microalgae. Although extensively studied, understanding this interaction remains hampered due to significant disparities in the biological responses reported in the literature. The result of this investigation has reawakened the need for further research. In addition, the literature review highlighted a non-uniformity in the experimental set-up as well as in the method of culture monitoring that would likely be at the origin of the discrepancy in the results obtained so far. In particular, two points emerge: the question of the strain acclimation to the light conditions as well as the PBR design used for the study (optical thinness and presence of optical aberrations).

The ambition of this PhD is to produce high quality biological data allowing a real extrapolation to the industrial scale. For this purpose, an experimental device has been developed to guarantee the same light history to all cells. It consists of a flat photobioreactor with a thin design and an instrumentation system allowing a continuous monitoring and a semi-automated operation of the bioreactors. Finally, to study the impact of light conditions on the growth of microalgae, the continuous culture mode was chosen to eliminate the self-shading of cells throughout the study. The characterization of this setup is the subject of the next chapter.

Introduction

The objective of this thesis is to better understand the influence of intermittent light conditions on the growth of microalgae. To this end, an experimental device was developed using a sequential methodology during the first two years of the thesis. This system includes a photobioreactor, its lighting device and the instrumentation developed for its operation in continuous mode. The following chapter is dedicated to their description and characterization. It includes five parts.

The first part presents the objectives to be reached by the experimental device before launching the test campaign. Specifically, this system requires exposing all cells within the culture medium to the same amount of light for a proper study.

To answer this need, a special culture vessel called the "ultra-thin flat panel" was developed. Its description is presented in the second part.

In addition to the design of the PBRs, the lighting system of the culture is crucial. It must provide a stable and homogeneous light to the culture and be adjustable in intensity and frequency. The developed lighting system, coupled with an instrumentation system, allows continuous monitoring of the culture. Thus, its description, its characterization, and its operation are the subject of the third part of this chapter.

A fourth part is devoted to the systems delivering the culture medium and gas to the culture. Although less decisive in the design of the experimental set-up, these systems are nonetheless essential. Indeed, they support the needs in nutrients of the culture.

Finally, once the device has been fully designed and developed, the question of its operation arises. The latter is also of great importance for the quality of the results. For this work, the choice was made to operate the device in turbidostat mode. Its operation is detailed in the last part of this chapter.

Objectives

As mentioned in the previous chapter, microalgae tend to self-shade as they grow, restricting access to light to other microalgae present in the same environment. This event results in the creation of a light gradient within the culture. Coupled with strirring flow, this leads to the perception of different light patterns by cells within the same culture. Consequently, not all cells perceive the same amount of light. In studies on the influence of light on microalgae's response, this phenomenon can be a significant source of bias. Indeed, as not all cells are exposed to the same light pattern, the result is the average combination of different light conditions. Therefore, in understanding and studying the influence of light on microalgae growth, it is essential that the experimental device guarantees an uniform exposure of the cells to light. However, to date, such a device does not exist on the market. With this objective in mind, an experimental device was designed and developed to 3.2. PBR design description 91 produce high quality biological data under carefully controlled conditions. This device must meet specific criteria to ensure an identical light history for all cells in the culture:

• The geometry of the PBR must allow a good distribution of the light and avoid as much as possible optical distortions,

• A thin design along the light path of the PBR is required to limit the light attenuation,

• The light source used must have a spectrum that triggers photosynthesis,

• The lighting device must provide light uniformly on the surface of the PBR,

• The light signal must not show any intensity and/or spectral variations in time,

• In spite of the thin design, the cell concentration must be kept low to avoid the self-shading phenomenon.

PBR design description

In designing an efficient PBR, light penetration and distribution inside the PBR are essential parameters to assess the impact of light upon microalgae growth. Along these lines, a special culture vessel called the "ultra-thin flat panel" photobioreactor was developed to expose all cells to the same light pattern. Its flat design allows, among other things, to eliminate the optical aberrations associated with angled or curved geometries.

The final set-up consists of a photobioreactor, its lighting system, and the instrumentation developed for its operation. The whole system is placed in a chamber (80 cm x 80 cm x 80 cm) to avoid the disturbance of external lighting (Figure 3.1). In total, two chambers were built, each containing three PBRs to work in triplicate, under the same experimental conditions.

Taking a closer look at the ultra-thin flat PBR design shows that it consists of two compartments (Figure 3.2). The first compartment is used to grow the microalgae, while the second compartment acts as a cooling jacket to regulate the culture's temperature. Each section is obtained by stacking two glass sheets held together by a 316L stainless steel skeleton. Between each glass sheet and steel layer, a silicone seal is placed to guarantee the water tightness and the harmlessness of the PBR. Each compartment has a thickness of 6 mm. With such thinness, it is essential to ensure that the shear stress imposed on the culture is not too high, but sufficient to allow a good stirring. In the literature, the critical value above which growth is impeded is defined at 0.9 Pa for the Chlorella genus [START_REF] Wang | Effects of shear stress on microalgae -A review[END_REF]. In order not to exceed this criterion, a CFD simulation was used to compute the shear stress associated to different PBR thicknesses [START_REF] Jiang | Shear stress computation in a millimeter thin flat panel photobioreactor: Numerical design validated by experiments[END_REF]. According to this study, we moved towards a 6 mm culture vessel imposing a shear stress of 0.75 Pa on the cultures, which is below the critical value. This thickness represents a required trade-off between the PBR thickness, the shear stress and the quality of the mixing.

The total structure measures 47 cm high x 8 cm width and has a 135 mL working volume. Its V-shaped configuration has been designed to improve mixing within the culture and therefore avoid cell settling and limit biofilm formation. Furthermore, this PBR has been designed so that it can be operated either in batch or continuous mode.

To this end, various holes have been drilled in the structure to allow the injection or withdrawal of medium. Located at the V-shaped base, there are two opposite holes: one for injecting the medium in the case of continuous operation, the other for sampling and/or draining the PBR. An overflow is located in the upper part of the PBR allowing a continuous operation of the PBR. The latter leads to a sealed and closed jar in which the culture medium discharged during the culture process is collected. Finally, bubbling is ensured by a hole located at the base of the V-shape through which the air is injected for both gas supply and mixing purposes.

The originality of this PBR design lies into its thinness (6 mm thick) which, combined with a low density culture, ensures that the illumination is homogeneous throughout the culture. In addition, its flat shape allows to avoid as much as possible undesirable optical effects leading to heterogeneous lighting inside of the culture medium. In this way, all the microalgae cells will receive the same amount of light allowing for a proper study of the impact of lighting conditions on microalgal growth.

Lighting device description

The developed lighting device fulfills two main objectives. On the one hand, it must provide uniform illumination evenly spread on the surface of the three PRBs. On the other hand, the device must be controllable to provide light intermittently to the cultures for studies conducted in fluctuating and flashing light. To meet these criteria, Light-Emitting Diodes (LEDs) were chosen as the light source because of their ability to emit customizable light spectra, as well as their low heat generation, low power consumption and durability. In addition, when coupled with electronic switches, they allow precise and fast alternating

Light characteristics

The illumination of the culture vessels was provided by a LEDs panel developed especially for this work. The structure is made of plexiglass with a size of about 80 cm x 60 cm corresponding to the width of the chamber and going beyond the height of the PBRs, respectively. On this LEDs panel, ten LEDs strips are glued two by two to form five double rows of light. The arrangement has been designed so that the three central rows of LEDs are facing the 3 PBR, while the two remaining rows are at the ends of each side of the chamber (Figure 3.1b). Thus, each PBR receives the same amount of light, i.e., they are all illuminated by a central strip and receive light contribution from two other lateral strips. Yet the edge effect still exist but it is reduced thanks to the reflexion of the chamber walls.

LEDs spectrum

To induce photosynthesis, it is important to provide a light spectrum adapted to the culture of microalgae. Previously, we have seen that photosynthesis is triggered under an actinic lighting (PAR: 400 to 750 nm). In this range, microalgae absorb in specific regions of the visible spectrum corresponding to the pigments they contain. In green microalgae, the major pigments are chlorophylls a and b, and some carotenoids such as lutein and β-carotene. While chlorophylls a and b show two absorption peaks in the blue (430 and Chapter 3. Development and characterization of the experimental device 465nm, respectively) and red (663 and 645nm, respectively) regions, carotenoids absorb rather in the blue region of the spectrum (spectral range from 400 to 500 nm). Faced with this observation, the LEDs chosen to carry out this work (Liite-Liili 2400 2ft 4000K from CATS) have a light spectrum corresponding to a neutral white light. As can be seen in Figure 3.3, the spectrum of these LEDs covers the entire PAR whose composition is relatively balanced and uniform over the visible spectrum, with a peak near the blue region. The latter is relatively close to that of the light emitted by the sun, thus covering all the needs of microalgae for their growth.

Spatial distribution of light

Once the spectral component of the LEDs is defined, the spatial distribution of the light within the two measurement chambers is characterized. This parameter is all the more important as it allows to validate that the microalgae are subjected to a homogeneous light field, a major precaution in this work.

For this purpose, numerous measurement points of light intensity were recorded in both chambers using a light meter (LI-COR LI-250A). These measurements were performed at the PBR plane at different spatial positions. Following these measurements, a map of the spatial distribution of the light at the surface of the PBR was obtained for each chamber by a bilinear interpolation. This method calculates the value of any point from its four nearest neighbors in each direction. Thus, it proposes the spatial representation of the light to the dimensions of the chambers. The resulting maps are shown in Figure 3.4, where the measurement points appear as white dots. The X-axis represents the chamber width (x, in cm) whereas Y-axis accounts for its height (z, in cm). The glazed part of a PBR is mapped with a dashed black line. In addition to this mapping, the variation of the light intensity over the height of the photobioreactor is depicted. The displayed results show only the light field on a single PBR in each chamber for readability reasons. However, the results are processed over the entire chamber intensity data. To compare the results between the two chambers, the light intensities were normalized by the maximum intensity obtained for each chamber. Both chambers exhibit relatively similar light distribution over the PBR surface. One can see a higher light intensity in the center of the glass part than on the upper and lower parts of the PBR. However, over the entire glass surface of the PBR (from 15 to 55 cm in Z-axis), the light intensity varies between 84 and 100% of the maximum intensity for both chambers. By taking a closer look at the dispersion of light intensities, chamber 1 has a coefficient of variation of 6 %, while chamber 2 exhibits a coefficient of variation of 9 %. In both cases, the intensity values' dispersion around the mean is less than 10 %. Consequently, the results suggest that the light field received at the PBR surface is relatively homogeneous in the two chambers. Based on this observation, the intensities given later in the manuscript correspond to the peak intensity.

Light stability

Ensuring the homogeneity of the light distribution on the surface of the photobioreactors is not enough to conduct studies on understanding the relationship between light and microalgae response. It is also necessary to ensure that the spectrum provided is stable over time and does not vary with the electrical power supply. Indeed, as photosynthetic organisms, microalgae are sensitive to the slightest variations in light intensity and/or quality.

To this end, light spectra were recorded continuously for 48 hours, the acquisition time corresponding to a complete culture in the device. The procedure was repeated twice at two different electrical powers: the minimum and maximum applicable. These measurements were performed on both chambers and gave similar results. Only the results of chamber 1 are displayed to avoid redundancy. They are reported in Figures 3.5 and 3.6.

Considering first the whole spectrum, one can see that the shape of the spectrum is preserved between the minimum and maximum power (Figure 3.5). However, a slight loss of the peak in the blue region of the spectrum at maximum power is notable. This decrease has been quantified at 14 % compared to the peak observed at minimum power, which is a minimal loss. Therefore, the spectrum is not distorted in response to the applied power. Then, by looking at the temporal stability, one can note that regardless of the power used, the spectra are perfectly superimposed between 0 and 48 hours. From these results, it can be concluded that no modification of the spectrum occurs over time. To go further in the analysis, the stability over 3 wavelengths in time was also explored. All the chosen wavelengths have a biological meaning. The first, at 430 nm, corresponds to the absorption peak of chlorophylls in the blue region of the visible light spectrum. The second wavelength (550 nm) is the one corresponding to the absorption of the cell walls, outside the pigment absorption zone. Finally, the last wavelength, 663 nm corresponds to the absorption of chlorophylls in the red region of the spectrum. Like the spectrum, the stability of these three wavelengths was followed over 48 hours, corresponding to the culture time in the device. The results are graphed in Figure 3.6. One can notice that, at maximum power, the time to reach the steady-state is a little longer than at minimum power (about 1 hour and instantly, respectively). Once the steady-state is reached, the signals are perfectly stable over time.

These results demonstrated that in addition to being homogeneous on the PBR surface, the light source has a spectrum that does not vary over time and does not distort with applied power. Consequently, the light set up for the biological experiments is ideal for studying the effect of light on microalgae's response without introducing biases induced by the experimental setup.

Control and monitoring

An instrumentation and metrology system has been developed to allow the control of the luminous device as well as the continuous and semi-automated monitoring of the cultures.

The control part is ensured by Arduino cards connected to the computer. They allow adjusting both the intensity and the frequency of the studied light cycles. In this way, the multiplexer recovers the signals on a single fiber for 2 minutes, and then switches on another fiber. This process is repeated in a loop throughout the culture. These light signals are then routed to a spectrophotometer (OceanOptics FlAME-T-XR1-ES -wavelength range: 200 to 1025 nm). The latter converts the data received into spectra of transmitted light over a range of 400 to 700 nm covering the spectrum of LEDs (3 on Figure 3.7). These spectra are recorded directly on the computer throughout a culture. Thanks to the relationship between transmittance and absorbance, the data are finally converted in real-time into optical density values. For more details on the monitoring procedure of the cultures from light spectra, refer to Sub-section 3.5.1.

Liquid and gas utilities

In addition to light, cultures need nutrients for their growth. This supply is ensured by a fluidic and a gaseous system for nutrients and carbon source intake, respectively. This system is continuously controlled by a computer (Figure 3.7).

Liquid supply

The nutrient supply is provided by a sterilized 10 L bottle of culture medium. This bottle is pressurized with filtered air and connected to a system of valves, themselves connected to the PBRs. The computer automatically controls these valves to inject the culture medium when the cell concentration is too high. The operation of this system is described in more detail in the Section 3.5.

Gas supply

Contrary to the supply of culture medium, which is likely to vary according to the light conditions, the gas supply is provided fixedly and constantly during the whole experimental campaign.

The gas is supplied to the cultures through a 0.2 µm filter. It consists of a mixture of air and CO 2 . The air comes from the laboratory's compressed air network, while the carbon dioxide comes from a pressurized bottle with 100% CO 2 . Downstream of these two pieces of equipment, a pressure regulator is placed to regulate the pressure to protect the mass flow controllers. Each of the gases passes through their respective mass flow controllers (Brooks Instrument -SLA5850 -flow range: 0.003 to 50 L/min) set at the desired rate. They are then collected in a mixing chamber to compose the final gas with the desired CO 2 concentration that will feed the PBR. At the outlet of the mixing chamber, there is an overflow valve that acts as a safety valve in too high pressure. The gas mixture is then led to a 6-way manifold whose function is to allow a uniform distribution of the incoming gas to the mass flow controllers of the 6 PBRs. Once the mass flow controllers are crossed, the gas mixture is routed to each of the PBRs, where it is injected at the V-shape.

Mode of operation

The cultivation mode chosen to operate the PBRs during the experimental campaign is the turbidostat. Such a system allows feeding the cells with fresh medium in a continuous way. Maintaining a constant and non-limiting supply of nutrients is a way to avoid the contribution of a nutrient deficiency. Indeed, a nutrient undersupply can influence microalgae's response in conjunction with light, and therefore be a source of bias. Besides, this cultivation mode allows controlling the cells' concentration and maintains it constantly throughout the culture. By choosing to maintain a low cell density, the cells' mutual shading can be considered negligible. Under these conditions, all cells within the PBR are exposed to the same light pattern and experience the same light history.

Description of the turbidostat operation

The culture device is equipped with an automated dilution system based on the principle of feedback between the optical density (OD) of the culture and the dilution rate. This operating system is commonly called turbidostat. In practical terms, this means that the cell concentration is kept fixed throughout the culture by dynamic adjustment of the 100 Chapter 3. Development and characterization of the experimental device dilution rate.

To ensure that the cells are exposed to the same amount of light within the culture, it is essential to keep the cell concentration low. For this purpose, the setpoint triggering the dilution order was set at a transmitted light corresponding to 80% of the incident light or an optical density value of 0.1 (Eq. 3.1). The dilution stops once the transmitted light value is back above 80%. Consequently, in this configuration, all cells grow under a light intensity ranging between 100 and 80% of the incident value.

A = log 10 I 0 I = log 10 100 80 0.1 (3.1)
From a practical point of view, in the developed device, the monitoring is realized by an optical density measurement which is related to the transmittance by the Equation 3.2. However, as microalgae do not absorb light uniformly over the whole spectrum, the question of the wavelength for optical density monitoring arises. By performing the monitoring on the wavelength corresponding to the maximum absorption peak of the spectrum (463 nm, chlorophyll a), it ensures that the absorption of light is less on the other regions of the spectrum.

A = -log(T ) (3.2)
However, the cells are not the only components able to absorb light. This is also the case of some minerals present in the culture medium and of the glass walls constituting the PBR or the plexiglas walls protecting the LEDs panel. To get rid of their contribution, which is fixed in time, a blank measurement is necessary. Before launching each new culture, the PBR is filled with culture medium, and the optical density is acquired for about 1 hour. This value (about 30000 counts on a 6536 counts full scale) is then subtracted from all OD readings of the spectrophotometer.

Finally, for all cultures, the first parameter to be evaluated is the growth rate since it indicates the cells' well-being. In turbidostat mode, the growth rate is equivalent to the dilution rate in steady state. The latter can be obtained simply by weighing the mass of the culture drawn off. In practice, once the culture is acclimated, a pot of known mass (m 1 ) is placed at the PBR overflow outlet at a time t 1 . At a time t 2 , generally after 24 hours, the pot is removed, and its mass (m 2 ) is weighed. It is then put back in place. This cycle continues until the measurements have stabilized. In this way, the growth rate is calculated by first converting the weighed mass into volume (Eq. 3.3). Since the culture has a density close to water, the density of water (ρ) is used for the calculation. Then, this volume is reduced to the volume capacity of a PBR (0.135 L) and divided by the time interval of the measurement (t 2 -t 1 ) (Eq. 3.4).

V = (m 2 -m 1 ) ρ (3.3) D = µ = V t 2 -t 1 × 1 V P BR (3.4)
3.5. Mode of operation 101

Measurement accuracy

The uncertainty in the measurement of the growth rate associated with the device itself has been characterized. It is given by Equation 3.5, where:

Err = 1 t × µ ln DO 1 DO 0 + ∆V P BR V P BR (3.5)
• t is the time interval of a growth rate measurement in hours,

• µ is the growth rate in h -1 ,

• DO 0 and DO 1 represent the set monitoring optical density values (0.10 and 0.11, respectively)

• and ∆V P BR V P BR is the working volume of the PBR with an estimated error of 1 %.

The demonstration of the calculation is given in Appendix D The graphical representation of the uncertainty of measurement as a function of the growth rate is given in Figure 3.8. At first sight, the correlation between uncertainty and growth rate is negative. The higher the growth rate, the lower the associated measurement uncertainty. Above a growth rate of 0.049 h -1 , the associated error is less than 10%. Therefore, growth rate results above this value are considered reliable. In contrast, the lower the growth rate is, the more cautiously the results can be considered reliable.

Conclusion

The aim of this chapter was to describe and characterize the experimental device developed during this thesis. It includes three distinct parts:

• a culture device, with a thin and flat design allowing to limit as much as possible the optical disturbances/distortions,

Introduction

This chapter aims at describing the processing methods developed to explore the response of microalgae cells under different lighting conditions. It can be divided into four parts.

The first part focuses on the strain used for this work. Firstly, it explains the means used to maintain it in both liquid and solid cultures. Secondly, it details the methodology used to define the culture medium adapted to the strain for this study.

In a second part, the cultivation strategy to carry out this study is described. It includes the methodology used to acclimatize the cells prior to culture under the studied conditions as well as the test campaign listing all the experimental conditions explored in this work.

A third part is devoted to the analytical procedures deployed on the cultures. Two types of analyses are described: pigment profiling and evaluation of the status of the photosynthetic apparatus. This section demonstrates how, from these measurements, information on the response of cells to light conditions can be recovered.

Finally, the last part discusses the statistical testing procedure deployed at different levels in this work. These tests allow to ensure both the quality of the final dataset and to evaluate the significance of the light patterns influence.

Microalgal strain and maintenance of cultures

Subculturing

Chlorella vulgaris SAG 211-11b was purchased axenic from the Culture Collection of Algae at Göttingen University (SAG), Germany. The strain was maintained in liquid culture over B3N medium in 250 mL flasks in an incubator (INFORS HT Minitron) at 25°C on an orbital shaker (100 rpm). The culture was exposed to continuous low light intensity (30 µmolPhotonPAR/m 2 /s) and air-enriched with 1 % CO 2 . Once a week, 500 µL of the culture was resuspended into 50 mL fresh medium. Liquid culture was also dispatched on agar medium plate both for conservation reasons and to verify the absence of contamination.

Growth medium screening

Before cultivating the strain and starting the experiments, the first approach is to identify the optimal growth medium for the strain. In this regard, a screening of different culture media was carried out. Based on the literature, BG-11 Medium (BG11), Bold's Basal Medium (BBM), 3NBBM medium (B3N) which is BBM enriched with three times the nitrate, Sueoka's medium (SUEOKA), Volvox Medium (VOLVOX) and Jaworski's Medium (JM) were selected as they are indicated for Chlorophyta phylum growth [START_REF] Barsanti | Algae: anatomy, biochemistry, and biotechnology[END_REF][START_REF] Andersen | Algal culturing techniques[END_REF].

For the sake of not to bias the screening by the former subculturing, Chlorella vulgaris was maintained on each medium for three weeks. Every week, the cultures were passaged onto fresh medium (1/100 sampling). These flasks were kept on an orbital shaker (100 4.1. Microalgal strain and maintenance of cultures 105 rpm) at 18-20°C under a cold white LED spectrum light of 75 µmolPhotonPAR/m 2 /s. The cultures were conducted in triplicates. The screening was carried out on the fourth week. Cell growth was monitored twice daily using optical density at 750 nm as a proxy of cell concentration.

Media screening results are presented in Figure 4.1. Graph 4.1a depicts, for each medium, the growth rates as well as their standard deviation at the end of the screening, while graph 4.1b represents an example of a triplicate growth curve obtained during the screening of one media, here B3N. As one can see, standard deviations are quite low compared to averages (CV < 10 %). Although the media BG11 and BBM are statistically different, this statement cannot be made for the media B3N and SUEOKA and for VOLVOX and JM. The highest growth rate was obtained with BG11 (0.073 ± 0.001 h -1 ) followed by BBM (0.071 ± 0.001 h -1 ), B3N (0.064 ± 0.004 h -1 ), SUEOKA (0.063 ± 0.006 h -1 ), VOLVOX (0.053 ± 0.002 h -1 ) and JM (0.051 ± 0.004 h -1 ). Based on the screening procedure, the BG11 medium was discarded since it produced foam. Next came the BBM and B3N media. Between these two media, B3N was chosen because of the amount of nitrate available in this medium. Indeed, the BBM and B3N media have the same composition (Table 4.1). The only difference is the nitrate content which is three times higher in the B3N medium than in the BBM medium. Yet nitrogen is the third essential nutrient for microalgae growth, especially for the synthesis of proteins and pigments, after light and carbon dioxide. As a matter of fact, Jeanfils et al. [START_REF] Jeanfils | Effect of high nitrate concentrations on growth and nitrate uptake by free-living and immobilizedChlorella vulgaris cells[END_REF] demonstrated that the growth of Chlorella vulgaris increased with nitrate concentration up to 12 mM. Above this concentration, a slow down is observed, a sign of inhibition due to a too high concentration. In our case, B3N has a concentration of 9 mM (3 mM for BBM), which is not toxic for the cells. Moreover, it has been shown that nitrate uptake is affected by light intensity and periodicity [START_REF] Meseck | Photoperiod and light intensity effects on growth and utilization of nutrients by the aquaculture feed microalga, Tetraselmis chui (PLY429)[END_REF]. Thus, to avoid potential interference with a nitrogen limitation during the test campaign, B3N medium was chosen over BBM, although the latter achieved a 10% more efficient performance under the screening's culture conditions. Finally, visually, cultures grown on B3N medium appeared greener Chapter 4. Materials and methods than those grown on BBM, suggesting a higher production of chlorophyll. Once the culture medium is chosen, it is important to ensure that during the operation of the PBRs, it does not cause nutrient deficiencies that could interfere with the study. For this purpose, cultures were grown under continuous light at 320 µmolPhotonPAR/m 2 /s in the experimental setup. After three days of culture in turbidostat mode, the PBRs were emptied. The cultures were centrifuged, and the supernatant was kept for analysis by ion chromatography (ICS-5000+ Ion Chromatography system -Thermo Fisher Scientific) coupled with a conductivity detector. This technique allows the content of cationic and anionic species present in the culture medium to be quantified. The results are presented in Figure 4.2.

These graphs present the concentration obtained after chromatographic analysis according to the ionic species, before (in dark blue) and after cultivation (in light blue). For comparison, the theoretical concentration is also displayed (in purple). One can see that the concentration of ionic species in the culture medium before cultivation is quite close to the theoretical concentration of the B3N medium. Only Mg 2+ and Ca 2+ cations appear a little lower than the expected values. A natural precipitation of these ions can explain this result [START_REF] Abbona | The initial phases of calcium and magnesium phosphates precipitated from solutions of high to medium concentrations[END_REF]. When looking at the ionic concentrations after several days in the culture device, the results show a nitrate consumption of about 18%. However, the remaining concentration (7.8 mM) prevents a potential nitrate shortage. In addition to nitrate, Ca 2+ and Mg 2+ concentrations are significantly different from those of the formulated medium. Again, this is a consequence of the precipitation of these ions. Bubbling in CO 2 -enriched air slightly acidifies the pH (6.67 and 6.49 before and after bubbling, respectively), which exacerbates these ions' precipitation. Although they partially precipitate, their presence once the culture has reached a steady-state means that the microalgae do not totally consume the non-precipitated part.

In short, these results allow us to conclude that the chosen medium, coupled with a turbidostat operation, does not deficient the microalgae in nutrients throughout their cultivation. Hence, with CO 2 supplementation, we only get the effect of light.

Strategy of culture

During the entire test campaign, all the cultures were prepared and analyzed using the same protocols and techniques. Cells were cultured in the previously described experimental setup. The culture strategy deployed is broken down into different steps. First, the cells must be acclimated to the desired light conditions studied. Then, the growth rate is measured until stabilization, i.e. no significant difference, supported by a statistical test. Finally, the cultures are drained and will be further analyzed. All these procedures are detailed below.

Culture acclimation

One of the major conditions for the cultures' success is the acclimation state of the cells to the light patterns to be studied. Indeed, cells have two categories of regulation/protection mechanisms against excessive light: short-term (seconds to minutes) and long-term (hours to days). These mechanisms involve dynamic modifications of the photosynthetic apparatus (pigment contents, electron flow etc...). Therefore, not acclimating the cells is equivalent to measuring the transient state of a culture and not its steady state in a given light condition.

Faced with this observation, the first step of the experimental procedure consists in the acclimation of the culture. Preliminary results showed that chambers 1 and 2 gave the same culture performance for the same conditions. For instance, when the microalgae were grown at 160 µmolPhotonPAR/m 2 /s under air-enriched with 0.5% CO 2 , chambers 1 and 2 gave a growth rate of 0.049 ± 0.005 h -1 and 0.050 ± 0.004 h -1 , respectively. Therefore, and for time-saving reasons, one chamber was used to acclimate the cells, while the other was used to measure culture performance.

In practical terms, the acclimation process takes place as follows. Chamber 2 has been designated as the acclimation chamber. Each of the PBRs pre-filled with culture medium receives a volume fraction of the preculture. After seeding, the cultures are grown for 48 hours. After this time, they are transferred to chamber 1 for monitoring. As the swapping may result in a slight loss of volume, the cultures were given an additional 24 hours to grow in the new chamber. Then the weighing jars were placed and the growth rate was measured once a day, for each PBR, until it stabilized (about 48 to 72 hours depending on conditions).

Growth parameters

Before starting a culture, the culture vessel is sterilized and rinsed with water. Then, the gas is turned on and the lights are adjusted according to the test campaign. Once set, the blank measurement with culture medium is acquired for one hour before inoculating the cells.

For each culture, all abiotic parameters except light were kept constant throughout the experiment. In that respect, the strain was cultured over B3N medium and maintained at 20°C using a heater-chiller. Aeration was set at 1.5 vvm, with 2.5% CO 2 -enriched air for mixing and growth purposes.

Test campaign

First, the response to continuous illumination was investigated over a range from 25 to 800 µmolPhotonPAR/m 2 /s (Table 4.2). These experimental points allowed us to obtain the light response curve (Figure 2.2). From this curve, the different key zones were determined:

• the photolimitation zone,

• the photosaturation zone, also called the plateau zone,

• the transition zone between the two previous regimes of photolimitation and photosaturation.

The determination of these zones allowed us to target the experimental points to be investigated in the intermittent light regimes. Thus, three specific points were retained:

• a first point corresponding to the end of the photolimitation zone,

• a second point corresponding to the entry in the plateau zone,

• and a last point corresponding to the middle of the plateau zone.

Strategy of culture 109

We would have liked to study also a point at the end of the plateau zone (i.e. in the photoinhibition zone) but it was not possible with the current experimental device.

By transposing these key points into incident light intensities (Io), this corresponds to 200, 400 and 800 µmolPhotonPAR/m 2 /s (Table 4.2). For each of these intensities, three frequencies were explored: 1 Hz, 10 Hz and 100 Hz. The first two frequencies correspond to fluctuating light frequently encountered for cultures grown in PBR. The last frequency corresponds to flashing light and coincide with the characteristic times involved in photosynthesis. Moreover, these three intermittent light regimes are commonly studied in the literature, making it easier to compare our results.

All L/D cycles present a ratio of the light phase over the cycle duration (duty cycle, ε) maintained at 50%. Thus, the light perceived by microalgae during a cycle can be expressed as:

I avg = I 0 × ε (4.1)
These averaged intensity values are presented in blue brackets in the Table 4.2.

The results obtained in intermittent mode are then compared to the performances obtained in continuous light for the same amount of light.

Light pattern

Incident intensity (µmolPhotonsPAR/m /s)

Light exposure time (ms)

Duty cycle (-)

Continuous 25 ∞ 1 50 ∞ 1 80 ∞ 1 120 ∞ 1 160 ∞ 1 200 ∞ 1 240 ∞ 1 280 ∞ 1 320 ∞ 1 360 ∞ 1 400 ∞ 1 500 ∞ 1 600 ∞ 1 700 ∞ 1 800 ∞ 1
Fluctuating -1 Hz

Analytical procedures

At the end of each run, in addition to growth rates, two types of analysis were conducted on the cultures: determination of the photosynthetic status of the cells and quantification of the pigment profile. These two analyses give us information on the accllimation strategies of the cell to different light patterns.

Photosynthetic apparatus status

AquaPen AP 110-C PAM fluorometer was used to qualify photosynthetic apparatus status. Before starting these analyses, cells were harvested and prepared. For this purpose, the cultures were drained from the PBRs and centrifuged (4°C -11000 rpm -10 min).

The supernatant was discarded, and the pellet was resuspended in Milli-Q water. The procedure was repeated twice. In the last washing, the samples were diluted with the amount of water necessary to obtain an optical density of 0.5 at 750 nm. This procedure allows standardizing all samples before the analysis of the status of the photosynthetic apparatus. From each sample, 3 mL are put in a cuvette and dark-adapted for 15 minutes to be then analyzed by the fluorometer. To run the analyses, the aquapen parameters were set as follows: measuring color: 630 nm, measuring flash pulse: 900 µmolPhotonPAR/m 2 /s, saturating pulse: 1500 µmolPhotonPAR/m 2 /s, measuring pulse: 1000 µmolPhotonPAR/m 2 /s, actinic light: 300 µmolPhotonPAR/m 2 /s. Two types of measurement were conducted.

Non-Photochemical Quenching (NPQ) protocol

First, a quantification of the non-photochemical quenching is performed. This measurement requires a dark-adapted sample. Basically, the measurement starts by exposing the sample to measuring light to acquire the minimum level of fluorescence (F 0 ). A short flash of saturating light is then applied to reduce the plastoquinone pool and measure the maximum fluorescence (Fm) in the dark-adapted state. After a short dark relaxation, the sample is exposed to actinic irradiation for 200 seconds. Simultaneously, a sequence of 10 saturating flashes (20 seconds apart) is applied in addition to the actinic light to probe the maximum fluorescence in light-adapted state (Fm L ). The schematic representation of the protocol is displayed in Figure 4.3.

From this protocol, two parameters can be calculated: the Fv/Fm ratio (Eq. 4.2) and the NPQ (Eq. 4.3). The former can be seen as an indicator of light damage. Although quick and informative, the Fv/Fm ratio can be considered an overly simplistic description of photosynthetic apparatus. To fill this gap, the NPQ measurement provides more detailed information about the cell's management of actinic light. It is a measurement reflecting

F v/F m = (F m -F o) F m (4.2) N P Q = (F m -F m L ) F m L (4.3)

Light Curve (LC) protocol

After NPQ quenching quantification, the light curve protocol is applied to acquire the parameters needed to construct the Light Response Curve relating the rate of photosynthesis to photon flux density. To this end, the sample is exposed to a stepwise increase of light intensity. A total of seven intensities of continuous light are explored: 10, 20, 50, 100, 300, 500, 1000 µmolPhotonPAR/m 2 /s. Each light phase lasts 60 seconds during which the instantaneous fluorescence (Ft L ) during light adaptation and the maximum fluorescence (Fm L ) in light adaptation state are measured based on a saturating pulse modulated fluorometry (PAM). With these values, the effective quantum yield of photosynthesis can be determined for each phase of light according to the Equation 4.4.

QY L = (F m L -F t L ) F m L (4.4)
Once obtained, the light response curve describes how the photosynthesis rate varies according to the light. Besides, it provides information on the maximum photosynthetic capacity, the quantum yield, the light compensation point, and the radiation utilization efficiency of the cells. To extract these values, we use a model that best describes the experimental points and makes sense biologically. Here, the Han model is used (4.4) [START_REF] Han | Effect of photoinhibition on algal photosynthesis: a dynamic model[END_REF]. This model describes three different dynamic states of the cell: • State B: The unit has captured a photon and processes its energy with a characteristic time τ . Once the photon has been processed, the units returns to the open state. If, while processing a photon, the unit is hit by another photon the unit has a chance (k d ) to enter the damaged state. Therefore, the mathematical description of this state involves the A and C states (Eq. 4.6). In steady state, this model gives :

dB dt = Iσ P SII A - B τ + C τ r -k
A(I) = 1 1 + aI + bI 2 B(I) = aI 1 + aI + bI 2 C(I) = bI 2 1 + aI + bI 2 (4.8)
with:

a = σ P SII τ b = σ 2 P SII k d τ r τ
Assuming the growth rate to be proportional to B τ , and τ being independant of light, one obtains the expression for PI:

P I(I) = c × aI 1 + aI + bI 2 (4.9)
where c is a scaling factor for the photosynthetic rate.

A processing routine was developed to identify the parameter groups in Equation 4.9 by minimizing the deviation (sum of squared differences) between the identified curve and the experimental points. With these parameters, one can draw the curve identified (Figure 4.5). By its mathematical form, the key points of the curve can be deduced through the analytical expression. Thus, for each culture, the following parameters can be obtain as follows:

• P max , the maximum rate of photosynthesis:

P max = c × a 2 √ b + a (4 .10)
• I P max , corresponds to the intensity for which the photosynthesis rate is maximum:

I P max = 1 √ b (4.11)
• I k , the light intensity corresponding to the intersection point between the theoretical linear relationship and P max : • P ik , the photosynthetic rate at the intensity I k :

Ik = P max C × a = 1 2 √ b + a (4.12)
P Ik = a -c 2a + 2 √ b + b 2 √ b + a (4.13)
• I i , the intensity at which the depression of photosynthesis occurs defined when P = P Ik in the descending phase of the curve:

I i = 1 2b × (-(a - ac P ik ) + √ ∆) (4.14) with ∆ = (a - ac P ik ) 2 -4b
From the light curves and the identified key points, the antenna sizes can be approximated as follows:

Antenna size = P Ik Ik P max (4.15)
The numerator of the Equation 4.15 represents the slope of the light curve in the photolimitated phase, or the capacity to collect light. It can be used as a proxy for the amount of chlorophyll. The steeper the slope, the greater the amount of chlorophyll. The maximum photosynthetic rate P max is used as a proxy for the number of photosystems. The higher this number is, the higher the number of photosystems. The ratio of these two quantites allows evaluating the number of chlorophyll molecules per photosystems also known as the antenna size.

Pigment extraction

After the analysis of the photosynthetic status, the remaining part of the twice-washed culture is centrifuged one more time (4°C -11000 rpm -10 min) to assess and quantify the pigment profile of the cultures. Then, the pellet is frozen before being freeze-dried (1 day primary drying, 2 days secondary drying, Christ alpha 1-2 LD +). Approximately 2 to 3 mg of dried microalgae powder are weighted and homogenized in 5 mL pure methanol using MP Biomedicals FastPrep42 bead miller. In order to enhance the extraction, the samples are then left in 60 °C water bath for 20 minutes. Finally, the supernatant is filtered (0.22 µm -PP) and stored in dark vials at 4°C before quantification.

Quantification of pigments were carried out on an Ultima 3000 HPLC (Thermo Fisher Scientific) coupled with an UV Detector. Separation was achieved on an Acclaim Polar Advantage II C18 column (4.6 x 150 mm, 3 µm, 120 Å) from Thermo Fisher Scientific. Column temperature was maintained at 30 °C. Pure methanol was the mobile phase, the flow rate was 0.5 mL/min and the elution set in isocratic mode. Injection volume was 5 µL and the total run analysis was 40 minutes. Compounds were identified by comparison of their retention time and of their UV-Vis spectra with standard solutions (Table 4.3). UV-Vis spectra were recorded from 200 nm to 700 nm, absorbance was recorded at 400, 450, 500 and 650 nm. Chlorophyll a and b quantifications were done at 650 nm while carotenoids were assessed at 450 nm. It was achieved using the area of the peaks in external calibration, which concentrations ranged from 0.0185 to 18.905 mg/L (Table 4.3). Pigment standards and methanol were purchased from Sigma-Aldrich. Standards had a purity greater than 97%.

Statistical testing

A statistical testing procedure was employed to ensure the reliability of our work. Although most people use the ANOVA as a reference test, in our case, we turned to another test because we were not sure that we could satisfy all the assumptions that this test implies, in particular the one of normality. Thus, we chose the Kruskal-Wallis statistical significance test instead. This is a non-parametric test that compares the distribution of small samples (n=3, in our case). This statistical test was performed at two levels:

• During a run, to determine the growth rate difference between one day and the next until its stabillization,

• Between tested conditions, to evaluate the significance of the light patterns influence.

In addition, we still performed ANOVA testing. The results confirmed the conclusions obtained by Kruskal-Wallis statistical significance test. For the sake of simplicity, only the Kruskal-Wallis p-values are reported in the manuscript.

Conclusion

This chapter presented the materials and methods used in this work. Chlorella vulgaris cells are cultured in the experimental device. Before each measurement, the cultures are acclimatized to the light conditions according to the test campaign. The other abiotic parameters are kept constant. The growth rate is acquired over 24-hour periods until its stabilization, verified by statistical testing. On these acclimatized cultures, two methods of analysis are used. On the one hand, the photosynthetic status of the cells is performed by fluorometry on fresh samples. On the other hand, the pigment profiling is completed by chromatographic method on dried samples. All these analyses provide information on the mechanisms used by the cells to cope with the different light patterns.

Introduction

Given the large number and the multiparametric aspect of the results, a progressive approach is adopted for their analysis. In this chapter, the results obtained under a continuous illumination of increasing intensity are analyzed. These results will serve as a reference for the comparison of the results obtained under intermittent lighting. The latter will be the subject of the next chapter.

The following chapter is broken down into three parts, each dealing with a specific aspect of the results. The ordering of the results allows a progressive description from the culture scale to the cell scale. Throughout this chapter, each value presented is the average of three biological replicates that were acclimated for several days to the same light condition.

The first part deals with the growth rate obtained under different intensities of continuous light. This is the first macroscopic result of biotechnological interest. Indeed, this parameter is used as an indicator of the general well-being of a culture. On an industrial scale, it allows the process productivity to be assessed.

Then, to move closer to photosynthesis, the pigment profile is analyzed. This includes the absolute pigment concentration as well as the evolution of their ratio as a function of light intensity. This part is divided into two sub-sections, each one dealing with a different pigment category: photocollecting pigments (chlorophylls) on one side and photoprotective pigments (carotenoids) on the other.

Finally, the last part presents the results obtained at the most microscopic scale: the state of the photosynthetic apparatus in relation to the applied light intensity.

Growth rate

The first level of description of the relationship between light and the response of microalgae is the growth rate. Figure 5.1 reports the growth rates as a function of the light intensity under which the cultures were grown. On this graph, one can identify three zones:

• From 25 to 120 µmolPhotonPAR/m 2 /s: the photolimitation zone or affinity phase.

In this zone, light is delivered in a limited way. Therefore, the growth rate increases linearly with light.

• From 120 to 200 µmolPhotonPAR/m 2 /s: a transition zone from light-limited to light-saturated photosynthesis.

• From 200 to 800 µmolPhotonPAR/m 2 /s: the photosaturation zone or plateau. The growth rate is no longer limited by light but by the reaction time speed of some internal reactions. It reaches a maximum (about 0.088 h -1 ) and is maintained, although the light intensity is increased. In this zone, there is no significant difference in the growth rate (p-values > 0.05).

The response of Chlorella vulgaris to light intensity has been extensively studied by scholars. Reported growth rates under different operating conditions range from 0.025 h -1 [START_REF] Forrest | Light and growth medium effect on Chlorella vulgaris biomass production[END_REF][START_REF] Grudzinski | Strong-light-induced yellowing of green microalgae Chlorella: A study on molecular mechanisms of the acclimation response[END_REF] for the least successful cultures to 0.100 h -1 [START_REF] Liao | Simultaneous enhancement of Chlorella vulgaris growth and lipid accumulation through the synergy effect between light and nitrate in a planar waveguide flat-plate photobioreactor[END_REF][START_REF] Sun | Application of growth-phase based light-feeding strategies to simultaneously enhance Chlorella vulgaris growth and lipid accumulation[END_REF] for the fastest cultures, on average. In the photolimitation zone, particularly between 25 and 50 µmolPhotonPAR/m 2 /s, the measured growth rates are relatively low (0.003 ± 0.001 h -1 and 0.009 ± 0.004 h -1 , respectively). In literature, studies conducted at such intensities have reported higher growth rates, typically between 0.025 and 0.034 h -1 at 37.5 µmolPhotonPAR/m 2 /s [START_REF] Seyfabadi | Protein, fatty acid, and pigment content of Chlorella vulgaris under different light regimes[END_REF][START_REF] Amini Khoeyi | Effect of light intensity and photoperiod on biomass and fatty acid composition of the microalgae, Chlorella vulgaris[END_REF], for comparison. Such a difference can be explained by a very high coefficient of variation of the measurement point (47 %). This could be the consequence of the error associated with the measurement of the growth rate by the experimental device which is about 50 % for growth rates of 0.009 h -1 . Applying a 50 % correction, the growth rate would take a value of 0.014 h -1 . Although compared to the literature, this number still appears to be a little low, it fits well with the other measurement points in the linear photolimitation zone. In the photosaturation zone, the growth rate is relatively stabilized around 0.088 h -1 , a value in agreement with the literature. This means that our strain is in a good physiological state and does not suffer from nutrient limitation. Therefore, the cellular machinery is running at its maximum capacity.

Finally, looking at the general appearance of the curve, the profile obtained is the one found in the classical light curve schemes of the literature. However, in our case, no inhibition phase is observed (classically reported around 500 µmolPhotonPAR/m 2 /s [START_REF] Ho | Characterization and optimization of carbohydrate production from an indigenous microalga Chlorella vulgaris FSP-E[END_REF]). Contrary to the standard light curve, we reason in growth rate, not in instantaneous photosynthesis rate. Thus, the growth rates values presented are the result of acclimation of the cells to different intensities. Cultures above 500 µmolPhotonPAR/m 2 /s take qualitatively much longer to acclimatize and reach the plateau than cultures at lower intensities. Yet the acclimatized response achieved the same level. This phenomenon shifts the curve to the right and thus delays the appearance of the photoinhibition zone. As our experimental device is limited to intensities up to 800 µmolPhotonPAR/m 2 /s, the Chapter 5. Experimental results under continuous light inhibition zone was not observed.

Pigment profile

In order to get more insight on the cellular response of microalgae to light regimes, a pigment profile exploration was performed. Indeed, pigments are responsible for light collection, but also are key players in the photoprotection and photoacclimation mechanisms. A rearrangement of the pigment content provides insight into how the cell reacts to the perceived light. In this work, we monitored and quantified the two categories of pigments found in Chlorella vulgaris species: chlorophylls and carotenoids. First, the absolute values of the concentrations are analyzed for each pigment and then special attention is paid to their ratio.

Chlorophylls content and ratio

Chlorophylls play a central role in photosynthesis for light harvesting. Figure 5.2 describes the total chlorophyll content as a function of light intensity. Chlorophyll a and b contents separately are also plotted. At first glance, the overall trend is for a down-regulation of chlorophyll a and b expression as light intensity increases. This diminution seems to qualitatively follow an inverse function. In the photolimitation zone, between 25 and 200 µmolPhotonPAR/m 2 /s, the total chlorophyll content decreases by 69 %, and in the photosaturation zone by 67 %. When considering the total chlorophylls, the content ranges from 26.52 ± 1.02 mg/gDW at 25 µmolPhotonPAR/m 2 /s to 2.26 ± 0.45 mg/gDW at 800 µmolPhotonPAR/m 2 /s.

Pigment profile 121

These results are consistent with what is reported in the literature. Depending on the environmental conditions and the strain, the total chlorophyll content may fluctuate between 0.5 to 4 % of dry weight [START_REF] Spolaore | Commercial applications of microalgae[END_REF][START_REF] Harun | Bioprocess engineering of microalgae to produce a variety of consumer products[END_REF]. In our case, this amount varies between 0.2 and 2.7 %. The lower limit may seem a bit low compared to what is reported. This is simply because very few studies have explored the pigment content of acclimated cultures at such high intensities. Therefore, the present study that proposed measurement of acclimated cultures up to 800 µmolPhotonPAR/m 2 /s provide original data that confirms that the strain continues to limit the collecting pigments as part of the strategy to face high light intensities.

Looking at each of the chlorophylls separately, one can notice that the trend is the same. Chlorophyll a varies from a maximum of 17.83 ± 0.56 mg/gDW at 25 µmolPhotonPAR/m 2 /s to 1.42 ± 0.23 mg/gDW at 800 µmolPhotonPAR/m 2 /s, which corresponds to a variation of 1.78 to 0.14 % of dry weight. These values are a bit lower than what is usually reported (between 0.3 and 3 % depending on the conditions [START_REF] Edward | Phycology[END_REF]). As before, this is due to the acclimation status of the culture at high intensities. As for chlorophyll b, its content fluctuates between 8.69 ± 0.51 mg/gDW and 0.84 ± 0.00 mg/gDW at 25 and 800 µmolPhotonPAR/m 2 /s, respectively.

The downregulation of chlorophyll production as intensity increases is a mechanism of adjustment of the cell to the amount of light perceived. Indeed, to carry out photosynthesis, microalgae have constant light requirements to fulfill. In conditions of limiting light (affinity phase), the cell upregulates its chlorophyll production to capture as much light as possible. Da Silva Ferreira et al. [START_REF] Da | Impact of culture conditions on the chlorophyll content of microalgae for biotechnological applications[END_REF] reported this phenomenon in their review on the impact of growing conditions on chlorophyll content. Conversely, in saturated light conditions, microalgae do not need as much chlorophyll to collect the amount of light necessary for photosynthesis. Thus, they reduce the chlorophyll production as a protective mechanism to avoid an excess of energy entering the electron transport chain that may damage the photosynthetic apparatus.

To go further, the ratio chlorophyll b/chlorophyll a as a function of light intensity is analyzed (Figure 5.3). This ratio gives information on the adaptation strategy of microalgae to light. It generally fluctuates between 0.3 and 0.5 depending on the light regime [START_REF] Edward | Phycology[END_REF]. In our case, this ratio remains constant around 0.5 regardless of the light intensity of culture meaning that the cells have kept the same pigment proportion while down-regulating their production (p-values > 0.25). This result is somewhat surprising. One would expect that under light-limiting conditions, the cell would increase its production of chlorophyll b to absorb more light (ratio around 0.5). Conversely, under saturating light conditions, one would expect a decrease in chlorophyll b to limit light capture (ratio rather around 0.3) [START_REF] Grudzinski | Strong-light-induced yellowing of green microalgae Chlorella: A study on molecular mechanisms of the acclimation response[END_REF]. However, microalgae exposed between 200 and 800 µmolPhotonPAR/m 2 /s exhibit the same growth performance (plateau zone). This suggests that the photosynthetic apparatus functioning is not disturbed even at the saturating intensities explored in this work. Thus, the cell does not need to modulate the chlorophyll composition at high intensities, but only to reduce the number of photosynthetic units to collect less light while maintaining the same performance.

Carotenoids content and ratio

In addition to chlorophylls, particular attention has been paid to carotenoids. Indeed, carotenoids are accessory pigments with a dual role. On the one hand, they broaden the absorption spectrum of chlorophyll a and transport the absorbed energy to the reaction center, where the chemical conversion of energy occurs. On the other hand, under certain conditions, they can also be used to protect the cell from the harmful effects of light through photoprotection and photoacclimation mechanisms [START_REF] Gong | Carotenoids from microalgae: A review of recent developments[END_REF][START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF].

For this work, the concentrations of lutein, violaxanthin and zeaxanthin were monitored. These three pigments are xanthophylls (oxygenated derivatives of carotenes) assumed to be involved either directly or indirectly in the non-photochemical quenching (NPQ) of excess light energy in the antenna of PSII. Figure 5.4 shows the evolution of the total carotenoids concentration as a function of the light intensity under which the cultures were grown. As one can see, the total carotenoids content changes from 2.68 ± 0.08 to 0.36 ± 0.08 mg/gDW between 25 and 800 µmolPhotonPAR/m 2 /s, respectively. As with chlorophylls, the dynamic is down-regulation of carotenoid production as intensity increases. To dive deeper into the results, a distinction was made between lutein and pigments belonging to the VAZ cycle.

Lutein

Lutein is the most abundant carotenoid pigment in Chlorella vulgaris, whose can be expressed up to 0.45 % DW [START_REF] Liu | Chlorella: Industrial Production of Cell Mass and Chemicals[END_REF][START_REF] Gouveia | Evolution of pigment composition in Chlorella vulgaris[END_REF]. In our study, the overall trend of this pigment drops with intensity (Figure 5.4). The maximum amount is obtained at 25 µmolPhotonPAR/m 2 /s with 2.44 ± 0.06 mg/gDW. This value represents 0.24 % of the total dry weight of Chlorella vulgaris and 91 % of the total carotenoids content. Conversely, the lowest lutein content is recorded for a light intensity of 800 µmolPhotonPAR/m 2 /s (0.21 ± 0.04 mg/gDW, i.e. 0.02 % DW and 58 % of total carotenoids). This sudden decline in lutein content with intensity may seem surprising at first glance. Indeed, lutein is a xanthophyll that serves different functions [START_REF] Pessarakli | Handbook of photosynthesis[END_REF][START_REF] Jahns | The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II[END_REF]. It plays:

• a structural role by allowing the stabilization of antenna proteins,

• a light-harvesting role by broadening the absorption spectrum of chlorophyll,

• a photoprotective role by dissipating the excess energy and detoxifying oxidants such as ROS.

Along these lines, one can logically expect an accumulation of lutein as the light intensity increases. To go beyond the simple observation of the absolute decrease in lutein content, it is interesting to see how its ratio evolves in relation to the total chlorophyll content (Figure 5.5). This ratio gives a more accurate picture of the pigment rearrangement within the cell according to the light intensity by evaluating the photoprotection capacity of the photosynthetic apparatus in relation to its light harvesting capacity. As can be seen, below 500 µmolPhotonPAR/m 2 /s, the ratio seems to be stabilized around an average value of 0.075. For higher intensities, it increases until it reaches its maximum value of 0.096 at 800 µmolPhotonPAR/m 2 /s, which corresponds to a 28 % increase. Although the absolute concentration decreases with intensity, the proportion of lutein to chlorophylls increases slightly for intensities above 500 µmolPhotonPAR/m 2 /s. This suggests that lutein has an interest in photoprotection although it is not a central pigment.

Yet the role of lutein as a photoprotective pigment is not very clear. Some studies report an increase in lutein content under high light conditions [START_REF] Niyogi | The roles of specific xanthophylls in photoprotection[END_REF][START_REF] Bonente | Analysis of LhcSR3, a protein essential for feedback de-excitation in the green alga Chlamydomonas reinhardtii[END_REF][START_REF] Schüler | Improved production of lutein and β-carotene by thermal and light intensity upshifts in the marine microalga Tetraselmis sp. CTP4[END_REF]. The authors explain this accumulation by the direct implication of lutein in the NPQ by quenching the chlorophyll in the singlet ( 1 Chl + ) and triplet ( 3 Chl + ) state [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Jahns | The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II[END_REF]. Another Chapter 5. Experimental results under continuous light possible explanation for the accumulation of lutein under high light is the presence of a second carotenoid cycle in addition to the VAZ cycle: the lutein epoxide cycle (Lx-L cycle) [START_REF] Esteban | Involvement of a Second Xanthophyll Cycle in Non-Photochemical Quenching of Chlorophyll Fluorescence: The Lutein Epoxide Story[END_REF]. In this cycle, Lx is de-epoxidized upon illumination, through the action of violaxanthin de-epoxidase (VDE), and converted to lutein. This cycle would play a role in photoprotection and in particular in the regulation of thermal energy dissipation. Nevertheless, it is only present in certain species, unlike the VAZ cycle which is very widespread. Other recent studies have demonstrated a decrease in lutein concentration with light intensity [START_REF] Ho | Enhancing lutein productivity of an indigenous microalga Scenedesmus obliquus FSP-3 using light-related strategies[END_REF][START_REF] Xie | Phototrophic cultivation of a thermotolerant Desmodesmus sp. for lutein production: Effects of nitrate concentration, light intensity and fed-batch operation[END_REF][START_REF] Ma | Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles[END_REF][START_REF] Ma | Two-stage bioprocess for hyper-production of lutein from microalga Chlorella sorokiniana FZU60: Effects of temperature, light intensity, and operation strategies[END_REF]. Two different hypotheses have been put forward to explain this phenomenon. The first is a reduction in the size of the light-harvesting antenna complex, where lutein is located [START_REF] Xie | Phototrophic cultivation of a thermotolerant Desmodesmus sp. for lutein production: Effects of nitrate concentration, light intensity and fed-batch operation[END_REF][START_REF] Ma | Two-stage bioprocess for hyper-production of lutein from microalga Chlorella sorokiniana FZU60: Effects of temperature, light intensity, and operation strategies[END_REF]. The second is a downregulation of lut1 gene expression under high light [START_REF] Ma | Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles[END_REF].

VAZ cycle

Turning now to the pigments of the VAZ cycle, as the antheraxanthin standard was missing, only violaxanthin and zeaxanthin concentrations were monitored. Figure 5.4 depicts the overall trend in the sum of the concentrations of these two pigments. Unlike lutein, the total concentration of the VAZ cycle pigments remained stable at around 0.17 mg/gDW regardless of the intensity applied. While all other pigments show lower concentrations at high light, the VAZ cycle pigments maintain their levels, suggesting photoprotective activity.

When looking at the ratio of each of these pigments to their total concentration (Figure 5.6), one can see that a clear interconversion between violaxanthin and zeaxanthin occurs. At low light intensities (below 160 µmolPhotonPAR/m 2 /s), violaxanthin is the majority pigment present in the membranes. However, as the intensity increases, violaxanthin is progressively converted into zeaxanthin. At high intensities (800 µmolPhotonPAR/m 2 /s), only zeaxanthin is detectable and quantifiable by HPLC. The moment when zeaxanthin overtakes violaxanthin corresponds to the entry in the plateau zone of the light response curve. In this zone, the photosynthetic apparatus operates at full capacity while the light energy is oversupplied and saturating. Yet carotenoids, and in particular zeaxanthin, play a fundamental role in reducing the overexcitation of reaction centers [START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Holt | Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants[END_REF][START_REF] Wormit | Excitation energy transfer and carotenoid radical cation formation in light harvesting complexes-A theoretical perspective[END_REF][START_REF] Jahns | The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II[END_REF]. Therefore, the appearance of zeaxanthin occurs concomitantly with the increase in light intensity and supersedes violaxanthin when light is provided in excess.

To illustrate the accumulation of zeaxanthin in cells acclimated to high light intensities, chromatograms are available in Figure 5.7. This figure depicts the chromatograms obtained from two cultures grown under opposite extreme intensities: 80 µmolPhotonPAR/m 2 /s (LL, Low Light) and 800 µmolPhotonPAR/m 2 /s (HL, High Light). The subtraction of these chromatograms shows that the only pigment accumulated at high intensity is zeaxanthin while the production of other pigments is only downregulated.

To take the analysis one step further, the ratios of these pigments on the chlorophyll content was evaluated (Figure 5.5). As for the lutein/total chlorophyll ratio, one can see a stabilization of the value around 0.019 for intensities below 500 µmolPhotonPAR/m 2 /s. Beyond that, the ratio increases and reaches the maximum value of 0.070 at 800 µmolPhotonPAR/m 2 /s. This evolution represents an increase of +268 %, compared to their stabilized average value for intensities below 500 µmolPhotonPAR/m 2 /s. This finding points to the pigments of the VAZ cycle as major contributors to photoprotection, in particular zeaxanthin. Indeed, these are the only pigments whose absolute concentration remained constant regardless of the light level while all the other pigments progressively dropped. Thus, as the light intensity increases, they become preponderant. This result is consistent with the literature since the VAZ cycle, particularly zeaxanthin, is an integral part of NPQ. Its prominent role is to dissipate the excess of energy at the reaction centers as heat.

The pigment rearrangement observed is a long-term photoacclimation mechanism taking place from a few hours to a few days. This response is greater for cultures grown at light intensities above 500 µmolPhotonPAR/m 2 /s, reflecting a longer acclimation time (stabilization of growth rate) for these cultures than for other cultures. Furthermore, with respect to the light intensity, the late shift in the ratio in favor of photoprotection over light collection (77 % increase in the total carotenoids/chlorophylls ratio between 500 and 800 µmolPhotonPAR/m 2 /s), highlights the importance of the acclimation state of the cultures. Although the light is provided overly, the photosynthetic apparatus manages the energy supply so as to maintain its performance (no photoinhibition observed). This implies that the cell's photoprotection mechanisms are sufficient to limit photodamage and maintain the photosynthetic activity at its maximum. This observation is in lines with the growth rates obtained at these intensities.

These results echo those found by Grudzinski et al. [START_REF] Grudzinski | Strong-light-induced yellowing of green microalgae Chlorella: A study on molecular mechanisms of the acclimation response[END_REF]. These authors studied the pigment response of Chlorella vulgaris at two intensities: 80 and 400 µmolPhotonPAR/m 2 /s. In their paper, they showed that under low light conditions, the cells exhibited a pigment profile rich in chlorophylls a and b, with a low amount of violaxanthin. In contrast, the response to high light conditions revealed overexpression of zeaxanthin (+1320 %). These authors demonstrated that the overaccumulation of zeaxanthin resulted from the increase in the violaxanthin pool (+116 %). This finding suggests that under high light conditions there was de novo synthesis of the xanthophyll cycle pigments. In contrast to their study, the present work only showed an accumulation of zeaxanthin without overexpression of the xanthophyll pigment pool.

Photosynthetic apparatus status

The final step in understanding the relationship between light and microalgal response is to characterize the status of the photosynthetic apparatus. Here we leave the world of biotechnology to dive into the photosynthesis mechanism.

The results presented below were obtained by fluorometry analysis on freshly harvested cultures using the AquaPen AP 110-C. A distinction is made between results obtained using the NPQ protocol (Fv/Fm and NPQ) and those obtained using the Light Curve protocol (P max , P ik , I k , I i ).

NPQ protocol

Starting with the NPQ protocol, Figure 5.8 presents the Fv/Fm and NPQ parameters as a function of cultivation light intensity. As one can see, the Fv/Fm ratio fluctuates more or less around 0.8. The maximum value (0.843 ± 0.006) is obtained at 50 µmolPhotonPAR/m 2 /s while the minimum value is 0.76 ± 0.02 at 800 µmolPhotonPAR/m 2 /s, a variation of 10 % of the maximum value). Looking more closely, two trends can be noticed. Between 25 and 280 µmolPhotonPAR/m 2 /s, the values of the ratio are relatively stabilized around 0.837 (p-values > 0.37). Above 280 µmolPhotonPAR/m 2 /s, the ratio drops slightly to 0.774 (p-values > 0.18). This points to a well-functioning photosynthetic apparatus. Indeed, a value of 0.8 indicates a fully photosynthetically capable culture, while the culture is still considered healthy down to 0.6 [START_REF] Masojídek | A closed solar photobioreactor for cultivation of microalgae under supra-high irradiance: basic design and performance[END_REF]. This is confirmed by the absence of a photoinhibition phase, even at high light intensities. Moving on to the NPQ results, one can notice that the trend is not the same. In the photolimitation zone (≤ 120 µmolPhotonPAR/m 2 /s), the NPQ value progresses with intensity starting from 0.164 ± 0.003 at 25 µmolPhotonPAR/m 2 /s to 0.417 ± 0.026 at 120 µmolPhotonPAR/m 2 /s. This result seems to suggest that at low light cultivation intensities, NPQ mechanisms are not activated. Accustomed to low light levels, when a saturating light flash is applied, the cells dissipate excess energy primarily through the unregulated pathway (formation of 3 Chl + , ROS, etc.). As the intensity increases to approach the saturation intensity of the photosynthetic apparatus, they gradually implement these mechanisms, resulting in an increase in the NPQ value. In the plateau zone between 160 and 500 µmolPhotonPAR/m 2 /s, the NPQ value is more or less stabilized around 0.3. Above 500 µmolPhotonPAR/m 2 /s, one can notice a collapse of this value down to 0.098 ± 0.078 at 800 µmolPhotonPAR/m 2 /s. This could suggest that the NPQ mechanism fails to keep pace with the excess energy at high light intensities. Therefore, the energy overage would be quenched by uncontrolled and potentially harmful mechanisms, such as the formation of chlorophyll in the triplet state or ROS. However, this explanation does not fit with the results obtained previously (growth rate, pigment profile).

The disparate character of the NPQ values with the rest of the results can simply be explained by the use of inappropriate settings of the measuring device. Indeed, the saturation pulses are expected to be of sufficient intensity (several orders of magnitude higher than the cultivation intensity) to simultaneously reduce all PSII reaction centers [START_REF] Logan | Context, Quantification, and Measurement Guide for Non-Photochemical Quenching of Chlorophyll Fluorescence[END_REF]. This ensures that the photons are not used for photochemical purposes. In our case, the device was set to a measurement flash intensity of 900 µmolPhotonPAR/m 2 /s. For cultures grown at low intensities (< 100 µmolPhotonPAR/m 2 /s), this has no impact. However, this flash is not sufficiently saturating for other cultures, leading to underestimating the NPQ value [START_REF] Logan | Context, Quantification, and Measurement Guide for Non-Photochemical Quenching of Chlorophyll Fluorescence[END_REF]. Unfortunatly, this misuse of the device makes the results meaningless and uninterpretable. It should be noted that this error does not affect the Fv/Fm or Light Curve results because these analyses are based on saturating flashes performed at 1500 µmolPhotonPAR/m 2 /s.

Light Curve protocol

After the determination of Fv/Fm and NPQ, each culture was subjected to the Light Curve protocol. For each of these cultures, a light response curve was plotted along with its identified curve for all three replicates. An example is shown in Figure 5.9.

From the identified curves, four parameters were extracted. Their evolution as a function of light intensity is shown in Figure 5.10. Looking first at the parameters related to photosynthetic yield, the maximum rate of photosynthesis (P max ) echoes the results obtained for growth rates. One can notice an increase from 102 ± 19 to 165 ± 4 between 25 and 160 µmolPhotonPAR/m 2 /s corresponding to the photolimitation zone. This is followed by stabilization at about 163 until 800 µmolPhotonPAR/m 2 /s (p-values > 0.13, excluding the values at 400 and 500 µmolPhotonPAR/m 2 /s). No decline of this number is observed, which confirms that the acclimation of the cultures allows maintaining the photosynthetic efficiency at its maximum despite high light intensities. The trend is the same for P ik . A positive progression is observed between 25 and 160 µmolPhotonPAR/m 2 /s (from 78.8 ± 4.0 to 117.0 ± 3.4) then stabilizes around 128 until 800 µmolPhotonPAR/m 2 /s (p-values > 0.12, excluding the values at 400 and 500 Generally speaking, it can be commented that the light response curve is affected by the light intensity to which the culture is acclimated while the general trend is maintained [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria 1[END_REF][START_REF] William | Measurement and Interpretation of Photosynthetic Light-Response Curves in Algae in the Context of Photoinhibition and Diel Changes[END_REF]. This can be observed in Figure 5.9 where the light curve of the culture grown under high light (here, 800 µmolPhotonPAR/m 2 /s) is higher and shifted to the right compared to the culture acclimated to low intensities (in the example, at 80 µmolPhotonPAR/m 2 /s). Besides, cultures acclimated to low light intensities exhibit a significant slope. This translation of the light response curve can simply be explained by the acclimation mechanisms set up by the cells according to the intensities under which the cultures were grown [START_REF] Grobbelaar | Variation in some photosynthetic characteristics of microalgae cultured in outdoor thin-layered sloping reactors[END_REF].

Let us first consider the case of cultures acclimated to low cultivation intensities. These cultures have high levels of chlorophyll a and accessory pigments (mainly chlorophyll b) that can transfer energy to the reaction centers. Therefore, these cultures can efficiently utilize low levels of irradiation, resulting in a higher slope. In addition, the onset of saturation and photoinhibition occurs at lower light intensities. This means that the transition between light-limited and light-saturated photosynthesis appears faster. This is mainly due to the low initial concentration of photoprotective pigments whose role is to protect the reaction centers from excessive excitation. Conversely, cultures grown at high light intensities can tolerate higher light intensities (higher P max ), thus delaying the onset of photoinhibition (higher I k and I i ). These cultures can better utilize high irradiance levels and generally have smaller antennae than low light-acclimated cells. They show a decrease in chlorophyll a and accessory pigments accompanied by a significant increase in photoprotective pigment content (mainly zeaxanthin). This accumulation of xanthophyll cycle pigments dissipates excess excitation energy via the NPQ mechanism. However, because these pigments absorb light without a concomitant increase in energy transfer to the reaction center, these cultures often exhibit lower maximum quantum yields at low light intensities (lower slope).

Antenna size

Thanks to the results obtained by the mathematical interpolation, we could deduce an approximation of the antenna size. Its evolution as a function of the light intensity is presented in Figure 5.11.

In this work, the size of an antenna is defined as the number of chlorophyll molecules per photosystem. In general, one can observe a decrease of the antenna size with increasing light intensity from 0.0046 ± 0.0002 ∝ chlorophyll/PSU to 0.0024 ± 0.0001 ∝ chlorophyll/PSU between 25 and 800 µmolPhotonPAR/m 2 /s. This decrease is faster in the photolimitation zone (-37 % between 25 and 200 µmolPhotonPAR/m 2 /s) than in photosaturation zone (-19 %). In this second zone, the value seems to stabilize gradually (p-values > 0.13 from 280 to 800 µmolPhotonPAR/m 2 /s, excluding the point at 400 µmolPhotonPAR/m 2 /s). This indicates that photosynthetic units contain less chlorophyll per photosystems at high intensities than at low intensities. Although it is only an approximation, this finding is very consistent with the pigment profile. This observation suggests a downregulation of the light harvesting capacity with increasing light intensity. This is a known long-term mechanism for acclimating to high intensity conditions [START_REF] Maxwell | Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (I. Light-Harvesting Complex II Abundance and Zeaxanthin Content in Chlorella vulgaris)[END_REF][START_REF] Patil | Reduced chlorophyll antenna mutants of Chlorella saccharophila for higher photosynthetic efficiency and biomass productivity under high light intensities[END_REF][START_REF] Levin | The desert green algae Chlorella ohadii thrives at excessively high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition[END_REF].

Cultures whose biotechnological performance is maintained at maximum at high light intensities are the consequence of a successful acclimation. This acclimation includes a variety of mechanisms whose objective is to adjust the amount of energy sent into the photosynthetic chain. In our study, we were able to follow only two of them: pigment rearrangement and decrease of the absolute pigment concentration. However, it should be kept in mind that there are others that probably took place-for example, the repair of the D1 protein and the accumulation of antioxidants.

Finally, in this work, only the steady-state response of photoacclimated cultures was studied. However, there are also transient photoprotection mechanisms that are put in place to protect the cells against the undesirable effects of exposure to intense light. These mechanisms have been highlighted in other studies. In this regard, acclimation of Chlorella vulgaris IPPAS C-1 cells to short exposure of high light has been shown to occur largely through modulation of the scattering cross-section of the cell rather than by altering their pigment content and composition [START_REF] Baránková | Light absorption and scattering by high light-tolerant, fast-growing Chlorella vulgaris IPPAS C-1 cells[END_REF]. Another work has highlighted a strong susceptibility of Chlorella fusca to the formation of large thylakoid aggregates (package effect) [START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF]. These aggregates are radiation-attenuating structures limiting the excitation energy from reaching the PSII reaction centers [START_REF] Raven | The cost of photoinhibition[END_REF]. In addition, they facilitate the dissipation of thermal energy by epoxidized xanthophylls. Other mechanisms may also take place. These include structural reorganization of the LCHII from trimeric to dimeric and monomeric forms [START_REF] Janik | Light-induced formation of dimeric LHCII[END_REF] and spatial reorganization of the LCHII, called state transitions [START_REF] Erickson | Light stress and photoprotection in Chlamydomonas reinhardtii[END_REF][START_REF] Kono | Long-term and short-term responses of the photosynthetic electron transport to fluctuating light[END_REF]. In the first case, dimeric and monomeric forms are preferred because they are less efficient in transferring excitation energy. In the second case, LHCIIs can dissociate from PSII to migrate and form quenching aggregates in the thylakoid membrane. These rearrangements allow reducing PSII quantum efficiency.

Conclusion

Chlorella vulgaris cultures were exposed to different intensities of continuous light until acclimation. Their responses both macroscopically (growth rate) and microscopically (pigment profile and status of the photosynthetic apparatus) were analyzed. Altogether, the results showed two different cellular responses depending on the light intensity to which the culture was acclimatized:

• From 25 to 200 µmolPhotonPAR/m 2 /s: cultures acclimated to limiting light levels exhibited linear light-dependent growth rates. As a result, the cells implemented strategies to collect as much light as possible. This resulted in relatively high levels of chlorophyll a and accessory pigments and low concentrations of photoprotective pigments.

• From 200 and 800 µmolPhotonPAR/m 2 /s: cultures acclimated to saturating light levels maintained the same growth dynamics but showed a very different pigment profile. Cells subjected to an energy flux exceeding the capacity of the photosynthetic apparatus downregulated their production of chlorophylls and accessory pigments (smaller antenna size). This strategy reduces the amount of harvested energy. At the same time, the cells accumulated zeaxanthin, a photoprotective pigment responsible for the dissipation of excess energy by NPQ. These photoprotective mechanisms allowed the cells to keep the machinery functioning at its maximum capacity, regardless of the light intensity, which resulted in a stabilized growth rate.

No photoinhibition phase was observed despite the increasing saturating light intensities applied. This absence, surprising at first sight, demonstrates the importance of the acclimation state of the culture which is an important output of the present work.

Finally, these results represent the long-term acclimated response of microalgae. It should be borne in mind that microalgae have other short-term mechanisms to protect themselves from intense light. However, with this experimental setup, these data could not be collected.

Introduction

This chapter is devoted to the analysis of the results obtained from cultures acclimated to intermittent light regimes. Three frequencies (1, 10, and 100 Hz) were explored at three different incident light intensities (I 0 =200, 400, and 800 µmolPhotonPAR/m 2 /s) for a total of 9 conditions. The duty cycle was kept at 0.5. The first two frequencies are defined as fluctuating light by scholars. They represent the frequencies commonly encountered in photobioreactors (10 to 0.1 Hz), depending on their designs and mixing rate. The exploration of this range of frequencies provides information on the long-term response of the cells grown in these devices, hence are of biotechnological interest for engineers. The last frequency is categorized as flashing light. It corresponds to the delivery of light pulses at characteristic times involved in photosynthesis. This light regime is investigated to understand the plasticity of the photosynthesis mechanism. For each light regime, the ratio between the light period and the dark period (ε) was set to 0.5. The results obtained are compared to the reference in continuous light for the same amount of average light perceived by the cultures (I avg = ε × I 0 ). The values presented are the mean value of three biological replicates of the same run.

As in the previous chapter, a stepwise approach is used to present the results. The chapter consists of three parts.

• The first part describes the growth rates obtained for all the intermittent regimes,

• The second part details the pigment content in chlorophylls and carotenoids for each light regime,

• The last part deals with the status of the photosynthetic apparatus.

Growth rate

Each frequency was studied at three levels of light intensity: a low, a moderate, and a high light level. The intensities were selected from the light curve obtained under continuous light (Figure 6.1). As a reminder, on this curve, three key areas were identified:

• a photolimitation zone from 25 to 120 µmolPhotonPAR/m 2 /s,

• a transition zone from 120 to 200 µmolPhotonPAR/m 2 /s,

• a photosaturation from 200 to 800 µmolPhotonPAR/m 2 /s.

Based on this observation, the following three specific points were selected:

• a first point corresponding to the end of the photolimitation zone -low light level with a 100 µmolPhotonPAR/m 2 /s average light intensity perceived by the culture (I 0 = 200 µmolPhotonPAR/m 2 /s),

• a second point corresponding to the entry in the plateau zone -moderate light level of average intensity of 200 µmolPhotonPAR/m 2 /s (I 0 = 400 µmolPhotonPAR/m 2 /s),

• and a last point corresponding to the middle of the plateau zone -high light intensity of an average intensity of 400 µmolPhotonPAR/m 2 /s (I 0 = 800 µmolPhotonPAR/m 2 /s).

The current experimental setup, limited to an incident light of 800 µmolPhotonPAR/m 2 /s, does not allow us to investigate the extreme point of the curve (end of the plateau zone) corresponding to an average perceived intensity of 800 µmolPhotonPAR/m 2 /s that would require an incident intensity of 1600 µmolPhotonPAR/m 2 /s. From this graph, one can notice that the measurement points in the intermittent regime seem to follow the same trend as that of continuous light. To facilitate the comparison of light regimes, a light-level approach is used.

Starting first with the low level (I avg = 100 µmolPhotonPAR/m 2 /s -photolimitation), it can be observed that the three intermittent light regimes are almost overlapping. No difference is observed between the different light regimes (p-value = 0.66). On average, they show a growth rate of 0.072 h -1 . In continuous light, the growth rate for the same amount of light is not known. However, assuming that this point is located in the photolimitation zone, one can calculate the growth rate's approximate value. For this, an affine regression was performed on the first three points of the light response curve under continuous illumination (red dotted line in Figure 6.1). Thanks to the equation, the growth rate can be estimated at about 0.072 h -1 for a continuous light exposure at 100 µmolPhotonPAR/m 2 /s (0.058 ± 0.002 h -1 at 80 µmolPhotonPAR/m 2 /s).

Considering then the moderate light (I avg = 200 µmolPhotonPAR/m 2 /s -entering the plateau zone), one can notice that the measurement points are a bit further apart from each other. While the light regime at 10 and 100 Hz are not significantly different (p-value = 0.12), the intermittent light at 1 Hz shows slight differences with that at 100 Hz (p-value = 0.046). However, when compared to the continuous light reference (0.085 ± 0.002 h -1 ), cultures grown under 100 Hz show the same performance (p-value = 0.49). In contrast, the 10 and 1 Hz regimes showed slight differences (p-values = 0.0495). The observed differences in performance can be quantified as -7 % and -11 % compared to the continuous light reference for the 10 Hz and 1 Hz regimes, respectively.

Finally, the results in high light (I avg = 400 µmolPhotonPAR/m 2 /s -middle of the plateau zone) are very similar to those obtained in moderate light at 200 µmolPhotonPAR/m 2 /s. The measurement points at 10 and 100 Hz show no differences (p-value = 0.10) while those at 1 Hz are slightly different from those at 100 Hz (p-value = 0.046). When the performances are compared with the reference in continuous light, no difference is observed for the 10 and 100 Hz regimes (p-values > 0.07). However, the intermittent regime at 1 Hz gives lower performance than the continuous light regime for the same amount of light (p-value = 0.0495). This decrease in performance was quantified at -6 %.

Given the small differences observed, these results do not provide any basis for concluding a potential effect of intermittent regimes on microalgal growth. Cultures grown under intermittent light at different frequencies and intensities performed the same as their reference under continuous light at equal amounts of light. This suggests that the combination of frequency and light levels tested does not affect the overall growth dynamic. Although the same level of performance is maintained, it is interesting to look at whether there were changes in the photosynthetic machinery under different light regimes and how it adapted to light flashes.

In the literature, many studies have been conducted on the impact of fluctuating and flashing light on the performance of microalgae. However, significant disparities have been reported in the biological responses. While some authors have reported a stimulating impact on productivity [START_REF] Janssen | Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/dark cycles[END_REF][START_REF] Janssen | Photosynthetic efficiency of Dunaliella tertiolecta under short light/dark cycles[END_REF][START_REF] Liao | A novel photobioreactor generating the light/dark cycle to improve microalgae cultivation[END_REF][START_REF] Nedbal | Microscopic green algae and cyanobacteria in high-frequency intermittent light[END_REF][START_REF] Janssen | Scale-up aspects of photobioreactors: effects of mixing-induced light/dark cycles[END_REF][START_REF] Sivakaminathan | Highthroughput optimisation of light-driven microalgae biotechnologies[END_REF], others have shown no effect [START_REF] Peter | Flashing light does not improve photosynthetic performance and growth of green microalgae[END_REF][START_REF] Janssen | Specific growth rate of Chlamydomonas reinhardtii and Chlorella sorokiniana under medium duration light/dark cycles: 13-87 s[END_REF][START_REF] Nedbal | Microscopic green algae and cyanobacteria in high-frequency intermittent light[END_REF][START_REF] Sivakaminathan | Highthroughput optimisation of light-driven microalgae biotechnologies[END_REF] or even deleterious effects [START_REF] Janssen | Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/dark cycles[END_REF][START_REF] Janssen | Photosynthetic efficiency of Dunaliella tertiolecta under short light/dark cycles[END_REF][START_REF] Peter | Flashing light does not improve photosynthetic performance and growth of green microalgae[END_REF][START_REF] Takache | Investigation of light/dark cycles effects on the photosynthetic growth of Chlamydomonas reinhardtii in conditions representative of photobioreactor cultivation[END_REF][START_REF] Nedbal | Microscopic green algae and cyanobacteria in high-frequency intermittent light[END_REF][START_REF] Sivakaminathan | Highthroughput optimisation of light-driven microalgae biotechnologies[END_REF][START_REF] Vejrazka | Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light[END_REF][START_REF] Chul | Energy-efficient cultivation of Chlamydomonas reinhardtii for lipid accumulation under flashing illumination conditions[END_REF][START_REF] Gris | Cultivation of Scenedesmus obliquus in Photobioreactors: Effects of Light Intensities and Light-Dark Cycles on Growth, Productivity, and Biochemical Composition[END_REF][START_REF] Combe | Long-term adaptive response to high-frequency light signals in the unicellular photosynthetic eukaryote Dunaliella salina[END_REF][START_REF] Brindley | Light regime optimization in photobioreactors using a dynamic photosynthesis model[END_REF]. The present work showed no effect of the intensity-frequency combination compared to the continuous light regime on growth rate for acclimatized cultures. This result is in agreement with the pioneer studies carried out. In this regard, Kok demonstrated that the photosynthetic activity of microalgae illuminated under intermittent light intensity approaches the same rate as an equivalent input of photons from constant light [START_REF] Kok | On the Efficiency of Chlorella Growth[END_REF]. Similar results were recently established [START_REF] Peter | Flashing light does not improve photosynthetic performance and growth of green microalgae[END_REF].

The conditions studied were not found to be discriminating. This is probably due to the combination of light level, frequency, and duty cycle applied. In a recent study, Schulze et al. [START_REF] Peter | Flashing light does not improve photosynthetic performance and growth of green microalgae[END_REF] looked at the combination of these three factors. The authors demonstrated that frequencies and duty cycle strongly affected the rate of photosynthesis while light level showed less action. This finding provides a new avenue of investigation for the duty cycle. In this work, the duty cycle was kept constant at 0.5. By varying this parameter, perhaps we could observe an effect on growth performance.

Pigment profile

Moving to the cell pigment content, the concentrations of chlorophyll and carotenoids pigments were monitored. As for the continuous light, the results are presented in a progressive way. For each class of pigments, the absolute concentrations are first given and then their ratios are analyzed. The results are compared to those obtained in continuous light for the same amount of light. Considering first the total chlorophyll content, the concentrations decrease with intensity for all the evaluated frequencies. Increasing the average light from 100 to 400 µmolPhotonPAR/m 2 /s, the concentrations change from 12.07 ± 0.41 mg/gDW to 7.66 ± 0.21 mg/gDW, from 11.76 ± 0.36 mg/gDW to 6.54 ± 0.09 mg/gDW, and from 13.52 ± 0.48 mg/gDW to 4.39 ± 0.3 mg/gDW for 1 Hz, 10 Hz, and 100 Hz, respectively. This corresponds to a decrease of -37 %, -44 % and -68 %, for each intermittent regime respectively. These values are consistent with the one assessed in continuous light between 120 and 400 µmolPhotonPAR/m 2 /s (-44 %). The downregulation appears just to be more pronounced for the 100 Hz regime. However, looking more closely at the data, the results point in different directions.

Chlorophylls content and ratio

While at 100 µmolPhotonPAR/m 2 /s, the 100 Hz regime shows a slightly higher amount of total chlorophyll than the other two intermittent light regimes (p-value = 0.0495), under average illumination at 200 µmolPhotonPAR/m 2 /s, the trend is different. The 1 Hz regime exhibits a more moderate decrease than the other two regimes (10.70 ± 1.15 mg/gDW, p-values = 0.0495), which give similar results (average concentration of 7.20 mg/gDW and p-value = 0.51). Finally, at 400 µmolPhotonPAR/m 2 /s, the absolute concentrations of total chlorophyll are all different from each other (p-values = 0.0495).

By looking then at the two pigments separately, the same down-regulation trend is unsurprisingly maintained. The concentrations of chlorophyll a and b respectively evolve in a very similar way. At 1, 10 and 100 Hz, the loss is quantified at -35 %, -42 % and -68 % for chlorophyll a, and at -41 %, -50 % and -68 % for chlorophyll b. In continuous light between 120 and 400 µmolPhotonPAR/m 2 /s, chlorophyll a and b contents were affected by -44 % and -42 %, respectively. Again, the drop is more severe for the 100 Hz regime. Interestingly, the latter is attributed to the concomitant downregulation of both chlorophyll a and b. Although higher in comparison to the other regimes, chlorophyll a and b concentrations are not so different from the continuous reference (p-values = 0.0495). As before, another remarkable point is the regime at 1 Hz under an average illumination of 200 µmolPhotonPAR/m 2 /s. This result seems to suggest a less severe downregulation compared to the other regimes (p-values = 0.0495). Considering the chlorophyll b/chlorophyll a ratio (Figure 6.3), the latter evolves between 0.4 and 0.5, all light regimes combined. The regimes at 1 Hz and 10 Hz follow the same trend (p-values > 0.51). They vary on average between 0.47 and 0.41 between 100 and 400 µmolPhotonPAR/m 2 /s. On the contrary, the regime at 100 Hz varies differently. At 100 and 400 µmolPhotonPAR/m 2 /s, it presents a ratio of 0.51 while at 200 µmolPhotonPAR/m 2 /s, it registers a drop to 0.39. This low value is attributed to a steeper drop in chlorophyll b concentration compared to the other regimes. Although the point at 200 µmolPhotonPAR/m 2 /s appears visually different from the continuous light reference, statistical tests did not highlight any significant difference (p-values > 0.28) between this regime and the continuous light reference. Considering the 1 and 10 Hz regimes, the ratio values are similar to the continuous reference between 100 and 200 µmolPhotonPAR/m 2 /s (p-values > 0.06). However, under 400 µmolPhotonPAR/m 2 /s, it is statistically lower than in continuous light (p-value = 0.0459). This seems to suggest that at an intensity of 400 µmolPhotonPAR/m 2 /s, cells favor chlorophyll a over b. However, as the ratio is close to the continuous reference (0.41 and 0.50, respectively), no conclusion can be drawn from these data. In summary, these results show a downregulation of chlorophyll concentration with intensity. The latter affected chlorophyll a and b in a similar way, confirmed by the ratio. This same phenomenon was reported for cultures grown in continuous light of increasing intensities. Therefore, even when cells are subjected to intermittent illumination, they use the same mechanism to regulate the amount of light absorbed. However, some small differences between the intermittent regimes and the continuous reference were detected. For instance, the 100 Hz regime presented a greater reduction in the total amount of chlorophyll compared to the other regimes (-24 % compared to the continuous light reference at 400 µmolPhotonPAR/m 2 /s). In addition, a more moderate drop in chlorophyll content was observed under the average 200 µmolPhotonPAR/m 2 /s intermittent light at 1 Hz. However, although these results show slightly different dynamics, nothing solid can be concluded. Therefore, the intermittent regimes studied do not seem to alter the light harvest of acclimatized cultures to any great extent.

Carotenoids content and ratio

As for the continuous light reference, carotenoids contents were followed for each intermittent light regime. First, the absolute concentration of total carotenoids and their ratio to total chlorophylls are analyzed. Then, to go further, a zoom is made on the different pigments. Figure 6.4 shows the evolution of total carotenoid content (lutein, violaxanthin, and zeaxanthin) according to the average light intensity of culture. As can be seen, the total carotenoids content decreases between 100 and 400 µmolPhotonPAR/m 2 /s, regardless of the applied frequency. The content goes from 1.23 ± 0.06 mg/gDW to 0.80 ± 0.15 mg/gDW (-35 %), 0.89 ± 0.05 mg/gDW to 0.74 ± 0.04 mg/gDW (-17 %) and 1.21 ± 0.06 mg/gDW to 0.48 ± 0.04 mg/gDW (-60 %) at 1, 10 and 100 Hz respectively. The same phenomenon of down-regulation with light intensity was observed for the reference in continuous light (-42 %). As for chlorophylls, the intermittent light regime at 100 Hz seems to accentuate the down-regulation in carotenoid pigments. However, this difference remains moderate. Moreover, one can notice that at an average light intensity of 200 µmolPhotonPAR/m 2 /s, the 1 Hz regime presents a higher carotenoid content of about +30 % compared to the continuous regime (p-value = 0.0495). The same phenomenon was observed with the total chlorophyll content.

To go further in the analysis, it is interesting to look at the carotenoid/chlorophyll ratio (Figure 6.4). Between 100 and 400 µmolPhotonPAR/m 2 /s, the ratio seems more or less stable, around 0.09. Analyzing by light level, at 100 µmolPhotonPAR/m 2 /s, although all regimes give different values (p-values = 0.0495), they are all around the reference in continuous light. The maximum difference of -17 % is reported for the 10 Hz regime compared to the continuous light reference.

The carotenoid/chlorophyll ratio value is similar to what was found for the reference in continuous light (0.8 between 25 and 500 µmolPhotonPAR/m 2 /s). Thus, at these light levels, the ratio remains in favor of chlorophylls, and thus of photocollection rather than photoprotection. This result suggests that the frequencies studied do not cause different pigment acclimation compared to what was obtained in continuous light for the same amount of light. At 200 µmolPhotonPAR/m 2 /s, although the absolute values of chlorophyll and carotenoids concentrations are higher at 1 Hz than for the other regimes, the ratio is identical to that obtained under continuous light (p-value = 0.83). Finally, under an average illumination of 400 µmolPhotonPAR/m 2 /s, no difference was reported for all regimes, including the reference to continuous light (p-value = 0.13). Thus, from these data, it can be concluded that cultures subjected to intermittent light and those subjected to continuous light for the same amount of light show a very similar pigment profile. This suggests that intermittent light at the frequencies and intensities studied does not induce a different pigment response compared to the continuous light. The cells use the same acclimation mechanisms. However, it would have been interesting to explore the response to higher light intensities. At such intensities (e.g. I 0 = 1600 µmolPhotonPAR/m 2 /s), one might have had the chance to observe an increase in the ratio in favor of carotenoids and thus of photoprotection.

Although the ratio of carotenoids to chlorophylls does not appear to be altered by the frequency, it is interesting to break down the carotenoid pigments to assess their distribution.

Lutein

.5 represents the absolute lutein concentration for all intermittent regimes as a function of average cultivation intensity. At first sight, the trend is the same as that found for the total carotenoid pigment concentration, which is not surprising. The concentrations between 100 and 400 µmolPhotonPAR/m 2 /s decrease by -43 %, -27.5 % and -66 % at 1, 10 and 100 Hz, respectively. Analyzing each light level against their continuous light reference, at 100 µmolPhotonPAR/m 2 /s, no significant difference is reported by the statistical test procedure (p-values > 0.06). At 200 µmolPhotonPAR/m 2 /s, the 10 and 100 Hz regimes give very similar results (p-value = 0.37). However, these results appear slightly different from the reference in continuous light (p-value = 0.0495). The regime at 1 Hz, on the other hand, is both different from the other two intermittent regimes but also from the reference in continuous light (p-values = 0.046). Finally, at 400 µmolPhotonPAR/m 2 /s, the 1 and 10 Hz regimes give very close lutein concentration values (p-value = 0.83) but a little higher from the reference in continuous light (p-values = 0.0495) whereas the 100 Hz regime is different from all the other regimes, including the reference (p-values = 0.0495). Two comments can be drawn from these results:

• The decline in lutein concentration with increasing light intensity was also reported for continuous light. Between 120 and 400 µmolPhotonPAR/m 2 /s, a drop of -50 % was recorded.

• As before, the same trend is maintained between chlorophylls and carotenoids. The frequency at 100 Hz records the greatest loss of pigments while the frequency at 1 Hz under an average intensity of 200 µmolPhotonPAR/m 2 /s shows a greater pigment content than the other regimes (p-value = 0.46). A possible explanation for this more moderate downregulation of carotenoids at 1 Hz -200 µmolPhotonPAR/m 2 /s would be attributed to the combination of light intensity and its solicitation time. Indeed, a light intensity of 200 µmolPhotonPAR/m 2 /s corresponds to the entry of the photosaturation phase. In this zone, the light starts to become potentially excessive for the photosynthetic apparatus. As a consequence, the cells put in place mechanisms to regulate the energy flux. This results, among other things, in the accumulation of photoprotective pigments. Among the intermittent regimes studied, the 1 Hz frequency has a much longer light phase (0.5 seconds) than the other two regimes (50 and 5 milliseconds). Therefore, light becomes potentially more harmfull compared to the other regimes, resulting in a greater need for lutein. However, this explanation does not fit with the results obtained for chlorophylls. In addition, under continuous light for the same amount of light, the lutein content is lower than the 1 Hz regime.

VAZ cycle

Turning to the pigments of the VAZ cycle, Figure 6.6 displays the evolution of the total concentration of VAZ cycle pigments (violaxanthin + zeaxanthin) as a function of the average culture intensity for the three intermittent light regimes. Overall, between 100 and 400 µmolPhotonPAR/m 2 /s, the concentration remains more or less stable around 0.16 mg/gDW for the 1 Hz regime and very close to the reference in continuous light (p-value = 0.66). On the contrary, the 10 and 100 Hz regimes show more important fluctuations. Between 100 and 400 µmolPhotonPAR/m 2 /s, the concentrations of violaxanthin and zeaxanthin successively goes from 0.10 ± 0.01 mg/gDW, to 0.07 ± 0.02 mg/gDW and 0.16 ± 0.02 mg/gDW for 10 Hz and from 0.16 ± 0.02 mg/gDW to 0.08 ± 0.03 mg/gDW and 0.12 ± 0.01 mg/gDW for 100 Hz. However, these variations remain centered around 0.11 mg/gDW and 0.12 mg/gDW for 10 Hz and 100 Hz respectively. Thus, the VAZ cycle pigments maintain their concentration regardless of the applied frequency and intensity, in the same way as the reference in continuous light. This suggests that the same acclimation mechanisms are at work and that the level of response is the same between continuous and intermittent light for the conditions studied.

Figure 6.6 also details the evolution of zeaxanthin concentration in relation to the average culture light intensity. At first glance, the concentration is relatively constant between 100 and 200 µmolPhotonPAR/m 2 /s and increases under an average illumination of 400 µmolPhotonPAR/m 2 /s, for the three regimes. This result is not surprising since it corresponds to the interconversion of violaxanthin to zeaxanthin as the light intensity increases. As can be seen, some differences are notable between the different intermittent regimes. The 10 and 100 Hz regimes present similar results (p-values > 0.06). The 1 Hz regime shows the same trend with a higher zeaxanthin content suggesting a greater need for photoprotection.

In general, the zeaxanthin concentration trend is upward oriented with intensity and follows that of the continuous light reference. However, the intensity at which zeaxanthin supplants violaxanthin shows some variation. In continuous light, this phenomenon occurs around 160 µmolPhotonPAR/m 2 /s. In the intermittent regime of 1 Hz, it takes place around 200 µmolPhotonPAR/m 2 /s while at 10 and 100 Hz, it takes place around 250 µmolPhotonPAR/m 2 /s (not shown on the graph for readability reasons). The delay of the interconversion could suggest that the introduced dark phases are rather favorable to the relaxation of the intense light excitation pressure. This hypothesis remains to be confirmed by fluorometry analyses, especially those of the NPQ (which are unfortunately not reliable in this work).

Photosynthetic apparatus status

After analyzing the biotechnological aspects of the cultures, let us dive deeper into the mechanism of photosynthesis. This is achieved by analyzing the status of the photosynthetic apparatus. As for the previous chapter, the results are categorized according to the fluorometry protocols: NPQ and Light Curve protocol. Starting with the Fv/Fm ratio, as can be seen, this ratio is relatively constant, around 0.813 for 1 Hz and 0.804 for 100 Hz. Between 100 and 400 µmolPhotonPAR/m 2 /s, the variation between the minimum and maximum value of the ratio is 6 % for both regimes. These values are very close to what was recorded for the continuous light reference. Between 120 and 400 µmolPhotonPAR/m 2 /s, the Fv/Fm ratio varied between 0.840 ± 0.010 and 0.777 ± 0.006, i.e. an average of 0.808 (8 % variation). This means that the frequencies and intensities tested for the two intermittent regimes at 1 Hz and 100 Hz do not induce any change in the functioning of the photosynthetic apparatus.

NPQ protocol

In contrast, the 10 Hz regime shows a slightly different pattern. While under 100 and 400 µmolPhotonPAR/m 2 /s, the ratio is the same as the other regimes, including the reference (p-value > 0.06), under 200 µmolPhotonPAR/m 2 /s, one can observe a small drop in this ratio (0.750 compared to 0.837 for the reference). However, despite this slight variation (12 %), the recorded Fv/Fm values indicate that the photosynthetic apparatus is healthy and still functioning properly (which is confirmed by the values of the maintained growth rates). The same trend of decreasing NPQ with intensity between 120 and 400 µmolPhotonPAR/m 2 /s is found for the continuous light reference. However, the NPQ values in intermittent light regimes are much lower than the continuous light reference. A low NPQ value could indicate that the cultures do not need to develop extensive physiological means to protect themselves by dissipating the excessive excitation energy into harmless heat. This hypothesis would support the view that the introduction of a dark phase is beneficial in reducing the excitation pressure on the photosynthetic chain, in spite of a doubled intensity during the light phase. However, it should be kept in mind that the NPQ results are highly questionable due to the misuse of the device.

Light Curve protocol

The cultures were then subjected to the Light Curve protocol. From the extracted light response curve, the identified light curves were obtained. In the same way, as for the continuous light reference, four parameters were extracted: P max , P ik , I k , and I i . Their evolution as a function of the cultivation light intensity for all the intermittent light regimes is shown in Figures 6.8 and 6.9.

Looking first at the parameters related to photosynthetic yield (Figure 6.8), no decay phase is reported. The maximum photosynthetic rate (P max ) of the regimes at 1 and 100 Hz follow perfectly the same trend as the reference in continuous light (p-values > 0.25). On the contrary, visually, the regime at 10 Hz decreases for an average intensity of 400 µmolPhotonPAR/m 2 /s (147 ± 21 compared to 166 ± 14 in continuous light with the same amount of light, i.e. a -13 % decrease). This result could suggest that the photosynthetic apparatus does not operate at its maximum capacity under this intermittent light regime at average intensities of 400 µmolPhotonPAR/m 2 /s. However, the growth rates obtained under this regime at these intensities do not suggest a decrease in photosynthetic activity. The decorrelation of photosynthesis and growth dynamics has also been observed for other types of stress, such as heavy metals [START_REF] Expósito | Performance of Chlorella Vulgaris Exposed to Heavy Metal Mixtures: Linking Measured Endpoints and Mechanisms[END_REF]. Nevertheless, these results are not significantly different from those found for the continuous reference (p-value = 0.12). The same trends are found for the P ik parameter. Considering then the parameters I k and I i (Figure 6.9), one can notice for all the intermittent regimes an increasing trend for both parameters with the light intensity. As before, the values obtained for all intermittent light regimes follow closely those of the reference in continuous light. No significant difference was reported between 100 and 400 µmolPhotonPAR/m 2 /s compared to the reference (p-values > 0.12). Thus, although the maximum photosynthetic activity at 400 µmolPhotonPAR/m 2 /s shows a 13 % drop compared to the continuous reference at 10 Hz, the I k and I i parameters are not affected. Furthermore, all the intermittent light regimes are similar to the continuous light reference. This suggests that the acclimated state of the cultures allows them to maintain the same performance as in continuous light.

Antenna size

Thanks to the previous results, the antenna size could be deduced for each of the intermittent regimes. Their evolution as a function of the average cultivation intensity is presented in Figure 6.10.

At first glance, the results show an expected negative correlation between antenna size and cultivation light intensity. The latter follows relatively well the same trend as the reference. No significant difference is reported between the all light regimes (p-values > 0.08). In continuous light from 120 to 400 µmolPhotonPAR/m 2 /s, the antenna size decreases from 0.0032 to 0.0027 ∝ chlorophyll/PSU, i.e. 18 % decrease. In the intermittent light regimes, the decrease is -28 %, -15 %, and -25 % for the 1 Hz, 10 Hz, and 100 Hz regimes, respectively. This indicates that photosynthetic units contain less chlorophyll per photosystem at high intensities than at low intensities. This finding is consistent with the pigment profile reported. Therefore, the mechanism of acclimation and regulation of energy flow involves the same mechanism as the continuous light reference. Besides, its response amplitude does not differ between both light regimes.

Conclusion

After assessing the biotechnological performance of cultures acclimated to constant illumination at different increasing intensities, Chlorella vulgaris cells were exposed to In general, the results indicate that the biotechnological performance is maintained at the same level as the regime under constant illumination of the same amount. At 200 and 400 µmolPhotonPAR/m 2 /s, a downward trend in growth rate is observed from continuous to 100 Hz, 10 Hz and finally 1 Hz, but the differences are either not statistically different or very slight (mostly less than 10 %). Therefore, these results demonstrate that there is no effect of intermittent light at the conditions tested for cultures that have been previously acclimatized. However, some differences with the continuous light regime could be noted:

• 1 Hz regime: cultures exposed to 200 µmolPhotonPAR/m 2 /s show a slightly higher pigment content in both chlorophylls and carotenoids,

• 10 Hz regime: cultures exposed to 400 µmolPhotonPAR/m 2 /s exhibit a slightly lower P max and P ik ,

• 100 Hz regime: cultures exposed to 200 µmolPhotonPAR/m 2 /s record a higher loss of chlorophylls and carotenoids content compared to the other intermittent regimes.

These differences do not seem to point to a consistent and/or known biological mechanism. Furthermore, compared to the reference in continuous light, the deviations are mostly less than 20 %. Thus, these differences are not significant enough to conclude on a real phenomenon. To go further and amplify a potential difference in response between continuous and intermittent lighting, it would be necessary to obtain a greater statistical strength by increasing the number of replicates. Furthermore, different parameters should be explored. Three avenues can be addressed. On the one hand, the most obvious would be to continue to explore the frequency range. On the other hand, one could evaluate different duty cycles to investigate the response when the illuminated phase is longer or shorter than the dark phase. Finally, the current experimental device could be evolved to expose cultures to even more extreme intensities until a photoinhibition phase appears.

Conclusion Conclusion

This work started from the observation that light is the key element to improve the economic viability of bioprocesses using microalgae. Indeed, the combination of light absorption by the organisms and agitation imposes intermittent light cycles that affect microalgae growth. Although the light integration by photosynthetic organisms is known to the scientific community, its biotechnological exploitation is still limited.

The collection of information in the scientific literature has revealed two things:

• Photosynthesis is a complex light-driven, finely regulated process that has both short-term and long-term regulatory mechanisms to protect itself and adapt to light fluctuations that may occur in their environment. Thanks to these mechanisms, they effectively counteract the energy overload of the photosynthetic apparatus and thus limit the occurrence of photodamage. By modulating light harvesting capacity and energy use, photosynthetic organisms can limit the damage to the photosynthetic system without interrupting the photosynthetic process and thus avoid the occurrence of photoinhibition as much as possible.

• Many studies have been devoted to studying the interaction between light and microalgae and how to manipulate them to improve the productivity of microalgae. However, although widely studied, the understanding of this interaction remains hampered by significant disparities in the biological responses reported in the literature. The result of this investigation has awakened the need for further research.

In addition, the literature review highlighted a variety of experimental designs and a non uniformity in the method of monitoring the cultures, which would probably be at the origin of the divergence of the results obtained so far. In particular, two points emerge: the question of the acclimation of the strain to the light conditions and the photobioreactor design used for the study (optical fineness and presence of optical aberrations).

Within these lines, it is important to produce high quality biological data allowing a relevant extrapolation to the industrial scale. For this purpose, an experimental device has been developed to reproduce light intermittency encountered in bioprocesses. This device guarantees the same light history to all cells (homogeneous lighting) and allows measuring the acclimatized biological responses according to the light patterns. It consists of a flat photobioreactor with a thin design allowing to limit as much as possible the 150 Chapter 6. Experimental results under intermittent light optical disturbances/distortions and of an instrumentation system allowing a continuous follow-up and a semi-automatic operation of the bioreactors. Such a system fulfills two functions. On the one hand, the culture is maintained at a low cell density, which ensures that light is delivered homogeneously to the entire culture. On the other hand, it keeps all abiotic factors (temperature, pH, nutrient concentration, etc.) fixed during the whole culture. In this way, light remains the only parameter that can influence the cellular response of microalgae from one culture to another. All these precautions were essential for the production of high-quality biological data.

The experimental campaigns were conducted in two stages. First, Chlorella vulgaris cultures were exposed to continuous illumination of varying intensities from 25 to 800 µmolPhotonPAR/m 2 /s. These results serve as a reference for comparison. Then, intermittent illumination was studied (1 Hz, 10 Hz and 100 Hz, illuminated fraction: 0.5). The first two frequencies explored correspond to the fluctuating light frequently encountered in photobioreactors. The last frequency corresponds to a flashing light and coincides with the characteristic times of photosynthesis. At the end of each cycle, the cultures were analyzed. Three parameters were monitored. The first parameter is the growth rate. This is an indicator widely used in biotechnology to describe both the general well-being of a culture and the productivity of the process. The pigment profile in chlorophylls and carotenoids was also measured. Finally, to get closer to photosynthesis, the status of the photosynthetic apparatus was quantified. The data obtained allow us to understand how microalgae adapt to light conditions by setting up photoprotection and photoacclimation mechanisms.

Under constant illumination, the cultures displayed two different cellular responses depending on the culture intensity to which the culture was acclimated:

• From 25 to 200 µmolPhotonPAR/m 2 /s: cultures acclimated to limiting light levels exhibited linear light-dependent growth rates. As a result, the cells implemented strategies to collect as much light as possible. This resulted in relatively high levels of chlorophyll a and accessory pigments and low concentrations of photoprotective pigments.

• From 200 and 800 µmolPhotonPAR/m 2 /s: cultures acclimated to saturating light levels showed a very different pigment profile. Cells subjected to an energy flux exceeding the capacity of the photosynthetic apparatus down-regulated their production of chlorophylls and accessory pigments (smaller antenna size). This strategy reduces the amount of energy harvested. At the same time, the cells accumulated zeaxanthin, a photoprotective pigment responsible for dissipating excess energy through NPQ. These photoprotective mechanisms allowed the cells to keep the machinery running at maximum capacity regardless of light intensity, resulting in a stabilized growth rate.

No photoinhibition phase was observed despite the increasing saturating light intensities applied. This absence, which may seem surprising at first glance, demonstrates the importance of the acclimation state of the culture on the results. Under intermittent illumination, the cultures maintained the same level of biotechnological performance as their references under constant illumination of the same amount of light. Therefore, these results demonstrate that there is no effect of intermittent light at the conditions tested for cultures that have been previously acclimated. The cells are therefore able to integrate light fluctuations and only perceive the averaged level. Thus, these experiments invalidate, at long times (those of continuous bioprocesses), the hypothesis that light modulation has an impact on the performance of microalgae.

To go further and amplify the few difference observed in response between continuous and intermittent lighting, it would be necessary to obtain greater statistical power by increasing the number of replicates. In addition, different parameters should be investigated. Three avenues can be explored. First, the frequency range could be extended. Second, different duty cycles could be evaluated to study the response when the light phase is longer or shorter than the dark phase. Third, we could evolve the current experimental set-up to expose the cultures to even more extreme intensities until the appearance of a photoinhibition phase. Finally, during the acclimation phase, cultures subjected to high intensity showed a longer stabilization time. Thus, in addition to the cellular response, it would be interesting to evaluate the acclimation dynamics of cultures exposed to different light patterns. Exported from [START_REF] Shen | Structure of a C2S2M2N2-type PSII-LHCII supercomplex from the green alga Chlamydomonas reinhardtii[END_REF] Table B.1: All data collected from studies conducted in fluctuating light with the growth rate (GR) as the output variable. The table lists the study microorganism, the experimental device used to measure the GR, the parameters of the L/D cycles as well as the experimental results with their coefficient of variation if known (N.A. if not available). The average light intensity (I avg ) is expressed in µmolE/m /s). The growth rate presented is weighted by the quantity of light. The reference to continuous light appears as CL

Studied microalga

Experimental device I avg τ c (s) ε (-) GR (h Grâce à ces mécanismes, les microalgues contrecarrent efficacement la surcharge énergétique en modulant la capacité de récolte de la lumière et l'utilisation de l'énergie. Ainsi, elles peuvent limiter les dommages subis par l'appareil photosynthétique sans interrompre le processus de photosynthèse et ainsi éviter autant que possible l'apparition de la photoinhibition.

• De nombreuses études ont été consacrées à l'étude de l'interaction entre la lumière et les microalgues et à la manière de les manipuler pour améliorer la productivité des microalgues. Cependant, bien que largement étudiée, la compréhension de cette interaction reste entravée par des disparités importantes dans les réponses biologiques rapportées dans la littérature. Le résultat de cette enquête a mis en évidence la nécessité de poursuivre les recherches. Par ailleurs, la revue de la littérature a mis en évidence une variété de plans expérimentaux et une non uniformité dans la méthode de suivi des cultures, qui seraient probablement à l'origine de la divergence des résultats obtenus jusqu'à présent. En particulier, deux points émergent : la question de l'acclimatation de la souche aux conditions lumineuses et le design du photobioréacteur utilisé pour l'étude (finesse optique et présence d'aberrations optiques).

Matériels et méthodes

Pour caractériser l'effet des fluctuations lumineuses sur les performances des microalgues vertes (taux de croissance, composition pigmentaire, état de l'appareil photosynthétique), il est important de produire des données biologiques de qualité permettant une extrapolation pertinente à l'échelle industrielle. Dans ce but, un dispositif expérimental a été développé pour reproduire l'intermittence de la lumière rencontrée dans les bioprocédés. Ce dispositif garantit le même historique lumineux à toutes les cellules (éclairage homogène) et permet de mesurer les réponses biologiques acclimatées en fonction des schémas lumineux. Il est constitué d'un photobioréacteur plan au design fin permettant de limiter au maximum les perturbations/distorsions optiques et d'un système d'instrumentation permettant un suivi continu et un fonctionnement semi-automatique des bioréacteurs. Un tel système remplit deux fonctions. D'une part, il maintient la culture à une faible densité cellulaire, ce qui garantit que la lumière est délivrée de manière homogène à l'ensemble de la culture. D'autre part, il maintient tous les facteurs abiotiques (température, pH, concentration en nutriments, etc.) fixes pendant toute la durée de la culture. Ainsi, la lumière reste le seul paramètre qui peut influencer la réponse cellulaire des microalgues d'une culture à l'autre. Toutes ces précautions étaient indispensables à la production de données biologiques de haute qualité.

Résultats expérimentaux

Tout d'abord, des cultures de Chlorella vulgaris ont été exposées à un éclairage continu d'intensité lumineuse allant de 25 à 800 µmolPhotonPAR/m 2 /s. Ces résultats servent de référence pour les comparaisons à venir. Ensuite, une illumination intermittente a été étudiée (1 Hz, 10 Hz et 100 Hz, fraction illuminée : 0,5). Les deux premières fréquences explorées correspondent à la lumière fluctuante fréquemment rencontrée dans les photobioréacteurs. La dernière fréquence correspond à une lumière flashée et coïncide avec les temps caractéristiques de la photosynthèse. A la fin de chaque cycle, les cultures ont été analysées. Trois paramètres ont été suivis. Le premier paramètre est le taux de croissance. Il s'agit d'un indicateur largement utilisé en biotechnologie pour décrire à la fois le bien-être général d'une culture et la productivité du procédé. Le profil pigmentaire en chlorophylles et en caroténoïdes a également été mesuré. Enfin, pour se rapprocher de la photosynthèse, l'état de l'appareil photosynthétique a été quantifié. Les données obtenues permettent de comprendre comment les microalgues s'adaptent aux conditions lumineuses en mettant en place des mécanismes de photoprotection et de photoacclimatation.

Sous un éclairage constant, les cultures ont présenté deux réponses cellulaires différentes en fonction de l'intensité de culture à laquelle la culture a été acclimatée :

• De 25 à 200 µmolPhotonPAR/m 2 /s : les cultures acclimatées à des niveaux de lumière limitants ont présenté des taux de croissance linéaires dépendant de la lumière. En conséquence, les cellules ont mis en place des stratégies pour collecter autant de lumière que possible. Il en résulte des niveaux relativement élevés de chlorophylle a et de pigments accessoires et de faibles concentrations de pigments photoprotecteurs.

• De 200 et 800 µmolPhotonPAR/m 2 /s : les cultures acclimatées à des niveaux de lumière saturants ont montré un profil pigmentaire très différent. Les cellules soumises à un flux énergétique dépassant la capacité de l'appareil photosynthétique ont régulé à la baisse leur production de chlorophylles et de pigments accessoires (taille d'antenne plus petite). Cette stratégie permet de réduire la quantité d'énergie récoltée. Dans le même temps, les cellules ont accumulé de la zéaxanthine, un pigment photoprotecteur chargé de dissiper l'énergie excédentaire par le biais du NPQ. Ces mécanismes de photoprotection ont permis aux cellules de maintenir la machinerie à sa capacité maximale, quelle que soit l'intensité lumineuse, ce qui a entraîné une stabilisation du taux de croissance.

Aucune phase de photoinhibition n'a été observée malgré les intensités lumineuses saturantes croissantes appliquées. Cette absence, qui peut paraître surprenante au premier abord, démontre l'importance de l'état d'acclimatation de la culture sur les résultats. Sous illumination intermittente, les cultures ont maintenu le même niveau de performance biotechnologique que leurs références sous illumination constante pour une même quantité de lumière.

Conclusion

Ces résultats démontrent qu'il n'y a pas d'effet de la lumière intermittente aux conditions testées pour des cultures qui ont été préalablement acclimatées. Les cellules sont donc capables d'intégrer les fluctuations lumineuses et ne perçoivent que le niveau moyen. Ainsi, ces expériences invalident, à des temps longs (ceux des bioprocédés continus), l'hypothèse selon laquelle la modulation de la lumière aurait un impact sur les performances des microalgues.

1

Overshoot day exported from [START_REF] Pozzobon | Effet de la photopériode sur la croissance des microalgues -Journée microalgues[END_REF]. Data from Global Footprint Network [START_REF]Supply and Demand[END_REF].

Figure 1 :

 1 Figure 1: Overshoot day exported from [2]. Data from Global Footprint Network [3].

  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Figure 1 . 1 :

 11 Figure 1.1: Micrographs of some microalgae of different phyla showing the diversity in shape, size, and color of microalgae. Images extracted from UTEX Culture Collection of Algae at The University of Texas at Austin [9]. A-Micrasterias hardeyii ; B-Volvox carteri ; C-Arthrospira platensis ; D-Colacium mucronatum ; E-Flintiella sanguinaria ; F-Glaucocystis nostochinearum.

Figure 1 . 2 :

 12 Figure 1.2: Major microalgae phyla distribution as per seven-kingdom classification scheme with major phyla distribution

Figure 1 . 4 :

 14 Figure 1.4: Schematic illustration of the cell organization of Chlorella vulgaris. Microscope observation from our lab, magnification x100 (image manipulation: contrast enhancement).

Figure 1 . 5 :

 15 Figure 1.5: Spectrum of the electromagnetic radiation

( a )

 a Figure 1.6: Chemical structures and absorption spectra of the five existing chlorophyll types

( a )

 a Figure 1.7: Chemical structures and absorption spectra of the most common carotenoids found in microalgae

Figure 1 . 8 :

 18 Figure 1.8: Schematic representation of a chloroplast structure inspired from[56]

Figure 1 . 9 :

 19 Figure1.9: Schematic representation of the distribution of protein complexes in the thylakoid membrane from[START_REF] Allen | Molecular recognition in thylakoid structure and function[END_REF]

Figure 1 . 10 :

 110 Figure 1.10: Schematic representation of a simplified structure of the PSII-LHCII supercomplex inspired from[START_REF] Heldt | Plant biochemistry[END_REF][START_REF] Xu | Functional organization of photosystem II antenna complexes: CP29 under the spotlight[END_REF]

  Side view (b) Top view from the stromal side. Membraneextrinsic subunits PsaC, PsaD and PsaE are omitted for clarity.

Figure 1 . 11 :

 111 Figure 1.11: PSI-LHCI supercomplex in green microalgae inspired from[START_REF] Pessarakli | Handbook of photosynthesis[END_REF][START_REF] Kubota-Kawai | Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii[END_REF]

Figure 1 . 12 :

 112 Figure 1.12: Schematic representation of the distribution of LHCII and LHCI in relation with the PS core inspired from[START_REF] Pessarakli | Handbook of photosynthesis[END_REF][START_REF] Kubota-Kawai | Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii[END_REF]

Figure 1 . 14 :

 114 Figure1.14: Schematic representation of the structure of the ATPase inspired from[START_REF] Heldt | Plant biochemistry[END_REF]

Figure 1 . 15 :

 115 Photosystem II Reaction center

Figure 1 . 17 :

 117 Figure 1.17: Trapping light and routing it to reaction centers placed energetically downhill

Figure 1 . 18 :1. 2 . 5 . 5

 118255 Figure 1.18: Schematic representation of the chemical transformation of the light energy through PSII inspired from[START_REF] Edward | Phycology[END_REF]

Figure 1 . 19 :

 119 Figure 1.19: Schematic representation of the Q cycle

Figure 1 . 20 :

 120 Figure 1.20: Schematic representation of the electron flow through PSI insprired from[START_REF] Heldt | Plant biochemistry[END_REF]

Figure 1 . 21 :

 121 Figure 1.21: Schematic representation of ATP regeneration through subunit conformational changes insprired from[START_REF] Heldt | Plant biochemistry[END_REF]

Figure 1 . 23 :

 123 Figure 1.23: Schematic representation of the Calvin-Benson cycle's reaction taking place in the stroma of the chloroplast

Figure 1 . 24 :

 124 Figure 1.24: Schematic representation of photorespiration pathway

Figure 1 . 25 :

 125 Figure 1.25: Schematic representation of the mechanisms involved during in the appearance of photodamages at the PSII level. The three main mechanisms are depicted with different colours: blue for the mechanism involving the donor side of the photochemistry, black for the mechanism involving the acceptor side and green for the triplet state chlorophyll. ROS formation appears in red. The compounds responsible for the degradation of protein D1 are shown in orange.

Figure 1 . 26 :

 126 Figure 1.26: Schematic representation of the VAZ cycle from[START_REF] Heldt | Plant biochemistry[END_REF]

Figure 1 . 27 :

 127 Figure 1.27: General diagram showing the mechanisms involved when qE is triggered by the pH gradient across the thylakoid membrane. The numbers represent the different possible interactions between the pigments during quenching. 1 -Electronic interaction between an excited chlorophyll and the PSII reaction center for photochemistry. In light overload, this path is jamed ; 2 -Dissipation by fluorescence of the excited chlorophylls in the singlet state to return to its ground state ; 3 -Electronic interaction between neighbouring excited chlorophyll molecules ; 4 -Formation of a carotenoid radical cation leading to dissipation by charge transfer ; 5 -Electronic interaction between chlorophyll and carotenoid molecules in the singlet state

Figure 1 . 28 :

 128 Figure1.28: Current model of the transition quenching state highlighting the dissociation of LHCII antennas in the form of aggregates, part of which is reassociated on PSI. Extracted from[START_REF] Finazzi | High Light Acclimation in Green Microalgae[END_REF]

51 Figure 1 . 29 :

 51129 Figure 1.29: Schematic representation of alternative electron pathways throught PSI. In purple, the two cyclic electron transport pathways, in orange, the water-water cycle and in green, chlororespiration.

Figure 1 . 30 :

 130 Figure 1.30: Schematic representation of a typical PAM fluorescence trace

CHAPTER 2 Light

 2 use in Biotechnology Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 2.1 Light and time patterns . . . . . . . . . . . . . . . . . . . . . . 62 2.1.1 Constant light intensity . . . . . . . . . . . . . . . . . . . . . . 63 2.1.2 Intermittent light . . . . . . . . . . . . . . . . . . . . . . . . . . 66 2.1.3 Light spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 2.2 PBR designs and light investigation . . . . . . . . . . . . . . . 75 2.2.1 Conventional designs . . . . . . . . . . . . . . . . . . . . . . . . 75 2.2.2 Reduced optical path designs . . . . . . . . . . . . . . . . . . . 82 2.2.3 Operation mode . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

  of the light on the depth of the PBR coupled with the trajectory of the cells Light pattern applied to a culture Time Time Light intensity Light intensity

Figure 2 . 1 :

 21 Figure 2.1: Schematic representation of a light pattern experience by the cells within a photobioreactor. The dark blue line represents the trajectory of the cells alternating between light and dark areas. At the surface, growth is often hindered by photoinhibition while in the reactor core it is limited by the absence of light.

Chapter 2 .Figure 2 . 2 :

 222 Photosynthesis

Time

  Relatively long light loads

Figure 2 . 3 :

 23 Figure 2.3: Schematic representation of different light integration responses by the cells according to the duration of the light loads. The dotted crenellations represent the periodicity of the light alternating between on and off phases. The continuous blue line represents the dynamic biological response of the cells to the integration of light. The long red dotted line represents the average cell growth in response to perceived light.

Figure 2 . 4 :

 24 Figure 2.4: Graphical representation of relative variations in data from fluctuating studies as a function of a selected L/D cycle parameter. Light blue rounds represent data obtained with the growth rate as the monitoring parameter while dark blue squares indicate photosynthetic rate studies.

Figure 2 . 5 :

 25 Figure 2.5: Boxplot of the percentage variation of the results of the studies carried out in fluctuating light (left) and flashing light (right) with respect to their reference in continuous light (CL). The results are presented from bottom to top as follows: growth rate (GR), photosynthesis rate (P O2 ) and the combination of growth rate and photosynthesis rate (GR and P O2 ). The solid line represents the median while the dashed line is the mean. Outliers have been excluded.

Figure 2 . 6 :

 26 Figure 2.6: Graphical representation of relative variations in data from flashing studies as a function of a selected L/D cycle parameter. Light blue rounds represent data obtained with the growth rate as the monitoring parameter while dark blue squares indicate photosynthetic rate studies.

Figure 2 . 7 :

 27 Figure 2.7: Schematic representation of the spectra of different light sources. The data were exported from the Lamp Spectral Power Distribution Database (LSPDD) [159]. The corresponding references to the plotted spectra are as follows: LED-SWIVLED12-domestic-use-warmwhite-color-Dalsbrand-3.2W-12V-94Lum-3000K-50000H (LED) ; FL-T8G13-domestic-use-coolwhite-color-GE-brand-32W-4000K (Fluorescent lamp) ; and INC-A19-domestic-use-softwhite-color-Rona-brand-100W-130V-2450K (Incandescent lamp).

Figure 2 . 8 :

 28 Figure 2.8: Schematic representation of the different designs of open pond systems exported from[START_REF] Shen | Microalgae Mass Production Methods[END_REF]

Figure 2 . 9 :

 29 Figure2.9: Schematics of several types of column photobioreactors exported from[START_REF] Md | Bioreactor for Microalgal Cultivation Systems: Strategy and Development[END_REF]

Figure 2 . 10 :

 210 Figure 2.10: Schematic representation of the different classes of airlift photobioreactors exported from [187]. (A) typical internal loop, (B) inverse internal loop, (C) split plate internal loop, (D) external loop.

( a )

 a Aerial plastic bag[START_REF] Huang | Design of Photobioreactors for Mass Cultivation of Photosynthetic Organisms[END_REF] (b) Plastic bag immersed in a pool[START_REF] Fret | Implementation of flocculation and sand filtration in medium recirculation in a closed microalgae production system[END_REF]

Figure 2 . 12 :

 212 Figure 2.12: Schematic of different type of plastic bag photobioreactors

Figure 2 . 13 :

 213 Figure 2.13: Schematics of different flat panel photobioreactors from[START_REF] Md | Bioreactor for Microalgal Cultivation Systems: Strategy and Development[END_REF]

  (a) Photography (b) Schematic diagram

Figure 2 . 14 :

 214 Figure 2.14: Photography and schematic diagram of a thin-layer cascade exported from [202]. 1retention tank ; 2 -planes ; 3 -pump ; 4 -gas supply ; 5 -pH stat system ; 6 -filter

3 92 3. 3 . 1 93 3. 3 . 2 97 3. 4 97 3. 4 . 1 99 3. 5

 3923193329749741995 Lighting device description . . . . . . . . . . . . . . . . . . . . . Light characteristics . . . . . . . . . . . . . . . . . . . . . . . . Control and monitoring . . . . . . . . . . . . . . . . . . . . . . Liquid and gas utilities . . . . . . . . . . . . . . . . . . . . . . . Liquid supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3.4.2 Gas supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mode of operation . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3.5.1 Description of the turbidostat operation . . . . . . . . . . . . . 99 3.5.2 Measurement accuracy . . . . . . . . . . . . . . . . . . . . . . . 101 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Figure 3 . 1 :

 31 Figure 3.1: Photograph of the experimental device. A single chamber containing the light panel as well as three PBRs. 1-Culture device ; 2-Optical fibers ; 3-Gas and liquid injections ; 4-LEDs panel ; 5-Aluminum chamber to protect from external light.

Figure 3 . 2 :

 32 Figure 3.2: Different views of the PBR design: composition and size. 1-Microalgae culture ; 2 -Thermal control

Figure 3 . 3 :

 33 Figure 3.3: Representation of the LEDs spectrum used in this work superimposed on the main pigments spectra found in green microalgae

2 Figure 3 . 4 :

 234 Figure 3.4: Light distribution over one of the PBR in each chamber. On each light map, the silhouette of the PBR is represented in black dotted lines. The white points are the coordinates of the measurement points. On the right of the map, the spatial distribution of light as a function of the height of each chamber is shown.

Figure 3 . 5 :

 35 Figure 3.5: Spectrum light stability over time and as a function of electrical power in the chamber 1

Figure 3 . 6 :

 36 Figure 3.6: Wavelength light stability for different wavelengths over time and as a function of electrical power in the chamber 1

  The monitoring part is based on the conversion of a light signal into an optical density value (Figure3.7). Concretely, each PBR has an optical fiber at the back to collect a light signal (1 on Figure 3.7). The collected signals are transmitted to a multiplexer (Fiberoptic Multiplexer OceanOptics MPM-2000) whose function is to switch on the same transmission channel the various light signals coming from the optical fibers of all the PBR (2 on Figure 3.7).

Figure 3 . 7 :

 37 Figure 3.7: Representation of the operation and control of the experimental device. For readability reasons, only one chamber with one PBR has been represented. 1 -Optical fibers ; 2 -Multiplexer ; 3 -Spectrophotometer

Figure 3 . 8 :

 38 Figure 3.8: Representation of valve flush uncertainty as a function of growth rate

( a )

 a Average growth rate of Chlorella vulgaris according to different growth media in replicates (n=3) (b) Growth curves of Chlorella vulgaris obtained in triplicate for the B3N medium screening

Figure 4 . 1 :

 41 Figure 4.1: Screening of different media for the growth of Chlorella vulgaris

  Anionic species (b) Cationic species

Figure 4 . 2 :

 42 Figure 4.2: Graphical representation of the ionic concentration of the different species found in the B3N medium. In purple, the theoretical results calculated from the theoretical concentrations of the medium.In dark blue, the results obtained by ion chromatography after formulation of the medium. In light blue, the ionic concentrations at the end of the turbidostat culture (n=3).

Table 4 . 2 :

 42 Summary of the light conditions studied in this work. The blue values in brackets are the average intensities perceived by the microalgae for the intermittent light regimes.

Figure 4 . 3 :

 43 Figure 4.3: Schematic representation of a typical analysis using the NPQ protocol

Figure 4 . 4 :

 44 Figure 4.4: Han's model with I: light intensityσ P SII : antenna size -k d : damage probabilityτ r : repair time constant and τ : time constant for reopenning the PSII

Figure 4 . 5 :

 45 Figure 4.5: Determination of the Han model parameters by analytical expression of the different key points of the PI curve. In dark blue, the experimental curve. In dashed light blue, the identified curve.

4. 4 .

 4 Statistical testing 115

Figure 5 . 1 :

 51 Figure 5.1: Growth rate in relation to light intensity under continuous light. Each value represents mean ± SD (n = 3).

Figure 5 . 2 :

 52 Figure 5.2: Evolution of the pigmentary composition in chlorophylls according to the light intensity under continuous light. Each value represents mean ± SD (n = 3).

Figure 5 . 3 :

 53 Figure 5.3: Evolution of the chlorophylls ratio according to the light intensity under continuous light. Each value represents mean ± SD (n = 3).

5. 2 . Pigment profile 123 Figure 5 . 4 :

 212354 Figure 5.4: Evolution of the pigmentary composition in carotenoids according to the light intensity under continuous light. Each value represents mean ± SD (n = 3).

Figure 5 . 5 :

 55 Figure 5.5: Evolution of the carotenoids ratio according to the light intensity under continuous light. Each value represents mean ± SD (n = 3).

Figure 5 . 6 :

 56 Figure 5.6: Evolution of the pigmentary composition in VAZ cycle pigments according to the light intensity under continuous light. Each value represents mean ± SD (n = 3).

Figure 5 . 7 :

 57 Figure 5.7: Chromatograms obtained by HPLC analysis of two samples. Low Light (LL): 80 µmolPhotonPAR/m 2 /s and High Light (HL): 800 µmolPhotonPAR/m 2 /s.

Figure 5 . 8 :

 58 Figure 5.8: Evolution of the Fv/Fm ratio and NPQ according to the light intensity under continuous light. Each value represents mean ± SD (n = 3). These results were obtained by aquapen using the NPQ protocol. Saturating pulse: 1500 µmolPhotonPAR/m 2 /s, measuring pulse: 900 µmolPhotonPAR/m 2 /s, actinic light: 300 µmolPhotonPAR/m 2 /s.

(a) 80 Figure 5 . 9 :

 8059 Figure 5.9: Results of a light response curve obtained by Aquapen at two different intensities.

Figure 5 . 10 :

 510 Figure 5.10: Evolution of the calculated parameters extracted from the light curve according to the light intensity under continuous light. Each value represents mean ± SD (n = 3). These results were obtained by the use of the aquapen. Saturating pulse: 1500 µmolPhotonPAR/m 2 /s, actinic pulse: 300 µmolPhotonPAR/m 2 /s.

Figure 5 . 11 :

 511 Figure 5.11: Evolution of the antenna size according to the light intensity under continuous light. Each value represents mean ± SD (n = 3).

Figure 6 . 1 :

 61 Figure 6.1: Growth rate in relation to light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3).

Figure 6 .

 6 Figure 6.1 depicts the results of growth rates for each intermittent light regime. The light blue line shows the results obtained in continuous light for recall. The points in the intermittent light regime are displayed according to the average light perceived by the culture. This makes it possible to compare, for the same amount of light energy supplied to the culture, continuous and intermittent light.

Figure 6 .

 6 Figure 6.2 displays the absolute concentrations of total chlorophylls as well as the respective concentrations of chlorophylls a and b for each intermittent light regime. The continuous light reference is also plotted to ease the comparison.

Figure 6 . 2 :

 62 Figure 6.2: Evolution of the pigmentary composition in chlorophylls according to the light intensity under intermittent light. Each value represents mean ± SD (n = 3).

Figure 6 . 3 :

 63 Figure 6.3: Evolution of the chlorophylls pigments ratio according to the light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3).

Figure 6 . 4 :

 64 Figure 6.4: Evolution of the pigmentary composition in carotenoids and its ration according to the light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3).

Figure 6 . 5 :

 65 Figure 6.5: Evolution of the lutein content according to the light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3).

Figure 6 . 6 :

 66 Figure 6.6: Evolution of the pigmentary composition in VAZ cycle pigments according to the light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3).

Figure 6 .

 6 Figure 6.7 presents the Fv/Fm and NPQ parameters for all intermittent light regimes as a function of cultivation light intensity.

Figure 6 . 7 :

 67 Figure 6.7: Evolution of the Fv/Fm ratio and NPQ according to the light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3). These results were obtained by aquapen using the NPQ protocol. Saturating pulse: 1500 µmolPhotonPAR/m 2 /s, measuring pulse: 900 µmolPhotonPAR/m 2 /s, actinic light: 300 µmolPhotonPAR/m 2 /s.

Figure 6 . 8 :

 68 Figure 6.8: Evolution of the calculated parameters extracted from the light curve according to the light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3). These results were obtained by the use of the aquapen. Saturating pulse: 1500 µmolPhotonPAR/m 2 /s, actinic pulse: 300 µmolPhotonPAR/m 2 /s.

Figure 6 . 9 :

 69 Figure 6.9: Evolution of the calculated parameters extracted from the light curve according to the light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3). These results were obtained by the use of the aquapen. Saturating pulse: 1500 µmolPhotonPAR/m 2 /s, actinic pulse: 300 µmolPhotonPAR/m 2 /s.

Figure 6 . 10 :

 610 Figure 6.10: Evolution of the antenna size according to the light intensity under intermittent light. The reference in continuous light is shown in light blue. Each value represents mean ± SD (n = 3).

Figure A. 1 :

 1 Figure A.1: Overall structure of a PSII-LHCII supercomplex from Chlamydomonas reinhardtii with a 3.37 Å resolution. A -Top view from the stromal side ; B -Side view along the membrane plane ; C -The arrangement of 13 small membrane intrinsic proteins in each monomer viewed from the stromal side.Exported from[START_REF] Shen | Structure of a C2S2M2N2-type PSII-LHCII supercomplex from the green alga Chlamydomonas reinhardtii[END_REF]

1. 1 209 2. 2

 12092 Micrographs of some microalgae of different phyla showing the diversity in shape, size, and color of microalgae. Images extracted from UTEX Culture Collection of Algae at The University of Texas at Austin [9]. A-Micrasterias hardeyii ; B-Volvox carteri ; C-Arthrospira platensis ; D-Colacium mucronatum ; E-Flintiella sanguinaria ; F-Glaucocystis nostochinearum. . 1.2 Major microalgae phyla distribution as per seven-kingdom classification scheme with major phyla distribution . . . . . . . . . . . . . . . . . . . . . 1.3 Diagram showing the production chain and valorization of microalgae from culture to applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Schematic illustration of the cell organization of Chlorella vulgaris. Microscope observation from our lab, magnification x100 (image manipulation: contrast enhancement). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Spectrum of the electromagnetic radiation . . . . . . . . . . . . . . . . . . 1.6 Chemical structures and absorption spectra of the five existing chlorophyll types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 Chemical structures and absorption spectra of the most common carotenoids found in microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8 Schematic representation of a chloroplast structure inspired from [56] . . . 1.9 Schematic representation of the distribution of protein complexes in the thylakoid membrane from [57] . . . . . . . . . . . . . . . . . . . . . . . . . 1.10 Schematic representation of a simplified structure of the PSII-LHCII supercomplex inspired from [44, 60] . . . . . . . . . . . . . . . . . . . . . . . . . 1.11 PSI-LHCI supercomplex in green microalgae inspired from [67, 68] . . . . . 1.12 Schematic representation of the distribution of LHCII and LHCI in relation with the PS core inspired from [67, 68] . . . . . . . . . . . . . . . . . . . . 1.13 Schematic representation of the structure of the cytochrome b 6 /f complex inspired from [44]. For the sake of simplicity, only one copy of each subunits is presented. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.14 Schematic representation of the structure of the ATPase inspired from [44] 1.15 Z scheme summarizing the reactions taking place during the light phase of photosynthesis inspired from [44] . . . . . . . . . . . . . . . . . . . . . . . Schematic representation of excitation sates of chlorophyll a and their return to ground state inspired from [44]. The different levels of rotational and vibration energy of electrons are represented by thin lines and the ground states by solid lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.17 Trapping light and routing it to reaction centers placed energetically downhill 1.18 Schematic representation of the chemical transformation of the light energy through PSII inspired from [47] . . . . . . . . . . . . . . . . . . . . . . . . 1.19 Schematic representation of the Q cycle . . . . . . . . . . . . . . . . . . . . 1.20 Schematic representation of the electron flow through PSI insprired from [44] 1.21 Schematic representation of ATP regeneration through subunit conformational changes insprired from [44] . . . . . . . . . . . . . . . . . . . . . . . 1.22 General diagram summarising the main reactions taking place during light reactions of photosynthesis insprired from [78] . . . . . . . . . . . . . . . . 1.23 Schematic representation of the Calvin-Benson cycle's reaction taking place in the stroma of the chloroplast . . . . . . . . . . . . . . . . . . . . . . . . 1.24 Schematic representation of photorespiration pathway . . . . . . . . . . . . 1.25 Schematic representation of the mechanisms involved during in the appearance of photodamages at the PSII level. The three main mechanisms are depicted with different colours: blue for the mechanism involving the donor side of the photochemistry, black for the mechanism involving the acceptor side and green for the triplet state chlorophyll. ROS formation appears in red. The compounds responsible for the degradation of protein D1 are shown in orange. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.26 Schematic representation of the VAZ cycle from [44] . . . . . . . . . . . . . 1.27 General diagram showing the mechanisms involved when qE is triggered by the pH gradient across the thylakoid membrane. The numbers represent the different possible interactions between the pigments during quenching. 1 -Electronic interaction between an excited chlorophyll and the PSII reaction center for photochemistry. In light overload, this path is jamed ; 2 -Dissipation by fluorescence of the excited chlorophylls in the singlet state to return to its ground state ; 3 -Electronic interaction between neighbouring excited chlorophyll molecules ; 4 -Formation of a carotenoid radical cation leading to dissipation by charge transfer ; 5 -Electronic interaction between chlorophyll and carotenoid molecules in the singlet state . . . . . . . . . . 1.28 Current model of the transition quenching state highlighting the dissociation of LHCII antennas in the form of aggregates, part of which is reassociated on PSI. Extracted from [108] . . . . . . . . . . . . . . . . . . . . . . . . . . 1.29 Schematic representation of alternative electron pathways throught PSI. In purple, the two cyclic electron transport pathways, in orange, the waterwater cycle and in green, chlororespiration. . . . . . . . . . . . . . . . . . 1.30 Schematic representation of a typical PAM fluorescence trace . . . . . . . . 2.1 Schematic representation of a light pattern experience by the cells within a photobioreactor. The dark blue line represents the trajectory of the cells alternating between light and dark areas. At the surface, growth is often hindered by photoinhibition while in the reactor core it is limited by the absence of light. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Figures Schematic representation of light-response curve of photosynthesis inspired from [141]. α is the maximum photosynthetic efficiency ; P max , the maximum photosynthetic rate ; R d , the dark respiration rate ; I c , the light compensation point ; I k , the transition light intensity from light limited to light saturated photosynthesis ; and I i , the light intensity above which photoinhibition occurs. The open arrows indicate the high light acclimated response direction and the filled arrows the low light acclimated direction. . 2.3 Schematic representation of different light integration responses by the cells according to the duration of the light loads. The dotted crenellations represent the periodicity of the light alternating between on and off phases. The continuous blue line represents the dynamic biological response of the cells to the integration of light. The long red dotted line represents the average cell growth in response to perceived light. . . . . . . . . . . . . . . 2.4 Graphical representation of relative variations in data from fluctuating studies as a function of a selected L/D cycle parameter. Light blue rounds represent data obtained with the growth rate as the monitoring parameter while dark blue squares indicate photosynthetic rate studies. . . . . . . . . 2.5 Boxplot of the percentage variation of the results of the studies carried out in fluctuating light (left) and flashing light (right) with respect to their reference in continuous light (CL). The results are presented from bottom to top as follows: growth rate (GR), photosynthesis rate (P O 2 ) and the combination of growth rate and photosynthesis rate (GR and P O 2 ). The solid line represents the median while the dashed line is the mean. Outliers have been excluded. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Graphical representation of relative variations in data from flashing studies as a function of a selected L/D cycle parameter. Light blue rounds represent data obtained with the growth rate as the monitoring parameter while dark blue squares indicate photosynthetic rate studies. . . . . . . . . . . . . . . 2.7 Schematic representation of the spectra of different light sources. The data were exported from the Lamp Spectral Power Distribution Database (LSPDD) [159]. The corresponding references to the plotted spectra are as follows: LED-SWIVLED12-domestic-use-warmwhite-color-Dalsbrand-3.2W-12V-94Lum-3000K-50000H (LED) ; FL-T8G13-domestic-use-coolwhitecolor-GE-brand-32W-4000K (Fluorescent lamp) ; and INC-A19-domesticuse-softwhite-color-Rona-brand-100W-130V-2450K (Incandescent lamp). . 2.8 Schematic representation of the different designs of open pond systems exported from [169] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 Schematics of several types of column photobioreactors exported from [186] 2.10 Schematic representation of the different classes of airlift photobioreactors exported from [187]. (A) typical internal loop, (B) inverse internal loop, (C) split plate internal loop, (D) external loop. . . . . . . . . . . . . . . . . 2.11 Schematics of several configurations of tubular photobioreactors . . . . . . 2.12 Schematic of different type of plastic bag photobioreactors . . . . . . . . . 2.13 Schematics of different flat panel photobioreactors from [186] . . . . . . . . 2.14 Photography and schematic diagram of a thin-layer cascade exported from [202]. 1 -retention tank ; 2 -planes ; 3 -pump ; 4 -gas supply ; 5 -pH stat system ; 6 -filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 1 .

 1 1: The energy content and the electrochemical potential of photons of different wavelengths

Light color Wavelength (nm) Energy content (kJ/ mol photons) Electrical potential (eV)

	Red	700	171	1.77
	Bright red	650	184	1.91
	Orange	620	193	2.00
	Yellow	600	199	2.07
	Green	560	214	2.21
	Blue green	500	239	2.48
	Blue	440	272	2.82
	Violet	400	299	3.10

Table 1 . 2 :

 12 Absorption peaks and the occurrence of different types of chlorophylls, data from[START_REF] Edward | Phycology[END_REF][START_REF] Chen | A red-shifted chlorophyll[END_REF]

		Absorption peaks (nm)	Occurrence
	Chlorophyll a	430 and 663	Universal
	Chlorophyll b	465 and 645	Mainly Euglenophyta and Chlorophyta
	Chlorophyll c1	440, 583 and 634	Mainly Heterokontophyta
	Chlorophyll c2	452, 586 and 635	Mainly Cryptista and Heterokontophyta
	Chlorophyll d	400, 456 and 696	Some Cyanobacteria
	Chlorophyll f	406 and 706	Some Cyanobacteria

PC 2+ 2 PC + LUMEN STROMA 4 photons P700 A0 Fx FA, FB 2 NADP + + 2H + 2 NADPH A1 QB CP 47 2 H + 2 Q Q QH2 2 QH 2 2 H 2O O2 + P680 QA Pheo 4 Mn Tyr 4 photons 2 H + 14 H + 14 H + FD FNR 4 H + 3 ADP + Pi 3 ATP 1 -2 ms 3 -5 ms 150 -550 µs 4 H +

  Light reactions generate essential energy intermediates for the incorporation of CO 2 during carbohydrate synthesis. The production of carbohydrates is essential since it serves two

		Chapter 1. Background
	CP	CP
	43	47
	2 Figure 1.22: General diagram summarising the main reactions taking place during light reactions of
	photosynthesis insprired from [78]

  •- 2 by demutualizing it into O 2 and H 2 O 2 thus preventing the possible formation of OH • . CAT is the enzyme responsible for the catalysis of H 2 O 2 into H 2 O and O 2 . APX fulfills essentially the same role as CAT in reducing H 2 O 2 . However, the end products are not the same. In this case, H 2 O 2 is reduced to H 2 O and DHA, using ascorbic acid as the reducing agent.

Table 4 . 1 :

 41 Composition of the B3N medium

	Component	Concentration (M)
	Macronutrients
	NaNO 3	8.82 x 10 -3
	CaCl 2 . 2H 2 O	1.70 x 10 -4
	MgSO 4 . 7H 2 O	3.04 x 10 -4
	K 2 HPO 4	4.31 x 10 -4
	KH 2 PO 4	1.29 x 10 -3
	NaCl	4.28 x 10 -4
	Alkaline EDTA Solution
	EDTA	1.71 x 10 -4
	KOH	5.53 x 10 -4
	Acidified Iron Solution
	FeSO 4 . 7H 2 O	1.79 x 10 -5
	H 2 SO 4	1.87 x 10 -5
	Boron Solution
	H 3 BO 3	1.85 x 10 -4
	Trace Metals Solution
	ZnSO 4 . 7H 2 O	3.07 x 10 -5
	MnCl 2 . 4H 2 O	7.28 x 10 -6
	MoO 3	4.93 x 10 -6
	CuSO 4 . 5H 2 O	6.29 x 10 -6
	Co(NO 3 ) 2 . 6H 2 O	1.68 x 10 -6

Table 4 . 3 :

 43 Standards concentration range and retention time for pigment quantification by HPLC

	Standards	Chlorophyll a Chlorophyll b	Lutein	Violaxanthin	Zeaxanthin
	Concentration range (mg/L)	0.2038 -4.076	0.4726 -18.905 0.0421 -0.842 0.0224 -0.559 0.0185 -0.4625
	Retention time (min)	15.9	21.3	11.3	8.5	11.8

Table B .1 continued from previous page

 B TableB.2: All data collected from studies conducted in fluctuating light with the photosynthesis rate (P O2 ) as the output variable. The table lists the study microorganism, the experimental device used to adapt the culture and measure the P O2 , the parameters of the L/D cycles as well as the experimental results with their coefficient of variation if known (N.A. if not available). The average light intensity (I avg ) is expressed in µmolE/m /s).The photosynthesis rate presented is weighted by the quantity of light. The reference to continuous light appears as CL. (a) Oxygen evolution rate in gO 2 /g/h ; (b) Oxygen evolution rate in µmolO 2 /g/s ; (c) Oxygen evolution rate in mgO 2 /g/h ; (d) Oxygen evolution rate in molO 2 /g/s and (e) Normalized oxygen evolution rate: fraction of the oxygen evolution rate in flashing light on the continuous one.

		Table B.1 continued from previous page Table B.1 continued from previous page
		Cultivation mode: turbidostat (set point: 70% of the maxi-	187 345	3.53 3	0.85 0.8	0.031 0.048		N.A. <10 %	-24 -62	[155]
	Chlamydomonas reinhardtii CC 1690 wild type 21 gr mt + Chlamydomonas reinhardtii CC 1690 wild type 21 gr mt + Chlamydomonas reinhardtii wild type strain coded 21 gr Chlorella sp. Dunaliella 11_H5 Chlamydomonas reinhardtii WT strain CC125 Chlamydomonas reinhardtii Studied microalga Chlamydomonas reinhardtii Chlamydomonas reinhardtii CC-124 Chlorella vulgaris SAG 12A CC 1690 wild type 21 gr mt + Chlamydomonas wild type mt-137c Chlorella sorokiniana (CCAP 211/8k) Chlorella vulgaris reinhardtii coded 137 AH Dunaliella salina CCAP 18/19 Chlamydomonas reinhardtii CC-124 wild type mt-137c	PBR design: rectangular PBR (70 mL working volume) Optical light path: 3 cm Light source: halogen lamp Illumination protocol: culture illuminated with a 16/8 h day-night cycle. During the 16 h period, the cells are exposed to different L/D cycles Cultivation mode: batch (final OD 680nm <0.25) Measurement: OD difference after 24 h for 2 or 3 days (after 3 days of acclimation in a turbidostat) PBR design: rectangular PBR (525 mL working volume) Optical light path: 1.45 cm Light source: halogen lamp Illumination protocol: culture illuminated with a 16/8 h day-night cycle. During the 16 h period, the cells are exposed to different L/D cycles Cultivation mode: semi-continuous (0.15 <OD 680nm <0.30) 773 650 325 325 325 520 1153 Measurement: OD difference for 24 h after 2 to 3 days of cultivation 397 PBR design: glass air-lift loop PBR (0.6 L working volume) 240 Light source: fluorescent light tubes Illumination protocol: PBR placed in a closed cabinet. The dark period obtained with a part of the PBR covered with 200 Cultivation mode: semi-continuous (OD 750nm <0.1) 345 PBR design: rectangular PBR (1 L working volume) 660 aluminum foil mal flux without algae) Measurement: dilution rate for 66 days 158 PBR design: microwell plate (150 µL working volume) 172 Optical light path: 5 mm 172 Light source: white LEDs 172 Illumination protocol: cells gradually acclimated to light intensity enclosed in a chamber 172 Cultivation mode: semi-continuous (OD 750nm <0.1) 345 Measurement: 3-hourly OD measurements 345 345 345 689 689 PBR design: flat panel PBR (200 mL working volume) 100 345 Light source: LEDs 100 345 Illumination protocol: culture enclosed in a black box 100 Measurement: 3-hourly OD measurements 689 Cultivation mode: batch mode with cell concentration al-100 689 689 Subculturing Monitoring device I avg ways lower than 1.2 g/L at the end of the culture Measurement: OD difference using a correlation with dry weight 200 689 200 PBR design: flasks (250 mL working volume) 215 PBR design: rectangular PBR Oxygen monitor set-up: 650 200 Light source: white LEDs 215 (70 mL working volume) small cylindrical stirred 200 PBR design: flat panel PBR (360 mL working volume) 100 Illumination protocol: culture enclosed in a black box 215 vial Cultivation mode: batch (final cell concentration <1 g/L) 215 Measurement: dry weight after 1 week of 215 Optical light path: 3 cm Light source: halogen 325 lamp Optical light path: 25 mm Light source: blue-red LEDs (450 and 630 nm) cultivation 215 Light source: halogen lamp Protocol: 10 min of 325 689 689 PBR design: torus PBR considered as flat panel (1.5 L working volume) 110 Optical light path: 4 cm 110 Light source: white LEDs 110 Cultivation mode: turbidostat (full light absorption and no 110 Cultivation mode: turbidostat (light transmission kept at 60 %) Measurement: biomass concentration difference for 6 h intervals after 72 h of steady state 100 PBR design: glass bubble PBR (380 mL working volume) 630 Light source: halogen lamp 473 PBR design: column PBR (30 mL working volume) 1000 Optical light path: 1.8 cm Light source: red LEDs (645 nm) 1000 Cultivation mode: batch (culture diluted <20 µM chl a) 1000 Measurement: dry weight increase in the exponential phase 1000 dark adaptation, Illumination protocol: culture il-325 then the sample is luminated with a 16/8 h day-exposed for 20 min night cycle. During the 16 h pe-to increasing light riod, the cells are exposed to dif-intensities ferent L/D cycles for 4 h Cultivation mode: turbidostat (set point : 70% of the maximal flux without algae) for 1 or 2 weeks 58 PBR design: cylindrical PBR (500 mL working volume) 400 Cultivation mode: turbidostat 520 Optical light path: 7.7 cm 400 (0.17 <OD680nm <0.25) dark zone) Measurement: dilution rate 110 110 110 110 110 Measurement: dilution rate over 3 -5 days 44 PBR design: microwell plate (150 µL working volume) 172 Optical light path: 5 mm 172 Light source: white LEDs 400 PBR design: flat PBR (375 mL Oxygen monitor set-up: 58 Illumination protocol: PBR enclosed in a chamber 400 working volume) consists of 3 chambers (2 Cultivation mode: turbidostat (2x10 5 culture <3x10 5 cell-400 water jackets and 1 mea-Light source: white LEDs 172 Illumination protocol: cells gradually acclimated to light intensity enclosed in a chamber 172 s/mL) Measurement: cell count difference at 24 h 400 surement chamber at the middle) intervals in the exponential phase 400 Optical light path: 25 mm Optical light path: 15 mm 58	CL 6.1 14.5 24.3 15.2 CL 15 15 CL CL CL CL 12.9 CL 3 10 20 CL 3 10 20 CL 3 CL 10 0.2 20 2 CL 20 3 10 τ c CL 20 (ms) 0.2 CL CL 2 1 20 CL 2 4 120 6.1 240 14.5 10 20 CL 1 2 6 0.2 CL 40 CL 1.2 1.4 2 24.3 CL CL 15.2 0.2 12 40 120 360 720 40 CL 3 4 CL 30 60 10 20 3 5 0.2	1 0.5 0.5 0.5 0.8 1 0.67 0.33 1 1 1 1 0.66 1 0.8 0.8 0.8 1 0.8 0.8 0.8 1 0.8 1 0.8 0.5 0.8 0.5 1 0.5 0.8 0.8 ε (-) 1 0.8 0.5 1 1 0.5 0.5 0.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0.8 0.8 1 0.5 0.5 0.5 0.1 1 0.75 1 0.5 0.5 0.5 0.5 1 1 0.8 0.5 0.5 0.5 0.5 0.5 0.5 0.75 1 0.8 0.5 1 0.5 0.5 0.8 0.8 0.67 0.4 0.05	0.116 0.090 0.090 0.067 0.123 0.110 0.127 0.100 0.160 0.041 0.128 0.0440 0.167 0.092 0.073 0.061 0.066 0.067 0.097 0.078 0.066 0.112 0.126 0.073 0.067 0.042 0.094 0.068 0.027 0.057 0.052 0.062 P O2 0.095 0.117 0.074 0.062 0.943 0.076 0.070 0.071 0.072 0.072 0.060 0.052 1.284 0.048 1.270 0.166 0.122 0.032 0.011 0.011 0.011 0.040 0.270 0.240 0.040 0.046 0.036 0.030 1.322 0.077 0.0429 1.078 0.0388 0.011 0.008 0.008 0.009 0.009 0.080 0.074 0.084 0.0304 0.31 0.0183 0.0104 0.088 0.093 0.0325 0.0271 0.22	-1 )	Exp. CV (%) N.A. N.A. N.A. N.A. N.A. <10 % <10 % <10 % >10 % N.A. <10 % <10 % >10 % <10 % <10 % <10 % <10 % <10 % <10 % <10 % <10 % >10 % <10 % N.A. <10 % N.A. <10 % N.A. <10 % N.A. <10 % <10 % Exp. N.A. >10 % CV (%) N.A. <10 % <10 % N.A. <10 % N.A. 10 % <10 % <10 % 10 % <10 % 10 % <10 % >10 % <10 % N.A. N.A. N.A. N.A. >10 % N.A. N.A. N.A. N.A. N.A. N.A. <10 % N.A. <10 % <10 % <10 % N.A. N.A. N.A. N.A. N.A. N.A. >10 % >10 % <10 % 10 % <10 % >10 % >10 % >10 % <10 % <10 % <10 %	η (%) Ref. -[138] -22 -22 -42 6 -[236] 15 -9 -[154] -[245] [237] --4.3 -[237] -21 -34 -28 -44 17 -2 --48 -[239] -26 -43 --7 91 -22 η (%) Ref. 127 327 ----22 13 -20 -25 -[238] 16 [138] (a) [246] -3 -16 36 -23 35 13 49 9 -[245] -66 -66 -66 -44 -[154] -[235] 15 -10 -25 40 -11 -14 --10 [155] -66 -74 -74 -73 -73 4 --29 -[241] -57 -76 13 18 -24 [247] (b) 26 -37 -29
	tertiolecta CCAP								[236]
	19/6B

Table B .2 continued from previous page

 B TableC.1: All data collected from studies conducted in flashing light with the growth rate (GR) as the output variable. The table lists the study microorganism, the experimental device used to measure the GR, the parameters of the L/D cycles as well as the experimental results with their coefficient of variation if known (N.A. if not available). The average light intensity (I avg ) is expressed in µmolE/m /s). The growth rate presented is weighted by the quantity of light. The reference to continuous light appears as CL

				594	12.9		0.66	409	N.A.	-9
		PBR design: bubble column PBR	Oxygen monitor set-up:	63	CL		1	4.617E-07	N.A.	-
	Studied microalga	(1.8 L working volume) Experimental device transparent glass tank	I avg	Freq.	ε		GR	Exp.	η (%) Ref.
		Optical light path: 8 cm	Optical light path: 1 cm	63	10 (Hz)		0.05	2.142E-06 (h -1 )	N.A. CV (%)	-77
	Chlamydomonas	Light source: fluorescent light tubes PBR design: flat PBR (360 mL working volume) Light source: white LEDs 63 100 Optical light path: 25 mm	1 CL	1	0.05	6.290E-06 0.072	N.A. 10 %	-	-32
	reinhardtii CC-124 wild type mt-137c	Light source: blue-red LEDs (450 and 630 nm)	67.8 100	CL 10	1 0.1	4.963E-07 0.059	N.A. 10 %	--18	[238]
		Cultivation mode: turbidostat (light transmission kept at	67.8 100	10 50	0.1 0.1	2.052E-06 0.062	N.A. <10 %	-59 -14
		60 %)		101.05 CL		1	7.324E-07	N.A.	-
		intervals after 72 h of steady state Measurement: biomass concentration difference for 6 h	101.05 1 100 100	0.05 0.1	7.150E-06 0.082	N.A. <10 %	-51 14
		PBR design: horizontal tubular PBR	101.05 1 120 CL	1	0.05	6.242E-06 0.006	N.A. N.A.	-	-57
	Scenedesmus almeriensis CCAP 276/24 Chlorella pyrenoidosa	Optical light path: 5 mm ing Light source: fluorescent lamps Illumination protocol: black paint periodically shaded tub-	126 60 135.6 126 126 180 90	CL 100 CL 10 1 CL 100	1 0.5 1 0.1 0.1 1 0.5	9.048E-07 0.013 9.697E-07 1.782E-06 3.082E-06 0.008 0.018	N.A. N.A. N.A. N.A. N.A. N.A. N.A.	-111 --80 -66 -118	[242] (d) [196]
		Cultivation mode: batch (final dry weight <1 g/L) Protocol: cell Cultivation mode: semi-continuous (C=1.8 g/L) concentration of 0.1 Measurement: dry weight g/L variation within a period	135.6 240 135.6 120 202.1 300	10 CL 1 100 CL CL	0.2 0.2 0.5 1 1 1	1.723E-06 0.010 3.435E-06 0.021 1.386E-06 0.009	N.A. N.A. N.A. N.A. N.A. N.A.	-65 -29 117 ---
				202.1 150	10 100	0.1 0.5	2.262E-06 0.020	N.A. N.A.	-84 125
		PBR design: column PBR (30 mL working volume)	202.1 1000	1 CL	1	0.1	4.675E-06 0.040	N.A. N.A.	-	-66
	Chlorella vulgaris	Optical light path: 1.8 cm		252 1000	CL 12.5	1 0.5	1.640E-06 0.044	N.A. N.A.	-10	[235]
		Light source: red LEDs (654 nm)		252 1000	10 16.7	0.2 0.5	1.801E-06 0.041	N.A. N.A.	3	-78

Table C .1 continued from previous page

 C

		Cultivation mode: batch (culture diluted <20 µM chl a)	1000	25	0.5	0.040	N.A.	0
		Measurement: dry weight increase in the exponential phase	1000	33.3	0.5	0.040	N.A.	0
		for 4 h
	Scenedesmus obliquus SAG 276.7	PBR design: flat PBR (150 mL working volume) Optical light path: 1.2 cm Light source: LEDs (CL under fluorescent lamp)	150 150	CL 10	1 0.1	0.036 0.014	>10 % >10 %	--61	[240]
		Cultivation mode: batch (initial OD750nm = 0.2)	150	15	0.15	0.015	>10 %	-58
		Measurement: exponential phase	150	10	0.1	0.023	>10 %	-36
		PBR design: column PBR (30 mL working volume)	1000	CL	1	0.041	N.A.	-
		Optical light path: 1.8 cm	1000	25000	0.5	0.041	N.A.	0
	Scenedesmus quadricauda	Light source: red LEDs (654 nm)	1000	10000	0.5	0.044	N.A.	7	[235]
		Cultivation mode: batch (culture diluted <20 µM chl a)	1000	7143	0.5	0.046	N.A.	12
		Measurement: dry weight	1000	33	0.5	0.046	N.A.	12
		increase in the exponential phase for 4 h	1000	25	0.5	0.051	N.A.	24
			1000	12.5	0.5	0.047	N.A.	15

Table C .2 continued from previous page

 C

				67.8	CL	1	4.963E-07	N.A.	-
				67.8	10	0.1	6.227E-07	N.A.	26
				67.8	20	0.1	6.788E-07	N.A.	37
				67.8	50	0.1	7.197E-07	N.A.	45
				101.05 CL	1	7.356E-07	N.A.	-
				101.05 10	0.05	5.130E-07	N.A.	-30
				101.05 50	0.05	8.844E-07	N.A.	20
				126	CL	1	9.048E-07	N.A.	-
	Scenedesmus almeriensis CCAP	Cultivation mode: semi-continuous (C=1.8 g/L)	Protocol: cell concentration of 0.1 g/L	126 135.6	20 CL	0.1 1	9.572E-07 9.697E-07	N.A. N.A.	6 -
	276/24			135.6	10	0.2	9.904E-07	N.A.	2
				135.6	50	0.2	1.265E-06	N.A.	31
				202.1	CL	1	1.386E-06	N.A.	-
				202.1	10	0.1	2.220E-06	N.A.	60
				202.1	50	0.1	1.487E-06	N.A.	7
				252	CL	1	1.640E-06	N.A.	-
				252	10	0.2	1.376E-06	N.A.	-16
				252	50	0.2	1.679E-06	N.A.	2
				339	CL	1	1.911E-06	N.A.	-
				339	10	0.5	1.422E-06	N.A.	-26
				339	50	0.5	1.796E-06	N.A.	-6
				404.2	CL	1	2.007E-06	N.A.	-
				404.2	10	0.2	1.263E-06	N.A.	-37
				404.2	20	0.2	1.355E-06	N.A.	-33

Table C .2 continued from previous page

 C

	630	CL	1	2.119E-06	N.A.	-
	630	10	0.5	2.091E-06	N.A.

In this manuscript, the cellular structure as well as the mechanism of photosynthesis are focused on microalgae belonging to the phylum Chlorophyta. In other phyla, discrepancies in cellular composition (organelles, their number and spatial arrangement) are reported.

Acknowledgments

I would like to thank my arms for always being by my side. My legs for always supporting me and finally my fingers, because I could always count on them.

Chapter 3. Development and characterization of the experimental device • a lighting device with flashing capabilities, ensuring a homogeneous lighting, stable in time and insensitive to the applied electric power,

• an aluminum chamber containing the first two devices in order to protect them from external light.

This device, combined with a semi-automated instrumentation, allows continuous monitoring in turbidostat mode. Such a system serves two functions. On the one hand, the culture is maintained at a low cell density ensuring that light is delivered homogeneously to the whole culture. On the other hand, it keeps all abiotic factors (temperature, pH, nutrients concentration, etc.) fixed for the culture duration. In this way, the light remains the only parameter that can influence microalgae's cellular response from one culture to another. All these precautions are essential for the production of high quality biological data.

APPENDIX B

Fluctuating light studies 159

where:

• V is the volume that has been flushed over time ∆t,

• ∆t the time interval of the growth rate measurement,

• and V P BR the PBR working volume.

To evaluate the measurement uncertainty associated with the experimental setup, the uncertainty of each term of the equation must be determined:

Measurements are performed on a 24-hour basis. The total flush volume over this period is weighted. Keeping in mind that a lab balance has a typically relative accuracy of 10 -6 and the density is very close to 1000kg.m -3 , the relative accuracy of ∆V /V is better than 1 %. The volume of the PBR (V P BR ) is known with a good certainty: its error is estimated at 1 %.

Finally, one has to evaluate the accuracy of the term ∆t/t of equation D.1. The problem is that, even though the time interval between two measurements of V can be measured with excellent accuracy (some seconds over 25 hours, hence of the order of 10 -4 ), flushes occur as discrete pulses, based on the OD evolution. The actual time interval is therefore known with an uncertainty equal to the averaged time interval between two successive flushes ∆t f lush :

Finally, the relative uncertainty of the growth rate can be expressed as:

∆t f lush depends on the growth rate. In order to evaluate its value, we assume an exponential growth between two flushes. Figure D.1 depicts the evolution of the optical density, as a proxy of cell concentration, between two flushes (over ∆t f lush ).

Thanks to the previous assumption, we can express the growth dynamics of the microalgae in the device to determine ∆t f lush :

From this expression, one can deduce:

By replacing this expression in Equation D.4, one can deduce the final expression of the relative uncertainty associated with the experimental setup:

Consistently, the error decreases with the growth rate.

APPENDIX E French extended abstract

Introduction L e changement climatique ainsi que l'épuisement des ressources fossiles, poussent l'humanité à trouver des alternatives aux commodités pétro-sourcées. Les microalgues photosynthétiques émergent comme une des solutions. En effet, leur production peut répondre à de larges besoins (alimentaires, cosmétiques, pharmacologiques) et s'accompagne de bénéfices environnementaux importants (séquestration du CO 2 , assainissement de l'eau, biocarburant). De plus, leur culture peut être menée sur des terres non-arables, préservant ainsi les cultures vivrières. Cependant, la production de microalgues photosynthétiques n'est, à ce jour, pas économiquement viable. Elle est limitée par la ressource lumineuse. En plus de la quantité totale, la façon dont cette lumière est délivrée (intensité, temporalité, spectre) est considérée comme ayant un impact sur les performances de la culture. Le comprendre pour le manipuler pourrait permettre d'augmenter les rendements de culture.

Etat de l'art

Ce travail est parti du constat que la lumière est l'élément clé pour améliorer la viabilité économique des bioprocédés utilisant des microalgues. En effet, la combinaison de l'absorption de la lumière par les organismes et de l'agitation impose des cycles lumineux intermittents qui affectent la croissance des microalgues. Bien que l'intégration de la lumière par les organismes photosynthétiques soit connue de la communauté scientifique, son exploitation biotechnologique est encore limitée.

La collecte d'informations dans la littérature scientifique a révélé deux choses :

• La photosynthèse est un processus complexe, finement régulé par la lumière, qui dispose de mécanismes de régulation à court et à long terme pour se protéger et s'adapter aux fluctuations lumineuses qui peuvent survenir dans leur environnement.

Pour aller plus loin et amplifier les quelques différences observées dans la réponse entre éclairage continu et intermittent, il serait nécessaire d'obtenir une plus grande puissance statistique en augmentant le nombre de réplicats. De plus, différents paramètres devraient être étudiés. Trois pistes peuvent être explorées. Premièrement, la gamme de fréquences pourrait être étendue. Deuxièmement, différents duty cycles pourraient être évalués pour étudier la réponse lorsque la phase claire est plus longue ou plus courte que la phase sombre. Troisièmement, nous pourrions faire évoluer le dispositif expérimental actuel pour exposer les cultures à des intensités encore plus extrêmes jusqu'à l'apparition d'une phase de photoinhibition. Enfin, pendant la phase d'acclimatation, les cultures soumises à des intensités élevées ont montré un temps de stabilisation plus long. Ainsi, en plus de la réponse cellulaire, il serait intéressant d'évaluer la dynamique d'acclimatation des cultures exposées à différents schémas lumineux. List of Tables Title : Biotechnological performances of acclimated green microalgae cultures: effect of light and its modulations Keywords : Biotechnology, Green microalgae, Photosynthesis, Light, Acclimation Abstract : Microalgae are emerging today as part of the answer to the major challenges of this century: climate change, population growth, and depletion of fossil fuel reserves. However, their autophototrophic production is limited by light resource. In addition to the total quantity, the way this light is delivered (intensity, temporality, spectrum) is considered to impact the performance of the culture. Understanding this to manipulate it could lead to increased culture yields. To characterize the effect of light fluctuations, an original experimental device was designed. This device allows ensuring homogeneous lighting of all the culture cells over long periods. In this way, only acclimated biological responses are measured. First, continuous illumination was studied for intensities from 25 to 800 µmolPhotonsPAR/m²/s. The results allowed to delimit a photo-limitation zone from 25 to 200 µmolPhotonsPAR/m²/s and a photo-saturation zone from 200 to 800 µmolPhotonsPAR/m²/s. The absence of photoinhibition suggests that the cells' photoprotection mechanisms are sufficient to protect them and maintain the same performance (µ = 0.088 h -1 on average). Then, intermittent illumination was investigated (1 Hz, 10 Hz, and 100 Hz, duty cycle: 0.5). The results showed no difference from the continuous regime. These experiments highlight the importance of the acclimation state of the cultures. Thus, they invalidate, at long times (those of continuous bioprocesses), the hypothesis that light modulation impacts microalgae performance.