Christos Nitsos
email: christos.nitsos@centralesupelec.fr

Rayen Filali
email: rayen.filali@centralesupelec.fr

Behnam Taidi
email: behnam.taidi@centralesupelec.fr

Julien Lemaire
email: julien.lemaire@centralesupelec.fr

Current and novel approaches to downstream processing of microalgae: A review

Keywords: Biotechnology, microalgae, harvesting, downstream processing, cell wall disruption, high value molecule extraction, bioenergy

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Since CO2 has been identified as a potent greenhouse gas, reduction of anthropogenic CO2 emissions has become a focal point of both policy and research initiatives. The extended use of fossil hydrocarbon reserves to produce heat, electricity, transportation fuel and precursors for the chemical industry, have unbalanced the natural carbon cycle leading to increased CO2 levels in the atmosphere. This has been linked to the increase in the average global temperature. Several world agreements have promoted the replacement of traditional fossil derived energy with renewable solar, wind, water, geothermal and biomass alternatives [START_REF] Chu | The path towards sustainable energy[END_REF][START_REF] Demirbas | Potential contribution of biomass to the sustainable energy development[END_REF]. More specifically, the Paris Agreement has set the ambitious goal to reduce CO2 emissions to a level that will limit average global temperature rise to less than 2 o C [START_REF] Krug | Accounting of GHG emissions and removals from forest management: a long road from Kyoto to Paris[END_REF]. In this context, not only reducing anthropogenic CO2 generation, but also CO2 capturing technologies have become important.

Microalgae ability to fix CO2 and convert it into useful molecules via photosynthesis makes them attractive for the emerging bio economy. Microalgae can be divided into prokaryotic blue and green algae (cyanobacteria), and eukaryotic microalgae that are further divided into Chlorophyta, Phaecophyta, and Chrysophyceae based on the type of their photosynthetic pigments (green, brown and gold, respectively) [START_REF] Masojídek | Photosynthesis in Microalgae[END_REF]. The production of biomass and useful metabolic products from a readily available and free carbon source is not the only advantage of microalgae. Their ability to utilize inorganic nutrients such as nitrogen and phosphorus, allows their use for tertiary and quaternary effluents treatment from secondary wastewater treatment process plants [START_REF] Abdel-Raouf | Microalgae and wastewater treatment[END_REF]. Organic carbon assimilation from microalgae in heterotrophic or photomixotrophic growth metabolism is also possible, enhancing their wastewater treatment potential [START_REF] Neilson | The uptake and utilization of organic carbon by algae: an essay in comparative biochemistry[END_REF] and potential valorization on the anaerobic co-digestion process. Coupling wastewater treatment with CO2 capture and biomass production may serve as an elegant and efficient solution of current environmental and economic challenges, for example by integrating CO2 from industrial flue gasses to microalgal wastewater treatment [START_REF] Molazadeh | The Use of Microalgae for Coupling Wastewater Treatment With CO2 Biofixation[END_REF][START_REF] Razzak | Integrated CO2 capture, wastewater treatment and biofuel production by microalgae culturing-A review[END_REF]. Although this type of an integrated approach has not yet reached maturity, the All-gas project is an example of municipal wastewater treatment, CO2 fixation and biogas production that has reached the demonstration scale [START_REF] Maga | Life cycle assessment of biomethane produced from microalgae grown in municipal waste water[END_REF].

As with higher plants, microalgae require land for their production, although the use of coastal waters are increasingly suggested. Microalgae, however, can give higher productivity compared to terrestrial crops due to their continuous cultivation mode, fast growth rate, short cycle doubling time, year-round operation and higher content in target molecules [START_REF] Eing | Pulsed Electric Field Treatment of Microalgae-Benefits for Microalgae Biomass Processing[END_REF]. By extrapolating results from laboratory scale microalgae cultivation to the commercial scale it was estimated that several Chlorophyta achieved much higher oil yields compared to land crops [START_REF] Nascimento | Microalgae Versus Land Crops as Feedstock for Biodiesel: Productivity, Quality, and Standard Compliance[END_REF]. For example, Chlamydomonas sp. and Chlorella vulgaris oil yield was 10.4 m 3 ha -1 year -1 and 44.4 m 3 ha -1 year -1 , respectively. Palm oil productivity on the other hand was estimated at only 6 m 3 ha -1 year -1 [START_REF] Nascimento | Microalgae Versus Land Crops as Feedstock for Biodiesel: Productivity, Quality, and Standard Compliance[END_REF]. Biomass yields of microalgae is also higher and in the range of 70-100 t DW ha -1 year -1 (metric tones DW per hectare per year), compared to 20 t DW ha -1 year -1 of miscanthus energy culture [START_REF] Eing | Pulsed Electric Field Treatment of Microalgae-Benefits for Microalgae Biomass Processing[END_REF][START_REF] Lewandowski | Delayed harvest of miscanthus-influences on biomass quantity and quality and environmental impacts of energy production[END_REF], where DW is the dry weight of the sample. Both open and closed systems have been used for microalgae culture. Open systems mainly consist of raceway ponds with paddle wheels [START_REF] Filali | Optimization of a raceway pond system for wastewater treatment: a review[END_REF] and open cascade systems [START_REF] Lee | Microalgal mass culture systems and methods: Their limitation and potential[END_REF] and are characterized by design and construction simplicity, lower capital cost, natural light and temperature control and easier maintenance. Their disadvantages include microbial contamination, high area demand, dependence on environmental conditions, insufficient mixing and mass transfer [START_REF] Ugwu | Microalgal Culture Systems: An Insight into their Designs, Operation and Applications[END_REF][START_REF] Acién | 1 -Photobioreactors for the production of microalgae[END_REF][START_REF] Eing | Pulsed Electric Field Treatment of Microalgae-Benefits for Microalgae Biomass Processing[END_REF]. Closed culture systems (photobioreactors) on the other hand require less space, enable better operational parameters control, allow axenic culture conditions, and good culture mixing, but at higher construction and operation cost. This makes them suitable for the cultivation of more microalgae species, including less robust species, or when the targeted product purity is essential [START_REF] Acién | 1 -Photobioreactors for the production of microalgae[END_REF]. Harvesting and concentration costs of microalgal biomass is a major inconvenience of the overall biorefinery process in both reactor types.

Microalgae cultivation requires significant area and land availability can be an important limiting factor of a microalgae-based bioeconomy. The low DW biomass productivity of photosynthetic microalgae cultures means that large volume of water is required to produce relatively low biomass amounts. Available alkaline or saline water reservoirs valorisation can partially cover the water requirements of microalgae cultivation [START_REF] Hannon | Biofuels from algae: challenges and potential[END_REF]; such a solution, however, would be limited to a small number of microalgae species adapted to survive in harsh media. Industrial process water, with or without supplements, can also serve as a growth medium for microalgae cultures.

Light is the main limiting factor in microalgae cultures [START_REF] Carvalho | Light requirements in microalgal photobioreactors: an overview of biophotonic aspects[END_REF]. Photosynthetic biomass growth and productivity relies on CO2 assimilation, and the flux of light energy the cells are exposed to [START_REF] Clement-Larosiere | Carbon dioxide biofixation by Chlorella vulgaris at different CO2 concentrations and light intensities[END_REF]. Additionally, exposure to light is not homogeneous inside photobioreactors, and depends on the geometrical configuration and the culture systems hydrodynamic properties. The cells auto-shading and formation of biofilms on the photobioreactor walls or on the culture's surface also have a strong impact on light penetration. The drop in light intensity is very steep even a few millimetres from the photobioreactor surface. Indeed, photobioreactors are naturally divided into light and dark zones, with efficient photosynthetic activity occurring only in the light zones. Depending on the bioreactor design parameters, such as tube diameter or pond depth, the dark areas can constitute the majority of the reactor volume [START_REF] Carvalho | Light requirements in microalgal photobioreactors: an overview of biophotonic aspects[END_REF]. Light limitation is a major reason for the low productivity of photoautotrophic cultures compared to heterotrophic fermentations.

Temperature effects can also significantly affect production efficiency and may require temperature control increasing investment and operation costs [START_REF] Ras | Temperature effect on microalgae: a crucial factor for outdoor production[END_REF]. Typical biomass concentration for photoautotrophic cultures is in the region of 0.5 to 2.0 g L -1 , whereas in heterotrophic cultures biomass concentrations of 18 g L -1 [START_REF] Cheng | Biodiesel production from Jerusalem artichoke (Helianthus Tuberosus L.) tuber by heterotrophic microalgae Chlorella protothecoides[END_REF] and 24 g L -1 [START_REF] Chen | Biomass and lipid production of heterotrophic microalgae Chlorella protothecoides by using biodiesel-derived crude glycerol[END_REF] are possible. For traditional industrial fermentations utilizing yeast, concentrations of greater than 50 or 60 g L -1 can be achieved [START_REF] Di Serio | A kinetic and mass transfer model to simulate the growth of baker's yeast in industrial bioreactors[END_REF][START_REF] Paredes-López | Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture[END_REF]. In turn, the low cell densities of microalgae cultures make downstream processing, such as cell concentration and extraction of valuable molecules, critical to the success of industrial microalgae valorization [START_REF] Gayen | Sustainable Downstream Processing of Microalgae for Industrial Application[END_REF].

Microalgae in the context of biorefinery

Deriving value from microalgae through a biorefinery operation (Fig. 1) can appear rather attractive as the carbon nutrient (CO2) is not only free but its removal from the atmosphere is desired. The use of microalgae, however, presents several challenges: the growth of microalgae requires significant quantities of carbon, nitrogen and phosphorus. The inorganic carbon required for photosynthesis can come either from CO2 or from dissolved bicarbonate ions in the medium [START_REF] Gonçalves | A review on the use of microalgal consortia for wastewater treatment[END_REF]. Inorganic nitrogen (nitrate, nitrite, ammonium, ammonia, molecular nitrogen) uptake is species dependent [START_REF] Hellebust | Regulation of Nitrogen Assimilation in Green Microalgae 16[END_REF]. Organic compounds such as urea can also be used as a nitrogen source [START_REF] Kim | Effects of nitrogen sources on cell growth and biochemical composition of marine chlorophyte Tetraselmis sp. for lipid production[END_REF]. Phosphorus, on the other hand, is assimilated mainly as phosphate ions, with the ability of organic phosphorus uptake also shown [START_REF] Singh | Modelling phosphorus uptake in microalgae[END_REF]. Most microalgae have the ability to uptake more phosphorus than is required for growth and store it in the form of inorganic polyphosphate. This natural adaptation to phosphorus poor environments is termed luxury phosphorus uptake [START_REF] Solovchenko | Luxury phosphorus uptake in microalgae[END_REF]. The use of industrial fertilizers as sources of nitrogen and phosphorus can be a cheaper alternative to growth medium formulation increasing the economic sustainability of microalgae culture and valorisation. Magnesium (Mg 2+ ), an important component of the photosynthetic apparatus, is also critical for microalgae growth and biomass productivity is related to its concentration in the growth medium [START_REF] Amor-Ben Ayed | Magnesium Uptake by the Green Microalga Chlorella vulgaris in Batch Cultures[END_REF], 2015). Coupling microalgae biorefinery with process water treatment and carbon dioxide biofixation is economically attractive for sustainable microalgae culture. Process water is rich in inorganic content that is expensive to remove in traditional tertiary process water treatment operations. Their use as growth media in a biorefinery would reduce the cost of both process water treatment and microalgae culture. In addition, the process water would provide a readily available source of water, and organic and/or inorganic compound necessary for microalgae growth. The ability of microalgae to remove nitrogen [START_REF] Delgadillo-Mirquez | Nitrogen and phosphate removal from wastewater with a mixed microalgae and bacteria culture[END_REF] magnesium [START_REF] Ben Ayed | The Use of Chlorella Vulgaris to Accumulate Magnesium under Different Culture Conditions[END_REF] and excess phosphorus -through luxury phosphorus uptake- [START_REF] Solovchenko | Luxury phosphorus uptake in microalgae[END_REF] from their growth medium, makes them suitable for such water depollution applications (Fig. 1). The biomass produced can be used as a fertilizer or converted to renewable fuels and energy and increase the operation's sustainability. This could be an interesting solution despite the problem of light penetration into the culture caused by the secondary effluent's turbidity and colour. Lack or limitations of nutrients that are necessary for growth in the medium can affect the composition of the microalgae biomass. Such limitations are often desirable and are induced as a stress factor; can be accomplished by formulation of the growth medium. For example, nitrogen limitation in the latter stages of microalgae growth can induce lipid production as an energy storage mechanism, and therefore increase the value of the produced biomass [START_REF] Juneja | Effects of Environmental Factors and Nutrient Availability on the Biochemical Composition of Algae for Biofuels Production: A Review[END_REF]. The effects of limitation of major nutrients in the growth medium can be seen in Table 1.

Figure 1 Table 1 The low dry weight content of microalgae cultures (typically around 1 g L -1 ) makes harvesting and concentration an expensive unit operation of any biorefinery. Harvesting and dewatering are the main downstream processes required for any applications where the whole microalgae biomass can be used directly as the final product, such as for animal and fish feed or food supplements (e.g. Arthrospira platensis tablets and powder). Biomass concentration can be achieved by the combination of some techniques such as settling, decantation, centrifugation and filtration; all followed by an operatory process of microalgae conditioning by drying of the concentrated pastes [START_REF] Raja | Applications of microalgal paste and powder as food and feed: An update using text mining tool[END_REF]. Harvesting and dewatering processes may also include membrane filtration, shaking separators, flotation and flocculation, or pressing.

The various molecules of interest in the microalgae biomass can be classified into structural e.g. cell wall carbohydrates and proteins and cell membrane lipids; and non-structural e.g. storage lipids and carbohydrates, vitamins, polyunsaturated fatty acids and pigments [START_REF] Cuellar-Bermudez | Extraction and purification of highvalue metabolites from microalgae: essential lipids, astaxanthin and phycobiliproteins[END_REF][START_REF] Shannon | Enzymatic extraction of fucoxanthin from brown seaweeds[END_REF]. These may be the final target molecules, as in the case of ω-3 or ω-6 fatty acids and β-carotene that can be used in food supplements; or used as feedstock for fuels and chemical products, as in the case of carbohydrates and lipids. Such diversity in the chemical composition of microalgae, requires a fractionation step of the target molecule. The fractionation process must be selective in order to maximize the desired molecule's yield and minimize formation of unwanted and inhibitory products.

Cell disruption can be achieved by physical (Ultrasonication, High Pressure Homogenization, Bead Milling, Pulsed Electric Field) chemical (Acid, Alkaline and Oxidation), thermal (Hydrothermal, Steam Explosion) and biological methods (Enzymatic treatment) (Soo Youn Lee et al., 2017). During cell wall disruption, intracellular molecules can be extracted with organic solvents [START_REF] Araujo | Extraction of lipids from microalgae by ultrasound application: Prospection of the optimal extraction method[END_REF], ionic liquids (Y. Zhang et al., 2018), supercritical CO2 [START_REF] Reyes | Effect of high-pressure compaction on supercritical CO2 extraction of astaxanthin from Haematococcus pluvialis[END_REF][START_REF] Cheng | Comparative study of lipid extraction from microalgae by organic solvent and supercritical CO2[END_REF] or supercritical mixtures of CO2 and solvents [START_REF] Obeid | Supercritical carbon dioxide extraction and fractionation of lipids from freeze-dried microalgae Nannochloropsis oculata and Chlorella vulgaris[END_REF]. Cell-wall components fractionation, may require their further depolymerization via hydrothermal [START_REF] Lorente | Microalgae fractionation using steam explosion, dynamic and tangential cross-flow membrane filtration[END_REF], acid [START_REF] Hernández | Saccharification of carbohydrates in microalgal biomass by physical, chemical and enzymatic pretreatments as a previous step for bioethanol production[END_REF] or enzymatic hydrolysis [START_REF] Sierra | Enzymatic cell disruption of the microalgae Chlamydomonas reinhardtii for lipid and protein extraction[END_REF]. The targeted molecules separation and purification can be performed with chromatography [START_REF] Bousquet | Characterization and purification of polyunsaturated fatty acids from microalgae by gas chromatography-mass spectrometry and countercurrent chromatography[END_REF][START_REF] Fábryová | Purification of lutein from the green microalgae Chlorella vulgaris by integrated use of a new extraction protocol and a multi-injection high performance counter-current chromatography (HPCCC)[END_REF] and membrane technology [START_REF] Giorno | Purification of triacylglycerols for biodiesel production from Nannochloropsis microalgae by membrane technology[END_REF].

2 Microalgal cell wall recalcitrance

The bulk production of fuels such as biogas [START_REF] Córdova | Physical Pretreatment Methods for Improving Microalgae Anaerobic Biodegradability[END_REF] or bio-oil through hydrothermal liquefaction [START_REF] Hu | Investigation of an alternative cell disruption approach for improving hydrothermal liquefaction of microalgae[END_REF] are common methods of deriving value from microalgae biomass. In such cases, cell disruption does not facilitate extraction or fractionation of molecules but enhances the conversion reactions towards the desired end products (Mahdy et al., 2014a;[START_REF] Zhang | Bio-oil production from hydrothermal liquefaction of ultrasonic pre-treated Spirulina platensis[END_REF]. Cell wall disruption may be essential for the efficient extraction of intracellular molecules such as pigments and oils. Cell wall components may themselves be target molecules, requiring disruption for their efficient fractionation e.g. carbohydrates for bioethanol production [START_REF] Kim | Bioethanol production from micro-algae, Schizocytrium sp., using hydrothermal treatment and biological conversion[END_REF], or hydrocarbons from the Botryococcus extracellular matrix [START_REF] Ciudad | Performance of an enzymatic extract in Botrycoccus braunii cell wall disruption[END_REF] Cell wall structure (Fig. 2) and composition has been extensively reviewed elsewhere [START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF]. Cell wall thickness and chemical composition have been identified as the most important factors determining the cell wall strength (Zhipeng Duan et al., 2017). Cell wall robustness can vary related to microalgae species [START_REF] Montsant | Presence of a Nonhydrolyzable Biopolymer in the Cell Wall of Vegetative Cells and Astaxanthin-Rich Cysts of Haematococcus pluvialis (Chlorophyceae)[END_REF]. Isochrysis Galbana completely lacks a cell wall [START_REF] Throndsen | Chapter 5 -The Planktonic Marine Flagellates[END_REF], making it very fragile, whereas Chlorella sp. have a glucosamine rigid cell wall and a hemicellulose-like cell wall layer mainly of galactose and mannose sugars [START_REF] Rodrigues | Evaluation of Chlorella (Chlorophyta) as Source of Fermentable Sugars via Cell Wall Enzymatic Hydrolysis[END_REF]. Haematococcus pluvialis has a very thick cell wall composed of cellulose-like polysaccharides and a very resistant polymer, algaenan [START_REF] Hagen | Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation[END_REF]. Algaenan is one of the most inert microalgae cell wall components and very resistant to physical, chemical and enzymatic pretreatment [START_REF] Mendes-Pinto | Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability[END_REF].

The cell wall of the blue-green alga A. platensis is composed of four layers one of which is a more robust peptidoglycan layer [START_REF] Van Eykelenburg | On the morphology and ultrastructure of the cell wall of Spirulina platensis[END_REF]. Tisochrysis lutea has a cell wall consisting of two-layered plates [START_REF] Orlova | Ultrastructural Changes in Marine Microalgae from Different Taxonomic Groups during Batch Cultivation[END_REF]. A comparative study of cell disruption of various species with ultrasonication has correlated cell wall composition and structure to resistance to physical damage (shear stress) (Zhipeng Duan et al., 2017). From the treatment of green algae and cyanobacteria the following suggested order of cell wall robustness can be proposed for a few species: Chlorella pyrenoidosa > Synechococcus elongatus > Microcystis aeruginosa > Chlamydomonas reinhardtii. The more resistant cell wall of C. pyrenoidosa is composed mainly of cellulose rich polysaccharides, the cyanobacteria of a peptidoglycan layer with an extracellular mucilage layer and a further, serrated external layer (S-layer) and finally, of the rupture prone C. reinhardtii of hydroxyproline-rich glycoproteins (Zhipeng Duan et al., 2017). The cell wall of Chlorella strains also contains a chitin-like layer [START_REF] Kapaun | A chitin-like glycan in the cell wall of a Chlorella sp. (Chlorococcales, Chlorophyceae)[END_REF].

Besides the organic layers some microalgae cell walls have inorganic components, like the cell wall of the diatom Phaeodactylum tricornutum, that contains a silica shell [START_REF] Gügi | Diatom-Specific Oligosaccharide and Polysaccharide Structures Help to Unravel Biosynthetic Capabilities in Diatoms[END_REF].

Similar indirect suggestions for a possible correlation between cell wall composition and robustness exist in other articles. In a study of rupture of Tetraselmis suecica., Chlorella sp. and Nannochloropsis sp, by High Pressure Homogenization the pressure required to rupture 50% of the cells was determined at 170 bar, 1060 bar and 1380 bar, respectively [START_REF] Spiden | Quantitative evaluation of the ease of rupture of industrially promising microalgae by high pressure homogenization[END_REF]. The order of cell wall robustness of these species therefore can be proposed as: Nannochloropsis sp. > Chlorella sp. > T. suecica.

Although the cell wall composition of these species was not measured in the study, it is noted from the literature that they are made up from polymers resistant to mechanical rupture, polysaccharides and glycoproteins, and carbohydrate scales, for Tetraselmis suecica., Chlorella sp. and Nannochloropsis sp, respectively [START_REF] Spiden | Quantitative evaluation of the ease of rupture of industrially promising microalgae by high pressure homogenization[END_REF]. In a similar approach, cell fragility at 1750 bar was studied (Taleb A. 2016) from which the order of cell wall robustness can be proposed for the following species: Scenedesmus sp. UTEX 1589 > Nannochloropsis salina 537 CCMP > Scenedesmus obliquus 393 UTEX > Nannochloropsis gaditana 527 CCMP > Parachlorella kessleri 2229 UTEX. Again, the cell wall composition of the species was not provided.

A second level of recalcitrance is presented by microalgae organized in coenobial communities, held together by extracellular structures. For example, Botryococcus strains have thick cell walls, rich in hemicellulose-like carbohydrates predominantly of arabinose and galactose composition, an algaenan layer, and form colonies held together with a hydrocarbon extracellular matrix. Equally complex is the cell wall of Scenedesmus, containing cellulose-like carbohydrate layers, algaenan and an external coenobial seath when the cells are grown in colonies.

Figure 2 Modifications during the different stages of microalgae growth can also significantly affect the recalcitrance of the cell wall. For example, H. pluvialis vegetative cells are susceptible to breakage at moderate pressures (4000 to 10,000 psi), whereas cysts with thicker algaenan-rich cell walls required very high pressure (20,000 psi) for complete cell disruption [START_REF] Montsant | Presence of a Nonhydrolyzable Biopolymer in the Cell Wall of Vegetative Cells and Astaxanthin-Rich Cysts of Haematococcus pluvialis (Chlorophyceae)[END_REF]. It has also been shown that growth rate can also affect cell wall robustness, as cultures grown at higher rates produce cells with thinner walls [START_REF] Němcová | Cell wall development, microfibril and pyrenoid structure in type strains of Chlorella vulgaris, C. kessleri, C. sorokiniana compared with C. luteoviridis (Trebouxiophyceae, Chlorophyta)[END_REF]. In general, the thickness and chemical composition of the cell wall can vary a lot even in the same species depending on the growth conditions [START_REF] Liu | Optimization of Parameters for Isolation of Protoplasts from the Antarctic Sea Ice Alga Chlamydomonas Sp. ICE-L[END_REF]. Concerning the enzymatic methods, the multilayered structure of a cell wall poses an additional challenge as each layer may require enzymatic treatment by a different class of enzymes, increasing the enzymatic cocktail's complexity and cost, or requiring multi-stage pretreatment. In electricity-based pretreatment methods, such as Pulsed Electric Field, the small size of the microalgae cell also enhances resistance to disruption.

Structural modifications induced from the specie's growth state, energy requirements, pretreatment costs and price of the desired products must be carefully considered in order to select an appropriate cell disruption method. Sensitivity of the targeted molecules to degradation during pretreatment must also be considered in order to minimize losses in the final product yield and biological activity. Some interesting microalgae species, their cell wall composition, targeted molecules, and their respective prices, are summarized in Table 2.

Table 2 2. Harvesting

Harvesting is the solid-liquid separation of cells from the growth medium. The microalgae cells are the main culture product, whereas the nutrient depleted medium is a valuable water source that can be recycled into the bioreactor directly or supplemented with nutrients, if required. The very low microalgae biomass concentrations make harvesting a critical operation unit for a biorefinery from the economical point of view. Further downstream processing steps such as cell disruption and extractionfractionation cannot be performed at low solids content as the high diluted solutions would not be treated. The typically low biomass yield in microalgae production systems poses severe economic and energetic restrictions to the harvesting and consequently to the whole biorefinery process. Operating cost cannot exceed the value of final products, and since at least some of the biorefinery end products will be fuels, the harvesting energy should not exceed the biomass energy content. The relatively small size of microalgae cells -typically between 2 and 25 μm as well as the negative surface charge pose additional challenges to the harvesting step. Harvesting can be divided into preconcentration, aiming to increase the initial biomass content from 0.5-1.0% to around 3% and dewatering that can lead to a very concentrated microalgal biomass, up to 25% in dry weight [START_REF] Muylaert | 5 -Harvesting of microalgae: Overview of process options and their strengths and drawbacks[END_REF]. In the case of larger size filamentous microalgae like A. platensis the biomass can be efficiently harvested using vibrating sieves, a relatively simple technology [START_REF] Shelef | Microalgae harvesting and processing: a literature review[END_REF].

Sedimentation

Natural sedimentation of microalgae cells is a very simple and low-cost harvesting method. The speed of sedimentation depends on cell diameter and the density difference between the cell and the growth medium, according to Stoke's equation, commonly used as a first approximation:

𝑉 𝑠 = 𝑔𝑑 2 (𝜌 𝑐 -𝜌 𝑚 ) 18𝜇
where, Vs is sedimentation velocity, g accelertion of gravity, d cell diameter, ρc microalgae cells density, ρm medium density, and μ the medium viscosity. The Stoke's equation can describe sedimentation of round shaped cells with good approximation, however its use in not universal and for different cell shapes the equation cannot be applied [START_REF] Peperzak | Phytoplankton sinking rates in the Rhine region of freshwater influence[END_REF]. Indeed, when the shape of the microalgae is not spherical or where there are formations like thorns on the surface of the cell the correlation of cell size to settling rate can be negative [START_REF] Peperzak | Phytoplankton sinking rates in the Rhine region of freshwater influence[END_REF]. A negative correlation between size and sedimentation rate has also been observed in the case of some microalgae colonies [START_REF] Peperzak | Phytoplankton sinking rates in the Rhine region of freshwater influence[END_REF]. It seems that cell density -or the difference between cell and medium density-is more significant and higher cell densities will result in higher sedimentation rates [START_REF] Peperzak | Phytoplankton sinking rates in the Rhine region of freshwater influence[END_REF]. Sedimentation velocity can vary several orders of magnitude among microalgae species depending on cell size, density and shape. As a general rule small diameter and low-density microalgae tend to sediment very slowly compared to larger and denser cells [START_REF] Mathimani | A comprehensive review on harvesting of microalgae for biodiesel -Key challenges and future directions[END_REF].

In mixed microalgae cultures, sedimentation by gravity is an efficient harvesting method if the colony is dominated by species with large settling velocities. When slow settling microalgae become dominant the harvesting efficiency is reduced even at prolonged settling periods of 24h [START_REF] Park | Recycling algae to improve species control and harvest efficiency from a high rate algal pond[END_REF]. Natural sedimentation has been proposed as a harvesting method or pre-concentration step even for slow settling microalgae like Monoraphidium sp. that could be harvested after 24 h with a 98% yield [START_REF] Yu | Isolation of a novel strain of Monoraphidium sp. and characterization of its potential application as biodiesel feedstock[END_REF].

The filamentous cyanobacterium A. platensis with a fast sedimentation velocity of 0.64 m h -1 and accumulation of carbohydrates during nitrogen starvation could be concentrated 15 times by spontaneous sedimentation that achieved 93% removal of water [START_REF] Depraetere | Harvesting carbohydrate-rich Arthrospira platensis by spontaneous settling[END_REF]. The diatom Amphora with the impressive sedimentation velocity of 2.91 m h -1 , can be harvested by a simple low cost and fast gravity sedimentation [START_REF] Chtourou | Characterization of Amphora sp., a newly isolated diatom wild strain, potentially usable for biodiesel production[END_REF]. It also exhibits relatively fast growth rate and significant fat accumulation. Microalgae with such properties have a great advantage compared to slowly settling ones in terms of harvesting efficiency. Their valorization could be prioritized over other species as they may be an economically viable solution to a truly efficient microalgae biorefinery.

Nitrogen starvation in Nannochloropsis salina and Chlorella sp. cultures led to reduction in the cell density and settling velocity by as much as an order of magnitude at the latter culture stages [START_REF] Baroni | The effect of nitrogen depletion on the cell size, shape, density and gravitational settling of Nannochloropsis salina, Chlorella sp. (marine) and Haematococcus pluvialis[END_REF]. This could be a disadvantage for harvesting as nitrogen starvation is typically related to lipid accumulation, and the lipid rich cells could be harder to separate. A compromise might be necessary between biomass, lipid yields and settling velocity if unassisted gravity sedimentation is being considered as the sole method of harvesting. However, as gravity sedimentation can seldom induce the required biomass dewatering, its use for pre-concentration followed by another method is preferable. For example, gravity sedimentation coupled with centrifugation can reduce the harvesting cost and energy by a factor of 30 compared to centrifugation alone [START_REF] Badvipour | Process evaluation of energy requirements for feed production using dairy wastewater for algal cultivation: Theoretical approach[END_REF].

Coagulation-Flocculation

Coagulation and flocculation are conventional technologies used in water treatment to promote the separation of small particles by sedimentation. Both methods involve the addition of compounds which cause the agglomeration of particles into larger flocs to increase their sedimentation velocity, according to Stoke's equation. Coagulation is a chemical technique based on the neutralization of charge of the microalgae surface to disrupt the repulsive forces and promote cell aggregation. Flocculation is a physical method that uses compounds on which cells can clump together without involving surface charge neutralization. There is a subtle difference between the two phenomena but in the scientific literature most authors only refer to flocculation.

Coagulation can occur with the addition of inexpensive multivalent salts such as Al2(SO4)3 that could remove more than 85% of N. salina in 62 min, although at a relatively high dosage -229 mg L -1 [START_REF] Chatsungnoen | Continuous flocculation-sedimentation for harvesting Nannochloropsis salina biomass[END_REF]. Coagulation of C. reinhardtii at elevated pH values was induced by ferric (FeCl3), calcium (CaCl2), and magnesium chlorides (MgCl2). Even at very low concentrations (>5 mM) more than 90% biomass harvesting could be achieved making this an attractive and inexpensive method [START_REF] Fan | Flocculation of Chlamydomonas reinhardtii with Different Phenotypic Traits by Metal Cations and High pH[END_REF].

Coagulation with multivalent cations can be improved by pH adjustment to basic values. Indeed, its efficiency can be related to the cells zeta potential, a pH depended phenomenon [START_REF] Fan | Flocculation of Chlamydomonas reinhardtii with Different Phenotypic Traits by Metal Cations and High pH[END_REF].

Adjustment of pH to 9.51 induced coagulation of Chaetoceros calcitrans and 89% of cells were harvested with a sedimentation rate of 0.125 m h -1 and a concentration factor of 4 in 10 min (Şirin et al., 2015). Adjustment of pH to 10.2 with addition of sodium or potassium hydroxide led to high harvesting efficiency (> 90%) of C. calcitrans biomass. At increased polyelectrolyte dosages the sedimentation rate was greatly improved without loss of cell viability or harvesting yield [START_REF] Harith | Effect of different flocculants on the flocculation performance of microalgae, Chaetoceros calcitrans, cells[END_REF]. C. vulgaris biomass required a pH higher than 11 for coagulation efficiency above 90% with magnesium hydroxide for reuse [START_REF] Vandamme | Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications[END_REF]. Microalgae biomass contamination by the coagulation reagents is the biggest process disadvantage as they can reduce the extracted biomolecules quality or interfere with other downstream processing operations [START_REF] Vandamme | Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications[END_REF]. This can be overcome by re-suspending the coagulation reagent after harvesting. For example in the coagulation of C. vulgaris and Phaeodactylum tricornutum with Mg(OH)2, a mild acidification of the microalgae slurry could dissolve 95% of the precipitated magnesium hydroxide [START_REF] Vandamme | Reversible Flocculation of Microalgae using Magnesium Hydroxide[END_REF].

The use of inorganic reagents can be less effective when used for the harvesting of marine microalgae due to the shielding of cells by ions in the medium that lead to inhibition of floc formation [START_REF] Vandamme | Flocculation as a low-cost method for harvesting microalgae for bulk biomass production[END_REF]. Organic polymers on the other hand can be effective flocculants for both marine and freshwater microalgae (Table 3). Organic polymers with positive charges also neutralize the negative surface charge of microalgae cells but can act on several cells simultaneously leading to a bridging effect (Figure 3) and floc formation. Reduced harvesting efficiencies have sometimes been observed at increased dosages of cationic polymers [START_REF] Lam | Harvesting and cell disruption of microalgae[END_REF]. This has been explained by a protection of the cells by the excess flocculant leading to surface charge reversal and repulsive forces from the cationic polymers inhibiting floc formation (Figure 3).

Figure 3 To achieve flocculation, charged organic polymers can be used such as, chitosan a natural-based cationic biopolymer, which is efficient at low concentrations. At 20 mg L -1 and pH 8 a 90% harvesting efficiency and 80% cell viability of C. calcitrans is possible [START_REF] Harith | Effect of different flocculants on the flocculation performance of microalgae, Chaetoceros calcitrans, cells[END_REF]. Although alginate has been shown to not induce flocculation in some cases [START_REF] Zhu | Effect of Processing Parameters on Flocculation of <Emphasis Type="Italic">Picochlorum oklahomensis</Emphasis>[END_REF], its addition with chitosan that has an opposite charge, creates a polyelectrolyte complex that can enable harvesting of microalgae by flocculation at ppm range concentrations. Scenedesmus obliquus biomass was harvested with an efficiency of 86%, with this way [START_REF] Matter | Harvesting of microalgae Scenedesmus obliquus using chitosan-alginate dual flocculation system[END_REF]. Cationic starch is another cationic biopolymer that can induce flocculation of Chlorella protothecoides at 40 mg L -1 and achieves an extraction efficiency of 98% at near neutral and alkaline pH values [START_REF] Letelier-Gordo | Effective harvesting of the microalgae Chlorella protothecoides via bioflocculation with cationic starch[END_REF]. Polyacrylamide is another type of organic polymer that has been extensively used for harvesting of microalgae by flocculation. They achieved very high flocculation efficiencies between 90% and 100% for a variety of microalgae species. [START_REF] Van Haver | Polyelectrolyte flocculants in harvesting microalgal biomass for food and feed applications[END_REF]. The charge of the polymers seems to play a more important role in the case of marine microalgae. For example, in the marine microalgae N. salina using polyacrylamide polymers of medium, high, and very high charge -with all other parameters kept the same-improved the harvesting efficiency from 73% to 88% to 94%, respectively (Table 3). This effect was not observed for the freshwater microalgae C. vulgaris, where the harvesting efficiency remained almost 100% irrespectively of the charge of the polymer [START_REF] Van Haver | Polyelectrolyte flocculants in harvesting microalgal biomass for food and feed applications[END_REF]. This could be caused by the presence of salt ions in the marine microalgae medium that can partially neutralize or counteract the charge of the polymer; or it could be related to the surface charge of the individual species. In both cases it is worth noting that the very promising harvesting results were achieved with very low polyacrylamide dosages; 3 mg L -1 for N. salina and 1.66 mg L -1 for C. vulgaris [START_REF] Van Haver | Polyelectrolyte flocculants in harvesting microalgal biomass for food and feed applications[END_REF]. Despite these promising results, polyacrylamides are potentially hazardous chemicals and their presence can contaminate the harvested biomass, making it unsuitable for pharmaceutical, cosmetic and food applications, and limiting their use to biofuels production.

Tannins is a class of phenolic biomolecules -present in plant biomass-that are very efficient flocculants (Table 3). POLYSEPAR ® CFL25, a tannin quaternary ammonia salt with low molecular weight and high charge showed 95 % harvesting efficiency of Chlorella sp. cells at a dosage of 30 mg L -1 (Van Haver and Nayar, 2017). Tanfloc SL, another tannin polymer of low molecular weight and low to medium charge could harvest 100% of C. vulgaris cells at a dose of 5 mg L -1 [START_REF] Van Haver | Polyelectrolyte flocculants in harvesting microalgal biomass for food and feed applications[END_REF]. A lower cost bio flocculant has been produced by fermentation of rice bran with Bacillus agaradhaerens C9. The bioflocculant is possibly a hemicellulose oligomers hydrolysate as xylanase activity was detected in the fermentation broth. It achieved around 90% harvesting efficiency at a 60 mg L -1 concentration [START_REF] Liu | Valorization of untreated rice bran towards bioflocculant using a lignocellulose-degrading strain and its use in microalgal biomass harvest[END_REF]. Other bioflocculant polymers such as poly γ-glutamic acid produced by Bacillus Licheniformis have been successfully used for the flocculation of Desmodesmus brasiliensis at > 98 % efficiency. Anionic and non-ionic polymers showed no or poor flocculation efficiency in most cases, or had inferior performance compared to cationic ones [START_REF] Tilton | The flocculation of algae with synthetic polymeric flocculants[END_REF][START_REF] Udom | Harvesting microalgae grown on wastewater[END_REF][START_REF] Uduman | Marine microalgae flocculation and focused beam reflectance measurement[END_REF]. This could be caused by the fact that the surface charges of microalgae are usually negative [START_REF] Danquah | Dewatering of microalgal culture for biodiesel production: exploring polymer flocculation and tangential flow filtration[END_REF] and their neutralization requires positively charged flocculants or combination with coagulants. Another interesting approach is thermal flocculation without the addition of a flocculating agent. For example, thermal treatment of C. vulgaris and S. obliquus induced the secretion of organic matter that acts as a flocculating agent and causes the formation of flocs. The authors have hypothesized that the organic matter can be polysaccharides and/or protein [START_REF] Xue | Optimization of thermal preflocculation treatment for effective air flotation harvesting of microalgae[END_REF] As harvesting can represent up to 30% of the total microalgae processing cost, coagulant or flocculant price can have a negative impact on biorefinery economics [START_REF] Wu | Evaluation of several flocculants for flocculating microalgae[END_REF]. Prices can vary significantly between the less expensive coagulant salts such as Al2(SO4)3 (0.2 $ kg -1 ) and the more expensive flocculant: chitosan (19.6 $ kg -1 ) [START_REF] Wu | Evaluation of several flocculants for flocculating microalgae[END_REF]. It has been deduced that coagulationflocculation can be only marginally cheaper than centrifugation; auto flocculation, bioflocculation and electroflocculation methods were suggested as more economically viable alternatives [START_REF] Vandamme | Flocculation as a low-cost method for harvesting microalgae for bulk biomass production[END_REF]. The use of polyelectrolytes as economical flocculants has also been proposed, as the doses required are up to two orders of magnitude less than salts or chitosan and their cost is comparable to the inexpensive salt coagulants. [START_REF] Granados | Evaluation of flocculants for the recovery of freshwater microalgae[END_REF].

Table 3 2.3 Centrifugation Centrifugation process simply increases the g force accelerating settlement as stated by Stokes equation.

Strain morphology can play an important role in the centrifugation efficiency. For example, linear A. platensis filaments could be harvested at around 85% efficiency at 4000 x g for 10 min, in a laboratoryscale fixed angle rotor centrifuge. In contrast helical A. platensis filaments showed poor harvesting yield (around 50%) even at 9000 x g [START_REF] Cuellar-Bermudez | Harvesting of Arthrospira platensis with helicoidal and straight trichomes using filtration and centrifugation[END_REF]. S. obliquus cells could be removed from the culture by centrifugation at 3000 x g for 10 min, in a 750W TD5A centrifuge, with a harvest efficiency of 99.3% [START_REF] Wang | Microalgae harvest influences the energy recovery: A case study on chemical flocculation of Scenedesmus obliquus for biodiesel and crude bio-oil production[END_REF]. Various types of centrifuges can be used to harvest and concentrate microalgae, such as, spiral plate centrifuge [START_REF] Collet | Life-cycle assessment of microalgae culture coupled to biogas production[END_REF], decanter centrifuge [START_REF] Adesanya | Life cycle assessment on microalgal biodiesel production using a hybrid cultivation system[END_REF], disk stack centrifuge [START_REF] Milledge | Disc stack centrifugation separation and cell disruption of microalgae: A technical note[END_REF], and hydrocyclone [START_REF] Amaro | 16 -Microalgal fatty acids-From harvesting until extraction[END_REF].

Although centrifugation is a very efficient dewatering method, it has some of the highest energy requirements [START_REF] Guldhe | An innovative electrochemical process to alleviate the challenges for harvesting of small size microalgae by using non-sacrificial carbon electrodes[END_REF]. Especially when microalgae are used to produce biofuels, the overall energy consumption should ideally be lower than the biofuel energy content. When producing biogas from microalgae, it was shown that neither centrifugation nor flocculation or sedimentation as a standalone harvesting method can meet this criterion as the energy input was greater than the energy content of the produced biogas. On the other hand, a positive energy balance can be achieved when centrifugation is combined with pre-concentration by sedimentation or flocculation, [START_REF] Milledge | Energy Balance of Biogas Production from Microalgae: Effect of Harvesting Method, Multiple Raceways, Scale of Plant and Combined Heat and Power Generation[END_REF]. Flocculation is used to preconcentrate the microalgal biomass or to reduce the energy costs of harvesting by producing large size flocs that can be centrifuged more efficiently than single cells [START_REF] Collotta | Comparative LCA of Flocculation for the Harvesting of Microalgae for Biofuels Production[END_REF][START_REF] Fan | Flocculation of Chlamydomonas reinhardtii with Different Phenotypic Traits by Metal Cations and High pH[END_REF]. In the harvesting of Chlorella sorokiniana cells by centrifugation, with or without preflocculation, the benefits of preflocculation were clearly shown. Preflocculation of the cells was performed with a dosage of 10 mg chitosan g -1 dry algae weight more than 99% clarification efficiency. The performance of centrifugation with and without flocculation was similar [START_REF] Xu | Chitosan flocculation to aid the harvesting of the microalga Chlorella sorokiniana[END_REF]. However, preflocculation decreased the volume of the algae suspension between 20 and 50 times, with more than 95% of the water being removed in the latter case, leading to significant reduction in the energy for dewatering [START_REF] Xu | Chitosan flocculation to aid the harvesting of the microalga Chlorella sorokiniana[END_REF]. Preconcentration by natural settling can be another option for energy reduction of centrifugation. It was estimated, for example that the energy consumption for concentration of C. vulgaris cells by spiral plate centrifugation could be as low as 0.042 kWh kg -1 of biomass if preconcentrated by natural settling [START_REF] Collet | Life-cycle assessment of microalgae culture coupled to biogas production[END_REF].

Disc stack centrifuges, on the other hand have a much higher energy consumption [START_REF] Milledge | Disc stack centrifugation separation and cell disruption of microalgae: A technical note[END_REF] that can represent almost up to half of the total energy requirements of a biorefinery [START_REF] Mata | Sustainability and economic evaluation of microalgae grown in brewery wastewater[END_REF]. Hydrocyclone centrifugation is a low-cost and low energy option for harvesting microalgae cells, however, as this method can achieve low cell concentrations (below 0.4% suspended solids) its usefulness is limited to preconcentration [START_REF] Amaro | 16 -Microalgal fatty acids-From harvesting until extraction[END_REF]. Decanter centrifuges can achieve high solid concentrations (approx. 20% DW) in continuous mode with pulsed discharges of the concentrated biomass. Its use is suggested together with flocculation [START_REF] Ramos Tercero | Autotrophic production of biodiesel from microalgae: An updated process and economic analysis[END_REF].

Flotation

Flotation can also be considered a gravity based harvesting method, but the strategy is opposite to the processes discussed above. The aim of this technique is to transfer the microalgae from suspension to the medium surface where they can be easily harvested by skimming. This is achieved by the introducing gas bubbles that attach to the cells and lift them. The microalgae cell attached to the gas bubble has much lower density compared to the medium. According to Stoke's equation, this creates a negative settling velocity or a rising velocity.

Several variations of flotation harvesting exist. In dissolved air flotation (DAF), pressure reduction of air saturated water leads to bubble formation. When optimized it can lead to high biomass harvesting yields. A 91% harvesting efficiency was achieved with dissolved air flotation in the pilot scale with a floatation tank of 3.8 L [START_REF] Niaghi | Optimization of dissolved air flotation technique in harvesting microalgae from treated wastewater without flocculants addition[END_REF]. DAF is usually more expensive due to the cost of water pressurization. In dispersed air flotation, air bubbles are created by gas injection into the medium through a diffuser or sparger, often assisted by mechanical agitation for more efficient dispersion.

Bubble sizes typically fall within the 60 to 655 μm range with a concentration of 2 10 5 bubbles cm -3 . The process efficiency depends on the formed bubbles stability as well as the proper bubble size (optimally below 500 μm) [START_REF] Alhattab | Dispersed air flotation and foam fractionation for the recovery of microalgae in the production of biodiesel[END_REF]. Dispersed air flotation has been used to harvest Chlorella saccharophila [START_REF] Alhattab | Dispersed air flotation of Chlorella saccharophila and subsequent extraction of lipids -Effect of supercritical CO2 extraction parameters and surfactant pretreatment[END_REF]. Flotation is often combined with flocculation to enable harvesting of concentrated flocs. This is an efficient harvesting method with reported harvesting yields of 93.6 % for S. obliquus by thermal flocculation and air flotation [START_REF] Xue | Optimization of thermal preflocculation treatment for effective air flotation harvesting of microalgae[END_REF]; or 80% harvesting of Dunaliella salina by combining NaOH-induced coagulation and DAF in the pre-industrial scale [START_REF] Besson | Flocculation-flotation harvesting mechanism of Dunaliella salina: From nanoscale interpretation to industrial optimization[END_REF]. Flotation has also been combined with bioflocculation either by inducing exopolysaccharides production in A. plantensis with more than 90% harvesting efficiency [START_REF] Vergnes | Bicarbonate Concentration Induces Production of Exopolysaccharides by Arthrospira platensis That Mediate Bioflocculation and Enhance Flotation Harvesting Efficiency[END_REF]; or bioflocculant produced from Cobetia marina for the flocculation-flotation of C. vulgaris also with higher than 90% efficiency [START_REF] Lei | Effective harvesting of the microalgae Chlorella vulgaris via flocculation-flotation with bioflocculant[END_REF].

Besides air, flotation can also be achieved by other gases such as ozone. The harvesting efficiency reported for an ozone dosage of 0.23 mg mg -1 of dried biomass was 79.6%. Although not so efficient, ozonoflotation simultaneously pretreats the microalgae cells and increases the lipid extraction yields [START_REF] Velasquez-Orta | Microalgae harvesting using ozoflotation: Effect on lipid and FAME recoveries[END_REF]. Ozone reduces the microalgae carbohydrate and protein content due to cell lysis and partial release of these molecules in the medium. Denaturation of these proteins leads to foaming that acts as a surfactant and aids flotation [START_REF] Nava Bravo | Bio-crude oil production using catalytic hydrothermal liquefaction (HTL) from native microalgae harvested by ozone-flotation[END_REF]. Indeed, the addition of surfactants like cetyl trimethyammonium bromide (CTAB), has verified the beneficial effect of foaming on flotation. Foam facilitates microalgae cell attachment to the air bubbles by modifying the hydrophobicity of the bubble surface. The surfactant's introduction could modify the extracted lipids chemical composition, possibly due to increased extraction of lipids from the cell membrane's lipid bilayer [START_REF] Coward | Harvesting microalgae by CTAB-aided foam flotation increases lipid recovery and improves fatty acid methyl ester characteristics[END_REF]. The heat-induced flotation of Scenedesmus dimorphus at 85 o C has been proposed as a method for the utilization of heat from off-gases of industrial processes, with harvesting efficiency around 80% [START_REF] Laamanen | Development of heat-aided flocculation for flotation harvesting of microalgae[END_REF].

Membrane filtration

Membrane filtration, batch or continuous, is a very common harvesting method that can be employed as dead-end with vertical flow across the membrane, or as tangential filtration where the flow is parallel to the membrane surface [START_REF] Hung | Microfiltration of microalgae in the presence of rigid particles[END_REF]. Membrane material can be ceramic [START_REF] Jana | Energy efficient harvesting of Arthrospira sp. using ceramic membranes: analyzing the effect of membrane pore size and incorporation of flocculant as fouling control strategy[END_REF] or polymer [START_REF] Zhao | Comparison of axial vibration membrane and submerged aeration membrane in microalgae harvesting[END_REF], and the process can be microfiltration [START_REF] Kim | Turbulent jet-assisted microfiltration for energy efficient harvesting of microalgae[END_REF] or ultrafiltration (Zhang et al., 2019), with membrane pore sizes 10 μm -0.1 μm and 0.1 μm -0.01 μm, respectively. Molecule retention rate depends not only on membrane pore size but also on their chemical nature (polymeric, ceramic etc.).

Nylon membrane filtration is a very common filtration method. Gravity-driven dead-end filtration through 5 μm nylon membranes was enough to achieve > 90% harvesting efficiency of A. platensis cultures. Pumped filtration additionally increased the filtration flux up to 20 m 3 m -2 h -1 enabling a faster process [START_REF] Cuellar-Bermudez | Harvesting of Arthrospira platensis with helicoidal and straight trichomes using filtration and centrifugation[END_REF]. The flux of typical membrane filtration processes can be considerably smaller. For example, in the harvesting of Arthrospira sp. with ceramic microfiltration and ultrafiltration membranes, fluxes of 230 L m -2 h -1 and 93 L m -2 h -1 were reported, respectively [START_REF] Jana | Energy efficient harvesting of Arthrospira sp. using ceramic membranes: analyzing the effect of membrane pore size and incorporation of flocculant as fouling control strategy[END_REF].

For marine microalgae, growth medium salinity can have detrimental effects on harvesting efficiency by tangential-flow membrane filtration. Small increases in salinity of Picochlorum sp., and Tetraselmis sp. growth medium significantly increased membrane fouling and reduced the permeate flux and microalgae concentration factor, at the same time increasing the process energy requirements [START_REF] Das | The effect of culture salinity on the harvesting of microalgae biomass using pilot-scale tangential-flowfilter membrane[END_REF]. Membrane filtration performance is also influenced by the culture's growth phase. For example, harvesting of Scenedesmus acuminatus at the latter growth stages through ultrafiltration membranes increased flux from 97 L m -2 h -1 , to 131 L m -2 h -1 leading to faster processing by 36 %. This was caused by the reduction of the average molecular weight of algogenic organic matter (AOM) secreted by the cells in the latter stages. The high molecular weight content of AOM (higher than 50 kDa) decreased from 50% in the exponential phase to 42% in the stationary and 26% in the declining phase. As the high molecular weight fraction of AOM has the greatest membrane fouling potential its reduction led to minimized membrane fouling and increased flux. However, algogenic organic matter negatively impacted the process efficiency through enhanced fouling at all stages of ultrafiltration [START_REF] Ye | Influence of growth phase on the harvesting of Scenedesmus acuminatus using ultrafiltration[END_REF]. Fouling is the major problem of membrane harvesting as it can negatively impact the process performance, reducing flux and increasing energy requirements for efficient harvesting (Elcik and Cakmakci, 2017). Regarding the effect of different types of AOM on fouling, it was found that polysaccharides excreted by the microalgae cells in the medium were related to reversible membrane fouling, whereas deposition of excreted protein led to irreversible membrane fouling [START_REF] Jiang | Impact of transmembrane pressure (TMP) on membrane fouling in microalgae harvesting with a uniform shearing vibration membrane system[END_REF].

Other soluble microbial metabolism products such as humin-like substances also contribute to irreversible membrane fouling [START_REF] Wu | Probing the fouling process and mechanisms of submerged ceramic membrane ultrafiltration during algal harvesting under sub-and super-critical fluxes[END_REF][START_REF] Zhao | Increasing the vibration frequency to mitigate reversible and irreversible membrane fouling using an axial vibration membrane in microalgae harvesting[END_REF].

Fouling can be dealt with by adding surfactants like CTAB that remove hydrophilic and high molecular weight foulants and additionally lead to electrostatic microalgae cells neutralization allowing for more efficient dewatering [START_REF] Taghavijeloudar | The effects of surfactants (sodium dodecyl sulfate, triton X-100 and cetyl trimethyl ammonium bromide) on the dewaterability of microalgae biomass using pressure filtration[END_REF]. Alternatively, it can be prevented by the designing of antifouling membranes (Elcik et al., 2017), e.g. containing novel materials such as carbon nanotubes that improve membrane hydrophilicity [START_REF] Khairuddin | Harvesting Nannochloropsis sp. using PES/MWCNT/LiBr membrane with good antifouling properties[END_REF]. Another approach is the introduction of local turbulence. This was achieved by a membrane module with a cylinder with holes at its center that created turbulent jets ejecting the feed into the membrane surface in a perpendicular direction. When used for the microfiltration of C. vulgaris, at a speed of 7 L min -1 fouling was reduced by 126% compared to conventional type modules such as hollow fiber membrane [START_REF] Kim | Turbulent jet-assisted microfiltration for energy efficient harvesting of microalgae[END_REF]. Application of a shearing vibration is also able to reduce both reversible and irreversible fouling [START_REF] Zhao | A uniform shearing vibration membrane system reducing membrane fouling in algae harvesting[END_REF][START_REF] Zhao | Increasing the vibration frequency to mitigate reversible and irreversible membrane fouling using an axial vibration membrane in microalgae harvesting[END_REF]. Cake layer formation of the filtered microalgae cells also contributes to fouling and increased membrane resistance [START_REF] Wu | Probing the fouling process and mechanisms of submerged ceramic membrane ultrafiltration during algal harvesting under sub-and super-critical fluxes[END_REF]. Introduction of rigid particles both in dead-end and crossflow filtration, as in the case of Chlorella sp. harvesting, can reduce the cake resistance and increase the flux and the membrane performance [START_REF] Hung | Microfiltration of microalgae in the presence of rigid particles[END_REF]. Introduction of flocculants during the ceramic membrane filtration (both micro-and ultra-) of Arthrospira sp. also reduced fouling. This is caused as flocculation leads to the formation of larger size particles and reduces their ability to enter the membrane matrix [START_REF] Jana | Energy efficient harvesting of Arthrospira sp. using ceramic membranes: analyzing the effect of membrane pore size and incorporation of flocculant as fouling control strategy[END_REF]. Indeed, membrane filtration is often used in combination with flocculation due to the synergy of the two methods [START_REF] Sahoo | Sustainable dewatering and drying of self-flocculating microalgae and study of cake properties[END_REF][START_REF] Shi | Synergy of flocculation and flotation for microalgae harvesting using aluminium electrolysis[END_REF].

Table 4 3. Pretreatment of microalgae & Extraction of target molecules 3.1 Physical methods

Milling

Milling is one of the most promising methods for cell disruption of microalgae. Although it has been studied in various setups and sizes such as lab scale vortex milling [START_REF] Araya | Evaluation of the simultaneous production of lutein and lipids using a vertical alveolar panel bioreactor for three Chlorella species[END_REF] and pilot scale ball milling [START_REF] Balasundaram | A low energy process for the recovery of bioproducts from cyanobacteria using a ball mill[END_REF], bead milling has been the most studied method. Typically, bead milling uses discs or rings to set in motion the grinding elements inside the milling chamber (Günerken et al., 2015a). Several disruption mechanisms are possible during bead milling. Shear stress due to acceleration of beads towards the mill wall, shear due to centrifugal acceleration of the mill wall, and due to collision of cells with beads due to differences in velocities [START_REF] Kwade | Determination of the most important grinding mechanism in stirred media mills by calculating stress intensity and stress number[END_REF]. The main mechanism of cell disruption is the third, ie cell disruption occurs through beads collisions because of velocity gradients in tangential direction near the stirring disks [START_REF] Balasundaram | A low energy process for the recovery of bioproducts from cyanobacteria using a ball mill[END_REF][START_REF] Hennart | Identification of the grinding mechanisms and their origin in a stirred ball mill using population balances[END_REF][START_REF] Kwade | Determination of the most important grinding mechanism in stirred media mills by calculating stress intensity and stress number[END_REF] leading to combinations of cleavage, fracture and abrasion of cells or particles depending on particle size [START_REF] Hennart | Identification of the grinding mechanisms and their origin in a stirred ball mill using population balances[END_REF]. For large particles of around 15 μm particles disintegration occurs through cleavage and fracture, for medium size particles of around 0.8 μm through cleavage and abrasion, and for small particles only through cleavage [START_REF] Hennart | Identification of the grinding mechanisms and their origin in a stirred ball mill using population balances[END_REF]. Indeed, the disintegration of Nannochloropsis sp. in a bead mill, has revealed that larger size cell fractions are disintegrated via impact and compression -correlated to fracture and cleavage-and smaller fractions via shear forces more related to abrasion (Figure 4) [START_REF] Pan | Disintegration of Nannochloropsis sp. cells in an improved turbine bead mill[END_REF].

Table 5 Figure 4 Since beads transfer energy to the cells their size plays a critical role in process efficiency. Decreasing the bead size from 1mm to 0.3 mm improved the protein release kinetics of N. oleoabundans and C. vulgaris but the effect on the protein yield was less profound [START_REF] Postma | Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates[END_REF]. In the same work the kinetics of carbohydrate release from C. vulgaris peaked at a bead size of 0.4 mm and an optimum carbohydrate release yield of approximately 65% was achieved. Bead size did not significantly affect cell disintegration, protein and carbohydrate release of Tetraselmis suecica in the same study, indicating a weaker cell wall [START_REF] Postma | Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates[END_REF]. Protein release of 99% for T. suecica with a bead size of 0.3 mm was achieved at 400 sec of processing time where a plateau is observed. Maximum carbohydrate release did not reach a plateau and was only observed at the end of the experiment, i.e. 900 sec of processing time [START_REF] Postma | Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates[END_REF]. Small bead size also improved the kinetics of Nanochloropsis pretreatment in a turbine mill [START_REF] Pan | Disintegration of Nannochloropsis sp. cells in an improved turbine bead mill[END_REF]. More than 90% cell disruption1 was achieved at 25 min with 0.3-0.4 mm beads, whereas 45 min were required for a similar result with 0.8-1.0 mm size. A further increase of bead size to 1.8-2.0 mm led to a dramatic drop in the mill performance (65% cell disruption at 55 min). Optimal bead sizes for the disruption of Chlorella strain P12 with a 4.3 μm cell diameter, depended on the type of instrument used [START_REF] Doucha | Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers[END_REF]). An optimal bead diameter of 0.3-0.5 mm was found for the Dyno-Mill and LabStar LS 1 bead mills, whereas the optimal bead diameter for the homogenizer MS 18 was 0.5-0.7 mm [START_REF] Doucha | Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers[END_REF]. However, 1.3 mm glass beads were optimum for the bead milling of P. cruentum leading to a cell disintegration efficiency of 50% [START_REF] Montalescot | Optimization of bead milling parameters for the cell disruption of microalgae: Process modeling and application to Porphyridium cruentum and Nannochloropsis oculata[END_REF]. It is clear from the above that bead size alone cannot efficiently describe the process; other factors, such as the bead density affect the disruption efficiency. Beads with higher densities has been shown to perform better because they can tranfer higher energy to the cells [START_REF] Hopkins | Physical and chemical cell disruption for the recovery of intracellular protein[END_REF].

The milling chamber fill ratio is another important parameter. For C. vulgaris,increasing the chamber filling ratio had a positive effect on milling efficiency. For example, when using ZrO2 beads of 0.3 mm diameter in homogenizer MS 18, increasing the filling ratio from 60% to 75% and 80% increased cell disintegration from 65% to 83% and 85% respectively [START_REF] Doucha | Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers[END_REF]. Similarly, when using ZrO2 beads 0.3-0.4 mm in the Dyno-Mill KD 20 S increasing fill ratio from 75% to 85% increased cell disintegration from 75% to 83% [START_REF] Doucha | Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers[END_REF]. For P. cruentum increasing the filling ratio from 35% to 65% not only improved cell disruption, but also led to a shift in the optimum bead size from 1.3 mm to 0.65 mm [START_REF] Montalescot | Optimization of bead milling parameters for the cell disruption of microalgae: Process modeling and application to Porphyridium cruentum and Nannochloropsis oculata[END_REF]. Continuous bead milling can be performed in single and multi-pass operation and the flow rate can also affect the performance. Usually increasing the flow rate reduces cell disintegration efficiency, by reducing the residence time in the milling chamber [START_REF] Doucha | Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers[END_REF][START_REF] Montalescot | Optimization of bead milling parameters for the cell disruption of microalgae: Process modeling and application to Porphyridium cruentum and Nannochloropsis oculata[END_REF]. For example, increasing the feed rate from 12 Kg h -1 to 18 Kg h -1 reduced cell disintegration from 92% to 70%. Energetically, it is evident that an optimized process with the shortest duration of treatment is desired and a compromise on the disruption ratio may need to be reached.

During bead milling cell disruption2 follows first-order kinetics [START_REF] Doucha | Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers[END_REF]. Disintegration rates of N. gaditana show a sharp increase in the initial minutes and a plateau of 95% disintegration is reached at around 20 min (Safi et al., 2017a). Protein release in the same work reached a 50% yield plateau at around 8 min, suggesting that full cell disruption is not always required for efficient extraction of targeted molecules. For T. suecica the kinetics of cell wall disruption and protein release are identical and reach a 99% plateau after 6.7 min. A similar carbohydrate release on the other hand is achieved after 15 min without reaching a plateau [START_REF] Postma | Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates[END_REF].

Increasing the solids content of the microalgae biomass slurry has a positive effect both on the yield of extraction and the process energy consumption up to a point. After that threshold the process becomes inefficient. For the extraction of lipids from Schizochytrium this limit was 50 g L -1 of biomass [START_REF] Byreddy | Bead milling for lipid recovery from thraustochytrid cells and selective hydrolysis of Schizochytrium DT3 oil using lipase[END_REF], and the disruption of Nannochloropsis cells became inefficient after a 18% volume of cell concentration [START_REF] Pan | Disintegration of Nannochloropsis sp. cells in an improved turbine bead mill[END_REF]. Assuming a near water cell density [START_REF] Mathimani | A comprehensive review on harvesting of microalgae for biodiesel -Key challenges and future directions[END_REF]) and a 10% biomass dry weight content, the 18% v/v can be estimated at around 2% DW, a surprising low concertation limit for the process. On the other hand, the extraction of water-soluble protein from Nannochloropsis gaditana, was energy efficient at 100 g L -1 but with around 50% protein yield (Safi et al., 2017a).

Besides bulk molecules such as lipids and carbohydrates, bead milling pretreatment can improve the yields of high-value molecules found in microalgae. In the lab scale, bead milling of A. platensis biomass in 4 cycles of 25 sec at 30 Hz, allowed almost 100% extraction of proteins and phycocyanin at 46 g 100 g -1 biomass, and 95 mg g -1 biomass, respectively [START_REF] Jaeschke | Extraction of valuable compounds from Arthrospira platensis using pulsed electric field treatment[END_REF]. Bead milling pretreatment has also been used in lutein extraction from Scenedesmus almeriensis. When the dry biomass was treated for 5min, followed by an alkaline treatment and hexane extraction around 50% of lutein extraction yield was achieved. Addition of alumina powder in the biomass at 1:1 ratio acted as an extra disintegrating agent and increased lutein yield to 98 % [START_REF] Cerón | Recovery of lutein from microalgae biomass: development of a process for Scenedesmus almeriensis biomass[END_REF]. Bead milling of C. vulgaris for 1 h allowed yields of 50% chlorophyll and 30% carotenoids extracted with supercritical CO2 and ethanol as a cosolvent [START_REF] Safi | Extraction of lipids and pigments of Chlorella vulgaris by supercritical carbon dioxide: influence of bead milling on extraction performance[END_REF]. B-phycoerythrin, a natural high-value pigment was obtained from Porphyridium cruentum at a yield of 1.35 mg g -1 of wet biomass after bead milling [START_REF] Ruiz-Ruiz | Scaling-up of a B-phycoerythrin production and purification bioprocess involving aqueous two-phase systems: Practical experiences[END_REF]. Although these results are promising, bead milling pretreatment was used in combination with other treatments, such as freeze drying of the biomass, alkaline pretreatment, and addition of alumina powder as a second disintegration agent. Such steps would increase complexity and cost of an industrial process and their economic and energetic viability would have to be assessed.

In the end, bead milling is a very complex process and many more parameters, like bead density, agitation speed, agitators design, and solid content of biomass can affect the outcome. The microalgae cell wall robustness is also a critical factor as it can vary between very weak like Porphyridium cruentum to very tough like Nannochloropsis oculata [START_REF] Montalescot | Optimization of bead milling parameters for the cell disruption of microalgae: Process modeling and application to Porphyridium cruentum and Nannochloropsis oculata[END_REF]. Therefore, optimization can be tedious but also very precise if all parameters are carefully considered. The applicability of statistical design of experiments to such optimisation is evident.

High-pressure homogenization (HPH)

High-pressure homogenization (HPH) is typically used for emulsification purposes but is also suitable for the large-scale disruption of microalgae cells (Günerken et al., 2015a). Typically, a cell suspension is pumped at high pressures through a valve, colliding with an impact ring and then exiting from the high-pressure area into the environment where a high-pressure drop occurs (Middelberg, 1995). Various mechanisms of cell breakage (Figure 5) have been proposed including: shear due to sudden pressure drop, shear stress, cavitation and impingement of the cells to the surface of the valve at high velocities (Middelberg, 1995). Several of these mechanisms of cell disruption can occurr simultaneously and which one is prevalent may also be species dependent (Middelberg, 1995). However, in most cases cell disruption is caused by shear stress and pressure drop between the valve and the vessel, as well as cell impingement onto the valve walls [START_REF] Halim | Microalgal cell disruption for biofuel development[END_REF]. The impingment stress was reported as being proportional to the operating relative pressure in some types of homogenisers [START_REF] Chisti | Disruption of microbial cells for intracellular products[END_REF]. As pressure drop is one of the main cell-breaking mechanisms, the system operating pressure is critical for process performance [START_REF] Balduyck | Influence of High Pressure Homogenization on Free Fatty Acid Formation in Nannochloropsis sp[END_REF][START_REF] Halim | Microalgal cell disruption for biofuel development[END_REF][START_REF] Onumaegbu | Optimization of Pre-Treatment Process Parameters to Generate Biodiesel from Microalga[END_REF][START_REF] Samarasinghe | Algal cell rupture using high pressure homogenization as a prelude to oil extraction[END_REF]. Increasing the pressure in the HPH of Chlorella sp. from 250 to 1400 bar increased cell rupture3 from 10% to 80% (measured by counting undisrupted cells before and after HPH in a Neubauer haemocytometer chamber) and subsequently the recovered lipids from 20% to 100% [START_REF] Yap | A mechanistic study of algal cell disruption and its effect on lipid recovery by solvent extraction[END_REF]. Similarly, in the HPH treatment of Desmodesmus sp. F51 increasing the pressure from 689.5 to 2758 bar increases the release of carotenoids from around 0.02 mg g -1 to around 0.28 mg g -1 and the release of chlorophylls from around 0.01 mg g -1 to almost 1.5 mg g -1 [START_REF] Xie | Disruption of thermo-tolerant Desmodesmus sp. F51 in high pressure homogenization as a prelude to carotenoids extraction[END_REF]. Complete disruption of N. gaditana cells occurred at 1500 bar. However, the protein release had already peaked at a much lower pressure (1000 bar), with a yield of 50%, indicating that partial cell breakage is enough for the sufficient extraction of desired compounds (Safi et al., 2017a). As with other disruption and extraction methods, the method's influence on the target compound degradation is an important quality factor to consider during a biorefinery process.

Figure 5 The number of passes also has a positive impact on HPH extraction yields [START_REF] Balduyck | Influence of High Pressure Homogenization on Free Fatty Acid Formation in Nannochloropsis sp[END_REF][START_REF] Samarasinghe | Algal cell rupture using high pressure homogenization as a prelude to oil extraction[END_REF]. Increasing the number of passes from 1 to 4 increased carotenoid release from Desmodesmus sp. F51 from around 0.28 mg g -1 to around 0.42 mg g -1 , and chlorophyll release from 1.5 mg g -1 to 2.4 mg g -1 [START_REF] Xie | Disruption of thermo-tolerant Desmodesmus sp. F51 in high pressure homogenization as a prelude to carotenoids extraction[END_REF]. Five passes were optimal for release of carbohydrates and proteins from dilute solutions of C. vulgaris treated at 1500 bar [START_REF] Carullo | Effect of pulsed electric fields and high pressure homogenization on the aqueous extraction of intracellular compounds from the microalgae Chlorella vulgaris[END_REF]. After five passes of Chlorococcum sp. cells more than 90% disruption was achieved [START_REF] Halim | Microalgal cell disruption for biofuel development[END_REF]. Increasing the number of passes to 6 had a positive effect on both the total protein and reducing sugars extraction yields from HPH treated N. oculata [START_REF] Shene | High pressure homogenization of Nannochloropsis oculata for the extraction of intracellular components: Effect of process conditions and culture age[END_REF]. Pretreatment of Nannochloropsis sp. with HPH at 150 MPa required 6 passes to maximize extraction of green and red pigments, as deduced from increases in spectral intensities of the extracts at 415 nm and 620 nm, respectively [START_REF] Grimi | Selective extraction from microalgae Nannochloropsis sp. using different methods of cell disruption[END_REF].

In general, HPH exhibits a first order decrease of cell disruption rate after each passage through the homogenizer [START_REF] Halim | Microalgal cell disruption for biofuel development[END_REF]. The rupture of Nannochloropsis sp., Chlorella sp. and T. suecica cells followed an exponential decay as a function of the number of passes [START_REF] Spiden | Quantitative evaluation of the ease of rupture of industrially promising microalgae by high pressure homogenization[END_REF].

Smaller effects on HPH performance were attributed to cell concentration and culture stress level [START_REF] Samarasinghe | Algal cell rupture using high pressure homogenization as a prelude to oil extraction[END_REF]. Increasing the cell concentration of Desmodesmus sp. F51 from 2 and up to 90 g/L did not affect the particle size distribution of HPH treated cells nor the release of carotenoids and chlorophylls [START_REF] Xie | Disruption of thermo-tolerant Desmodesmus sp. F51 in high pressure homogenization as a prelude to carotenoids extraction[END_REF]. Increasing the cell concentration of N. oculata from 1.78% up to 8% w/w had little effect on the yield of lipids extracted [START_REF] Shene | High pressure homogenization of Nannochloropsis oculata for the extraction of intracellular components: Effect of process conditions and culture age[END_REF]. Indeed, it was found for Nannochloropsis sp. that important processing parameters such as flow rate, power draw and disruption efficiency were independent of cell concentration up to 25% w/w [START_REF] Yap | Energy evaluation of algal cell disruption by high pressure homogenisation[END_REF]. As 25% is a typical concentration target of many harvesting/dewatering methods the ability to effectively break cells at these concentrations makes HPH a potential method for industrial microalgal cell disruption.

The method is also affected by the microalgae species biological properties. More specifically by cell wall toughness and its resistance to disruption. For example, only 170 bars are enough to break T. suecica cells, whereas Chlorella sp. cells require 1070 bar, and Nannochloropsis sp. 2000 bar [START_REF] Spiden | Quantitative evaluation of the ease of rupture of industrially promising microalgae by high pressure homogenization[END_REF].

Ultrasonication

Two major mechanisms of cell disruption are reported during ultrasonication. Generation of highpressure bubbles and their cavitation, which generates shock waves that cause high shear forces [START_REF] Gerde | Evaluation of microalgae cell disruption by ultrasonic treatment[END_REF]Günerken et al., 2015b;[START_REF] Lee | Cell disruption and lipid extraction for microalgal biorefineries: A review[END_REF]. The additional effect of free radical generation and oxidative degradation of cells has also been reported [START_REF] Gerde | Evaluation of microalgae cell disruption by ultrasonic treatment[END_REF]. Increasing ultrasonication power and treatment time have a positive effect on cell disruption [START_REF] Greenly | Ultrasonic cavitation for disruption of microalgae[END_REF][START_REF] Keris-Sen | An investigation of ultrasound effect on microalgal cell integrity and lipid extraction efficiency[END_REF][START_REF] Wang | Microalgal cell disruption in a high-power ultrasonic flow system[END_REF][START_REF] Zhang | Bio-oil production from hydrothermal liquefaction of ultrasonic pre-treated Spirulina platensis[END_REF]. Increasing the power increases bubble generation but reduces pressure inside the bubbles [START_REF] Wang | Modeling bubble dynamics and radical kinetics in ultrasound induced microalgal cell disruption[END_REF]. Beyond the optimum power and time values, undesirable effects on released products can occur, such as oxidative degradation [START_REF] Gerde | Evaluation of microalgae cell disruption by ultrasonic treatment[END_REF], and reduction of fatty acids chain size and double bond saturation [START_REF] Cheng | Fractal microstructure characterization of wet microalgal cells disrupted with ultrasonic cavitation for lipid extraction[END_REF]. Increasing the frequency of ultrasonication, positively affects cell disruption. Optimum sonication frequency is species depended and should be determined for each microalgae species [START_REF] Kurokawa | Effect of sonication frequency on the disruption of algae[END_REF]. Application of a pulsed mode improved protein extraction yield from C. vulgaris FSP-E [START_REF] Chia | Microalgal Protein Extraction From Chlorella vulgaris FSP-E Using Triphasic Partitioning Technique With Sonication[END_REF]. The pulsed mode has many advantages such as energy input reduction, lower heat generation during treatment, hence reduction in the extracted protein denaturation. This is important as temperature during ultrasonication can reach as high as 93 o C if not controlled [START_REF] De Farias Silva | Pretreatment of microalgal biomass to improve the enzymatic hydrolysis of carbohydrates by ultrasonication: Yield vs energy consumption[END_REF]. Ultrasonication efficiency seems to be independent of cell concentration at low to medium concentrations (1.5 to 14.1 g L -1 ) [START_REF] Gerde | Evaluation of microalgae cell disruption by ultrasonic treatment[END_REF]. At higher cell concentrations a significant reduction in disruption efficiency can occur [START_REF] Greenly | Ultrasonic cavitation for disruption of microalgae[END_REF][START_REF] Wang | Microalgal cell disruption in a high-power ultrasonic flow system[END_REF] and has been attributed either to increased viscosity or reduced energy input per cell (Meng Wang and Yuan, 2015). Cell disruption in ultrasonication follows first order kinetics. The constant of disruption rate is directly proportional to the power level and has a parabolic relationship to cell concentration [START_REF] Halim | Mechanical cell disruption for lipid extraction from microalgal biomass[END_REF].

Figure 6

Cell aggregate formation during flocculation adversely affects cell disruption by ultrasonication. Cell aggregation reduces the penetration of ultrasonic power and protects cells inside the aggregate from bubble generation and collapse (Wang et al., 2015). The formation of microalgae cell aggregates by flocculation can occur naturally or be induced during harvesting/concentration [START_REF] Muylaert | 5 -Harvesting of microalgae: Overview of process options and their strengths and drawbacks[END_REF].

As flocculation is one of the most efficient concentration methods of microalgae biomass, its negative effect on cell disruption by ultrasonication demonstrates that careful selection of all downstream processes is required when assembling multiple methods in the same process.

Ultrasonication performed poorly in many comparative studies of cell disruption methods. It was less effective compared to microwave, electroflotation and thermal treatment (Florentino de Souza Silva et al., 2014), microwave and thermal treatment [START_REF] Mcmillan | Evaluation and comparison of algal cell disruption methods: Microwave, waterbath, blender, ultrasonic and laser treatment[END_REF], High-Pressure Homogenization, sulfuric acid and bead beating [START_REF] Halim | Microalgal cell disruption for biofuel development[END_REF], Pulsed Electric Field, and High-Pressure Homogenization [START_REF] Grimi | Selective extraction from microalgae Nannochloropsis sp. using different methods of cell disruption[END_REF]. For example, ultrasound was not effective when applied in the pretreatment of microalgae with a very resistant cell wall such as H. pulvialis cysts. Even at 600 W for 30 min at 20 kHz frequency and pulses 1 sec on 3 sec off a very low 12% astaxanthin yield was observed [START_REF] Liu | The efficiency and comparison of novel techniques for cell wall disruption in astaxanthin extraction from Haematococcus pluvialis[END_REF]. In another study 80% extraction efficiency of astaxanthin was achieved only when ultrasound pretreatment of the H. pluvialis biomass was performed in a 2M sodium hydroxide solution [START_REF] Haque | Intensified green production of astaxanthin from Haematococcus pluvialis[END_REF]. Although careful optimization of all these methods is required before safe conclusions can be drawn, it seems that ultrasonication is not a very effective method for cell disruption. It also suffers from the inability to handle large cell concentrations. Despite that, it can be useful in combination with other methods as it can enhance the disruption efficiency of other pretreatment methods. This will be discussed in following sections.

Pulsed Electric Field

Pulsed Electric Field is based on the exposure of microalgae cells to an intense electric field for very short durations (pulses). When the applied electric field is above a threshold it can induce reversible or irreversible pores creation (electroporation) on the cell membranes. Irreversible electroporation can facilitate intracellular material extraction (Figure 7) [START_REF] Luengo | Influence of the treatment medium temperature on lutein extraction assisted by pulsed electric fields from Chlorella vulgaris[END_REF]. The microalgae suspension must not contain ions in order to be non-conductive. The medium of marine microalgae that contains significant salt concentrations, must be removed before PEF treatment, for example by electrodialysis, or cell washing and resuspension, increasing process steps, cost and energy. PEF is therefore more suitable for freshwater microalgae treatment (Günerken et al., 2015b). The cell membranes permeabilization (electroporation) is dependent on the electric field energy [START_REF] Carullo | Effect of pulsed electric fields and high pressure homogenization on the aqueous extraction of intracellular compounds from the microalgae Chlorella vulgaris[END_REF]. The main effect of PEF is pore creation in the cell membranes and does not lead to total cell disruption and cellular debris generation [START_REF] Carullo | Effect of pulsed electric fields and high pressure homogenization on the aqueous extraction of intracellular compounds from the microalgae Chlorella vulgaris[END_REF]. Temperature increase during PEF treatment is generally small [START_REF] Picart | 18 -Pulsed electric fields[END_REF], for example during PEF treatment of C. vulgaris the sample temperature increased from 20 o C to 38 o C depending on the operating parameters; the effect of this temperature increase on the yield of extracted proteins and their functionality were not investigated [START_REF] Scherer | Pulsed electric field (PEF)-assisted protein recovery from Chlorella vulgaris is mediated by an enzymatic process after cell death[END_REF]. When very high treatment intensities were applied, e.g. higher than 35 kWh m -3 in the PEF treatment of Synechocystis PCC 6803, it led to limited cell disruption (approx.10%), while the cell majority (around 87%) remained intact but with damaged membranes [START_REF] Sheng | Evaluation of Cell-Disruption Effects of Pulsed-Electric-Field Treatment of Synechocystis PCC 6803[END_REF].

Figure 7 PEF efficiency is affected by the microalgae culture's growth state and cell size. Larger cells are more susceptible to electroporation, whereas smaller cells are more resistant (Safi et al., 2017a). The large size of H. pulvialis cells (20 μm) may, therefore, have aided PEF treatment, as a 96% astaxanthin extraction efficiency, using ethanol, was reported [START_REF] Martínez | Use of pulsed electric field permeabilization to extract astaxanthin from the Nordic microalga Haematococcus pluvialis[END_REF]. In contrast, the small cell size (2-8 μm) of C. sorokiniana [START_REF] Azaman | A comparison of the morphological and biochemical characteristics of Chlorella sorokiniana and Chlorella zofingiensis cultured under photoautotrophic and mixotrophic conditions[END_REF] may have a negative impact on PEF treatment. For example, a yield of 3.25 mg g -1 DW of pigments was achieved after PEF treatment, an improvement of 14% compared to the untreated sample [START_REF] Leonhardt | Biorefinery of Chlorella sorokiniana with pulsed electric field pre-treatment[END_REF]. Both species have rigid cell walls; H. pulvialis with a resistant algaenan layer, and C. sorokiniana a glucosamine rich and chitin-like layer.

Cell wall rigidity can lead to inefficient extraction of molecules even at very high intensities and number of pulses (Safi et al., 2017a). This has been confirmed by comparing the PEF efficiency between a wild type strain of C. reinhardtii with cell wall and a mutant without cell wall (T [START_REF] Lam | Harvesting and cell disruption of microalgae[END_REF]. A dramatic increase on protein extraction yields was observed in the cell wall free mutant even at very low power intensities. The effect could be repeated when the wild strain's cell wall was removed by enzymatic digestion.

Due to its relatively mild effect on cell integrity, PEF is often described as a mild or selective pretreatment method that can specifically target intracellular molecules, such as low molecular weight proteins and carbohydrates [START_REF] Carullo | Effect of pulsed electric fields and high pressure homogenization on the aqueous extraction of intracellular compounds from the microalgae Chlorella vulgaris[END_REF]Safi et al., 2017a;[START_REF] Lam | Mild and Selective Protein Release of Cell Wall Deficient Microalgae with Pulsed Electric Field[END_REF]. Although a significant increase in the release of water-soluble intracellular components is induced by PEF, their yields are relatively low (around or below 10%) even at relatively high intensities indicating only partial success of PEF as a pretreatment method. In all these studies the treated microalgae had a full cell wall, that may explain the low yields obtained.

The low levels of lipids released with PEF from Auxenochlorella protothecoides cells is possibly because the generated pores are more permeable to smaller water-soluble molecules rather than the larger lipid droplets [START_REF] Eing | Pulsed Electric Field Treatment of Microalgae-Benefits for Microalgae Biomass Processing[END_REF]. This can enable an easier separation of carbohydrates and proteins from lipids that could be sequentially extracted by organic solvents [START_REF] Eing | Pulsed Electric Field Treatment of Microalgae-Benefits for Microalgae Biomass Processing[END_REF]. PEF treatment of wet Synechocystis PCC 6803 biomass increased the lipid yields and lowered the solvent to biomass ratio from 10 to 5 as it allowed the penetration of isopropanol through the pores to extract lipid molecules [START_REF] Sheng | Evaluation of Cell-Disruption Effects of Pulsed-Electric-Field Treatment of Synechocystis PCC 6803[END_REF]. The kinetics of lipid extraction from Ankistrodesmus falcatus with the green solvent ethyl acetate were relatively slow and around 2h were required for the process to plateau at a yield of around 2500 μg L -1 . After PEF treatment the lipid extraction was almost time independent as increasing extraction time between 0 and 2 h lead to a marginal increase in lipids from 9000 μg L -1 to almost 9800 μg L -1 [START_REF] Zbinden | Pulsed electric field (PEF) as an intensification pretreatment for greener solvent lipid extraction from microalgae[END_REF]. Although a yield increase is also shown, extraction experiments with and without PEF treatments were performed with different microalgae samples, and therefore the effect of PEF on the yield remains inconclusive [START_REF] Zbinden | Pulsed electric field (PEF) as an intensification pretreatment for greener solvent lipid extraction from microalgae[END_REF]. Similarly, the PEF treatment of A. protothecoides increased lipid extraction with 100% ethanol from around 30 to around 220 mg g -1 of dry weight biomass [START_REF] Eing | Pulsed Electric Field Treatment of Microalgae-Benefits for Microalgae Biomass Processing[END_REF].

Drying

Drying is the decrease of microalgae biomass moisture content to around 10%. It is a post harvesting and dewatering process aims at improving the stability of the final product. The main challenges of drying are the long time and high energy input required and the associated cost increase. The simplest drying technology is sun drying as it requires low capital and operating cost [START_REF] Ansari | Evaluation of various cell drying and disruption techniques for sustainable metabolite extractions from microalgae grown in wastewater: A multivariate approach[END_REF]. However, long process time is needed for efficient water removal from the sample, even as high as 72 h [START_REF] Guldhe | Efficacy of drying and cell disruption techniques on lipid recovery from microalgae for biodiesel production[END_REF]. Sun drying can decrease the extraction yield of biomolecules from microalgae compared to untreated biomass [START_REF] Ansari | Evaluation of various cell drying and disruption techniques for sustainable metabolite extractions from microalgae grown in wastewater: A multivariate approach[END_REF] possibly due to degradation reactions or due to consumption from microbial contamination during the long drying times. Convection drying utilizes hot air to remove moisture from the biomass. It has shorter process times, up to 12h and leads to less biomolecule degradation compared to sun drying [START_REF] Ansari | Evaluation of various cell drying and disruption techniques for sustainable metabolite extractions from microalgae grown in wastewater: A multivariate approach[END_REF]. The lipids extracted from oven dried biomass has slightly lower saponification and acid values compared to sun dried biomass, indicating lower free fatty acid values.

Freeze drying, also called lyophilization, is a very mild drying method that does not destroy the biomolecules of the biomass samples [START_REF] Ansari | Evaluation of various cell drying and disruption techniques for sustainable metabolite extractions from microalgae grown in wastewater: A multivariate approach[END_REF]. It is based on the sublimation of ice from a frozen sample into the vapor phase inside a vacuum. Freeze-drying can have long process time, usually around 24h. Due to the requirement for deep freezing of the sample, and maintaining a vacuum and a negative temperature in the freeze-drier, it is the most energy consuming and expensive method, typically 1 or 2 orders higher than the rest (Table 6). Its long process time, high energy requirement and high cost should limit application of freeze-drying for very sensitive and high-value products. Spray drying is based on heating of the microalgae slurry as it passes through a spraying nozzle which leads to the formation of a dry powder as the moisture rapidly evaporates from the sample droplets. |Samples of freeze-dried P. tricornutum cells were found more prone to lipolysis, and spray dry lipolysis ed samples were more sensitive to oxidation caused by the reduction of the antioxidant carotenoid content [START_REF] Ryckebosch | Influence of drying and storage on lipid and carotenoid stability of the microalga Phaeodactylum tricornutum[END_REF]. Drum drying is a mature drying technology with applications in the food industry. It has lower energy requirements and costs compared to spray drying (Table 6) but can process only up to 1000 Kg h -1 of sample compared to the 10000 Kg h -1 maximum upper limit for spray drying. This big difference in processing capacity if favor of spray drying can compensate for the slightly higher energy and operational costs, and make spray drying an attractive drying method [START_REF] Fasaei | Techno-economic evaluation of microalgae harvesting and dewatering systems[END_REF].

Drying microalgae before extraction of valuable components clearly adds to the treatment cost while greatly increasing the extraction efficiency. Recent technology on extraction from wet biomass is promising but the cross-over point where the lower extraction efficiency of wet extraction can be accepted has not been reached. [START_REF] Xiao | A solar-driven continuous hydrothermal pretreatment system for biomethane production from microalgae biomass[END_REF] and steam explosion [START_REF] Lorente | Steam Explosion and Vibrating Membrane Filtration to Improve the Processing Cost of Microalgae Cell Disruption and Fractionation[END_REF]. In all these variations two main mechanisms are thought responsible for the microalgae cell disruption; cell wall rupture due to internal pressure buildup from the heating, and hydrolysis of cell wall components. Due to this dual effect the method can be classified both as physical and chemical. At lower temperatures where hydrolytic reactions of the cell wall components are not yet favoured disruption due to internal pressure build-up is the prevailing mechanism [START_REF] Mcmillan | Evaluation and comparison of algal cell disruption methods: Microwave, waterbath, blender, ultrasonic and laser treatment[END_REF]. Even at such mild treatment conditions, e.g. 90 o C, for 20 min, extensive disruption4 of 87% for N. oculata cells was observed. At higher temperatures the hydrolysis reactions of the microalgae structural components such as the cell wall polysaccharides becomes significant, leading to the production of monomeric sugars and the reduction of cell wall integrity [START_REF] Xiao | A solar-driven continuous hydrothermal pretreatment system for biomethane production from microalgae biomass[END_REF]. In the steam explosion variation of the method the microalgae slurry is heated under pressure with saturated steam to the desired temperature, and at the end of the reaction the slurry undergoes rapid decompression (explosion) through a release valve [START_REF] Lorente | Microalgae fractionation using steam explosion, dynamic and tangential cross-flow membrane filtration[END_REF]. The rapid change in pressure can create an additional cell disruption effect.

The hydrothermal method has been extensively used to pretreat lignocellulosic biomass, in order to remove hemicellulose [START_REF] Garrote | Hydrothermal processing of lignocellulosic materials[END_REF] and enhance the enzymatic digestibility of cellulose (Nitsos et al., 2013), and to subsequently increase the bioethanol [START_REF] Ruiz | Bioethanol production from hydrothermal pretreated wheat straw by a flocculating Saccharomyces cerevisiae strain -Effect of process conditions[END_REF] and biomethane [START_REF] Antwi | Anaerobic digestion and the effect of hydrothermal pretreatment on the biogas yield of cocoa pods residues[END_REF] production yields. The method can also be applied to microalgae that have a carbohydrate cell wall composition. For example, it is very efficient in the release of carbohydrates from microalgae. Carbohydrate yields of 44 % of total carbohydrate content from N. gaditana [START_REF] Lorente | Steam explosion as a fractionation step in biofuel production from microalgae[END_REF], 60% [START_REF] Fu | Hydrothermal hydrolysis pretreatment of microalgae slurries in a continuous reactor under subcritical conditions for large-scale application[END_REF] and 80 % [START_REF] Xiao | A solar-driven continuous hydrothermal pretreatment system for biomethane production from microalgae biomass[END_REF] from C. pyrenoidosa, 97 % from C. vulgaris [START_REF] Xiaojian | Medium-low temperature hydrothermal hydrolysis kinetic characteristics of concentrated wet microalgae biomass[END_REF] have been achieved. At prolonged pretreatment times the released biomolecules can further react to form degradation products [START_REF] Córdova | Physical Pretreatment Methods for Improving Microalgae Anaerobic Biodegradability[END_REF], such as furans and organic acids from the dehydration [START_REF] Horvat | Mechanism of levulinic acid formation[END_REF][START_REF] Mussatto | Alternatives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review[END_REF][START_REF] Palmqvist | Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification[END_REF], or from Maillard reactions [START_REF] Fu | Hydrothermal hydrolysis pretreatment of microalgae slurries in a continuous reactor under subcritical conditions for large-scale application[END_REF] of the carbohydrates with amine containing groups. Bioethanol production from hydrothermally treated Schizocytrium sp. slurry after enzymatic hydrolysis reached 90% of the theoretical maximum ethanol yield [START_REF] Kim | Bioethanol production from micro-algae, Schizocytrium sp., using hydrothermal treatment and biological conversion[END_REF]. Hydrothermal pretreatment increased the yield of biogas from the anaerobic digestion of microalgae biomass by 11% (Martín [START_REF] Juárez | Effect of pretreatments on biogas production from microalgae biomass grown in pig manure treatment plants[END_REF] and 28% [START_REF] Passos | Comparing pretreatment methods for improving microalgae anaerobic digestion: Thermal, hydrothermal, microwave and ultrasound[END_REF] for mixed microalgae biomass and 57% for pure C. pyrenoidosa [START_REF] Xiao | A solar-driven continuous hydrothermal pretreatment system for biomethane production from microalgae biomass[END_REF]. The production of toxic compounds due to the released molecules degradation during steam explosion led to a reduction of biomethane yield due to anaerobic digestion inhibition [START_REF] Córdova | Physical Pretreatment Methods for Improving Microalgae Anaerobic Biodegradability[END_REF].

Table 7

Two stages can be observed during hydrothermal treatment of microalgae based on the temperature regimes. Initially and up to 100 o C the release of high molecular weight intracellular components such as starch and protein occurs; the gelatinization of starch and denaturation of proteins can lead to an increase in the medium viscosity; at 140-160 o C the macromolecules are hydrolyzed into oligomers of lower molecular weight and the viscosity decreases (Chen et al., 2019a;[START_REF] Mcmillan | Evaluation and comparison of algal cell disruption methods: Microwave, waterbath, blender, ultrasonic and laser treatment[END_REF]. The microalgae slurries produced from hydrothermal pretreatment, exhibited shear thinning behavior, suggesting non-Newtonian fluid behaviour (Chen et al., 2018). The ability of viscosity reduction by increasing the temperature or the shear rate can be valuable as it would allow the processing of high biomass concentrations in downstream unit operations.

A kinetic study showed that the treatment temperature, time and pressure were the most important parameters of hydrothermal pretreatment in terms of carbohydrate release, whereas temperature was the main factor that controlled protein release [START_REF] Xiaojian | Medium-low temperature hydrothermal hydrolysis kinetic characteristics of concentrated wet microalgae biomass[END_REF]. This dependence of carbohydrate release on time and temperature is similar in the hydrothermal pretreatment of lignocellulosic biomass, due to the hydrolytic reaction that dominates it. To express the combined effect of time and temperature the severity factor (logRo) has been established in the hydrolysis of lignocellulosic materials, that can be calculated from the equation 𝑅 𝑜 = 𝑡 * 𝑒𝑥𝑝 ( 𝑇 (𝑡) -100 14.75 )

where, t is the pretreatment time and T(t) the pretreatment temperature for holding time t.

It is possible that due to similarities in the chemical composition of carbohydrates in plants and microalgal cell walls, the equation can correlate the effects of microalgae pretreatment with time and temperature. Some evidence towards that can be seen in Table 7. Although the data points correspond to yields from different studies, with different microalgae species, e.g. C.vulgaris [START_REF] Mendez | Enhancing methane production of Chlorella vulgaris via thermochemical pretreatments[END_REF], N. gaditana [START_REF] Lorente | Steam explosion as a fractionation step in biofuel production from microalgae[END_REF] C. pyrenoidosa [START_REF] Xiao | A solar-driven continuous hydrothermal pretreatment system for biomethane production from microalgae biomass[END_REF], different mixing and heating régimes, e.g. stirring in reactor [START_REF] Qu | Hydrothermal cell disruption of Nannochloropsis sp. and its influence on lipid extraction[END_REF], lack of mechanical mixing in steam explosion [START_REF] Lorente | Steam explosion as a fractionation step in biofuel production from microalgae[END_REF] and biomass concentrations ranging from 16 g L -1 [START_REF] Mendez | Enhancing methane production of Chlorella vulgaris via thermochemical pretreatments[END_REF] up to 20% w/w [START_REF] Qu | Hydrothermal cell disruption of Nannochloropsis sp. and its influence on lipid extraction[END_REF]), the increase of biomolecules release or extraction yield with increasing severity can be clearly seen.

Significant protein release from hydrothermally treated microalgae biomass is also possible; 47 % [START_REF] Xiao | A solar-driven continuous hydrothermal pretreatment system for biomethane production from microalgae biomass[END_REF] and 83% [START_REF] Fu | Hydrothermal hydrolysis pretreatment of microalgae slurries in a continuous reactor under subcritical conditions for large-scale application[END_REF] of protein was isolated from C. pyrenoidosa, and 76% from C. vulgaris [START_REF] Xiaojian | Medium-low temperature hydrothermal hydrolysis kinetic characteristics of concentrated wet microalgae biomass[END_REF]. Lower protein recovery (approx. 20%) is observed at lower severity factor (logRo) values (approx. 2). As with carbohydrates, protein release is also well correlated to the treatment's severity factor. Cell wall disruption also allowed the subsequent extraction of lipids, with yields of 60% from N. salina (Lee and Han, 2015a), 68% from C. vulgaris [START_REF] Xiaojian | Medium-low temperature hydrothermal hydrolysis kinetic characteristics of concentrated wet microalgae biomass[END_REF] and 84% from Nannochloropsis sp. [START_REF] Qu | Hydrothermal cell disruption of Nannochloropsis sp. and its influence on lipid extraction[END_REF] reported. After the hydrothermal pretreatment of C. vulgaris it was shown that the lipids remained in the biomass and were not detected in the pretreatment liquid that was rich in carbohydrates and protein [START_REF] Xiaojian | Medium-low temperature hydrothermal hydrolysis kinetic characteristics of concentrated wet microalgae biomass[END_REF]. This may allow the selective separation of water-soluble molecules and lipids, in comparison to other cell disruption methods like bead beating that generate difficult to separate water-oil emulsions.

Other applications include the extraction of carotenoids from H. pluvialis at yields higher than 85% [START_REF] Mendes-Pinto | Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability[END_REF]. Near complete extraction of astaxanthin was shown by hydrothermal disruption of H. pluvialis cells at 200 o C for 10 min (logRo 4) [START_REF] Cheng | Hydrothermal disruption of algae cells for astaxanthin extraction[END_REF]. The chemicals released from hydrothermal treatment of H. pluvialis at 200 o C for 20 min (logRo 4.2) showed significant antioxidant and antimicrobial activity. The antimicrobial activity was attributed to extracted short chain fatty acids, while the antioxidant activity was to vitamin E, penolics, as well as carbohydrate degradation products [START_REF] Rodríguez-Meizoso | Subcritical water extraction and characterization of bioactive compounds from Haematococcus pluvialis microalga[END_REF]. The extraction of phenolic compounds from Chlorella sp. by mild hydrothermal treatment is also possible. Treatment of Chlorella sp. biomass at a concentration of 20 wt. % for 5 min at 163 o C (logRo 2.6), yielded 58.73 mg of gallic acid equivalent g -1 that exhibited significant antioxidant activity (inhibiting by 68.5% the DPPH radical) [START_REF] Zakaria | Subcritical Water Technology for Extraction of Phenolic Compounds from Chlorella sp. Microalgae and Assessment on Its Antioxidant Activity[END_REF].

Temperature had a significant effect on extraction yield. Increasing the temperature from 100 o C to 170 o C increased both the total phenolic content and the antioxidant activity of the extracts. Further increases of the temperature up to 250 o C led to a decrease of the phenolic content and antioxidant activity by almost 50%, to levels comparable to those at 100 o C. [START_REF] Zakaria | Subcritical Water Technology for Extraction of Phenolic Compounds from Chlorella sp. Microalgae and Assessment on Its Antioxidant Activity[END_REF]. This was attributed to thermal degradation of the extracted phenolics. Increase of time from 5 to 20 min decreased phenolic content and antioxidant activity of the extracts by approximately 10% [START_REF] Zakaria | Subcritical Water Technology for Extraction of Phenolic Compounds from Chlorella sp. Microalgae and Assessment on Its Antioxidant Activity[END_REF]. Increasing the biomass concentration from 5 wt. % to 20 wt. % increased phenolic content by approx. 50%. Antioxidant activity, however, peaked at 12.5 wt. % and remained almost constant up to 20 wt. % of biomass concentration, but no explanation for this phenomenon was offered [START_REF] Zakaria | Subcritical Water Technology for Extraction of Phenolic Compounds from Chlorella sp. Microalgae and Assessment on Its Antioxidant Activity[END_REF].

Hydrothermal treatment of biomass at very high temperatures (above 230 o C -supercritical water) can lead to cellular structure disintegration through biomass carbonization, that can also be beneficial for lipid extraction [START_REF] Kröger | Hydrothermal Disintegration and Extraction of Different Microalgae Species[END_REF]. At even higher temperatures (e.g. 260, 300, 340 °C) the biomass undergoes hydrothermal liquefaction (HTL) and is directly converted to hydrothermal bio-oil that can be used as a biofuel (B. Zhang et al., 2018). This is no longer a pretreatment method but a direct conversion method of microalgae biomass into the final product. Its main advantages are that water removal is not required and that it is a single step process, but it is limited to biofuel production, and therefore maybe more suited for lower value microalgae or microalgae debris after the extraction of valuable molecules.

Overall hydrothermal pretreatment has many advantages including lack of reagents other than water, ability to operate at high solids content, ability to pretreat a variety of microalgae species and ease of post treatment separation and fractionation of compounds. Careful optimization is required, to minimize degradation of valuable products due to the high temperatures employed. Another disadvantage is the energy input required for the reaction to take place, however, significant energy can be recovered by heat exchange (Chen et al., 2019b) and even solar heated hydrothermal reactors showed significant extraction yields minimizing energy requirements of the process [START_REF] Xiao | A solar-driven continuous hydrothermal pretreatment system for biomethane production from microalgae biomass[END_REF].

Acid treatment

The pretreatment of microalgae with dilute solutions of inorganic acids can be considered as a hydrothermal pretreatment variation. The rate of hydrolytic reactions at elevated temperatures in water, are enhanced by the acid catalyst's addition. High carbohydrate, lipid and protein yields can be achieved at lower temperatures and shorter reaction times. Acid concentration employed is usually between 1-5 % and temperatures between 110 o C and 150 o C have been reported; but the specific conditions must be tailored to the target molecules to be extracted. Sulfuric acid is most commonly used for acid pretreatment due to its low cost and efficiency. Other acids such as nitric acid have also been tested, but significant reduction in the yield has been observed at higher acid concentrations [START_REF] Lee | Hydrothermal nitric acid treatment for effectual lipid extraction from wet microalgae biomass[END_REF]Lee and Han, 2015b). This is probably a result of the nitric acid's oxidative action that would degrade the released molecules. Hydrochloric acid gave superior saccharification results compared to sulfuric acid and nitric acid at concentrations ranging from 0.2 M to 2M, in the hydrolysis of Hindakia tetrachotoma ME03 biomass [START_REF] Onay | Bioethanol production via different saccharification strategies from H. tetrachotoma ME03 grown at various concentrations of municipal wastewater in a flatphotobioreactor[END_REF].

A modified version of the severity factor (logRo) from the hydrothermal pretreatment can be expressed in terms of time (t), temperature (T) and acid catalyst concentration (related to pH). The Combined Severity Factor (CSF) is given by the equation:

𝐶𝑆𝐹 = 𝑙𝑜𝑔 [𝑡 exp ( 𝑇 -100 14.75 )] -𝑝𝐻

A good correlation between the process CSF and the carbohydrate release or the lipid extraction yields can be obtained (Table 8). For N. gaditana a carbohydrate yield of 50 % was achieved at a CSF 0.4 and increased to 59%, 63% and 89.7% for CSFs of 0.75, 1.0 and 1.4, respectively [START_REF] Lorente | Steam explosion as a fractionation step in biofuel production from microalgae[END_REF]. Released carbohydrates from microalgae have been used to produce bioethanol with increased yield compared to the untreated biomass [START_REF] Choi | Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production[END_REF][START_REF] Ngamsirisomsakul | Enhanced bioethanol production from Chlorella sp. biomass by hydrothermal pretreatment and enzymatic hydrolysis[END_REF]. The degradation products generated from acid hydrolysis can lead to inhibition of fermentation-based microalgae valorisation processes (Martín [START_REF] Juárez | Effect of pretreatments on biogas production from microalgae biomass grown in pig manure treatment plants[END_REF]. Pretreatment liquid neutralization may also be required as the low pH values can significantly reduce microbial growth.

Table 8 Lipid extraction yields remain relatively low (< 30%) below CSF values of 1.4, whereas lipid yields up to 88% are reported at CSF of 1.9 [START_REF] Lee | Hydrothermal nitric acid treatment for effectual lipid extraction from wet microalgae biomass[END_REF]. This indicates that relatively high cellular wall degradation is required to enable efficient intracellular lipids solvent extraction Although the degree of cell disruption is not reported by the authors, it is possible that at these higher CSF' values where lipid extraction is efficient, extensive cell wall disruption has occurred. Optimal carbohydrate-hydrolysis and lipid-extraction yields seemed to occur in a very narrow parameter range (CSF values between 1.4 and 2). This can lead to severe degradation of the released products even with minute changes in the process control. Moderate protein release around 50% is also possible with CSF values of approximately 2 [START_REF] Lorente | Microalgae fractionation using steam explosion, dynamic and tangential cross-flow membrane filtration[END_REF].

Acid treatment has also showed promising results in extraction of high-value molecules. Astaxanthin extraction from H. pluvialis by treatment with 4M HCl at 70 o C for 60 min (CSF 1.5) achieved a yield of 80% [START_REF] Liu | The efficiency and comparison of novel techniques for cell wall disruption in astaxanthin extraction from Haematococcus pluvialis[END_REF]. Lutein extraction yield of 3.5 mg g -1 C. pyrenoidosa biomass was achieved with 16% HCl treatment for 41 min [START_REF] Arun | Ultrasound assisted enhanced extraction of lutein ( β , ε-carotene-3 , 3 '-diol ) from Mircroalga ( Chlorella pyrenoidosa ) grown in wastewater : Optimization through Response Surface Methodology[END_REF].

In general, dilute acid pretreatment is a very fast and efficient pretreatment method for the extraction of carbohydrates and lipids from microalgae. Application for extraction of high value molecules may also become possible in the future. However, due to the acids aggressive nature, careful pretreatment conditions optimization is required to avoid the extracted molecules degradation, especially the proteins, antioxidants, and pigments.

Alkaline treatment

Alkaline treatment involves biomass exposure to a basic aqueous medium at elevated temperature. Like acid hydrolysis, it can also be considered as a hydrothermal pretreatment variation with addition of a basic catalyst. A fundamental difference concerns the operatory condition of theses pre-treatments, especially adapted for lower temperatures, (below 120 o C), and longer treatment time that can span up to several days (Mahdy et al., 2014a). Alkaline pretreatment can target the hydrolysis of ester bonds and hydrogen bonds between polysaccharide and non-polysaccharide cell wall components, especially at lower temperatures [START_REF] Costa | Plant Cell Wall, a Challenge for Its Characterisation[END_REF]. For this reason, it has been extensively used for the extraction of hemicellulose from lignocellulosic biomass. At low temperature microalgae alkaline pretreatment induces a mercerization-like effect accompanied by cell wall swelling and an improvement of protein extraction yields [START_REF] Phong | Improving cell disruption efficiency to facilitate protein release from microalgae using chemical and mechanical integrated method[END_REF]. At higher temperatures intramolecular glycoside bonds are also affected leading to the hydrolysis of cell wall and intracellular carbohydrates.

Like acid pretreatment the combined effect of time (t), temperature (T) and base catalyst concentration (pOH), can be expressed by a modified Combined Severity Factor (CSF') that is given by the equation:

𝐶𝑆𝐹′ = 𝑙𝑜𝑔 [𝑡 exp ( 𝑇 -100 14.75 )] -𝑝𝑂𝐻

The CSF' can describe the effect of pretreatment on the hydrolysis of carbohydrates, release of protein and lipid extraction from microalgae. Again, a narrow range of CSF' values (between 1.2 and 2.5) provides the optimal carbohydrate hydrolysis yields (80 to 100 %) that have been reported. Approximately 80% of total carbohydrates were recovered in the hydrolysates of H. tetrachotoma ME03 (Onay, 2019) and a mixed microalgae culture [START_REF] Shokrkar | Bioethanol production from acidic and enzymatic hydrolysates of mixed microalgae culture[END_REF] at CSF' 2.4 and 2.5, respectively. Complete carbohydrate recovery was possible for C. vulgaris ESP6 at a much lower CSF' of 1.3 [START_REF] Liu | Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock[END_REF]. Protein release yields from a mixed extraction yield was moderate at 32%, however very low CSF' values were tested (CSF'<-2.9). These values were obtained by pretreatment temperatures up to 75 o C and prolonged pretreatment times of up to 48h; CaO (lime) was used as the alkaline catalyst. Similar results were found for the pretreatment of C. vulgaris with 42% protein yield at CSF' -1.8 [START_REF] Mendez | Enhancing methane production of Chlorella vulgaris via thermochemical pretreatments[END_REF]. The extraction of lipids by enzymatic hydrolysis of alkaline pretreated Nannochloropsis sp. was comprehensively studied for a variety of temperature, time and pH pretreatment conditions (Wu et al., 2017). Alkaline treatment was followed by an enzymatic hydrolysis step with a cocktail of commercial enzymes (cellulase, protease, lysozyme, and pectinase). The results show a very good correlation with CSF'. Maximum lipid extraction yields were obtained at low CSF' values, between -0.9 and -0.3, much lower compared to those required for maximum carbohydrate release shown above. They are also lower compared to the CSF values required for quantitative lipid extraction by acid pretreatment. This could be due to differences in cell wall resilience between species. Also, the results of this study are not directly comparable to the rest alkaline pretreatment studies as it employs an extra enzymatic hydrolysis process step, after alkaline treatment, leading to increased cell wall degradation and easier lipid extraction. Further increasing the severity beyond -0.3 leads to a reduction in the lipid yield, probably due to lipid degradation.

Alkaline pretreatment is also used to enhance the production of bio-methane from microalgae anaerobic digestion. The pretreatment of C. vulgaris and Scenedesmus sp. at low CSF' values led to marginal increases in the methane yield (20% improvement at the base case) (Mahdy et al., 2014a). Similar results were obtained in the alkaline pretreatment of mixed microalgae culture at low CSF' (0.24) that led to a 25% improvement of methane yield [START_REF] Solé-Bundó | Enhancement of microalgae anaerobic digestion by thermo-alkaline pretreatment with lime (CaO)[END_REF]. The NaOH pretreatment of C. vulgaris at CSF' -1.8 gave a 68.8% methane yield [START_REF] Mendez | Enhancing methane production of Chlorella vulgaris via thermochemical pretreatments[END_REF]. NaOH pretreatment of mixed microalgae biomass significantly increased methane production from approximately 162 mL CH4 g -1 of volatile solids (VS) to 377 mL CH4 g -1 VS, leading to 91% of biodegradability [START_REF] Juárez | Effect of pretreatments on biogas production from microalgae biomass grown in pig manure treatment plants[END_REF]. The enhanced extraction of the carotenoid lutein from Scenedesmus almeriensis by alkaline pretreatment has also been studied [START_REF] Cerón | Recovery of lutein from microalgae biomass: development of a process for Scenedesmus almeriensis biomass[END_REF]. The recovery yield can be related to CSF' over a very narrow range, between -2 and 0 with the maximum recovery of 90% achieved at -0.8 The rapid lutein yield decrease shows that optimization of parameters is critical for extraction of sensitive molecules to avoid loss through degradation reactions.

3.2.4 Oxidative pretreatment 3.2.4.1 Ozonolysis.

Exposure to oxidative agents can be a very aggressive treatment of biological materials. It has been traditionally used in the pulp and paper industry for pulp bleaching, as a more environmentally friendly alternative to traditional chlorine-based processes [START_REF] Rounsaville | Evolution of ozone for the bleaching of paper pulps[END_REF]. Bleaching is achieved by removing the residual and recalcitrant lignin that remains after the pulping process. It has also been applied as a pretreatment method of lignocellulosic biomass to enhance enzymatic hydrolysis [START_REF] García-Cubero | Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw[END_REF], and as a cell disruption method for treatment of microalgae (Keris-Sen and Gurol, 2017). As with pretreatment of lignocellulosic biomass, ozonolysis of microalgae cells can increase the enzymatic cell wall saccharification leading to 81% glucose yields. Pretreatment efficiency increases with the ozone dose administered up to a maximum and then gradually decreases. Low glucose yields at low ozone doses is attributed to insufficient cell wall disruption, whereas low yields at high ozone dosages is the result of carbohydrates degradation (Keris-Sen and Gurol, 2017). Thus, an ozone dose optimization is to be determined in order to improve the yield of glucose.

Exposure of microalgae cells to ozone could be performed by conventional bubbling of ozone or under pressure assisted ozonation with compression and decompression cycles [START_REF] Huang | Comparison of cell rupturing by ozonation and ultrasonication for algal lipid extraction from Chlorella vulgaris[END_REF].

Ozonolysis leads to cell rupture and the release of intracellular material in the medium that is accompanied by measurable increases of total organic compounds, nitrogen and phosphorus. Increasing the ozone pressure as well as the number of compression and decompression cycles is beneficial for the lipid extraction; optimization of the two parameters led to 27% lipid yield (g g -1 biomass) [START_REF] Huang | Comparison of cell rupturing by ozonation and ultrasonication for algal lipid extraction from Chlorella vulgaris[END_REF]. Two mechanisms of pretreatment were identified. Under conventional ozonation, oxidation increases cell wall permeability and cytoplasm release. In the pressure assisted system, compression increases dissolved gas inside the cytoplasm and during decompression gas expansion occurs. Repetition of the compression decompression cycle induces additional stress to the cells and increases disruption efficiency and lipid extraction yields [START_REF] Huang | Comparison of cell rupturing by ozonation and ultrasonication for algal lipid extraction from Chlorella vulgaris[END_REF].

Ozonation of the filamentous cyanobacteria A. platensis has been investigated in various ozone doses. At low ozone concentrations (5-15 mg L -1 ) the dominant effect observed was the filament's shear into smaller fragments. At higher ozone doses (up to 510 mg L -1 ) an increase in filament transparency accompanied by the release of intracellular organic material was observed; these cellular material subsequently formed clusters [START_REF] Akao | Effect of ozonation on Spirulina platensis filaments by dynamic imaging particle analysis[END_REF]. Similar effects were observed in the ozonation of mixed microalgae cultures for the production of methane through anaerobic digestion [START_REF] Cardeña | Enhancement of methane production from various microalgae cultures via novel ozonation pretreatment[END_REF]. At optimal ozonation pretreatment conditions a 66% increase in the methane yield was obtained. Under these conditions cells exhibited fragmentation in the cell walls due to oxidation leading to cell lysis and better access of hydrolytic enzymes from the anaerobic bacteria to the intracellular material [START_REF] Cardeña | Enhancement of methane production from various microalgae cultures via novel ozonation pretreatment[END_REF]. Ozonation pretreatment leads to significant increases in the oil extraction yield up to 100% in some cases [START_REF] Lin | A new processing scheme from algae suspension to collected lipid using sand filtration and ozonation[END_REF].

Hydrogen peroxide

H2O2 is an alternative oxidizing agent for the pretreatment of microalgae with a similar mechanism to ozonation. Oxidation of mixed microalgae culture with H2O2 leads to biomass solubilization of 23% and a 173% increase in the methane production yield corresponding to 67% biodegradability [START_REF] Juárez | Effect of pretreatments on biogas production from microalgae biomass grown in pig manure treatment plants[END_REF]. Astaxanthin extractability of 54.5 % from H. pluvialis was possible by treatment with 2M of H2O2 [START_REF] Haque | Intensified green production of astaxanthin from Haematococcus pluvialis[END_REF].

An alternative method to increase oxidation capacity is to combine the H2O2 with iron ions in order to induce Fenton-like reactions. The two chemicals react to create hydroxyl radicals (‧OH -) that attack the microalgae's organic molecules. The combined use of H2O2 with FeSO4 significantly increased lipid extraction efficiency from C. vulgaris to 78% of total lipids compared to 41% when H2O2 was used alone, [START_REF] Steriti | A novel cell disruption technique to enhance lipid extraction from microalgae[END_REF]. A Fenton-like reaction with FeCl3 as the cell disruption reagent for lipid extraction from Chlorella sp. KR-1, was improved by simultaneous UV-irradiation of the reaction mixture. The unaided Fenton-like reaction yielded 80% of total lipids at 0.5% H2O2. The UV-Fentonlike reaction experiment improved the lipid yield to 85% at a reduced 0.3% H2O2 consumption (Seo et al., 2015a). The substitution of H2O2 with the persulfate ion (S2O8 2-) was also investigated. The persulfate ion reacted with Fe 3+ to generate SO4 •-and S2O8 •-radicals instead of the ‧OH -radicals of the typical Fenton reaction [START_REF] Seo | Lipid extraction from microalgae cell using persulfate-based oxidation[END_REF]. The persulfate reagent at 2mM concentration led to improved lipid extraction yield 95%, compared to 80% for 0.5% (v/v) H2O2 at the same pretreatment conditions.

The aggressive nature of all the oxidative pretreatment reactions leads to chemical alterations of the targeted biomolecules. Most affected are the sensitive pigments like chlorophyll. The partial or complete destruction of chlorophyll in the oxidation pretreatment of microalgae samples has been reported for ozonation [START_REF] Huang | Comparison of cell rupturing by ozonation and ultrasonication for algal lipid extraction from Chlorella vulgaris[END_REF][START_REF] Kamaroddin | In-situ disinfection and a new downstream processing scheme from algal harvesting to lipid extraction using ozone-rich microbubbles for biofuel production[END_REF] and Fenton-like reaction pretreatments (Seo et al., 2015a, p.). Additionally, significant chemical changes are observed in the composition of the extracted lipids. These changes are mainly the significant increase of saturated and monounsaturated fatty acids and the reduction in polyunsaturated fatty acids. The Fenton pretreatment of C. vulgaris increased the saturated, monounsaturated and C18:2 fatty acids in the extracted lipid from 35 % (w/w) in the untreated sample to 80 % (w/w) after pretreatment. At the same time the content of polyunsaturated fatty acids in the cells pretreated by Fenton reaction was dramatically decreased from 60 % to 20 % (w/w) (Concas et al., 2015a). In another study of C. vulgaris pretreated by Fenton reaction the polyunsaturated fatty acids content decreased from 21 % (w/w) to 4.75 % (w/w) (Concas et al., 2015b). Similarly, the ozonation of C. vulgaris decreased the concentration of saturated fatty acids to very small or non-detectable amounts, and even decreased the content of some saturated fatty acids, probably due to very severe oxidation conditions [START_REF] Huang | Comparison of cell rupturing by ozonation and ultrasonication for algal lipid extraction from Chlorella vulgaris[END_REF]. The sensitivity of polyunsaturated fatty acids to oxidation was also shown in the ozonation of D. salina where accumulation of saturated fatty acids and even some hydrocarbons were detected in the extracted lipids [START_REF] Kamaroddin | In-situ disinfection and a new downstream processing scheme from algal harvesting to lipid extraction using ozone-rich microbubbles for biofuel production[END_REF].

The reduction in chlorophyll content is considered beneficial when the isolated lipids are transformed into biodiesel as chlorophyll deactivates the esterification catalyst (Seo et al., 2015a). Equally beneficial is the reduction in polyunsaturated fatty acids and the increase in saturated, as the corresponding saturated fatty acid methyl esters (biodiesel) are more resistant to oxidation and degradation from oxygen, heat, light etc. (Concas et al., 2015a;[START_REF] Steriti | A novel cell disruption technique to enhance lipid extraction from microalgae[END_REF]. However, if the targeted products are high added value molecules such as unsaturated fatty acids (e.g. ω3 fatty acids) pigments (e.g. chlorophyll, carotenoids) and antioxidants (e.g. astaxanthin) that are sensitive to oxidation, such pretreatment methods may be detrimental to the product quality and/or yield. If optimization of oxidative processes to minimize product degradation is not possible, they should be exclusively used for the production of bulk products such as fermentable sugars (Keris-Sen and Gurol, 2017), biogas [START_REF] Juárez | Effect of pretreatments on biogas production from microalgae biomass grown in pig manure treatment plants[END_REF] and biodiesel [START_REF] Steriti | A novel cell disruption technique to enhance lipid extraction from microalgae[END_REF].

Enzymatic methods

Enzymatic treatment is even more species specific than the methods described above. Microalgal cell walls are chemically complex and may contain several carbohydrates (cellulose, hemicellulose, pectin or chitin), protein or other organic polymers such as sporopollenin and algaenan. They are also structurally complex as these polymers are often organized in layers. Cell wall structure and composition varies between species and as a result the enzymatic activities on cell walls of different species will differ. Successful enzymatic disruption of such complex structures requires combinations of the necessary enzymatic activities. Indeed, the treatment of N. gaditana with Alkalase -a protease with esterase activity for peptide and amino acid esters-showed only 35% protein release (Safi et al., 2017b).

Although significant hydrolysis of protein can be achieved, cell wall integrity was not compromised and cell breakage was not detected, because the remaining structural components remain intact. Another disadvantage of the protease is the reduction of the released protein functionality due to their hydrolytic fragmentation (Safi et al., 2017a).

Similarly, treatment of Scenedesmus sp. with lysozyme -an enzyme that hydrolyses cell wall peptidoglycan by specifically degrading N-acetylglucosamine-increased the lipid extraction yield by only 16.6% compared with cellulase treatment alone [START_REF] Taher | Effective extraction of microalgae lipids from wet biomass for biodiesel production[END_REF]. Lysozyme treatment on C. vulgaris thinned and delaminated the outer wall and removed hair-like fibers from the cell's surface [START_REF] Gerken | Enzymatic cell wall degradation of Chlorellavulgaris and other microalgae for biofuels production[END_REF]. This study compared many enzymes (chitinase, chitosanase, b-glucuronidase, pectolyase, trypsin, laminarinase, lyticase, phospholipase A1, sulfatase, b-glucuronidase and sulfatase) and showed that lysozyme was the only enzyme able to increase cell permeability when applied alone. When lysozyme and sulfatase were combined the permeabilization of 96 % of the cell population was achieved. In other experiments lysozyme treatment, enabled high protein and pigments extraction from Chlorella sp., Scenedesmus sp. and Chlamydomonas sp. [START_REF] Al-Zuhair | Enzymatic pre-treatment of microalgae cells for enhanced extraction of proteins[END_REF]. This required a 30fold increase in the pretreatment time (16h) compared to the previous study [START_REF] Taher | Effective extraction of microalgae lipids from wet biomass for biodiesel production[END_REF].

The combination of a protease with a cellulase is a typical enzymatic treatment strategy as it can target the two main cell wall components, proteins/peptides and cellulose and/or other carbohydrates. In the treatment of C. reinhardtii and C. vulgaris Alkalase and Viscozyme (a multienzyme with b-glucanase, arabinase, hemicellulase, xylanase and other activities) led to almost complete protein and carbohydrate solubilization (Mahdy et al., 2014b). Cellulase, pectinase and lysozyme were used together for lipid extraction from Nannochloropsis sp. with a 22% recovery yield [START_REF] Chen | Influence of a facile pretreatment process on lipid extraction from Nannochloropsis sp. through an enzymatic hydrolysis reaction[END_REF]. In some cases, the cellulase-protease combination performed poorly. For example, treatment of H. pluvialis by combined protease K and drierase (a cellulase, hemicellulase and pectinase mix) did not enhance the extraction efficiency of astaxanthin. This was attributed to the enzymes inability to attack the cell wall sporopollenin, a very recalcitrant polymer [START_REF] Mendes-Pinto | Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability[END_REF]. This underlines the importance of selecting the appropriate mix of enzymatic activities according to the microalgae specie.

Combination of carbohydrate hydrolyzing enzymes is another strategy for cell wall disruption. The combined use of cellulase and pectinase at 1:1 ratio and 7000 U mL -1 total enzyme dosage enabled 71% astaxanthin extraction [START_REF] Ye | Mechanisms of breakdown of Haematococcus pluvialis cell wall by ionic liquids, hydrochloric acid and multi-enzyme treatment[END_REF]. The combined treatment of Nannochloropsis sp. with Cellulyve ® 50LC (cellulase) and Feedlyve ® GMA (Endo-β-1,4-mannanase) led to a lipid extraction yield of 90% [START_REF] Zuorro | Optimization of enzyme-assisted lipid extraction from Nannochloropsis microalgae[END_REF]. Treatment with carbohydrase cocktails has also been performed in tandem, when the enzymes have different temperature optima simultaneous treatment would lead to denaturation and loss of some enzymatic activity. For example, treatment of mixed cultures with βglucosidase/cellulase at 65 o C, followed by α-amylase at 95 o C and an amyloglucosidase at 55 o C yielded 95% of total sugars [START_REF] Shokrkar | Bioethanol production from acidic and enzymatic hydrolysates of mixed microalgae culture[END_REF]. A similar total sugar yield of 92% was achieved by the same treatment strategy in the case of H. tetrachotoma ME03 [START_REF] Onay | Bioethanol production via different saccharification strategies from H. tetrachotoma ME03 grown at various concentrations of municipal wastewater in a flatphotobioreactor[END_REF]. Fermentation of the sugar hydrolysates gave approximately 90% of theoretical bioethanol yield. It is also worth noting that the enzymatic reaction sequence initially targeted the cell wall structural carbohydrates (cellulase and b-glucosidase activities) followed by the intracellular storage carbohydrates (a-amylase and amyloglucosidase activities). This enzyme specificity towards the targeted molecules could be useful in hydrolysis products separation e.g. carbohydrates and proteins. This can be achieved by sequential treatment, first with proteases to enable protein release, followed by carbohydrate hydrolysing enzymes to remove sugars from the microalgae biomass. A variation of enzymatic pretreatment is autolysis. It occurs when the microalgae cells under stress produce cell wall degrading enzymes leading to cell lysis. When C. reinhardtii is exposed to nitrogen depletion stress the production of autolysin is induced. Autolysin is a protease that targets proline-rich areas in C. reinhardtii cell wall polypeptides. Its synthesis can be enough to enable extensive cell disruption and quantitative protein and lipid release [START_REF] Sierra | Enzymatic cell disruption of the microalgae Chlamydomonas reinhardtii for lipid and protein extraction[END_REF]. The main benefits of this strategy are the expensive enzyme redundancy and conservation of intracellular protein integrity.

Another strategy to reduce the high cost of enzymes is their in-situ production by coculturing the microalgae with an enzyme producing microorganism. For this reason, C. vulgaris was co-cultured with the bacterium Flammeovirga yaeyamensis that could release amylase, cellulase, and xylanase in the growth medium [START_REF] Chen | Improving microalgal oil collecting efficiency by pretreating the microalgal cell wall with destructive bacteria[END_REF]. The collected microalgae cells showed damage to the cell walls induced from the enzymes during co-culture. The supernatant from the co-culture was used to pretreat C. vulgaris cells in a separate pretreatment process, significantly increasing the release of hydrolyzed sugars and lipids.

The non-specific enzymatic pretreatment of microalgae is also possible with the use of oxidizing enzymes. For example, manganese peroxidase (a lignin degrading enzyme using Mn 2+ ions as oxidizing mediators) was produced by the white-rot fungi Anthracophyllum discolor. The enzymatic extract was used for the pretreatment of Botryococcus braunii to enhance methane production [START_REF] Ciudad | Performance of an enzymatic extract in Botrycoccus braunii cell wall disruption[END_REF].

The enzymatic pretreatment increased methane production from approximately 320 mL CH4/g VS to 521 mL CH4/g VS that corresponds to 90% of biodegradability, compared to 60% biodegradability of the untreated microalgae cells. A complex enzymatic broth containing laccase (another lignin oxidizing enzyme) from the fungus Trametes versicolor exhibited similar improvement in methane production from the anaerobic digestion of pre-treated mixed microalgae cultures [START_REF] Hom-Diaz | Enzymatic pretreatment of microalgae using fungal broth from Trametes versicolor and commercial laccase for improved biogas production[END_REF].

Despite the promising results, high enzyme cost is the major hurdle to be overcome for application of enzymatic processes to biorefineries. Several strategies have been employed towards this end, such as co-cultivation and autolysis that were discussed above. Another strategy concerns the optimization of the enzyme production process to reduce production cost through increased productivity and yield. Reduction of cost through lowering the enzyme dosage has also been achieved by optimizing the enzymatic cocktail and by discovery of new more potent enzymes. Such approaches in bioethanol production from lignocellulosic biomass have reduced the enzyme production costs by a factor of ten (10) [START_REF] Dahiya | Chapter 28 -Reducing enzyme costs, novel combinations, and advantages of enzymes could lead to improved cost-effective biofuels' production[END_REF].

Table 9 3.4 Novel downstream processing concepts

Combinations of cell disruption methods

Combinations of disruption methods have been frequently investigated to increase pretreatment efficiency. One example is the combination of heat treatment together with High Pressure Homogenization. This led to an initial increase in viscosity due to increased solubilization of polymers, such as proteins and carbohydrates, and a subsequent reduction of viscosity from the HPH treatment caused by a decrease in the molecular weight due to increased shear thinning [START_REF] Bernaerts | Impact of different sequences of mechanical and thermal processing on the rheological properties of Porphyridium cruentum and Chlorella vulgaris as functional food ingredients[END_REF][START_REF] Bernaerts | Microalgal biomass as a (multi)functional ingredient in food products: Rheological properties of microalgal suspensions as affected by mechanical and thermal processing[END_REF]. The combination allowed the treatment of cells at high concentrations, e.g. Nannochloropsis sp pastes of 20-25% solids [START_REF] Olmstead | Low solvent, low temperature method for extracting biodiesel lipids from concentrated microalgal biomass[END_REF]. The thermal pretreatment increased cell wall susceptibility to mechanical disruption by HPH and improved lipid extraction yields [START_REF] Halim | The CIDES process: Fractionation of concentrated microalgal paste for co-production of biofuel, nutraceuticals, and highgrade protein feed[END_REF].

Ultrasonication is a pretreatment method with moderate efficiency, however, it can be easily combined with other methods to increase cell disruption capability. When used together with H2O2 oxidation a two-fold increase of C. pyrenoidosa cell lysis was achieved, compare to ultrasonication alone (Z. Duan et al., 2017). Combined with alkali pretreatment it could improve cell disruption, protein solubilization and protein yield from C. sorokiniana and C. vulgaris, compared to either pretreatment method on its own [START_REF] Phong | Improving cell disruption efficiency to facilitate protein release from microalgae using chemical and mechanical integrated method[END_REF]. Improved cell lysis was also observed when ultrasonication was combined with physical pretreatment methods such as pressure drop through nozzle spraying (M. Wang and Yuan, 2015). When Parachlorella kessleri was treated with HPH low protein and carbohydrate yields of 11% and 20%, were obtained, respectively. When HPH was preceded by ultrasonication these values increased to 89% and 74%, respectively (Zhang et al., 2019a). Ultrasonication in combination with a two-phase system of methanol/K3PO4 solution can effectively combine cell-lysis and partition of proteins from cellular debris in one step [START_REF] Phong | Extractive disruption process integration using ultrasonication and an aqueous two-phase system for protein recovery from Chlorella sorokiniana[END_REF]. Cell lysis and recovery of protein in a single step is also possible with the combination of ultrasonication in an ionic liquid, that led to 95.0% protein recovery (Sze Ying Lee et al., 2017). Despite these interesting concepts, the major drawback of ultrasonication remains its inability to process high cell concentration cell-pastes. Ultrasolication at 400 W has been used in combination with the ionic liquid cholinium 2-hydroxy-3morpholinopropanesulfonate to extract 95% of total protein from C. vulgaris cells. Cell disruption and protein extraction were performed in a single step as the ionic liquid (as a 50mM aqueous buffer) was a suitable solvent for protein extraction (Lee et al., 2017). The processing time was 30 min and biomass concentration was 6 g L -1 (Lee et al., 2017).

Novel cell disruption methods

CO2 explosion

Cell lysis via CO2 explosion is an interesting method for microalgae pretreatment. The method includes cell pressurization with CO2 that leads to increased intracellular gas concentration. It is followed by the rapid pressure release that causes expansion of intracellular gas and leads to cell disruption. It has already been shown to work for lipid extraction from the lipid producing yeast Rhodotorula glutinis, where CO2 explosion was performed at 35 bar, leading to significant cell breakage and release of intracellular lipids to the aqueous phase [START_REF] Howlader | Microbial cell disruption for improving lipid recovery using pressurized CO2: Role of CO2 solubility in cell suspension, sugar broth, and spent media[END_REF]. The method has also been used for the extraction of carotenoids and chlorophylls from B. braunii [START_REF] Uquiche | Enhancement of pigment extraction from B. braunii pretreated using CO2 rapid depressurization[END_REF]. Pretreatment at 21 o C and 130 bar increased the pigments extraction yield 10-fold compared with non-treated cells. CO2 explosion is similar to steam explosion, but the lack of heating reduces degradation reactions and could be a viable method for the extraction of valuable and sensitive intracellular molecules. Lately, a continuous CO2 explosion system was described that can effectively disrupt Neochloris oleoabundans UTEX 1185 cells. The continuous method reduced CO2 consumption between 2-4 fold, reduced the process time 3-9 fold, and increased biomolecule yields by more than 2 fold compared to the batch method [START_REF] Günerken | Disruption of microalgae with a novel continuous explosive decompression device[END_REF]. Other non-reactive gasses such as N2 can also be used for cell lysis through explosive decompression [START_REF] Simpson | Disruption of Cultured Cells by Nitrogen Cavitation[END_REF].

Electricity-based methods

High voltage electric discharges (HVED) utilizes electrodes of needle-plate geometry to deliver high voltage pulses to microalgae suspensions. Electroporation of cells similar to that hown for PEF is also present in HVED treatment. HVED additionally induces thermal and mechanical effects to the cells due to cavitation and shockwave formation (Zhang et al., 2019c). Application of HVED (40 kV cm -1 , 4 ms) to Nannochloropsis sp. allowed the selective release of ionic components and small molecular weight water soluble components. Aggregation of the cells as a result of the electric discharge was also observed [START_REF] Grimi | Selective extraction from microalgae Nannochloropsis sp. using different methods of cell disruption[END_REF]. These findings were also confirmed in the HVED treatment of 1% w/w Parachlorella kessleri suspensions at similar conditions (40 kV cm -1 , 8 ms) that led to release of ionic cell components and carbohydrates but protein yield was only 15% w/w (Zhang et al., 2019b). For this reason, HVED was proposed as a first treatment step for the selective release of low molecular weight and water-soluble molecules, followed by organic solvent extraction of chlorophylls, carotenoids and lipids [START_REF] Zhang | Two-step procedure for selective recovery of bio-molecules from microalga Nannochloropsis oculata assisted by high voltage electrical discharges[END_REF]. Application of HVED to B. braunii cell collonies allows the easy separation of the cells from their hydrocarbon extracellular matrix. Subsequantly cells sink to the bottom while the hydrocarbon matrix rises to the surface of the process vessel, allowing for easy separation and collection [START_REF] Guionet | The narrow window of energy application for oil extraction by arc discharge[END_REF].

Non-thermal plasma is another electricity-based method where a needle to plate electrode geometry is placed in an argon filled reactor. The application of high voltage (17 kV, 30 mA) between the electrodes generated an intense core plasma towards the microalgae biomas that led to 65-70% cell rupture of N. gaditana cells and 18.7% lipid extraction [START_REF] Matos | DISRUPTION OF Nannochloropsis gaditana (EUSTIGMATOPHYCEAE) RIGID CELL WALL BY NON-THERMAL PLASMA PRIOR TO LIPID[END_REF]. The method decreased the content of polyunsaturated ω-3 fatty acids from 31% to 11% making it more suitable to biofuels applications. Nanowire-assisted electroporation of Chlorella sp. with copper oxide nanowire-modified threedimensional copper foam electrodes (10 sec, 2V) led to complete inactivation and disruption of the cells and release of organic matter in the liquid phase (50 mg L -1 ) with a very low energy consumption 0.014 kWh kg -1 [START_REF] Bai | Efficient nanowire-assisted electroporation and cellular inclusion release of microalgal cells achieved by a low voltage[END_REF]. Osmotic shock of A. platensis at the same pretreatment conditions led to the highest lipid yield (8.9% extracted with chloroform/methanol 1:2 v/v) compared to ultrasonication, autoclave, acid and alkaline treatments [START_REF] Sumprasit | Biodiesel and biogas recovery from Spirulina platensis[END_REF]

Osmotic shock

Osmotic shock is a simple and effective microalgae pretreatment method. When microalgae cells are suspended in high concentration salt solutions, the density difference between cytoplasm and salt solution creates an osmotic pressure across the cellular membrane that can lead to cell disruption. Treatment of C. vulgaris cells in a 10% w/v NaCl solution for 48 h yielded 86% of total lipids. Microscopic observations showed that osmotic shock acts by creating holes in the cell membranes and does not lead to complete cell disintegration. The disruption effect treatment is also more homogeneous in the cell population [START_REF] Heo | An integrative process for obtaining lipids and glucose from Chlorella vulgaris biomass with a single treatment of cell disruption[END_REF]. Effectiveness of the osmotic shock treatment depends on the concentration of the salt solution. When 6% w/v solutions of NaCl and sorbitol were used for the pretreatment of C. reinhardtii, lipid yields were not increased compared to the untreated cells [START_REF] Yoo | Direct lipid extraction from wet Chlamydomonas reinhardtii biomass using osmotic shock[END_REF]. When cell wall-free mutants were treated at the same conditions, lipid yield for sorbitol and NaCl increased to 20% and 35%, respectively [START_REF] Yoo | Direct lipid extraction from wet Chlamydomonas reinhardtii biomass using osmotic shock[END_REF]. The type and concentration of salts, incubation time and concentration of biomass are all important factors affecting the efficiency of the method (Krishna [START_REF] Koyande | Integration of osmotic shock assisted liquid biphasic system for protein extraction from microalgae Chlorella vulgaris[END_REF]. Osmotic shock has been used for extraction of protein from C. vulgaris biomass. Treatment with magnesium sulphate at 100 g L -1 for 10 min resulted in a high (> 90%) protein recovery. Increasing the biomass concentration from 0.25 % w/w to 5 % w/w resulted in a slight decrease of protein yield to approximately 80% ( Krishna Koyande et al., 2020). Osmotic shock is an attractive cell disruption method due to its simplicity and promising yields in biomolecules extraction.

Although prohibitively high treatment times of 48h are often reported, low treatment times of 10 min ware also shown, indicating that optimization of the method can reduce treatment time to more realistic levels.

Ionic liquids

Ionic liquids are organic salts with low melting points below 100 o C. The cation contains heteroatoms in the organic structure, usually nitrogen. The cations can be imidazolium, pyridinium, ammonium, cholinium and phosphonium, among others [START_REF] Orr | Disruption and Wet Extraction of the Microalgae Chlorella vulgaris Using Room-Temperature Ionic Liquids[END_REF]. The anions can be either organic or inorganic. Many ionic liquids are found in liquid form at room temperature and are very stable chemically and thermally. (Nitsos et al., 2013). These useful properties have classified them as green solvents. Ionic liquids have the ability to form hydrogen bonds, which allows them to interact with biopolymers such as cellulose found in the cell wall of microalgae. The recalcitrance of cellulose is based on the large number of hydrogen bonds formed between the cellulose microfibrils. The hydrogen bond capacity of ionic liquids allows the disruption of these interfibrillar hydrogen bonds of cellulose and can dissolve the cellulose structure. This leads to compromised microalgae cell wall structures and allows the more efficient extraction of intracellular or cell wall components of the microalgae (Chen et al., 2018). The chemical hydrolysis of cellulose by ionic liquids has also been proposed [START_REF] Van Spronsen | Separation and recovery of the constituents from lignocellulosic biomass by using ionic liquids and acetic acid as co-solvents for mild hydrolysis[END_REF]. Interaction with other cell wall components like hemicelluloses and proteins is also possible, although the complexity of the cell wall composition and variation between species make the exact mechanism of action still unclear [START_REF] Orr | Disruption and Wet Extraction of the Microalgae Chlorella vulgaris Using Room-Temperature Ionic Liquids[END_REF].

Imidazolium based ionic liquids are very commonly used for microalgae pretreatment and are very effective at improving lipid extraction [START_REF] Orr | Disruption and Wet Extraction of the Microalgae Chlorella vulgaris Using Room-Temperature Ionic Liquids[END_REF]. For example, pretreatment of C. vulgaris with different types of imidazolium based ionic liquids (Table 10) led to complete cell lysis and extraction yields ranging from 45 to 98% of total lipids [START_REF] Teixeira | Energy-efficient extraction of fuel and chemical feedstocks from algae[END_REF]. However, employment of high temperature (140 o C) for 30 min increases the energy input of the method. The requirement of heating to relatively elevated temperatures is relatively common in ionic liquid treatments, which could potentially render them less energy efficient [START_REF] Orr | Disruption and Wet Extraction of the Microalgae Chlorella vulgaris Using Room-Temperature Ionic Liquids[END_REF]. For this reason, microalgae treatment with ionic liquids at room temperatures has attracted some attention. Disruption of C. vulgaris cells was achieved with a variety of ionic liquids at ambient temperature for 16 h. Extraction of lipids from the pretreated cells with hexane showed significant improvement compared to untreated biomass (5% g g -1 biomass lipid extraction yield). The best result (25 % g g -1 biomass lipid extraction) was shown for a phosphonium ionic liquid tributylmethylphosphonium propanoate [START_REF] Orr | Disruption and Wet Extraction of the Microalgae Chlorella vulgaris Using Room-Temperature Ionic Liquids[END_REF]. These results however were performed with freeze dried biomass, that can facilitate the easier extraction of lipids due to structural damage to the cell membranes. Depending on pretreatment conditions, milder effects of ionic liquids on the lipid extraction yields from wet microalgal biomass (20%-40% of total oils) compared to freeze dried biomass (80-90 % of total oils) have been observed (Zhang et al., 2018). In the same study optimization of the pretreatment and extraction process using the phosphonium salt tributylmethylphosphonium propanoate could increase the oil yield at 80% (Zhang et al., 2018). The use of butyrolactam, caprolactam, propylammonium and hydroxypropylammonium based ionic liquids in the treatment of wet Chlorella sp. and Chlorococcum sp. achieved very good cell disruption. The best results were demonstrated by butyrolactam hexanoate with efficiency of 76% and 84% for Chlorella sp. and Chlorococcum sp., respectively [START_REF] Shankar | Protic ionic liquid-assisted cell disruption and lipid extraction from fresh water Chlorella and Chlorococcum microalgae[END_REF]. These results were obtained at room temperature, but long treatment times of 24 hours were required. Pigment extraction has also been reported with ionic liquids. Extraction of 85% of total astaxanthin was achieved with the treatment of H. pluvialis with a 40% w/w aqueous solution of 1-Butyl-3-Methylimidazolium Chloride for 1 h at 60 °C. Astaxanthin was extracted with methanol [START_REF] Liu | The efficiency and comparison of novel techniques for cell wall disruption in astaxanthin extraction from Haematococcus pluvialis[END_REF].

Table 10.

Viral cell lysis

A very novel approach, appealing for its low energy requirement is the viral lysis of microalgae cell walls. For example, infection of Chlorella variabilis by the Chlorella-virus Paramecium bursaria led to almost complete cell lysis and lipid yields comparable to microwave and ultrasonication [START_REF] Kim | Chlorella virus-mediated disruption of microalgal cell wall for biodiesel production[END_REF]. The method also proved successful in the production of bioethanol from starchaccumulating C. variabilis when combined with amylase treatment of the released starch and fermentation [START_REF] Cheng | Virus infection of Chlorella variabilis and enzymatic saccharification of algal biomass for bioethanol production[END_REF]. Viral production and cell lysis efficiency was significantly influenced by the carbon to nitrogen ratio of the culture when viral infection was done in situ (Y.-S. Cheng et al., 2015). This approach allows for nitrogen content manipulation to optimize lipid or carbohydrate production and hence, cell lysis. Additionally, it reduces the processing times of viral cell lysis [START_REF] Kim | Chlorella virus-mediated disruption of microalgal cell wall for biodiesel production[END_REF].

Cell disruption by contact

Cell disruption of Aurantiochytrium sp. was achieved with a poly-dimethylaminomethylstyrene coated nylon membrane. The membrane was submerged in a culture of microalgale cells and the culture was shaken bringing the membrane and cells into contact. Contact of the negatively charged cell surface with the functionalized membrane's positive charged surface led to electrostatic interactions -possibly rearrangements in the phospholipid bilayer-and cell lysis. Although cell disruption efficiency was relatively low (26%) the method is very promising due to its simplicity and low energy requirements. [START_REF] Yoo | An effective, cost-efficient extraction method of biomass from wet microalgae with a functional polymeric membrane[END_REF].

A similar effect was noticed when C. vulgaris cells were contacted with silver nanoparticles [START_REF] Abdul Razack | Biosynthesis of silver nanoparticle and its application in cell wall disruption to release carbohydrate and lipid from C. vulgaris for biofuel production[END_REF]. Silver nanoparticles are known for their bacteriocidal properties that are promoted by a positive surface charge [START_REF] Abbaszadegan | The Effect of Charge at the Surface of Silver Nanoparticles on Antimicrobial Activity against Gram-Positive and Gram-Negative Bacteria: A Preliminary Study[END_REF], and therefore a similar cell lysis mechanism may be in action. The proposed method, however, included drying of the cells both pre and post treatment and its efficiency on wet microalgae could vary.

Combined harvesting and cell disruption

Among the most promising novel approaches are those combining harvesting with microalgae biomass pretreatment. Such methods if properly optimized can have significant benefits for both capital investment and operating costs as they reduce the number of downstream unit operations required. One such example is electroflotation, by alternating current (Florentino de Souza [START_REF] De Souza | Comparison of pretreatment methods for total lipids extraction from mixed microalgae[END_REF].

Introduction of the alternating current that breaks water molecules and creates oxidative species like O3, H2O2 and -OH. These in turn cause cell disruption through oxidative reactions. The method achieved very high harvesting efficiency of 99% at 140 min processing time. Lipid extraction yield with chloroform-methanol increased from 5% in untreated cells to 25% when pretreated with electroflotation.

In a similar approach ozonoflotation, combines the harvesting effect of gas bubbles with the oxidative pretreatment induced by the contact of ozone with the microalgae cell walls. The method could harvest 80% of the algal biomass and double the lipids yield (measured by extraction with chloroform-methanol 2:1 v/v from lyophilized biomass), indicating significant cell disruption effects [START_REF] Velasquez-Orta | Microalgae harvesting using ozoflotation: Effect on lipid and FAME recoveries[END_REF]. Ozonation could potentially achieve the simultaneous disinfection, harvesting and pretreatment of cultures as was demonstrated by the short time in situ ozonation of D. salina [START_REF] Kamaroddin | In-situ disinfection and a new downstream processing scheme from algal harvesting to lipid extraction using ozone-rich microbubbles for biofuel production[END_REF]. The study showed that ozonation of a mixed culture of the green alga D. salina and Halomonas, a Gram-negative bacterium representing bacterial contamination, with 8 mg L -1 of ozone for 10 min resulted 93% sterilization efficiency without harming the microalgae cells [START_REF] Kamaroddin | In-situ disinfection and a new downstream processing scheme from algal harvesting to lipid extraction using ozone-rich microbubbles for biofuel production[END_REF].

Ozone-flotation has been used for efficient harvesting and pretreatment of microalgae for biooil production through hydrothermal liquefaction [START_REF] Nava Bravo | Bio-crude oil production using catalytic hydrothermal liquefaction (HTL) from native microalgae harvested by ozone-flotation[END_REF]. One of the major disadvantages of open pond systems is the high contamination from bacteria and other microorganisms as they are exposed to the natural elements. This problem can be enhanced when process water -that often carries a high bacterial load-is used as the culture medium. Reduction of bacterial load during cultivation or during harvesting and pretreatment with ozonation can make open pond systems more attractive for microalgae biorefineries.

Combined harvesting and pretreatment can also be achieved by a flocculation variation. Cells are harvested by coagulation-flocculation with ferric chloride at 200 ppm and pH 3 with 90% efficiency after 20 min. In the second step, cells are pretreated with 0.5% H2O2 at 90 o C. Interaction of ferric ions with H2O2 forms a Fenton-like reagent with potent oxidation radicals leading to 80% lipid extraction efficiency. With oxidized pyrite (20 mM) as catalyst, 90% lipid extraction and esterification were achieved (Seo et al., 2015b). As has been described in the oxidative pretreatment section, such methods may not be suitable for the valorization of antioxidants, pigments, and unsaturated fatty acids due to the severe influence of the oxidative reagents.

Energy efficiency of downstream processing

Among the reviewed microalgae harvesting methods, dissolved air flotation [START_REF] Niaghi | Optimization of dissolved air flotation technique in harvesting microalgae from treated wastewater without flocculants addition[END_REF] and electroflocculation (Vandamme et al., 2011) appear to be the most energy efficient with 0.0025 kWh m - 3 and 0.15-1 kWh m -3 of sample, respectively. However, as these harvesting methods were performed at the laboratory scale, upscaling could alter the energy requirements of the processes. Centrifugation also shows a favorable energetic assessment with an energy range of 0.70-1.30 kWh m -3 required at high scale [START_REF] Fasaei | Techno-economic evaluation of microalgae harvesting and dewatering systems[END_REF]. The main advantage of centrifugation systems is their ability to deliver high harvesting efficiency (up to 99%) and high solids concentrations (up to 20%) at this energy range (Table 11). This makes centrifugation ideal as a dewatering process able to produce microalgae pastes. Its combination with other harvesting techniques therefore could establish efficient harvesting and dewatering operations [START_REF] Fasaei | Techno-economic evaluation of microalgae harvesting and dewatering systems[END_REF]. A scenario of combined membrane filtration and centrifugation for harvesting and dewatering required 0.84 kWh•kg -1 , which is inside the energy input range of centrifugation alone (Table 11). Flocculation combined with centrifugation in the same scenario considerably reduced the energy requirements to 0.06 kWh•kg -1 [START_REF] Fasaei | Techno-economic evaluation of microalgae harvesting and dewatering systems[END_REF]. A similar energy input (0.05 kWh•kg -1 ) was required for combined flocculation and membrane filtration. From these examples it is apparent that size increase induced through the formation of flocks greatly enhances the efficiency of both centrifugation and membrane filtration. On the other hand, the use of flocculants increases the cost to 0.97 € Kg -1 compared to the 0.30 € Kg -1 required for combined membrane filtration and centrifugation. Such scenarios provide good benchmarks for development of harvesting and dewatering systems tailored to the needs of specific microalgae biorefineries. Selection of the appropriate methods could depend on the application, for example value of isolated biomolecules and the energy content of the biomass for biofuel production.

The energy efficiency of High Pressure Homogenization cell disruption method has been extensively studied. Energy consumption is dependent on biomass concentration. Low cell densities clearly do not favor the treatment in terms of energy requirements. For example, 20 kWh kg -1 DW is required for cell disintegration of 1.2%, w/w C. vulgaris suspensions [START_REF] Carullo | Effect of pulsed electric fields and high pressure homogenization on the aqueous extraction of intracellular compounds from the microalgae Chlorella vulgaris[END_REF]. Increasing the concentration of Desmodesmus sp. F51413 from 2.0 g L -1 to 90 g L -1 reduced the specific energy consumption from 413 kJ g -1 to 9.19 kJ g -1 [START_REF] Xie | Disruption of thermo-tolerant Desmodesmus sp. F51 in high pressure homogenization as a prelude to carotenoids extraction[END_REF]. This solids content increase does not affect carotenoid extraction yields that remains unchanged throughout the whole 2.0 g L -1 to 90 g L -1 range. Efficient disruption (>95%) of a 10% suspension of N. gaditana could also be achieved at high energy efficiency (<0.5 kWh kg -1 biomass). The method is also cost efficient with a reported price range of 0.15-0.25 € kg -1 of isolated protein (Safi et al., 2017a). Energy requirements are also dependent on cell wall robustness. The energy required for the disruption of single of T. suecica cells was 5.88 x 10 -5 J cell ;1 for Chlorococcum sp., it was 6.43 10 -5 J cell -1 [START_REF] Halim | Mechanical cell disruption for lipid extraction from microalgal biomass[END_REF]. The cell walls of T. suecica are made up from polysaccharides such as mannans, together with 2-keto sugars and glycoproteins.

Chlorococcum sp. has a cell wall primarily of polysaccharides such as cellulose pectins, hydroxyprolinerich glycoproteins and arabinogalactan proteins. Correlation of cell wall strength to compositional differences are not easy. However, the authors have attributed the difference in energy disruption requirements to the thinner cell walls of T. suecica. For relatively fragile microalgae like Nannochloropsis sp. cell disruption and energy efficiency were independent of cell density up to 25% solids [START_REF] Yap | Energy evaluation of algal cell disruption by high pressure homogenisation[END_REF]; although this effect could be related with the ease of cell wall disruption it remains to be investigated. Treatment of the relatively tough B. braunii was also energy efficient (~0.04 kWh kg -1 ) when extraction of the hydrocarbon rich extracellular matrix was considered [START_REF] Tsutsumi | Mechanical cell disruption of microalgae for investigating the effects of degree of disruption on hydrocarbon extraction[END_REF].

Bead milling was less energy efficient compared to HPH for the disruption of N. gaditana cells for maximum protein release (0.43 kWh kg -1 and 0.32 kWh kg -1 , respectively) (Safi et al., 2017a). Specific energy requirements were, reported for maximum protein release by bead milling from T. suecica (0.49 kWh kg -1 ), Neochloris oleoabundans (0.55 kWh kg -1 ) and C. vulgaris (0.72 kWh kg -1 ), with experiments performed at 9% (w/v) solids [START_REF] Postma | Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates[END_REF]. It was also shown that energy efficiency can increase with the reduction of bead diameter, probably due to more efficient bead impacting. Maximum carbohydrate release was achieved at similar specific energy for T. suecica (0.48 kWh kg -1 ) and C. vulgaris (0.72 kWh kg -1 ) but higher energy input was required for N. oleoabundans (0.94 kWh kg -1 ).

These values indicate that both HPH and bead milling can perform below the maximum specific energy limit of 0.682 kWh kg -1 for efficient target molecules extraction in the biorefinery, as set by the National Algal Biofuels Technology Roadmap target of the US Department of Energy [START_REF] Postma | Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates[END_REF]. Bead milling treatment of B. braunii is also energy efficient (~0.08 kWh kg -1 ).

PEF pretreatment of 10% solids of N. gaditana cells was significantly less efficient compared to HPH and bead milling requiring an energy input of 10.42 kWh.kg -1 for a very low 10% protein yield, compared to 50% with the other methods at much lower energy inputs (Safi et al., 2017a). The very small size of N. gaditana cells (2-4 μm) could adversely affect PEF efficiency and PEF should be only considered for larger microalgae cells. Protein extraction from C. reinhardtii by PEF pretreatment was equally inefficient in terms of energy input. Even at high energy input of 26.5 kWh kg -1 protein yields did not exceed 15% (T [START_REF] Lam | Harvesting and cell disruption of microalgae[END_REF]. The average cell size of C. reinhardtii was 4-5 μm. In contrast disruption of the cell wall deficient mutant of C. reinhardtii, in the same study, was more energy efficient with 40% protein yield at 2.5 kWh kg -1 (T [START_REF] Lam | Harvesting and cell disruption of microalgae[END_REF]. This shows that the cell wall of C. reinhardtii causes its resilience to PEF and indicates that PEF may be less suitable for microalgae species with a recalcitrant cell wall. Even at low biomass concentrations (4 g L -1 ) PEF is still not as energy efficient as HPH and bead milling. Contradicting these results efficient extraction of molecules from A.

protothecoides with PEF at 10% solids was reported with a 0.42 kWh kg -1 specific energy input [START_REF] Eing | Pulsed Electric Field Treatment of Microalgae-Benefits for Microalgae Biomass Processing[END_REF]. Average cell size was larger at 7 μm and the studied microalgae is known to have a rigid cell wall consisting of a triple layer with an acetolysis-resistant material sandwiched between polysaccharides [START_REF] Kalina | The Spectroscopy of the Acetoresistant Cell-Wall Biopolymers of Auxenochlorella-Protothecoides, Scenedesmus-Quadricauda and Chlamydomonas-Geitleri Zygospores (chlorophyta)[END_REF]. These discrepancies demonstrate how careful optimization may be needed for a more conclusive evaluation of a process potential, and that process efficiency can be species specific.

Hydrothermal treatment methods also appear to be energy efficient. An 87% disruption of N. oculata cells could be achieved with water bath heating at 0.01 kWh L -1 energy input [START_REF] Mcmillan | Evaluation and comparison of algal cell disruption methods: Microwave, waterbath, blender, ultrasonic and laser treatment[END_REF].

Steam explosion of C. sorokiniana was very efficient as it enabled microalgal biomass solubilization in the range 65-73% with an energy input of 0.03-0.06 kWh kg -1 of volatile solids [START_REF] Córdova | Physical Pretreatment Methods for Improving Microalgae Anaerobic Biodegradability[END_REF]. These studies, however, lacked information on solids content of the treated sample and therefore the more relevant specific energy input per unit biomass could be significantly higher. An energy input of 1.58 kWh kg -1 for the autoclaving of S. obliquus (5 min at 121 o C) is a more accurate estimation of the energy requirements of hydrothermal treatment [START_REF] Ansari | Evaluation of various cell drying and disruption techniques for sustainable metabolite extractions from microalgae grown in wastewater: A multivariate approach[END_REF]. Even at these relatively mild pretreatment conditions five-fold higher energy is required compared to HPH and bead milling. Even higher energy input (2.26 kWh kg -1 ) was reported for the efficient disruption of Nannochloropsis oceanica at 10% solids by steam explosion. At these optimal pretreatment conditions extensive cell disruption and almost complete lipid recovery could be achieved (J. Cheng et al., 2015). Although the energy input is higher compared with previously discussed HPH and bead milling significant heat recovery is possible for hydrothermal and steam explosion pretreatment methods via heat exchange (Chen et al., 2019b).

Utrasound and osmotic shock treatments were in the same energy range as hydrothermal with 1.5 kWh kg -1 and 1.72 kWh kg -1 , respectively [START_REF] Ansari | Evaluation of various cell drying and disruption techniques for sustainable metabolite extractions from microalgae grown in wastewater: A multivariate approach[END_REF]. Enzymatic treatments are also very attractive and promising methods due to their low energy requirements. The treatment of a 10% (w/v) slurry of N. gaditana with Alkalase could be performed at a specific energy input of 0.34 kWh kg -1 (Safi et al., 2017a). Although soluble protein yield was lower compared to HPH and bead milling in the same study, enzymatic reaction optimization and use of complementary enzymatic activities can greatly improve the reaction efficiency, as was previously discussed.

Table 11 Table 12 4

. Conclusion and outlook

Although a significant amount of research on microalgae valorization has been conducted it cannot be considered exhaustive. The large number of available (as well as yet unexplored) microalgae species combined with the variety of downstream processing methods requires an even greater research and optimisation effort. A unification and standardization of reporting would allow the easier comparison of different studies and a more comprehensive understanding of the field. For example, the microalgae cell concentration in slurries is reported in various ways including number of cells per unit of volume, volatile solids per unit of volume and total solids per unit of volume, often hampering comparison of reported results between studies. Similarly, the pretreatment method efficiency has been variably evaluated by cell lysis, turbidity and product extraction yield. Between these methods cell counting is an objective way of reporting cell disruption efficiency. Turbidity is affected by cell size and agglomeration of cells or debris, and yield can be affected by the formation of degradation products [START_REF] Spiden | Quantitative evaluation of the ease of rupture of industrially promising microalgae by high pressure homogenization[END_REF]. Decrease of the cells dry weight (due to release of intracellular materials) and increase of free proteins and DNA in the supernatant are good indicators that the cell wall structure is compromised. These methods could be sufficient to express pretreatment efficiency, especially as partial cell wall disruption is not visible by cell counting (Safi et al., 2017a).

Energy efficiency is reported as energy input per unit of microalgal biomass, energy input per unit of harvested product, energy input per unit energy content of the microalgal biomass or its lipid products. The latter calculation methods are more important when biofuels and energy are the main microalgal biorefinery target products, as the overall process net energy ratio cannot exceed the energy content of the microalgae and its products. When the production of high value molecules such as antioxidants, pigments, and food supplements is the main goal of the biorefinery energy input is more related to process cost and therefore should be reported as energy input per unit mass of the biomass.

The effect of biomass concentration on the energy and the extraction efficiency of the various pretreatment methods, has not been thoroughly investigated yet. Few methods have demonstrated efficient cell disruption at truly high biomass concentrations such as 20%. Much of the works were conducted at cell concentrations around or below 10%. Indeed, it appears that 10% solids content represents a limit above which mixing, and mass transfer is not efficient in the valorization of lignocellulosic biomass [START_REF] Weiss | Enzymatic hydrolysis is limited by biomasswater interactions at high-solids: improved performance through substrate modifications[END_REF]. Determining the concentration limits for cell disruption methods is of critical importance to select operational conditions where energy input is minimal without compromising the extraction efficiency. This limit should also be correlated to the requirements of the harvesting method in order to reduce the energy input of biomass concentration and dewatering. In this respect, energy intensive dewatering operations to very high solids content may only be required for applications of microalgae for direct use as feed or as food supplements in the form of pills and powders.

Harvesting and dewatering of microalgae biomass can become more efficient by combination of methods. Initial preconcentration by simple and low-cost methods such as gravitation settling can be extremely beneficial in terms of cost and energy requirements. Therefore, selection of suitable microalgae strains with high sedimentation velocities and ability to accumulate the desired biomolecules is paramount. The initial microalgae species selection can provide considerable cost, energy and time savings, as these methods are much more efficient when applied to moderately weak cell walls. Flocculation and membrane filtration are also promising preconcentration methods and combined with efficient dewatering processes such as centrifugation can reduce energy and cost requirements. The effect of upscaling of the harvesting and dewatering operations on energy efficiency also has to be considered.

Among emerging microalgae pretreatment methods thermal such as hydrothermal and steam explosion, dilute acid and alkaline hydrolysis are interesting due to the ability for fast and efficient cell disruption and fractionation of biomass components. Optimization and easy correlation of yield and energy requirements with process parameters such as time, temperature and pH with a single severity factor is also attractive. Viscosity reduction through hydrolytic reactions of macromolecules can enable such methods to handle increased solids concentrations of microalgal biomass. Energy recovery through heat exchange is an additional benefit. Enzymatic pretreatment is very promising due to the low energy requirements selectivity and the non-destructive nature of the process that can preserve the bioactive properties of the isolated molecules. Explosive decompression of microalgal cells pressurized by a gas such as CO2 and N2 is also interesting, although it has not been yet extensively studied. The mode of action is similar to steam explosion, but the lack of heat may make the method viable for the isolation of sensitive molecules such as pigments and antioxidants.

Among the most interesting methods, however, are those combining harvesting with pretreatment such as electroflocculation, ozonoflotation, and chemical flocculation with iron and oxidation through Fenton reaction by addition of H2O2. Even if these methods fail to reach industrial maturity, the reduction of unit operation can provide significant benefits in terms of investment and operating costs. Similar strategies may prove critical for the implementation of a mature microalgae biorefinery in the future. a Bligh and Dyer method : chloroform-methanol (1:2 v/v); b modified Bligh and Dyer method ; c hexane-methanol (7:3 v/v); d diethyl ether e na : information not available:; f calculated as % of total monosaccharides measured in the pretreatment liquid; g retention time in a continuous system with a flow rate of 14 L h -1 ; h retention time in a continuous system with a flow rate of 40 L h -1 ; i calculated as % of total protein measured in the pretreatment liquid as total Kjeldahl nitrogen (TKN) x 5.95 ; j calculated as % of total protein measured in the pretreatment liquid with the Lowry method [START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF], D Phaeodactylum tricornutum from [START_REF] Gügi | Diatom-Specific Oligosaccharide and Polysaccharide Structures Help to Unravel Biosynthetic Capabilities in Diatoms[END_REF], E Arthrospira platensis [START_REF] Van Eykelenburg | On the morphology and ultrastructure of the cell wall of Spirulina platensis[END_REF] F Tisochrysis lutea [START_REF] Orlova | Ultrastructural Changes in Marine Microalgae from Different Taxonomic Groups during Batch Cultivation[END_REF].

CRediT author statement

Christos

  Nitsos: Investigation, Writing -Original Draft, Visualization, Rayen Filali: Conceptualization, Writing -Review & Editing, Visualization, Behnam Taidi: Conceptualization, Writing -Review & Editing, Supervision, Julien Lemaire: Conceptualization, Writing -Review & Editing

Figure 1 .

 1 Figure 1. Overview of microalgae biorefinery Figure 2. Cell wall structure of a: A Chlamydomonas biflagellate cell, B Desmodesmus four-celled coenobium, C mature Haematococcus lacustris cystm from[START_REF] Baudelet | A new insight into cell walls of Chlorophyta[END_REF], D Phaeodactylum tricornutum from[START_REF] Gügi | Diatom-Specific Oligosaccharide and Polysaccharide Structures Help to Unravel Biosynthetic Capabilities in Diatoms[END_REF], E Arthrospira platensis[START_REF] Van Eykelenburg | On the morphology and ultrastructure of the cell wall of Spirulina platensis[END_REF] F Tisochrysis lutea[START_REF] Orlova | Ultrastructural Changes in Marine Microalgae from Different Taxonomic Groups during Batch Cultivation[END_REF].

Figure 3 .Figure 4 .

 34 Figure 3. Typical mechanisms of microalgae flocculation (a) coagulation with bivalent cations (b)neutralization and bridging effect of cationic polymers (c) autoflocculation induced by excreted algogenic organic matter (d) detailed mechanism of flocculation with cationic polymers including "charge shielding" hypothesis for negative effect of high cationic polymer dosages (adapted from[START_REF] Lam | Harvesting and cell disruption of microalgae[END_REF] Figure4. Types of forces acting acting on microalgae cellss during bead milling. The diagram is not to scale and servesd for illustration purposes only.

Figure 5 .

 5 Figure 5. Mechanism of cell disruption during High Pressure Homogenization. The cell sizedigram is not shown to scale. Pressure drop is the main effect of the process causingcause of the cell disruption.

Figure 6 .

 6 Figure 6. Mechanism of cell disruption by ultrasound treatment Figure 7. Pore formation in the cell membrane and subsequent release of intracellular molecules as a result of PEF treatment of microalgae cells

Table 2 .

 2 Cell wall structures and high-value products of typical microalgae species

	Robustness	Species	Cell size	Cell wall composition	Reference	Main product/price a
	of cell wall		(μm)
	High	Scenedesmus sp.	5-7	Trilaminar sheath (algaenan), Inner cell wall	(Baudelet et al., 2017).	Carbohydrates
				(mannose, glucose), coenobial adhesive
	High	Haematococcus pluvialis	20	Trilaminar sheath (algaenan), primary,	(Baudelet et al., 2017).	Astaxanthin / 1,450-5075 € kg -1
				secondary and tertiary cell wall (mannose,
				other sugars)
	Medium	Phaeodactylum	25	Silica, sulfated α-glucurunomannan, proteins,	(Johansen, 1991) (Le	Fucoxanthin/ $ 200 kg -1 -4450 €g -1 b
		tricornutum		polyamines	Costaouëc et al., 2017)
	Medium	Arthrospira platensis	8	Peptidoglycan layer (Murein), two fibrillary	(Akao et al., 2019;	Phycocyanin/360€ kg -1
				layers, sheath of acidic polysaccharides	Kröger et al., 2018)
	Medium	Chloromonas nivalis	15	By-layered cell wall	(Remias et al., 2010)	Astaxanthin/ 1,450-5075 € kg -1
	Medium	Dunaliella salina	5-25	Double-layered rigid wall (hyaline, algaenan) (Baudelet et al., 2017)	β-carotene/ 215-2150 € kg -1
	Low	Isochrysis galbana	5-6	Lack of cell wall, plasma membrane	(Thu et al., 2015)	ω3 ω6/ 78-116 € kg -1
		a as reviewed in (Cuellar-Bermudez et al., 2015), b from (Shannon and Abu-Ghannam, 2018)

Table 3 .

 3 Flocculation process parameters for typical polyelectrolytes. Table adapted from Van Haver and Nayar, 2017

	Flocculant	Cell	Dosage	Volume	Time	Species	Harvesting	Reference
		concentration	(mg L -1 )	(L)	(min)		efficiency
							(%)
	Chitosan	-	20	0.5	240	C. calcitrans		Harith et al. 2009
	Magnafloc® LT 25	-	0.1	0.5	240	C. calcitrans		Harith et al. 2009
	Chitosan+Sodium alginate	1.24 a	30 ppm + 40 ppm	0.01	60	S. obliquus		Matter et al., 2018
	Zetag 7557,
		0.7 b	10	0.004	120	Neochloris oleoabundans		Lam, 2017
	Polyacrylamide
	Synthofloc 5080H,
		3.6 b	10	0.25	120	P. tricornutum		Lam, 2017
	Polyacrylamide
	Cationic starch	0.77 g L -1	40	-	30	C. protothecoides		Letelier-Gordo et al., 2014
	Zetag 63, Polyacrylamide	10 6 cells mL -1	10	1	30	Chlorella stimatophora		Van Haver and Nayar, 2017
	F04650, Polyacrylamide,
		0.7 g L -1	3	0.05	60	N. salina		Van Haver and Nayar, 2017
	Medium charge
	FO4800, Polyacrylamide,
		0.7 g L -1	3	0.05	60	N. salina		Van Haver and Nayar, 2017
	High charge

Table 4 .

 4 Process parameters and yields of typical microalgae harvesting processes

	Method	Species	Time	Initial biomass	Total	Harvesting	Reference
				concentration	volume	efficiency
	Gravitational settling	Monoraphidium sp. FXY-10	24 h	0.218 g L -1	-	98 %	Yu et al., 2012
	Gravitational settling	A. platensis	-	0.55 g L -1	1 L	85 %	Depraetere et al., 2015
	Coagulation	N. salina	2.5 h	0.5 g L -1	0.113 L	85 %	Chatsungnoen and Chisti, 2016
	Flocculation	C. reinhardtii	0.25 h	0.7 g L -1	0.1 L	90%	Fan et al., 2017
	Flocculation	C. calcitrans	0.17 h	0.095 g L -1	0.25 L	89 %	Şirin et al., 2015
	Flocculation	C. vulgaris	0.5 h	0.5 g L -1	0.1 L	90 %	Vandamme et al., 2012
	Flocculation	D. brasiliensis	0.017 h 0.5 g L -1	50 L	99.2 %	Ndikubwimana et al., 2016
	Centrifugation	A. platensis	0.17 h	0.41 g L -1	0.005 L	85%	Cuellar-Bermudez et al., 2019
	Centrifugation	S. obliquus	0.17 h	0.746 g L -1	70 L	99.3%	Wang et al., 2019
	Dissolved air flotation Mixed microalgae	-	0.545 g L -1	-	91%	Niaghi et al., 2015
	Flocculation-flotation S. obliquus	0.25 h	0.73x10 6 cells mL 1 1 L	93.6 %	Xue et al., 2019
	Βioflocculation	A. plantensis	0.25 h	1.4 g L -1	0.6 L	95%	Vergnes et al., 2019
	Βioflocculation	Mixed microalgae	0.083 h 0.419 g L -1	0.95 L	79.6%	Velasquez-Orta et al., 2014

Table 6 .

 6 Effect of different drying methods on extraction of biomolecules from S. obliquus. Table adapted from[START_REF] Ansari | Evaluation of various cell drying and disruption techniques for sustainable metabolite extractions from microalgae grown in wastewater: A multivariate approach[END_REF]

					Carbohydrate
	Drying		Lipid yield	Protein yield		Energy
		Process conditions			yield		Cost	Reference
	Method		(% w/w)	(% w/w)		(KWh kg -1 )
					(% w/w)
	Untreated							Ansari et al.,
		-	-	31.07	19.9	-	-
								2018
	Sun-drying	Natural sunlight (400-						Ansari et al.,
		1200 mmolm -2 s -1 ),	11.64	25.7	19.5	0.055	0.011 $ L -1	2018
		18-27 o C, 2-3 days
	Freeze-drying	Deep freeze -84 o C ,						Ansari et al.,
			18.45	31	21.3	21.81	1.189 $ L -1
		0.9 kW for 24 h						2018
	Oven-drying							Ansari et al.,
		Air oven, 60 o C 12 h	13	30.98	18.8	6.33	0.343 $ L -1
								2018
	Spray dryer							Fasaei et al.
		-	-	-	-	6.1	0.30 € kg -1
								2018
	Drum dryer
		-	-	-	-	5.1	0.25 € kg -1

Table 7 .

 7 Effect of process severity factor during hydrothermal pretreatment on biomolecule extraction yields

		Pretreatment	Reactor loading/ Biomass	Time	Temperature		Yield
	Species	method	Concentration	(min)	( o C)	logRo	(%)	Reference
			Lipids
	Chlorella sorokiniana	Steam explosion	100g / 20% w/w solids	5	120	1.29	18.4 a	Lorente et al.,
	Nannochloropsis gaditana Steam explosion	250g / 17% w/w solids	5	120	1.29	3.6 a	Nurra et al., 2014
	Nannochloropsis gaditana Steam explosion	100g / 20% w/w solids	5	120	1.29	17.9 a	Lorente et al.,
	Nannochloropsis gaditana Steam explosion	100g / 20% w/w solids	5	150	2.17	18.2 a	Lorente et al.,
	Chlorella vulgaris	Hydrothermal	30 mL/ 50 g L -1	120	150	3.60	67.7 c	Xiaojian et al., 2017
	Nannochloropsis oceanica Steam explosion	200 mL / 10% w/w solids	5	207.12	3.85	93.9 b	Cheng et al., 2015
	Nannochloropsis sp.	Hydrothermal	na e / 20 w/w solids	60	180	4.13	84.0 d	Qu et al., 2018
			Carbohydrates
	Nannochloropsis gaditana Steam explosion	100g / 20% w/w solids	5	120	1.29	43.7 f	Lorente et al.,
	Chlorella vulgaris	Hydrothermal	na e / 5g tCOD L -1	20	120	1.89	51.8 f	Mendez et al., 2013
	Nannochloropsis gaditana Steam explosion	100g / 20% w/w solids	5	150	2.17	44.4 f	Lorente et al.,
	Chlorella vulgaris	Hydrothermal	na e / 5g tCOD L -1	40	120	2.19	58.2 f	Mendez et al., 2013
	Chlorella pyrenoidosa	Hydrothermal	na e / 10 % w/w solids	10 g	160	2.77	46.9	Fu et al., 2018
	Chlorella pyrenoidosa	Hydrothermal	na e / 7 % w/w solids	15 h	155.2	2.80	80.5	Xiao et al., 2019

Table 8 .

 8 Effect of process severity factor during dilute acid pretreatment on biomolecule extraction yields Bligh and Dyer method : chloroform-methanol (1:2 v/v); b hexane; c total reducing sugar content of pretreatment liquid determined with dinitrosalicylic acid method

			Reactor loading/
			Biomass	Time	Temperature		Yield
	Species	Acid	Concentration	(min)	( o C)	CSF	(%)	Reference
				Lipids
	N. gaditana	H2SO4, 5% w/w wet basis	100g / 20% w/w solids	5	120	0.40	17.7 a	Lorente et al., 2015
	N. gaditana	H2SO4, 10 % w/w wet basis	100g / 20% w/w solids	5	120	0.75	17.8 a	Lorente et al., 2015
	N. gaditana	H2SO4, 5% w/w wet basis	100g / 20% w/w solids	5	150	1.02	18.5 a	Lorente et al., 2015
	N. salina	HNO3 0.57%, v/v	na/ 20 g L -1	30	120	1.21	24.4 b	Lee et al., 2014
	N. gaditana	H2SO4, 10 % w/w wet basis	100g / 20% w/w solids	5	150	1.40	18.5 a	Lorente et al., 2015
	N. gaditana	H2SO4, 5% w/w wet basis	100g / 20% w/w solids	5	150	1.87	79 a	E. Lorente et al., 2017
	N. salina	HNO3 0.5%, v/v	na/ 20 g L -1	60	120	1.94	88.4 b	Lee and Han, 2015
				Carbohydrates
	mixed culture	H2SO4 0.5 M	na	10	121	1.32	18 c	Shokrkar et al., 2017
	mixed culture	H2SO4 0.5 M	na	20	121	1.62	44 c	Shokrkar et al., 2017
	mixed culture	H2SO4 1 M	na	20	121	1.92	58 c	Shokrkar et al., 2017
	mixed culture	H2SO4 0.5 M	na	40	121	1.92	85 c	Shokrkar et al., 2017
	mixed culture	H2SO4 1 M	na	40	121	2.22	92 c	Shokrkar et al., 2017

a

Table 9 .

 9 Extraction yield achieved by different cell disruption methods

Table 12 .

 12 Energy and cost requirements of cell disruption methods

			Biomass	Energy	Cost
	Disruption method	Species				Reference
			concentration	(KWh kg -1 )
	HPH	N. gatidana	100 g L -1	0.32	0.15-0.25 a	Safi et al. 2017
	Bead milling	N. gatidana	100 g L -1	0.43	0.4-1 a	Safi et al. 2017
	PEF	N. gatidana	100 g L -1	10.42	2 -20 a	Safi et al. 2017
	Enzymatic	N. gatidana	100 g L -1	0.34	-	Safi et al. 2017
	Ultrasound	S. obliquus	-	1.5	0.26 b	Ansari et al.,
	Autoclave	S. obliquus	-	1.58	0.26 b	Ansari et al.,
	Steam explosion	N. oceanica	10 % w/w	2.26	-	Cheng et al.,
	Osmotic shock	S. obliquus	-	1.72	0.31 b	Ansari et al.,

a € Kg -1 of isolated protein b € L -1 of treated sample

As determined by microscopic counting, in a hemocytometer, of intact cells before and after disintegration

As measured by counting undisrupted cells before and after milling in a Bürker chamber

As measured by counting undisrupted cells before and after HPH in a Neubauer haemocytometer chamber

As measured by counting the number of intact cells treated after a specific time interval against those initially determined from the control, using a microscope

Acknowledgments

This study was carried out in the Centre Européen de Biotechnologie et de Bioéconomie (CEBB), supported by Région Grand Est, Département de la Marne, Grand Reims and the European Union. Authors are grateful to Pr. PERRE Patrick, director of the Chair of Biotechnology of CentraleSupélec for funding all the works of this article.

Figure 7

Method