Carl Safi
email: csafi@me.com

Bachar Zebib

Othmane Merah

Pierre-Yves Pontalier

Carlos Vaca-Garcia

Morphology, composition, production, processing and applications of Chlorella vulgaris: A review

Keywords:

E conomie and technical problems related to the redu ction of petroleum resources require the valorisation of renewable raw material. Re cently, microalgae emerged as promising alternative feedstock that represents an enormous biodiversity with multiple benefits exceeding the potential of conventional agricultural feedstock. Thus, this comprehensive review arti cle spots the light on one of the most interesting microalga Chlorella vulgaris. It assembles the history and a thorough des cription of its ultrastructure and composition a ccording to growth conditions. The harvesting te chniques are presented in relation to the nove! algo-refinery concept, with their te chnological advan cements and potential applications in the market.

Introduction

Microalgae have an ancient history that left a footprint 3.4 bil lion years aga, when the oldest known microalga, belonging to the group of cyanobacteria, fossilised in rocks of Western Australia. Studies confirmed that until our days their structure remains unchanged and, no matter how primitive they are, they still represent rather complicated and expertly organised forms of life [START_REF] Von Ditfurth | Anfang war der Wasserstoff[END_REF]. Nevertheless, other reports estimated that the actual time of evolution of cyanobacteria is thought to be doser to 2.7 billion years aga [START_REF] Brasier | Questioning the evidence for Earth's oldest fossils[END_REF][START_REF] Dalton | Microfossils: squaring up over ancient life[END_REF]. Hence, evolutionary biologists estimate that algae could be the ancestors of plants. Thus, through time algae gave rise to other marine plants and moved to the land during the Palaeozoic Age 450 millions years aga just like the scenario of animais moving from water onto land. However, evolutionists need to overcome multiple obstacles ( danger of drying, feed, reproduction, and protection from oxygen) to definitely confirm this scenario complemented with more scientific evidence.

Like any other phytoplankton, microalgae have a nutritional value. The first to consume the blue green microalga were the Aztecs and other Mesoamericans, who used this biomass as an important food source [START_REF] Venkataraman | Spirulina platensis (Arthrospira): physiology, cell Biology and biotechnology[END_REF]. Nowadays, these microscopie organisms are still consumed as food supplement such as Chlorella vulgaris and Spirulina platensis [START_REF] Fradique | Incorporation of Chlore/la vulgaris and Spirulina maxima biomass in pasta products. Part 1: preparation and evaluation[END_REF] and their products are also used for different purposes like dyes, pharmaceuticals, animal feed, aquaculture and cosmetics. For the last two decades, microalgae started to take a new course with increasing applications motivated by the depletion of fossil fuel reserves, the consequent increase in oil prices and the global warming concern. These dramatic thresholds are forcing the world to find global strategies for carbon dioxide mitigation by proposing alternative renewable feedstocks and intensifying researches on third-generation biofuels. In this context, microalgae are regarded nowadays as a promising sustainable energy resource due to their capacity to accumulate large quantities of lipids suitable for biodiesel production that performs much like petroleum fuel [START_REF] Gonzâlez-Fernândez | Impact of microalgae characteristics on their conversion to biofuel. Part I: focus on cultivation and biofuel production[END_REF][START_REF] Tran | Catalytic upgrading of biorefinery oil from micro-algae[END_REF]. They also proved to be a source of products such as proteins, carbohydrates, pigments, vitamins and minerais [START_REF] Mata | Microalgae for biodiesel production and other applications: a review[END_REF]. In addition, microalgae capture sunlight and perform photosynthesis by produ cing approximately half of atmospheric oxygen on earth and absorbing massive amounts of carbon dioxide as a major feed. Therefore, growing them next to combustion power plants is of major importance due to their remarkable capacity to absorb carbon dioxide that they convert into potential biofuel, food, feed and highly added value components [START_REF] Banerjee | Botryococcus braunii: a renew able source of hydrocarbons and other chemicals[END_REF][START_REF] Ghirardi | Microalgae: a green source of renewable H(2)[END_REF][START_REF] Lorenz | Commercial potential for Haematococcus microalgae as a natural source of astaxanthin[END_REF][START_REF] Singh | Bioactive compounds from cyanobacteria and microalgae: an overview[END_REF][START_REF] Spolaore | Commercial applications of microalgae[END_REF][START_REF] Walker | Microalgae as bioreactors[END_REF].

Microalgae can grow in bath fresh and marine water as well as in almost every environmental condition on earth from frozen lands of Scandinavia to hot desert soils of the Sahara [START_REF] Lee | Phycology[END_REF]. If production plants were installed in an intelligent way, microalgae would not compete with agricultural lands, there would be no conflict with food production [START_REF] Singh | Renewable fuels from algae: an answer to debatable land based fuels[END_REF] and especially would not cause deforestation.

Microalgae represent an enormous biodiversity from which about 40.000 are already described or analysed [START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF]. One of the most remarkable is the green eukaryotic microalga C. vulgaris, which belongs to the following scientific classification: Domain: Eukaryota, Kingdom: Protista, Divison: Chlorophyta, Class: Tre bouxiophyceae, Order: Chlorellales, Family: Chlorellaceae, Genus: Chlorella, Specie: Chlorella vulgaris. Hence, Martinus Willem Bei jerinck, a Dutch researcher, first discovered it in 1890 as the first microalga with a well-defined nucleus [START_REF] Beijerinck | Kulturversuche mit Zoochlorellen, Llchenengonidien und anderen niederen Algen[END_REF]. The name Chlorella cornes from the Greek word chloras (XÀ.rup6ç;), which means green, and the Latin suffix el/a referring to its microscopie size. lt is a unicellular microalga that grows in fresh water and has been present on earth since the pre-Cambrian period 2.5 billion years aga and since then its genetic integrity has remained constant [START_REF] Von Ditfurth | Anfang war der Wasserstoff[END_REF]. By the early 1900s, Chlorella protein content ( > 55% dry weight) attracted the attention of German scientists as an unconventional food source. In the 1950s, the Carnegie Institution of Washington [START_REF] Burlew | Alga! culture from laboratory to pilot plant[END_REF] took over the study and managed to grow this microalga on a large scale for CO 2 abatement. Nowadays, Japan is the world leader in consuming Chlorella and uses it for medical treatment [START_REF] Kitada | Supercritical C02 extraction of pigment components with pharmaceutical importance from Chlore/la vulgaris[END_REF][START_REF] Morris | Protein hydrolysates from the alga Chlore/la vulgaris 87 /1 with potentialities in immunonutrition[END_REF] because it showed to have immune-modulating and anti-cancer properties [START_REF] Justo | Effects of the green algae Chlore/la vulgaris on the response of the host hematopoietic system to intraperitoneal ehrlich ascites tumor transplantation in mice[END_REF][START_REF] Konishi | Antitumor effect induced by a hot water extract of Chlore/la vulgaris ( CE): resistance to Meth-A tumor growth mediated by CE-induced polymorphonuclear leukocytes[END_REF][START_REF] Morimoto | Anti-tumour-promoting glyceroglycolipids from the green alga, Chlore/la vulgaris[END_REF][START_REF] Singh | Inhibitory potential of Chlore/la vulgaris (E-25) on mouse skin papillomagenesis and xenobiotic detoxication system[END_REF][START_REF] Yasukawa | Inhibitory effects of sterols isolated from Chlore/la vulgaris on 12-0-tetra decanoylphorbol-13-acetate-induced inflammation and tumor promotion in mouse skin[END_REF]. After feeding it to rats, mice and rabbits in the form of powder, it showed protection properties against haema topoiesis [START_REF] De Souza Queiroz | Chlore/la vulgaris treatment ameliorates the suppressive effects of single and repeated stressors on hematopoiesis[END_REF] age-related diseases like cardiovascular diseases, hypertension and cataract; it lowers the risk of atherosclerosis and stimulates collagen synthesis for skin [START_REF] Sana | Effect of lipophilic extract of Chlore/la vulgaris on alimentary hyperlipidemia in cholesterol-fed rats[END_REF][START_REF] Sano | Effect of dried, powdered Chlore/la vulgaris on experi mental atherosclerosis and alimentary hypercholesterolemia in cholesterol fed rabbits[END_REF]. Furthermore, C. vulgaris is also capable of accumulating important amounts of lipids, especially after nitrogen starvation with a fatty acid profile suitable for biodiesel production [START_REF] Converti | Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlore/la vulgaris for biodiesel production[END_REF][START_REF] Zheng | Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves[END_REF].

The available reviews have focused so far on evaluating micro algae as an important source of lipids for biofuel production [START_REF] Chisti | Biodiesel from microalgae[END_REF][START_REF] Yamamoto | Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlore/la vulgaris (Chlorophyta, Trebouxiophyceae)[END_REF] and also explained in details the different production processes and harvesting techniques. The following review covers greater information about C. vulgaris, including not only produc tion and harvesting techniques already conducted on this micro alga, but also detailed information about its ultrastructure and chemical composition accompanied by cell wall breaking techni ques and extraction processes. The last section focuses on the multiple applications and potential interests of this microalga in different areas and not only on the production of fatty compounds.

Morphology

C. vulgaris is a spherical microscopie cell with 2-10 µm dia meter [START_REF] Yamamoto | Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlore/la vulgaris (Chlorophyta, Trebouxiophyceae)[END_REF][START_REF] Illman | Increase in Chlore/la strains calorific values when grown in low nitrogen medium[END_REF][START_REF] Yamamoto | Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxio phyceae)[END_REF] and has many structural elements similar to plants (Fig. 1 ).

Cell wall

The rigidity preserves the integrity of the cell and is basically a protection against invaders and harsh environment. It varies according to each growth phase. During its early formation in its autosporangia, the newly formed cell wall remains fragile, forming a 2 nm thin electron-dense unilaminar layer [START_REF] Yamamoto | Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlore/la vulgaris (Chlorophyta, Trebouxiophyceae)[END_REF][START_REF] Yvonne | Cell wall development, microfibril and pyrenoid structure in type strains of Chlore/la vulgaris, C. kessleri, C. sorokiniana compared with C. luteoviridis (Trebouxiophyceae, Chlorophyta)[END_REF]. The cell wall of the daughter cell gradually increases in thickness until it reaches 17-21 nm after maturation [START_REF] Yamamoto | Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlore/la vulgaris (Chlorophyta, Trebouxiophyceae)[END_REF][START_REF] Yamamoto | Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxio phyceae)[END_REF], where a microfibrillar layer is formed representing a chitosan-like layer composed of glucosamine [START_REF] Yvonne | Cell wall development, microfibril and pyrenoid structure in type strains of Chlore/la vulgaris, C. kessleri, C. sorokiniana compared with C. luteoviridis (Trebouxiophyceae, Chlorophyta)[END_REF][START_REF] Kapaun | A chitin-like glycan in the cell wall of a Chlore/la sp. (Chlorococcales, Chlorophyceae)[END_REF], which accounts for its rigidity. In the mature stage, cell wall thickness and composition are not constant because they can change according to different growth and environmental conditions. Furthermore, some reports [START_REF] Atkinson | John PCL Sporopollenin in the cell wall of Chlore/la and other algae: ultrastructure, chemistry, and incorporation of 14C-acetate, studied in synchronous cultures[END_REF][START_REF] Burczykj | The ultrastructure of the outer cell wall-layer of Chlore/la mutants with and without sporopollenin[END_REF] explained the rigidity of the cell wall by focusing on the presence of a sporopollenin layer, even though it is generally accepted that C. vulgaris has a unilaminar cell wall that Jacks sporopollenin, which is an extremely resistant polymerised carotenoid found on the cell wall of Haematococcus pluvialis [START_REF] Hagen | Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during apla nospore formation[END_REF] and Chlorellafusca [START_REF] Biedlingmaier | A correlation between detergent tolerance and cell wall structure in green algae[END_REF]. However, a contradictory study conducted on C. vulgaris by Martinez et al. [START_REF] Martînez | Morphometric and stereologic analysis of Chlore/la vulgaris under heterotrophic growth conditions[END_REF] reported the presence of sporopollenin by observing an outer trilaminar layer and by detecting resistant residues after being submitted to acetolysis.

Cytoplasm

It is the gel-like substance confined within the barrier of the cell membrane and it is composed of water, soluble proteins and minerais. It hosts the internai organelles of C. vulgaris such as mitochondria, a small nucleus, vacuoles [START_REF] Kuchitsu | Detection and characterization of acidic compartments (vacuoles) in Chlore/la vu/garis 11 h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques[END_REF], a single chloroplast and the Golgi body [START_REF] Salomon | Biology[END_REF].

Mitochondrion

Every mitochondrion contains some genetic materials, the respiratory apparatus and has a double-layer membrane; the outer membrane surrounds the whole organelle and is composed of an equal ratio of proteins and phospholipids. Nevertheless, the inner membrane is composed of thrice more proteins than phospholi pids; it surrounds the internai space called the matrix, which contains the majority of mitochondrial proteins [START_REF] Salomon | Biology[END_REF].

Chloroplast

C. vulgaris has a single chloroplast with a double enveloping membrane composed of phospholipids; the outer membrane is permeable to metabolites and ions, but the inner membrane has a more specific function on proteins transport. Starch granules, composed of amylose and amylopectin, can be formed inside the chloroplast, especially during unfavourable growth conditions. The pyrenoid contains high levels of ribulose-1,5-bisphosphate carbox ylase oxygenase (RuBisCO) and is the centre of carbon dioxide fixation. The chloroplast also stores a cluster of fused thylakoids where the dominant pigment chlorophyll is synthesised masking the colour of other pigments such as lutein. During nitrogen stress, lipid globules mainly accumulate in the cytoplasm and the chloroplast [START_REF] Lee | Phycology[END_REF][START_REF] Van Den Hoek | Algae: an introduction ta phycology[END_REF].

Reproduction

C. vulgaris is a non-motile reproductive cell (autospore) that reproduces asexually and rapidly. Thus, within 24 h, one cell of C. vulgaris grown in optimal conditions multiplies by autosporula tion, which is the most common asexual reproduction in algae. In this manner, four daughter cells having their own cell wall are formed inside the cell wall of the mother cell (Figs. 2 and3) [START_REF] Yamamoto | Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlore/la vulgaris (Chlorophyta, Trebouxiophyceae)[END_REF][START_REF] Yamamoto | Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxio phyceae)[END_REF]. After maturation of these newly formed cells, the mother cell wall ruptures, allowing the liberation of the daughter cells and the remaining debris of the mother cell will be consumed as feed by the newly formed daughter cells.

Production

Annual production of Chlore/la reached 2000 t (dry weight) in 2009, and the main producers are Japan, Germany and Taiwan [START_REF] Brennan | Biofuels from microalgae -a review of technologies for production, processing, and extractions of biofuels and co-products[END_REF]. This microalga has a rapid growth rate and responds to each set of growth condition by modifying the yield of a specific component. C. vulgaris is ideal for production because it is remarkably resistant against harsh conditions and invaders. On the one hand, lipid and starch contents increase and biomass productivity ceases or decreases [START_REF] Pfibyl | Production of lipids and formation and mobilization of lipid bodies in Chlore/la vulgaris[END_REF] during unfavourable growth conditions such as nitrogen and phosphorus limitation, high CO 2 concentration, excessive exposure to light [START_REF] Converti | Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlore/la vulgaris for biodiesel production[END_REF][START_REF] Lv | Enhanced lipid production of Chlore/la vulgaris by adjustment of cultivation conditions[END_REF][START_REF] Pribyl | Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlore/la vulgaris[END_REF][START_REF] Widjaja | Study of increasing lipid production from fresh water microalgae Chlore/la vulgaris[END_REF], excess of iron in the medium [START_REF] Liu | Effect of iron on growth and lipid accumulation in Chlore/la vulgaris[END_REF] or increase in temperature [START_REF] Richmond | Kopel R A new tubular reactor for mass production of microalgae outdoors[END_REF]. On the other hand, protein content increases during normal and managed growth conditions (nitrogen supplementation). Therefore, many growth techniques have been tested in order to voluntarily target biomass productivity, lipid, proteins, carbohydrates and pigments content. Open ponds are the most common way of production and are the cheapest method for large-scale biomass production. These systems are categorised into natural waters (lakes, lagoons and ponds) or wastewater or artificial ponds or containers. They are usually built next to power plants or heavy industry with massive carbon dioxide discharge where the biomass absorbs nitrogen from the atmosphere in the form of NO x . In order to allow easy exposure of ail the cells to sunlight, especially at the end of the exponential growth phase, the optimal pond depth is 15-50 cm [START_REF] Brennan | Biofuels from microalgae -a review of technologies for production, processing, and extractions of biofuels and co-products[END_REF][START_REF] Richmond | Kopel R A new tubular reactor for mass production of microalgae outdoors[END_REF]. On the other hand, open pond systems have some limitations because they require a strict environmental control to avoid the risk of pollution, water evaporation, contaminants, invading bacteria and the risk of growth of other algae species. In addition, temperature differences due to seasonal change cannot be controlled and C0 2 concentration and excess exposure to sun light are difficult to manage. Moreover, near the end of the exponential growth phase, some cells are not sufficiently exposed to sunlight because other cells floating near the surface cover them, leading to lower mass yields. Therefore, stirring of the medium is preferable and is currently practiced.

Closed photo-bioreactor

This technology was implemented mainly to overcome some limiting factors in the open pond systems, thus growing the biomass in a managed environment (pH, light intensity, tempera ture, carbon dioxide concentration) to obtain higher cell concen tration as well as products that are more suitable for the production of pure pharmaceuticals, nutraceuticals and cosmetics. In addition, these systems are more appropriate for sensitive strains that cannot compete and grow in harsh environment. Feeding the biomass with C0 2 cornes by bubbling the tubes. Fluorescent lights are used in case the tubes are not or not sufficiently exposed to sunlight. The tubes are generally 20 cm or less in diameter [START_REF] Chisti | Biodiesel from microalgae[END_REF] and the thickness of their transparent walls is few millimetres, allowing appropriate light absorption. Hence, multiple designs have been used and tested: fiat-plate photo bioreactor [START_REF] Qiang | Productivity and photosynthetic efficiency of Spirulina platensis as affected by light intensity, algal density and rate of mixing in a fiat plate photobioreactor[END_REF][START_REF] Zhang | Evaluation of a vertical fiat-plate photobior eactor for outdoor biomass production and carbon dioxide bio-fixation: effects of reactor dimensions, irradiation and cell concentration on the biomass productivity and irradiation utilization efficiency[END_REF], tubular photo-bioreactor [START_REF] Molina Grima | Recovery of microalgal biomass and metabolites: process options and economics[END_REF] and column photo-bioreactor [START_REF] Kojima | Growth and hydrocarbon production of microalga Botryococcus braunii in bubble column photobioreactors[END_REF]. Degen et al. [START_REF] Degen | A navel airlift photobioreactor with bailles for improved light utilization through the flashing light effect[END_REF] achieved 0.11 g L -1 h-1 dry biomass productivity after growing the cells of C. vulgaris in a fiat panel airlift photobioreactor under continuous illumination (980 µE m-2 ç 1 ). Nonetheless, the main disadvantages of a closed system are the cost of the sophisticated construction, small illumination area and sterilising costs [START_REF] Lee | Microalgal mass culture systems and methods: their limitation and potential[END_REF].

Heterotrophic growth

This technique does not require light and the biomass is fed with organic carbon source. Thus, microalgae are grown in a stirred tank bioreactor or fermenter where a higher degree of growth are expected as well as low harvesting cost due to the higher dry biomass productivity achieved ( up to 0.25 g L -1 d -1 ) and high accumulation of different components such as lipids 22-54 mg L -1 d-1 [START_REF] Martînez | Morphometric and stereologic analysis of Chlore/la vulgaris under heterotrophic growth conditions[END_REF][START_REF] Liang | Biomass and lipid productivities of Chlore/la vulgaris under autotrophic, heterotrophic and mixotrophic growth condi tions[END_REF][START_REF] Ogawa | Bioenergetic analysis of mixotrophic growth in Chlore/la vulgaris and Scenedesmus acutus[END_REF]. The carbon sources used for C. vulgaris are glucose, acetate, glycerol and glutamate with max imum specific growth rate obtained with glucose. Nevertheless, the major disadvantage of this system is the price and availability of sugars, which compete with feedstocks for other uses such as food and biofuel productions.

Mixotrophic growth

C. vulgaris is capable of combining both autotrophic and heterotrophic techniques by performing photosynthesis as well as ingesting organic materials such as glucose, which is the most appropriate for C. vulgaris [START_REF] Liang | Biomass and lipid productivities of Chlore/la vulgaris under autotrophic, heterotrophic and mixotrophic growth condi tions[END_REF][START_REF] Ogawa | Bioenergetic analysis of mixotrophic growth in Chlore/la vulgaris and Scenedesmus acutus[END_REF][START_REF] Mallick | Green microalga Chlore/la vulgaris as a potential feedstock for biodiesel[END_REF][START_REF] Patina | A study of the growth for the microalga Chlore/la vulgaris by photo-bio-calorimetry and other on-line and off-line techniques[END_REF][START_REF] Yeh | Effects of cultiva tian conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlore/la vulgaris ESP-31[END_REF]. Hence, the cells are not strictly dependent on light or organic substrate to grow. This technique competes favourably with autotrophic systems and according to Yeh and Chang [START_REF] Yeh | Effects of cultiva tian conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlore/la vulgaris ESP-31[END_REF] mixotrophic conditions showed high dry biomass productivity (2-5 g L -1 d-1 ) and lipids productivity (67-144 mg L -1 d-1 ). The main advantages of mixotrophic meta bolism are limiting the impact of biomass Joss during dark respiration and reducing the amount of organic substrates used for growing the biomass.

Other growth techniques

Growth of C. vulgaris can take an additional dimension by co immobilising it with plant growing bacterium Azospirillum brasi lense in alginate beads [START_REF] De-Bashan | Cultivation factors and population size contrai the uptake of nitrogen by the microalgae Chlore/la vulgaris when interacting with the microalgae growth-promoting bacterium Azospirillum brasilense[END_REF][START_REF] Gonzalez | Increased growth of the microalga Chlore/la vulgaris when coimmobilized and cocultured in alginate beads with the plant growth-promoting bacterium Azospirillum brasilense[END_REF]. This technique has been extra polated to C. vulgaris and other microalgae from the hypothesis that A brasilense promotes terrestrial plant growth performance by interfering with the host plant hormonal metabolism and provides 0 2 for the bacteria to biodegrade pollutants and then the microalga consumes C0 2 released from bacterial respiration [START_REF] Munoz | Algal-bacterial processes for the treatment of hazar dous contaminants: a review[END_REF]. Consequently, depending on the strain of C. vulgaris [START_REF] De-Bashan | Increased pigment and lipid content, lipid variety, and cell and population size of the microalgae Chlore/la spp. when co-immobilized in alginate beads with the microalgae-growth-promoting bacterium Azospirillum brasilense[END_REF] this technique has an impact on prolonging its life span, enhancing biomass production, increasing cell size (62% larger) and accumu lating pigments and lipids. Simultaneously, uptake of zinc, cad mium, phosphorus, nitrogen and other heavy metals from wastewater increases. On the other hand, growing C. vulgaris with its associative bacterium Phyllobacterium myrsinacearum also has a different impact by ceasing its growth or cell death [START_REF] Lebskyvk | Ultrastructure ofinteraction in alginate beads between the microalga Chlore/la vulgaris with its natural associative bacterium Phyllobacterium myrsinacearum and with the plant growth promoting bacteriumAzospirillum brasilense[END_REF]. Further more, mixing and shear stress have an effect on increasing the photosynthetic activity and growth of C. vulgaris. Thus, optimal conditions (tip speed of 126 cm ç 1 and friction velocity 2.06 cm s-1 ) increased the photosynthetic activity by 4-5% with 48-71% stronger growth compared to null tip speed or friction velocity. Nevertheless, higher tip speed and friction velocity decreased both photosynthetic activity and growth to the value of the unstirred condition and even lower [START_REF] Leupold | Influence ofmixing and shear stress on Chlore/la vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii[END_REF]. This process contributes to 20-30% of the total biomass production cost [START_REF] Molina Grima | Recovery of microalgal biomass and metabolites: process options and economics[END_REF]. The most common harvesting technique for C. vulgaris is centrifugation (5000 rpm, 15 min) [START_REF] Converti | Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlore/la vulgaris for biodiesel production[END_REF]70] because it is highly efficient (95% recovery), not time consuming, and treats large volumes. In addition, the morphology of C. vulgaris permits high centrifugai stress without damaging its structure during the process. Other techniques are also applied such as flocculation, flotation and filtration or by combining two techniques to max imise recovery of the biomass.

Flocculation

During the exponential growth phase, the algal cells have high negative surface charge and are difficult to neutralise, and thus the cells remain dispersed. After reaching the stationary or the declining phase, the negative charge decreases, allowing the cells to aggregate and to form lumps, thereby resulting in a process called auto-flocculation. This phenomenon is asso ciated with elevated pH due to C0 2 , nitrate and phosphate assimi lation [START_REF] Vandamme | Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications[END_REF]. Moreover, auto-flocculation can occur by interactions between algae and bacteria or excreted organic molecules or by simply cutting C0 2 supply; this method is Jess expensive but time consuming. In general, culture of microalgae is very stable and auto-flocculation probability is negligible and sometimes mislead ing. In order to accelerate coagulation, it is necessary to increase the pH by adding a base. The most effective is sodium hydroxide, which induces more than 90% flocculation at pH 11 and requires Jess quantity (9 mg of NaOH per gram of dry biomass) [START_REF] Vandamme | Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications[END_REF][START_REF] Wu | Evaluation of flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium[END_REF]. But on an industrial scale, lime seems to be the most cost-efficient. This mechanism is associated with Mg 2 + from hydrolysed Mg (OH) 2 , which precipitates attracting with it the negatively charged microalgal cells. Chitosan is also an interesting flocculating agent [START_REF] Divakaran | Flocculation of algae using chitosan[END_REF], which showed maximum efficiency at pH 7 with 90% microalgal recovery. Further on, using bioflocculants like Paeniba cillus sp. with the presence of a co-flocculant (CaCl 2 ) also showed an efficient flocculation (83%) at pH 11 [START_REF] Oh | Harvesting of Chlore/la vulgaris using a bioflocculant from Paenibacillus sp. AM49[END_REF]. Flocculation is some times considered as a pre-harvesting step in order to facilitate or complement other harvesting methods like centrifugation or filtration [START_REF] Chang | Coagulation-membrane filtration of Chlore/la vulgaris at different growth phases[END_REF][START_REF] Lee | Coagulation-membrane filtration of Chlore/la vulgaris[END_REF].

Flotation

To our knowledge, there is very limited evidence of its feasibility, but this method consists of trapping the cells using dispersed micro-air bubbles. Flotation can also occur naturally when the lipid content in microalgae increases. Cheng et al. [START_REF] Cheng | Dispersed ozone flotation of Chlore/la vulgaris[END_REF] induced effective flotation on C. vulgaris by using dispersed ozone gas (0.05 mg g-1 biomass). Thus, unlike flocculation, this method does not require synthetic chemicals, but its economic viability is not yet known, especially on an industrial scale.

Filtration

This method involves continuous passing of the broth with the microalga across a filter on which algal cells will concentrate constantly until it reaches a certain thickness. Due to the small size of C. vulgaris, conventional filtration is not an adequate method to be applied. Instead, ultrafiltration or microfiltration is more efficient. Fouling generated by soluble compounds like exopoly saccharides of some microalgae such as Porphyridium is one of the major limitations during the ultrafiltration process, but with Chlorella this phenomenon is negligible, and thus its structure provides more important permeation flux without the need of an additional unit operation like swirling while filtering [START_REF] Frappart | Influence of hydro dynamics in tangential and dynamic ultrafiltration systems for microalgae separation[END_REF][START_REF] Morineau-Thomas 0, Jaouen | The role of exopolysac charides in fouling phenomenon during ultrafiltration of microalgae ( Chlor e/la sp. and Porphyridium purpureum ): advantage of a swirling decaying flow[END_REF]. More over, microfiltration and ultrafiltration are affected by different parameters such as filter type; transmembrane pressure, flow velocity, turbulent cross-flow and growth phase, and therefore a compromise that takes into consideration these parameters should be made. Furthermore, they can be accompanied by another harvesting technique (flotation or flocculation) that improves the process [START_REF] Chang | Coagulation-membrane filtration of Chlore/la vulgaris at different growth phases[END_REF][START_REF] Lee | Coagulation-membrane filtration of Chlore/la vulgaris[END_REF][START_REF] Hung | Microfiltration for separation of green algae from water[END_REF].

Primary composition

Proteins

Proteins are of central importance in the chemistry and composition of microalgae. They are involved in capital raies such as growth, repair and maintenance of the cell as well as serving as cellular motors, chemical messengers, regulators of cellular activ ities and defence against foreign invaders [START_REF] Salomon | Biology[END_REF].

Total proteins content in mature C. vulgaris represents 42-58% of biomass dry weight [START_REF] Becker | Microalgae: biotechnology and microbiology[END_REF][START_REF] Morris | Utilisation of Chlore/la vulgaris cell biomass for the production of enzymatic protein hydrolysates[END_REF][START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF][START_REF] Servaites | A dye binding method for measurement of total protein in microalgae[END_REF][START_REF] Seyfabadi | Protein, fatty acid, and pigment content of Chlore/la vulgaris under different light regimes[END_REF], and varies according to growth conditions. Proteins have multiple raies, and almost 20% of the total proteins are bound to the cell wall, more than 50% are internai and 30% migrate in and out of the cell [START_REF] Berliner | Proteins in Chlore/la vulgaris[END_REF]. Their molecular weight revealed by SOS-PAGE comprises between 12 and 120 kDa, with the majority between 39 and 75 kDa after growing C. vulgaris under autotrophic or heterotrophic conditions. Nevertheless a higher intensity peak is observed for cells grown in autotrophic conditions [START_REF] Morris | Utilisation of Chlore/la vulgaris cell biomass for the production of enzymatic protein hydrolysates[END_REF][START_REF] Hanan | Comparative effects of autotrophic and heterotrophic growth on some vitamins, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scaven ging activity, amino acids and protein profile of Chlore/la vulgaris Beijerinck[END_REF].

Protein nutritional quality is determined by its amino acid profile [START_REF] Becker | Microalgae: biotechnology and microbiology[END_REF][START_REF] Safi | Evaluation of the protein quality of Porphyridium cruentum[END_REF], and like the majority of microalgae, the amino acid profile of C. vulgaris compares favourably and even better with the standard profile for human nutrition proposed by World Health Organisation (WHO) and Food and Agricultural Organisation (FAO), because the cells of C. vulgaris synthesise essential and non-essential amino acids (Table 1 ). Furthermore, regardless of the extraction procedure, C. vulgaris proteins showed excellent emul sifying capacity [START_REF] Ursu | Extraction, fractionation and functional properties of proteins from the microalgae Chlore/la vulgaris[END_REF] that is comparable and even better than the commercial ingredients. Results showed that the emulsifying capacity of C. vulgaris proteins extracted at pH= 7 reached 3090 ± 50 mL oil/g protein with a stability of 79 ± 1%. Therefore, proteins of C. vulgaris open the gate for additional valorisation options of this microalga in the market, especially in the food sector.

Protein extraction is technically the same for ail microalgae and is mainly conducted by solubilisation of proteins in alkaline solution [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF][START_REF] Bajguz | Effect of brassinosteroids on nucleic acids and protein content in cultured cells of Chlore/la vulgaris[END_REF][START_REF] Rausch | The estimation of micro-algal protein content and its meaning to the evaluation of algal biomass 1. Comparison of methods for extracting protein[END_REF]. Further purification can be followed by pre cipitating the solubilised proteins with trichloroacetic acid (25% ICA) [START_REF] Barbarino | An evaluation of methods for extraction and quantification of protein from marine macro-and microalgae[END_REF][START_REF] Oliveira | Purification of polysaccharides from a biofilm matrix by selective precipitation of proteins[END_REF] or hydrochloric acid (0.1 N HCI) [START_REF] Chronakis | The behaviour of protein preparations from blue-green algae (Spirulina platensis strain Pacifica) at the air/water interface[END_REF]. Another separation method could be applied by means of ultrafiltration. Indeed, this method is usually applied for harvesting the cells but considering the study conducted by Safi et al. [START_REF] Safi | A two-stage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption[END_REF], a two-stage ultrafiltration process was applied on the aqueous extract of Tetraselmis suecica containing solubilised molecules (starch, pro teins and low molecular weight polysaccharides). The first phase of the process completely retained starch molecules, and then the second phase completely retained proteins, allowing only small polysaccharides to be present in the filtrate of the second phase of the process. This process could be extrapolated to C. vulgaris with minor modifications of the eut-off of the ultrafiltration membranes [START_REF] Safi | A two-stage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption[END_REF].

Quantification is carried out by elemental analysis, Kjeldahl, Lowry assay, Bradford assay or the dye binding method. However, the first two analyses take into consideration total nitrogen present in the microalga, and multiplying it by the standard nitrogen to protein conversion factor (NTP) 6.25 may lead to overestimation or underestimation of the true protein quantity. Therefore, several studies calculated from an amino acid profile a [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF]. b [START_REF] Shaaban | Green microalgae water extracts as foliar feeding to wheat plants[END_REF][START_REF] Faheed | Effect of Chlore/la vulgaris as bio-fertilizer on growth parameters and metabolic aspects of lettuce plant[END_REF].

C [START_REF] Naik | Production of first and second generation biofuels: a comprehensive review[END_REF].

recommended a new NTP lower than the standard 6.25 [START_REF] Diniz | Gross chemical profile and calculation of nitrogen-to-protein conversion factors for five tropical seaweeds[END_REF][START_REF] Lopez | Protein measurements of microalgal and cyanobacterial biomass[END_REF][START_REF] Lourenço | Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds[END_REF][START_REF] Lourenço | Distribution of intracellular nitrogen in marine microalgae: calculation of new nitrogen to-protein conversion factors[END_REF][START_REF] Lourenço | Distribution of intracellular nitrogen in marine microalgae: basis for the calculation of specific nitrogen to-protein conversion factors[END_REF].

Nevertheless, a study conducted by Safi et al. [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF] correlated the evaluation of the NTP to the rigidity of the cell wall by evaluating the NTP of five crude microalgae including C. vulgaris and their protein extract, and concluded that no universal conversion factor could be recommended for multiple reasons such as cell wall rigidity, growth conditions, growth media and environmental uncertainty. Gonzalez-Lopez et al. [START_REF] Lopez | Protein measurements of microalgal and cyanobacterial biomass[END_REF] determined the NTP using a different technique that correlates protein content (Lowry assay) to total nitrogen content (Kjeldahl and elemental analysis) and also estimated that the I<jeldahl method correlates better with the Lowry assay. In addition, Servaites et al. [START_REF] Servaites | A dye binding method for measurement of total protein in microalgae[END_REF] quantified proteins of 12 different microalgae including C. vulgaris by staining the protein isolate with Coomassie brilliant blue R-250 {CBB) on a paper and then eluting the remaining stained proteins in 1% sodium dodecyl sulphate {SOS) followed by measuring the absor bance at 600 nm. This method gave almost similar results com pared to the Dumas method. On the other hand, the colorimetric method of Lowry [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF] was also considered as one of the most accurate methods to quantify proteins [START_REF] Peterson | Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall[END_REF], but with time this method showed to only quantify hydro-soluble proteins [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF][START_REF] Safi | Evaluation of the protein quality of Porphyridium cruentum[END_REF][START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF][START_REF] Peterson | Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall[END_REF][START_REF] Crossman | Determination of protein for studies of marine herbivory: a comparison of methods[END_REF][START_REF] Sriperm | Evaluation of the fixed nitrogen-to-protein (N:P) conversion factor (6.25) versus ingredient specific N:P conversion factors in feedstuffs[END_REF][START_REF] Yeoh | Protein contents, amino acid compositions and nitrogen-to-protein conversion factors for Cassava roots[END_REF], which represents the major part of proteins. The Lowry assay is more acceptable than the Bradford assay because the latter does not react with ail the amino acids present in the extract, thus giving lower protein concentrations [START_REF] Barbarino | An evaluation of methods for extraction and quantification of protein from marine macro-and microalgae[END_REF].

Lipids

Lipids are a heterogeneous group of compounds that are defined not by their structure but rather by the fact that they are soluble in non-polar solvents and relatively insoluble in water [START_REF] Bajguz | Effect of brassinosteroids on nucleic acids and protein content in cultured cells of Chlore/la vulgaris[END_REF]. During optimal growth conditions C. vulgaris can reach 5-40% lipids per dry weight of biomass [START_REF] Becker | Microalgae: biotechnology and microbiology[END_REF], and are mainly composed of glycolipids, waxes, hydrocarbons, phospholipids, and small amounts offree fatty acids [START_REF] Lee | Phycology[END_REF][START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF]. These components are synthe sised by the chloroplast and also located on the cell wall and on membranes of organelles ( chloroplast and mitochondrion mem branes). Nevertheless, during unfavourable growth conditions, lipids content (mainly composed of triacyglycerols) can reach 58% [START_REF] Mata | Microalgae for biodiesel production and other applications: a review[END_REF][START_REF] Becker | Microalgae: biotechnology and microbiology[END_REF][START_REF] Stephenson | Influence of nitrogen Iimitation regime on the production by Chlorella vulgaris of Iipids for biodiesel feedstocks[END_REF]. Unlike other lipids, triacylglycerols do not perform a structural role but instead accumulate as dense storage lipid droplets in the cytoplasm and in the inter-thylakoid space of the chloroplast [START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances[END_REF]. Liu et al. [START_REF] Liu | Effect of iron on growth and lipid accumulation in Chlore/la vulgaris[END_REF] optimised a method that detects the accumula tion of lipid droplets inside the cells of C. vulgaris after each growth phase. The method relies on staining the cells with Nile red dye and then observing the accumulation of lipids with fluorescence microscope by emitting blue light that reveals the lipid droplets, especially neutral lipids. This technique showed a correlation between the quantity of neutral lipids accumulated and fluorescence intensity. However, according to Chen et al. [START_REF] Chen | Microwave-assisted nife red method for in vivo quantification of neutral Iipids in microalgae[END_REF] without cell disruption, this method could be ineffective due to the presence of a thick cell wall of some microalgae that can prevent complete access of the reagent inside the cell. Thus, cell disruption is a necessity to prevent wrong measurements and quantification.

The extraction process of total lipids from C. vulgaris is generally conducted by the method of Bligh and Dyer (a mixture of chloroform and methanol), or by hexane, or petroleum ether [START_REF] Zheng | Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves[END_REF][START_REF] Pribyl | Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlore/la vulgaris[END_REF][START_REF] Liu | Effect of iron on growth and lipid accumulation in Chlore/la vulgaris[END_REF][START_REF] Lee | Microalgal mass culture systems and methods: their limitation and potential[END_REF][START_REF] Lee | Comparison of several methods for effective Iipid extraction from microalgae[END_REF][START_REF] Mercer | Developments in oil extraction from microalgae[END_REF][START_REF] Phukan | I<ataki R Microalgae Chlorella as a potential bio-energy feedstock[END_REF]. Quantification of total lipids is conducted gravimetrically after evaporating the extracting solvent; in addi tion, column chromatography is carried out in order to separate different lipid constituents followed by evaporating the solvent and then weighing the remaining lipid extract [START_REF] Olmstead | A quantitative analysis of microalgal Iipids for optimization of biodiesel and omega-3 production[END_REF]. Indeed, these solvents are not used on an industrial scale because they are harmful for the environment, toxic, highly flammable and con taminate the extract [START_REF] Mercer | Developments in oil extraction from microalgae[END_REF]. Total lipids are composed of three major fractions phospholipids (PL), glycolipids {GL) and neutral lipids {NL). These fractions are fractionated by sequential elution of chloroform and acetic acid for NL, acetone and methanol for GL, and methanol for PL recovery [START_REF] Olmstead | A quantitative analysis of microalgal Iipids for optimization of biodiesel and omega-3 production[END_REF]. Supercritical carbon dioxide {SC-C0 2 ) extraction has been identified as an alternative for a greener extraction since it gives pure extracts free of contamina tion. Moreover, in order to increase the yield of extraction, a co solvent to SC-C0 2 such as ethanol can be used or a preliminary cell disruption technique can be performed [START_REF] Dejoye | Combined extraction processes of Iipid from Chlorella vulgaris microalgae: microwave prior to supercritical carbon dioxide extraction[END_REF]. It is noteworthy that the addition of ethanol increases the extraction yield of total lipophilic molecules (lipids and pigments), but it could also bypass the energetic yet efficient cell disruption technique, and therefore the production cost could be significantly reduced [START_REF] Safi | Extraction of Iipids and pigments of Chlorella vulgaris by supercritical carbon dioxide: influence of bead milling on extraction performance[END_REF].

The fatty acid profile changes with respect to growth condi tions and is suitable for different applications. For instance, according to Yeh and Chang [START_REF] Yeh | Effects of cultiva tian conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlore/la vulgaris ESP-31[END_REF], the fatty acid profile of C. vulgaris grown under mixotrophic growth conditions can accumulate 60-68% saturated and monounsaturated fatty acids composed of palmitic acid C16:0, stearic acid C18:0 fatty acids, palmitoleic acid C16:1 and oleic acid C18:1 [START_REF] Zheng | Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves[END_REF]. Such a profile is more suitable for biodiesel production [START_REF] Yeh | Nitrogen starvation strategies and photobioreactor design for enhancing Iipid content and Iipid production of a newly isolated microalga Chlorella vulgaris ESP-31: implications for biofuels[END_REF]. On the contrary, if it is grown under favourable growth conditions, its fatty acid profile is unsuitable for biodiesel [START_REF] Stephenson | Influence of nitrogen Iimitation regime on the production by Chlorella vulgaris of Iipids for biodiesel feedstocks[END_REF] but more suitable for nutritional uses because it is more concentrated in polyunsaturated fatty acids such as linoleic acid C18:2, linolenic acid C18:3, and eicosapentaenoic acid C20:5 [START_REF] Chen | Microwave-assisted nife red method for in vivo quantification of neutral Iipids in microalgae[END_REF].

Carbohydrates

Carbohydrates represent a group of reducing sugars and poly saccharides such as starch and cellulose. Starch is the most abundant polysaccharide in C. vulgaris. It is generally located in the chloroplast and is composed of amylose and amylopectin, and together with sugars they serve as energy storage for the cells. Cellulose is a structural polysaccharide with high resistance, which is located on the cell wall of C. vulgaris as a protective fibrous barrier. In addition, one of the most important polysaccharides present in C. vulgaris is the p1 __.3 glucan [START_REF] Lordan | Marine bioactives as functional food ingredi ents: potential to reduce the incidence of chronic diseases[END_REF], which has multiple health and nutritional benefits.

Total carbohydrates are generally quantified by the sulphuric phenol method [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF][START_REF] Shi | Purification and identification of polysaccharide derived from Chlorella pyrenoidosa[END_REF], yielding simple sugars after hydrolysis at 110 °C, then quantification of the latter by HPLC ( especially HPIC). Starch quantification is much better using the enzymatic method compared to the acidic method [START_REF] Dragone | Nutrient limitation as a strategy for increasing starch accumulation in microalgae[END_REF][START_REF] Fernandes | Starch determination in Chlorella vulgaris -a comparison between acid and enzymatic methods[END_REF]. During nitrogen limita tion, total carbohydrates can reach 12-55% dry weight. [START_REF] Branyikova | Microalgae -nove! highly efficient starch producers[END_REF][START_REF] Choix | Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: 1. Autotrophic conditions[END_REF].

Moreover, C. vulgaris has a remarkably robust cell wall [START_REF] Janczyk | Nutritional value of Chlorella vulgaris: effects of ultrasonication and electroporation on digestibility in rats[END_REF], mainly composed of a chitosan like layer, cellulose, hemicellulose, proteins, lipids and minerais [START_REF] Abo-Shady | Chemical composition of the cell wall in some green algae species[END_REF][START_REF] Griffiths | The fine structure of autotrophic and heterotrophic cells of Chlorella vulgaris (Emerson strain)[END_REF][START_REF] Northcote | The chemical composition and structure of the cell wall of Chlorella pyrenoidosa[END_REF].

The sugar composition (Table 2) of the cell wall is a mixture of rhamnose, galactose, glucose, xylose, arabinose and mannose [START_REF] Takeda | Classification of Chlorella strains by means of the sugar compo nents of the cell wall[END_REF][START_REF] Takeda | Classification of Chlorella strains by cell wall sugar composition[END_REF][START_REF] Takeda | Sugar composition of the cell wall and the taxonomy of Chlorella (Chlorophyceae)l[END_REF][START_REF] Takeda | Chemical composition of cell walls as a taxonomical marker[END_REF][START_REF] Takeda | Studies on the cell wall of Chlorella V. Comparison of the cell wall chemical compositions in strains of Chlorella ellipsoidea[END_REF], rhamnose being the dominant sugar [START_REF] Takeda | Sugar composition of the cell wall and the taxonomy of Chlorella (Chlorophyceae)l[END_REF][START_REF] Blumreisinger | Cell wall composition of chlorococcal algae[END_REF][START_REF] Ogawa | A new trisaccharide, <X-o-glucopyranuronosyl (1 --+3)-<X-L-rhamnopyranosyl-(1--+ 2)-<X-L-rhamnopyranose from Chlorella vulgaris[END_REF].

Pigments

The most abundant pigment in C. vulgaris is chlorophyll, which can reach 1-2% dry weight and is situated in the thylakoids. C. vulgaris also contains important amounts of carotenoids (Table 3) that act as accessory pigments by catching light; P-carotene for instance is associated with the lipid droplets in the chloroplast, and primary carotenoids are associated with chlorophyll in thylakoids where they trap light energy and transfer it into the photosystem. However, as in terrestrial plants, some pigments act as photo protectors by protecting chlorophyll molecules from degradation and bleaching during strong exposure to radiation and oxygen [START_REF] Salomon | Biology[END_REF].

These pigments have multiple therapeutic properties, such as antioxidant activities [START_REF] Gouveia | Chlorella vulgaris and Haematococcus pluvialis biomass as colouring and antioxidant in food emulsions[END_REF], protective effect against retina degen eration [START_REF] Fernandez-Sevilla | Obtaining lutein-rich extract from microalgal biomass at preparative scale[END_REF][START_REF] Granado | Nutritional and clinicat relevance of lutein in human health[END_REF], regulating blood cholesterol, prevention from

Table 2

Simple sugars composition of the cell wall poly saccharides [START_REF] Takeda | Sugar composition of the cell wall and the taxonomy of Chlorella (Chlorophyceae)l[END_REF]. chronic diseases (cardiovascular and colon cancer) and fortifying the immune system [START_REF] Cha | Antiproliferative effects of carotenoids extracted from Chlorella ellipsoidea and Chlore/la vulgaris on human colon cancer cells[END_REF][START_REF] Tanaka | Augmentation of antitumor resistance by a strain of unicellular green algae, Chlorella vulgaris[END_REF]. Pheophytins are biochemically similar to chlorophyll but lacking Mg ++ ion; they can form after chlorophyll degradation during the growth of microalgal cells or during harsh extraction conditions. In addition, these pigments are lipophilic and their extraction is generally associated with lipid extraction.

Neutra! sugars

Rhamnose

Many studies worked on optimising the extraction process of pigments using solvents (dimethyl formamide, dichloromethane, acetone, hexane, and ethanol), soxhlet, ultrasound-assisted extrac tion [70,[START_REF] Gors | Quality analysis of commercial Chlorella products used as dietary supplement in human nutrition[END_REF][START_REF] Kong | Optimization of ultrasound-assisted extraction parameters of chlorophyll from Chlorella vulgaris residue after lipid separation using response surface methodology[END_REF][START_REF] Li | Isolation and purification of Iutein from the microalga Chlorella vulgaris by extraction after saponification[END_REF][START_REF] Maxwell | Photosystem II excitation pressure and development of resistance to photoinhibition (1. Iight-harvesting complex II abundance and zeaxanthin content in Chlorella vulgaris )[END_REF], and pressurised liquid extraction (PLE) that showed useful simultaneous extraction of carotenoids and chlor ophyll, and also minimised the formation of pheophytins [70,[START_REF] Cha | Effect of pressurized liquids on extraction of antioxidants from Chlorella vulgaris[END_REF] at high temperature ( > 110 °C). Moreover, SC-CO 2 extraction was also carried out to enhance carotenoids recoveries, and the best conditions were 35 MPa and 40-55 °C on crushed cells, and under these conditions the extract was golden and limpid unlike solvents extraction; thus by using SC-CO 2 , higher selectivity can be achieved [START_REF] Kong | Optimization of ultrasound-assisted extraction parameters of chlorophyll from Chlorella vulgaris residue after lipid separation using response surface methodology[END_REF][START_REF] Cha | Effect of pressurized liquids on extraction of antioxidants from Chlorella vulgaris[END_REF]. This hypothesis was confirmed by Kitada et al. [START_REF] Kitada | Supercritical C02 extraction of pigment components with pharmaceutical importance from Chlore/la vulgaris[END_REF], using different optimum conditions (50 MPa and 80 °C) because the study was conducted on whole cells; thus stronger conditions were required. In addition, co-solvent such as 5% ethanol has been added as a booster to increase the extraction yield. Analyses and quantification of pigments are conducted by high performance liquid chromatography (HPLC) and spectro photometry using specific equations [START_REF] Rj | Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents[END_REF] or by plotting the calibration curve for each pigment.

Minerais and vitamins

Minerais are determined after incinerating the biomass and then analysis by atomic absorption spectrophotometry (Table 4). They play important functional roles in humans [START_REF] Salomon | Biology[END_REF]. For instance, potassium cation is principal for human nutrition; it is associated with intracellular fluid balance, carbohydrate metabolism, protein synthesis and nerve impulses. In addition, it is used as chemical fertilizer in agriculture in the form of chloride (KCI), sulphate (K2SO4) or nitrate (KNO 3 ). Magnesium is important in maintaining normal and constant nervous activity and muscle contraction; hence magnesium deficiency in human organism can lead to depression and symptoms of suicidai behaviour. Zinc is an essen tial component of enzymes, which participates in many metabolic processes including synthesis of carbohydrates, lipids and proteins and it is also a cofactor of the superoxide dismutase enzyme, which is involved in the protection against oxidative processes and reducing the severity of strong diarrhoea.

Table 5

Vitamins profile of C. vulgaris.

Vitamins Content (mg 100 g-1 )

Maruyama Yeh Panahi et al. [START_REF] Maruyama | Application of unicellular algae Chlorella vulgaris for the mass-culture of marine rotifer Brachionus[END_REF] et al. [START_REF] Yeh | Nitrogen starvation strategies and photobioreactor design for enhancing Iipid content and Iipid production of a newly isolated microalga Chlorella vulgaris ESP-31: implications for biofuels[END_REF] et al. [START_REF] Panahi | Investigation of the effects of Chlore/la vulgaris as an adjunctive therapy for dyslipidemia: results of a randomised open-label clinical trial[END_REF] B1 Vitamins are classified as water-soluble (C and B) and fat soluble (A, D, E, and K). C. vulgaris has an important vitamin profile (Table 5) that are key elements for cell growth and differentiation in the human body (Vitamin A), and have antioxidant activity that acts as radical scavenger together with improving blood circula tion and controlling muscle functions (Vitamins E and C) [START_REF] Becerra | Supercritical fluid extraction and supercritical fluid chromatography of Vitamin E in pharmaceutical prepara tions[END_REF]. Vitamin B complex occupies the largest number in living organ isms and is a major actor for enzymes activity in metabolism [START_REF] Brown | Nutritional properties of microalgae for mariculture[END_REF], promotes red blood cells growth, reduces the risk of pancreatic cancer, and maintains healthy skin, hair and muscles. Vitamins profile is sensitive to growth conditions; thus the best concentra tion was achieved after 24 h autotrophic growth with 10% C0 2 , but during heterotrophic conditions vitamins content was higher than autotrophic due to the presence of glucose in the medium and used as carbon source to produce organic compounds [START_REF] Hanan | Comparative effects of autotrophic and heterotrophic growth on some vitamins, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scaven ging activity, amino acids and protein profile of Chlore/la vulgaris Beijerinck[END_REF]. Another possible explanation for the high content of vitamins may be the alterations in the ultrastructure of the photosynthetic apparatus which were found to be associated with changes in cellular components [START_REF] Ochiai | Studies on chlorophyll formation in Chlorella protothecoides I. Enhancing effects of light and added 6-aminolevulinic acid, and suppres sive effect of glucose on chlorophyll formation[END_REF].

Cell disruption techniques

C. vulgaris has a resistant cell wall, which is a major barrier for digestibility and extraction process of ail internai components.

Breaking the cell wall is an important challenge and a costly unit operation. Multiple techniques have been carried out on C. vulgaris (Table 6). Cooling the system during mechanical cell breaking is always required because the high-energy input overheats the broken microalga and jeopardises the integrity of target compo nents by damaging or oxidising them. Enzymatic treatment is a promising technique that requires a deep understanding of the ultrastructure and composition of the cell wall in order to select the appropriate enzyme and to reduce the enzyme concentration required to hydrolyse the cell wall. According to Lee et al. [START_REF] Lee | Comparison of several methods for effective Iipid extraction from microalgae[END_REF] and Zheng et al. [START_REF] Zheng | Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves[END_REF] the best cell disruption techniques with 30% dry weight lipid recovery of C. vulgaris grown under autotrophic conditions were autoclaving, microwave, enzymatic and grinding with liquid nitrogen. Nonetheless, the quality of the target mole cules is susceptible to be different with respect to the cell disruption method applied. Thus, the amino acid profile of proteins obtained after conducting an alkaline treatment on C. vulgaris is different from the amino acid profile obtained after high-pressure homo genisation [START_REF] Safi | Release of hydro-soluble microalgal proteins using mechanical and chemical treat ments[END_REF].

The success of cell disruption techniques is generally assessed by conducting microscopie observations or by comparing the extracted yield of a component before and after applying the cell disruption.

Applications and potential interests

Biofuels

Dependency on energy sources is growing faster, especially with the exponential increase in demand, which is leading to more dramatic consequences for the environment. Third generation biofuel form algae or microalgae is considered as one of the alternatives to current biofuel crops such as soybean, corn, rape seed and lignocellulosic feedstocks because it does not compete with food and does not require arable lands to grow [START_REF] Singh | Renewable fuels from algae: an answer to debatable land based fuels[END_REF]. However, biofuel from microalgae is promising in the long term because it is now accepted that the production cost is still high and cannot yet compete with conventional fuel. But it competes favourably with crops by their potential of producing 10-20 times more oil [START_REF] Demirbas | Biofuels from algae for sustainable development[END_REF] within a shorter period of time. As mentioned previously, C. vulgaris has the potential to accumulate high amounts of lipids, especially while growing it under mixotrophic conditions. Its fatty acid profile showed to be suitable for biodiesel production with an oxidative stability after transforming it to biodiesel, and has properties [START_REF] Wang | Fast pyrolysis of microalgae remnants in a fluidized bed reactor for bio-oil and biochar production[END_REF] that comply with the US Standard (ASTM 6751), European Standard (EN 14214), Brazilian National Petroleum Agency (ANP 255) and Australian Standard for biodiesel [START_REF] Francisco | Microalgae as feedstock for biodiesel production: carbon dioxide sequestration, Iipid production and biofuel quality[END_REF] and also compared favourably with (ASTM and EN) an Indian biodiesel standard [START_REF] Mallick | Green microalga Chlore/la vulgaris as a potential feedstock for biodiesel[END_REF]. After lipid extraction the remaining residue is rich in proteins, carbohydrates and minor amounts of lipids. Thus, Wang et al. [START_REF] Wang | Fast pyrolysis of microalgae remnants in a fluidized bed reactor for bio-oil and biochar production[END_REF] applied fast pyrolysis on C. vulgaris remnants using an atmospheric-pressure fluidised bed reactor at 500 °C and obtained bio-oil and biochar representing 94% of energy recovery from the remnant, without forgetting the small amount of biogas recovered. However, the quality of bio-oil was poor due to the presence of nitrogen in significant amounts (12.8% dry weight). Besides, C. vulgaris has high starch content and algal starch proved to be a good source for bioethanol production. Hirano et al. [START_REF] Hirano | C02 fixation and ethanol production with microalgal photosynthesis and intracellular anaerobic fermentation[END_REF] extracted starch from C. vulgaris and achieved 65% ethanol-conversion rate after saccharification and fermenta tion with yeast. Hydrothermal liquefaction is another alternative route for biofuel production from microalgae. It involves the reaction of biomass in water at high temperature with or without the presence of a catalyst to obtain bio-crude [START_REF] Ross | Hydrothermal processing of microalgae using alkali and organic acids[END_REF]. The main advantage of this method is that it improved 10-15% the energetic Cumulative energy demand and energy production associated with the production of 1 MJ of biodiesel from C. vulgaris [START_REF] Sander | Life cycle analysis of algae biodiesel. !nt[END_REF].

taking into account ail the energetic debt for 1 MJ biodiesel production from C. vulgaris. The only positive balance obtained was 0.57 MJ for wet oil extraction with low nitrogen for cell growth (Table 7), and ail the other revealed negative balance. Hence, microalgal biofuel production still needs efficient improve ment to reduce energy input needed in order to reach competitive prices with petroleum in the market, and more important to be an overall sustainable production. value of C. vulgaris by acting on the whole biomass, suggesting that oil is also derived from carbohydrates and proteins [START_REF] Biller | Potential yields and properties of oil from the hydrothermal liquefaction of microalgae with different biochemical content[END_REF], and thus no need to stress the microalgae to increase lipid content. Hence, the best conditions applied on C. vulgaris in a batch reactor were 300-350 °C, with 150-200 bar in water or with the presence of an organic acid or heterogeneous catalysts, and the results indicate that bio-oil formation follows the trend lipids > proteins > carbo hydrates [START_REF] Ross | Hydrothermal processing of microalgae using alkali and organic acids[END_REF][START_REF] Biller | Potential yields and properties of oil from the hydrothermal liquefaction of microalgae with different biochemical content[END_REF][START_REF] Biller | Catalytic hydrothermal processing of microalgae: decomposition and upgrading of lipids[END_REF]. Nowadays, algal biofuel is suffering from several drawbacks, jeopardising its commercialisation on an industrial scale due to high production cost that is far from being competitive with fossil fuel, and also questioning the sustainability of this production. Hence, different studies considered life cycle assessment analysis as an effective tool to identify the reasons leading to production deficit and exploring its environmental impact [START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Collet | Life-cycle assessment of microalgae culture coupled to biogas production[END_REF][START_REF] Jorquera 0 | Comparative energy life-cycle analyses of microalgal biomass production in open ponds and photobioreactors[END_REF][START_REF] Lam | Microalgae biofuels: a critical review of issues, problems and the way forward[END_REF][START_REF] Sander | Life cycle analysis of algae biodiesel. !nt[END_REF][START_REF] Singh | A critical review of biochemical conversion, sustainability and life cycle assessment of algal biofuels[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: a comparison of raceways and air-lift tubular bioreactors[END_REF][START_REF] Yang | Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance[END_REF]. There fore, it was agreed that the major costs corne from infrastructure, production set-up, fertilizers, harvesting, drying the biomass, transportation, water footprints, cell disruption and oil extraction process. For instance, Lardon et al. [START_REF] Lardon | Life-cycle assessment of biodiesel production from microalgae[END_REF] performed an analysis by 7.2. Human nutrition C. vulgaris is one of the few microalgae that can be found in the market as a food supplement or additive [START_REF] Fradique | Incorporation of Chlore/la vulgaris and Spirulina maxima biomass in pasta products. Part 1: preparation and evaluation[END_REF][START_REF] Li | Isolation and purification of Iutein from the microalga Chlorella vulgaris by extraction after saponification[END_REF], colourant (C. vulgaris after carotenogenesis) and food emulsion [START_REF] Fernandes | Starch determination in Chlorella vulgaris -a comparison between acid and enzymatic methods[END_REF]. These products corne in different forms such as capsules, tablets, extracts and powder [START_REF] Liang | Current microalgal health food R&D activities in China[END_REF][START_REF] Yamaguchi | Recent advances in microalgal bioscience injapan, with special reference to utilization of biomass and metabolites: a review[END_REF]. Nevertheless, despite ail the healthy benefits that C. vulgaris and other microalgae can provide, with their remarkable richness in proteins, lipids, polysaccharides, pigments and vitamins, they are rather considered as nutraceu ticals instead of food products due to the Jack of clear common official legislations in terms of quality and requirements regarding microalgae [START_REF] Grobbelaar | Quality Contrai and Assurance: crucial for the sustainability of the applied phycology industry[END_REF][START_REF] Gulati | Legislation relating to nutraceuticals in the European Union with a particular focus on botanical-sourced products[END_REF]. Moreover, C. vulgaris extract proved to have preservative activity higher than those obtained synthetically, i.e., butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) [START_REF] Rodriguez-Garcia 1 | Evaluation of the antioxidant activity of three microalgal species for use as dietary supplements and in the preserva tion of foods[END_REF].

Animal feed

It is estimated that about 30% of microalgal production is sold for animal feed purposes [START_REF] Becker | Micro-algae as a source of protein[END_REF] due to the increasing demand for food with natural composition instead of synthesised ingredients. This has triggered intensive research into finding natural ingre dients that improve the quality of animal food products [START_REF] Fernandes | Starch determination in Chlorella vulgaris -a comparison between acid and enzymatic methods[END_REF]. Thus, while stressing C. vulgaris, it accumulates important amount of carotenoids and after feeding it to animais such as fish and poultry it showed interesting pigmentation potential for fish flesh and egg yolk in poultry, together with enhancing health and increasing life expectancy of animais [START_REF] Yamaguchi | Recent advances in microalgal bioscience injapan, with special reference to utilization of biomass and metabolites: a review[END_REF][START_REF] Becker | Micro-algae as a source of protein[END_REF][START_REF] Chac6n-Lee | Microalgae for "Healthy" foodspossibilities and challenges[END_REF][START_REF] Gouveia | Pigmentation of gilthead seabream, Sparus aurata (L 1875), using Chlore/la vulgaris (Chlor ophyta, Volvocales) microalga[END_REF][START_REF] Gouveia | Potential use of a microalga (Chlore/la vulgaris) in the pigmentation of rainbow trout ( Oncorhynchus mykiss) muscle[END_REF][START_REF] Gouveia | Functional food oil coloured by pigments extracted from microalgae with supercritical C02[END_REF][START_REF] Gouveia | Chlore/la vulgaris used to colour egg yolk[END_REF]. Moreover, C. vulgaris showed a protective effect against heavy metals and other harmful compounds (lead, cadmium, and naphtalene) by reducing significantly the oxidative stress induced by these harmful com pounds, and increasing the antioxidant activity in the organisms of tested animais [START_REF] Shimjy | Protective effects of Chlore/la vulgaris on liver toxicity in cadmium-administered rats[END_REF][START_REF] Vijayavel | Antioxidant effect of the marine algae Chlore/la vulgaris against naphthalene-induced oxidative stress in the albino rats[END_REF][START_REF] Yun | Protective effect of Chlore/la vulgaris against lead-induced oxidative stress in rat brains[END_REF].

Wastewater treatment

Many studies demonstrated the remarkable potential of C. vulgaris in fixating up to 74% carbon dioxide when grown in a photobioreactor [START_REF] Keffer | Use of Chlore/la vulgaris for C0(2) mitigation in a photobioreactor[END_REF], and in absorbing 45-97% nitrogen, 28-96% phosphorus and in reducing the chemical oxygen demand (COD) by 61-86% from different type of wastewater such as textile, sewage, municipal, agricultural and recalcitrant [START_REF] Aslan | Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae[END_REF][START_REF] Feng | Lipid production of Chlore/la vulgaris cultured in artificial wastewater medium[END_REF][START_REF] Lau | Wastewater nutrients removal by Chlore/la vulgaris: optimization through acclimation[END_REF][START_REF] Lim | Use of Chlore/la vulgaris for bioremediation of textile wastewater[END_REF][START_REF] Silva-Benavides | Nitrogen and phosphorus removal through laboratory batch cultures of microalga Chlore/la vulgaris and cyanobacterium Planktothrix isothrix grown as monoalgal and as co-cultures[END_REF][START_REF] Valderrama | Treatment of recalcitrant wastewater from ethanol and citric acid production using the microalga Chlore/la vulgaris and the macrophyte Lemna minuscula[END_REF][START_REF] Yun | Carbon dioxide fixation by algal cultivation using wastewater nutrients[END_REF]. Micro algae provide a pathway for the removal of vital nutrients (nitro gen and phosphorus), carbon dioxide, heavy metals and pathogens present in wastewaters and necessary for their growth. In addi tion, saving and requirements for chemical remediation and possible minimisation of fresh water use for biomass production are the main drivers for growing microalgae as part of a waste water treatment process [START_REF] Brennan | Biofuels from microalgae -a review of technologies for production, processing, and extractions of biofuels and co-products[END_REF]. Thus, a faster growth rate accom panied by an elimination of water-contamination level is a promising and advantageous process. Furthermore, performance of C. vulgaris in synthesised wastewater was improved when co immobilised in alginate beads with microalgae growth-promoting bacteria, and removed 100% of ammonium (NH 4 +) during four consecutive cycles of 48 h, and 83% for phosphorus after one cycle of 48 h [START_REF] De-Bashan | Removal of ammonium and phosphorus ions from synthetic wastewater by the microalgae Chlore/la vulgaris coimmobilized in alginate beads with the microalgae growth promoting bacterium Azospirillum brasilense[END_REF]. Thus, C. vulgaris is considered as one of the best microalga for bioremediation of wastewater with an impressive potential to completely remove ammonium and sometimes mod est potential to eliminate phosphorus present in the medium [START_REF] Gonzalez | Efficiency of ammonia and phosphorus removal from a colombian agroindustrial wastewater by the microalgae Chlore/la vulgaris and Scenedesmus dimorphus[END_REF].

Agrochemical applications

Blue-green algal extract excretes a great number of substances that influence plant growth and development [START_REF] Ordog | Beneficial effects of microalgae and cyanobacteria in plant/soil systems, with special regard to their auxin-and cytokinin-like activity[END_REF]. These micro organisms have been reported to benefit plants by producing growth promoting regulators, vitamins, ami.no acids, polypeptides, antibacterial and antifungal substances that exert phytopathogen biocontrol, and polymers such as exopolysaccharides that improve plant growth and productivity [START_REF] De Mulé | Effect of cyanobacterial inoculation and fertilizers on rice seedlings and postharvest soi! structure[END_REF].

The bio-fertilisation effect using algae extract are recom mended for increasing the growth parameters of many plants [START_REF] Adam | The promotive effect of the cyanobacterium Nostoc muscorum on the growth of some crop plants[END_REF][START_REF] Saffan | Allelopathic effects of cyanobacterial exudates on some metabolic activities of Cynara cardunculus seeds during germination[END_REF]. This is due to the biochemical profile of algae extract rich in nitrogenase, nitrate reductase, and minerais, which are essential nutrients for plant growth. The effect of the aqueous extract of C. vulgaris as foliar feeding on nutrients status, growth, and yield of wheat plant (Triticum aestivum L. var. Giz 69) has been investigated [START_REF] Shaaban | Green microalgae water extracts as foliar feeding to wheat plants[END_REF]. Thus, this study found that a concentration of 50% (v/v) algae extract as one time foliar spray (25 days after sowing) increased the growth yield and weight gain by 140% and 40%, respectively. Moreover, another study showed the bio fertilisation impact of C. vulgaris on growth parameters and physiological responses of Lactuca sativa germination seeds in culture medium containing microalga grown for 3, 6, 9, 12 and 15 days [START_REF] Faheed | Effect of Chlore/la vulgaris as bio-fertilizer on growth parameters and metabolic aspects of lettuce plant[END_REF]. As a result, the addition of C. vulgaris to the culture medium or soil significantly increased fresh and dry weight of seedlings as well as pigments content. The best treatments were 2 and 3 g dry alga kg-1 soil. Ali these studies were conducted on the Iiquid extract of C. vulgaris as bio-fertilizer for plant growth.

Therefore, further studies should be carried out to estimate costs on a large scale of the algae cell extract as foliar fertilizer, compared to other commercial foliar fertilizers present in the market.

Algo-refinery concept

The concept of biorefinery has been inspired from the petro Ieum refinery concept. It reflects a platform that integrates a process to fractionate the components of a biomass [START_REF] Naik | Production of first and second generation biofuels: a comprehensive review[END_REF][START_REF] Singh | Commercialization potential of microalgae for biofuels production[END_REF] to produce multiple products, and thus a biorefinery takes advantage of the various components in the biomass in order to improve the value derived from each component and also generating its own power, which maximises profitability and preserve the environ ment Hence, C. vulgaris with ail its potential and richness in proteins, carbohydrates, lipids, pigments, minerais and vitamins described previously deserves to be completely refined (Fig. 4) without forgetting that every operation unit should take into account the next stage and preserve the integrity of ail compo nents of interest in the downstream process.

Conclusion

This review reflects a broader image about the potential benefits of C. vulgaris, and gives an insight about the technological advancements already conducted. C. vulgaris can easily be cultured with inexpensive nutrient regime and has faster growth rate as compared to terrestrial energy crops and high biomass produc tivity. However, production-processing cost remains too high to compete in the market. Indeed, this is the major problem facing the microalgal industry nowadays, but it should be recognised that much improvements have been achieved during the Iast decade and expectations are estimating that the nearest future of micro algal industry will be strongly competitive on different Ievels in the market. The remarkable values of C. vulgaris set the ground work to additional research for futuristic applications where it will be represented as a strong candidate for tomorrow's bio-industry.

5. 3 . 7 . 3 . 7 . 4 . 7 . 5 .

 3737475 Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 5.4. Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 5. 5. Minerais and vitamins .......................................................................................... 271 6. Cell disruption techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 7. Applications and potential interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 7. 1. Biofuels ...................................................................................................... 272 7. 2. Human nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3 Animal feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3 Wastewater treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4 Agrochemical applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27 4 8. Algo-refinery concept ................................................................................................ 275 9. Conclusion ......................................................................................................... 275

Fig. 1 .

 1 Fig. 1. Schematic ultrastructure of C. vulgaris representing different organelles.

Fig. 2 .

 2 Fig. 2. Drawings showing the different phases of daughter cell-wall formation in Chlore/la vulgaris: (a) early cell-growth phase; (b) late cell-growth phase; (c) chloroplast dividing phase; (d) early protoplast dividing phase; (e) late protoplast dividing phase; (f) daughter cells maturation phase and (g) hatching phase [35].

Fig. 3 .

 3 Fig.3. Newly formed cells emerging outside the cell wall of the mother cell after hatching[START_REF] Yamamoto | Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlore/la vulgaris (Chlorophyta, Trebouxiophyceae)[END_REF].

4. 1 .

 1 Autotrophic growth 4.1.1. Open pond systems

Fig. 4 .

 4 Fig. 4. Algo-refinery concept from production to valorisation.

Table 1

 1 Amino acid profile of Chlore/la vulgaris compared to other resources expressed in grams per 100 g of protein.

	Amino acids	C. vulgaris b	C. vulgaris"	C. vulgaris e
	Aspartic acid	9.30	10.94	9.80
	Tbreonine	5.30	6.09	5.15
	Serine	5.80	7.77	4.32
	Glutamic acid	13.70	9.08	12.66
	Glycine	6.30	8.60	6.07
	Alanine	9.40	10.90	8.33
	Cysteine	n.d	0.19	1.28
	Valine	7.00	3.09	6.61
	Methionine	1.30	0.65	1.24
	Isoleucine	3.20	0.09	4.44
	Leucine	9.5	7.49	9.38
	Tyrosine	2.80	8.44	3.14
	Phenylalanine	5.50	5.81	5.51
	Histidine	2.00	1.25	1.97
	Lysine	6.40	6.83	6.68
	Arginine	6.90	7.38	6.22
	Tryptophan	n.d	2.21	2.30
	Ornithine	n.d	0.13	n.d
	Praline	5.00	2.97	4.90

n.d: not detected; N/A: not available.

Recornrnendation from FAO/WHO b Eggs b Soya b

	N/A	11.00	1.30
	4.00	5.00	4.00
	N/A	6.90	5.80
	N/A	12.60	19.00
	N/A	4.20	4.50
	N/A	n.d	5.00
	3.50	2.30	1.90
	5.00	7.20	5.30
	N/A	3.20	1.30
	4.00	6.60	5.30
	7.00	7.00	7.70
	6.00	4.20	3.20
	N/A	5.80	5.00
	N/A	2.40	2.60
	5.50	5.30	6.40
	N/A	6.20	7.40
	1.00	1.70	1.40
	N/A	n.d	n.d
	N/A	4.20	5.30

Table 3

 3 Potential pigments content in C. vulgaris under different growth conditions.

	Pigments	µgg-1 (dw)

Table 4

 4 Minerais profile of C. vulgaris.Maruyama et al.[START_REF] Maruyama | Application of unicellular algae Chlorella vulgaris for the mass-culture of marine rotifer Brachionus[END_REF] Tokusoglu and Unal[START_REF] Toku�oglu | Biomass nutrient profiles of three microalgae: Spirulina platensis, Chlore/la vulgaris, and Isochrisis galbana[END_REF] Panahi et al.[START_REF] Panahi | Investigation of the effects of Chlore/la vulgaris as an adjunctive therapy for dyslipidemia: results of a randomised open-label clinical trial[END_REF]

	Minerais Minerai content (g 100 g-1 )
	Microelements
	Na	N/A	1.35	N/A
	K	1.13	0.05	2.15
	Ca	0.16	0.59	0.27
	Mg	0.36	0.34	0.44
	p	N/A	1.76	0.96
	Macroelements
	Cr	N/A	tr	tr
	Cu	N/A	tr	0.19
	Zn	N/A	tr	0.55
	Mn	N/A	tr	0.40
	Se	N/A	tr	N/A
	I	N/A	N/A	0.13
	Fe	0.20	0.26	0.68
	tr: traces; N/A: not available.

Table 6

 6 Different cell disruption techniques carried out on C. vulgaris.

	Cell disruption
	Acid treatment
	Alkaline treatment
	Autoclaving
	Head milling
	Electroporation
	Enzymatic lysis
	French press
	Manual grinding
	High pressure homogeniser
	Microwaves
	Osmotic shoclc
	Ultra-sonication
	N/A: not available .

Table 7 Time

 7

		Experimental set-up	References
	25min	Hot Ac20+H2S04 (9 :1, v-v)	[70]
	60min	2N NaOH	[83]
	5min	125 °C+ 1.5 MPa	[106]
	20min	Beads: 0.4-0.6 mm	[31]
		Rotational speed: 1500 rpm
	5min	Beads: 0.1 mm,	[106]
		Rotational speed: 2800 rpm
	2min	Beads: 1 mm	[59]
	N/A	Electric field: 3 kV/cm	[73]
	60min 10h	Electrode: 2 cm Snailase (5 mg L -1 ), 37 °C Cellulase or lysozyme ( 5 mg L -1 ), 55 "C	[31]
	N/A	4% Cellulase+ 1% others (w/v)	[199]
		25 mM sodium phosphate buffer
		pH 7.0
		0.5 M mannitol
	10h	4% Cellulase+ 1% macerozyme RlO+ 1% pectinase (w/v)	[90]
		pH 6.0
		25 mM phosphate buffer
		0.6 M sorbitol/mannitol ( 1: 1)
	24h	Cellulase 0.5 mg L	[200]
		0.5 M mannitol
	N/A	138MPa	[201]
	N/A	N/A	[78]
	1-lO min	With liquid nitrogen or quartz	[31]
	N/A	With dry ice	[169]
	N/A	N/A	[202]
	5min	100 °C, 2450 MHz	[31,106]
	5min	40-50 °C, 2450 MHz	[107]
	48 h	10% NaCI	[106]
	60min	2N NaOH	[83]
	6min	10W	[84]
	20min	600W	[31]
	5min	lOkHz	[106]
	15-60min	N/A	[50]

Oil Nitrogen for Energy Cumulative energy Yield extraction culture production (MJ) demand (MJ) (MJ)

	Dry	Sufficient	2.7	5.29	-2.59
	Wet	Sufficient	3.84	3.99	-0.15
	Dry	Low	1.57	2.32	-0.75
	Wet	Low	2.23	1.66	0.57

Table 3 -

 3

	Cosmetics
	-Neutraceuticals
	-Phannaceuticals
	-Food supplement
	-Dyes and paints
	-Bio-fertili zers
	-Antioxidants
	-Functional food

Acknowledgements ...................................................................................................... 275 References ............................................................................................................. 275

Acknowledgements

The authors would like to thank Agence Nationale de la Recherche (ANR) for the financial support, and Laboratoire de Chimie Agro industrielle (LCA) for providing ail the necessary tools and require ments to dress this review.