The Challenge of Ecophysiological Biodiversity for Biotechnological Applications of Marine Microalgae

In this review, we aim to explore the potential of microalgal biodiversity and ecology for biotechnological use. A deeper exploration of the biodiversity richness and ecophysiological properties of microalgae is crucial for enhancing their use for applicative purposes. After describing the actual biotechnological use of microalgae, we consider the multiple faces of taxonomical, morphological, functional and ecophysiological biodiversity of these organisms, and investigate how these properties could better serve the biotechnological field. Lastly, we propose new approaches to enhancing microalgal growth, photosynthesis, and synthesis of valuable products used in biotechnological fields, mainly focusing on culture conditions, especially light manipulations and genetic modifications.


Introduction
It is now well known that marine biotechnology (the so-called -blue biotechnology‖) can make a large contribution to the key societal challenges, in relation to the huge biological diversity populating marine ecosystems. As an alternative to higher plants, microbial organisms such as microalgae are biotin, folic acid and pantothenic acid) make microalgae an unconventional source for improving the food nutritional state and hence the health of humans and animals [13,66,71,72]. Furthermore, microalgae are considered to be of great interest in the biotechnological field because they are a precious source of lipophilic pigments such as chlorophyll (0.5% to 1% of dry weight), carotenoids (0.1% to 0.2% of dry weight on average and up to 14% of dry weight for β-carotene in some species, including Dunaliella sp.), xanthophylls (lutein, zeaxanthin and astaxanthin) and hydrophilic pigments, such as phycobiliproteins.
following sections). Excessive light or ultraviolet radiation in outdoor open systems might induce photoinhibition or can alter the healthy state of cells. Outdoor culturing for biomass production, with the exception of tropical areas, is strongly season dependent [91][92][93][94][95][96][97]. The transition periods of the year (e.g., end of spring and autumn) also show rapid climatological changes; a relevant temperature excursion between night and day might also affect physiological acclimation of microalgal biomass [98]. Other drawbacks, linked to the fact that cultures are open and not axenic, are correlated to the proliferation of bacteria, viruses, other microalgal species and predators that may decimate microalgal cultures [70]. Closed photo-bioreactors that can be set indoor or outdoor (either naturally or artificially illuminated), have been employed to axenically grow microalgae, such as cyanophytes [70,99,100]. For some diatoms, both outdoor and indoor cultivation systems have been established together with a generalised set of conditions [5]. The conditions and growth parameters can be set more accurately in indoor culture batches than in outdoor ones. Indoor culturing techniques prevent many environmental and biological causes of death or reduction in growth rate, and are more efficient for maintaining mass cultivation at high production rate. Section 4 of this review focuses on the possible ways to improve growth quantum yield of microalgal cultivation in indoor systems.

Exploring the Richness of Microalgal Biodiversity
Biodiversity is characterized by taxonomical metrics with distinct morphological traits, as well as other variables related to functional diversity such as ecology, physiology, and biochemistry of the species [101]. The cell functional traits regulate the ecophysiological requirements of the species, thereby shaping cell performances under specific conditions or along environmental gradients, as in nature. In this section, we aim to present the richness of microalgal biodiversity, which has been underexploited thus far in the biotechnological field.

Taxonomical Diversity
Biodiversity is huge in the microalgal realm, with almost 64 classes (phyla) and more than 70,000 species that could represent the basin to draw for a deep investigation of the microalgal world within a blue biotechnology aim [1]. The most recent and successful taxon group from an evolutionary and biogeochemical point of view is the diatoms' group [3,102] that has been estimated to have roughly 10 5 species [103]. Many diatoms comprise cryptic or hidden species not easily recognizable with classical microscopic tools [104,105]. For this reason in recent years, taxonomy has been using molecular tools, in addition to morphological identification to better decipher the richness of the microalgal realm [106] and the evolutionary trends of microalgae (e.g., [107][108][109][110]). Molecular tools allow for the investigation of small cell-sized species and uncultivable ones [111], as well as cryptic species [104,105,112]. As mentioned previously, for some taxa of phytoplankton, the known biodiversity does not take into account the hidden and cryptic species present in the groups [113]. Indeed, with the use of ribosomal or organelle markers as LSU, SSU, ITS, 5.8 S, RbcL and COX1 associated with the ultrastructure of a diatom cell wall, the silica frustule helps resolve cryptic species belonging to the following genera: Chaetoceros, Skeletonema and Pseudo-nitzschia [102,104,114] thereby enlarging the possibility of choosing the most appropriate species to the ad hoc cultivation system. For more information on taxonomical diversity and evolution of phytoplankton, we suggest the recent review papers [109,110].

Functional Diversity: Morphology and Size
Morphological variations amongst species lead cells to be more or less cultivable; these variations mostly imply changes in the surface: volume ratio, cell floatability and sinking rate. A cell's shape can vary from round to elongated or triangular, affecting interactions between cells and the surrounding environment and having consequences also on the assimilation of resources [115].
The main cellular morphology includes the presence of flagella (Table 2), such as in the dinophytes, cryptophytes and some haptophytes, in addition to the presence of spines, referred to as setae and found mostly in diatoms. The setae can have several functions, such as cell protection against predators, sedimentation by trapping air bubbles and generation of photosynthetic oxygen. Indeed, setae from some Chaetoceros species contain chloroplasts that are able to move up and down, probably in relation to the light they are exposed to [116].
Another morphological trait is represented by the possibility of forming a colony. Colony-forming species could be considered evolutionarily favoured due to the colony-formation implication in defence mechanism (Table 2), as well as in growth regulation and competition [117]. One example is represented by some species of Phaeocystis (haptophyte) that can be present in both colonies and single cells [118].
Chain-forming species (Table 2) are also widespread in an aquatic environment, and mostly belong to the diatom genus [119]. Chain formation confers resistance to predation [120,121], enhances nutrient fluxes [122] and can increase sexual reproduction if chains become entangled [102].
The morpho-functional traits listed above are strongly ecologically relevant, though they do not add any gain for biotechnological purposes in relation to the current cultivation techniques that use high nutrient content and shallowness of culture tanks.
Instead, cell size can be considered as a relevant functional trait (Table 2), which has many implications for the ecophysiological requirement of cells and on the ecology of phytoplankton [101,123]. Cells range from 0.3 μm to several millimetres [123]. The smallest photosynthetic organisms, belonging to picophytoplankton class (size ˂3 μm) are strongly adapted to grow and dominate in oligotrophic waters [124,125]. The tiny cell size confers important ecological and biological properties on picophytoplankton, such as the large surface area per unit of volume, minimal diffusion boundary layer (i.e., higher nutrient consumption rates), low sinking rate, low package effect and efficient light utilization [25,126,127]. Indeed, significant relationships between photosynthetic and growth rates were obtained in different picophytoplankton species, suggesting a direct and functional link between absorbed light [25,98] and metabolic processes. This could be related to a high physiological plasticity and intraspecific variability, as has been revealed in picophytoplankton [24,25,[128][129][130]. These features might be related to a potentially higher rate of speciation in these small organisms than in larger sized ones [128].  Little impact in shallow and oxygenated/mixed tanks Flagellates [131] Migration and motility Dinophytes, haptophytes and cryptophytes Little impact in shallow and oxygenated/mixed tanks Small cell size [127] Low nutrient requirements, high growth capacity, low sinking rate Picoeukaryotes High growth and production capacity and acclimation Benthic species [26,132] Growth on solid support (sediments, leaves), highly resistant species Some diatoms, cyanophytes Difficult to cultivate Toxic species [133] Defence mechanisms, highly competitive Cyanophytes, diatoms, dinophytes Discovery and selection of new molecules Sexual reproduction [134] Genetic recombination Some diatoms New strains selection with better fitness Diazotrophy [135] Atmospheric N 2 fixation Cyanophytes Low growth capacity  [102] Internal storage of nutrient Diatoms Long-term maintenance, decrease of dilution frequency Low light adapted [139] Growth under low light, photoinhibited under high light Deep chlorophyll maximum (DCM) species High growth rate under low light, high capacity of photoprotection Variable light adapted [24] Growth under low and high light, physiological plasticity Coastal species (some diatoms and haptophytes) High capacity of xanthophyll and antioxidant production, high growth rate Low iron requirement species [140] Growth in pelagic/oceanic ecosystems, photobiological and physiological adaptation Some pennate diatoms Little effect, Fe is provided in high quantity Oceanic Temperature zones [141,142] Low temperature growth Polar species (diatoms, haptophytes) Cost for low temperature maintenance of the cultures Temperature defence mechanisms, peculiar molecules for allowing photosynthesis and production at low temperature Calcareous microalgae [143] Species producing calcified scales around the cell Coccolithophorids Calcite production DMSP, DMS producer species [15] Antioxidant production (* DMS and ** DMSP) under environmental stresses

Prymnesiophytes, Diatoms, Dinophytes
Highly effective antioxidant systems, well-growing species Halophilic species [144] High salinity level, osmotic stress regulation Chlorophytes and cyanophytes Low growth capacity, costly culture management

Molecules of interest
Although there is not much knowledge of picophytoplankton despite its high biodiversity, in addition to the features discussed earlier, it is implied that exploring picophytoplankton for biotechnological purposes is challenging. The low consumption together with the high growth rate and low loss of energy between light harvesting and division processes might outweigh the scant biomass content per cell.

Functional Diversity: Uptake of Nutritional Resources
The diversity of culture media available for growth of marine and coastal microalgae (Table 3) reflects the physiological diversity of microalgal groups. The major natural seawater-enriched media are: the f/2 medium, widely used for coastal and diatom species [145]; the K medium for oceanic species; the Pro99 medium for cyanophytes and picoeukaryotes; and the MNK medium mainly used for coccolithophorids [146]. Table 3. The most commonly used culture mediums for growth of marine microalgae (see also [147]).

Name
Microalgae Specificities f/2 medium [148,149] Coastal microalgae, diatoms Enriched medium K medium [146,150] Oceanic microalgae Trace metals Pro99 [151] Prochlorococcus spp. and some picoeukaryotes High ammonia concentrations, No vitamin requirement MNK medium [152] Oceanic coccolithophores Enriched medium The macronutrients required by microalgae to perform photosynthesis and growth are nitrogen (nitrate, NO 3 , nitrite, NO 2 or ammonium, NH 4 ), phosphorus (phosphate, PO 4 ) and silicate (SiO 2 ), the latter being required only for diatoms and silicoflagellates. As reported in many studies [101,[153][154][155], the variability in uptake and the efficiency in using nutrients are high among phytoplankton groups, depending on evolutionary and ecological functional traits [156][157][158][159][160][161]. The most striking group corresponds to the diatoms, with the presence of a large central vacuole that can store nutrients and carbohydrates [102], which allows cells to maintain their growth during nutrient-depleted periods. This feature provides this group a competitive advantage over other groups.
Nitrogen can be provided as nitrate, nitrite or ammonium, which is a key variable for microalgal growth. It has been observed that different sources of nitrogen are differently metabolized, influencing the growth capacity and protein content [162], as observed in two Chaetoceros species [163]. Evidence from Meseck and collaborators [22] show that ammonium uptake efficiency in Tetraselmis chui PLY 429 significantly increased when carbon dioxide was added to the culture, while it decreased with a nitrate source. Experimental results obtained with the diatom Skeletonema marinoi grown in f/2 medium under different light conditions, revealed that after three or four days, ammonium concentration strongly decreased, while nitrite and nitrate concentrations remained high [164]. This result confirms that cells primarily use NH 4 , instead of NO 3 . Allen et al. [165] reported a decrease in growth rate of Phaedactylum tricornutum grown on nitrate as the only nitrogen source. In fact, the use of nitrate is more costly for cells than ammonium, because of the enzymes involved in its reduction into usable form [166,167]. However, high concentrations of ammonium have been shown to inhibit growth of some phytoplankton species, although the response is variable among the different groups [168]. Eppley et al. [169] did not show any differences on the half-saturation constants for nitrate and ammonium uptake among several coastal and oceanic species. Recently, the possible use of the intracellular NO 3 by the benthic diatom Amphora coffeaeformis as a possible survival mechanism in darkness and anoxia has been shown [170]. The same mechanism has also been reported in the pelagic Thalassiosira weissflogii [171].
Macronutrient limitation affects growth capacity of microalgae, as well as their physiological state. Some of the physiological variations induced by a reduction in nutrients might be used for biotechnological applications. Nitrogen starvation in many microalgal species is commonly linked to an enhancement of triglyceride accumulation [61,172]. By contrast, lipid content tends to decrease with phosphorus limitation [173]; the latter reduces the formation of phospholipids and triggers the production of triglycerides and other neutral lipids [174]. N-limitation in addition to P-limitation induces an increase in non-photosynthetic carotenoids, as for instance astaxanthin in Haematococcus pluvialis [175], or β-carotene in Dunaliella bardawil upon nitrogen limitation [176]. Generally, nutrient limitation negatively affects photosynthesis and growth rates of microalgae [177][178][179][180][181][182]. P-limitation seems to repress the carbon-concentrating mechanism (CCM) activity and leads to lower photosynthesis [183], while N-limitation may have a stimulatory effect on the activity of the CCM, as shown in Chlorella emersonii [183].
Micronutrients, such as iron, manganese, zinc, cobalt, copper, molybdenum and the metalloid selenium, are also essential for microalgal growth. They are present in insoluble forms and can be limiting in oligotrophic waters [184,185]. To cope with this feature, offshore species have evolved lower iron requirements to survive in iron-poor oceanic waters compared to coastal species [186]. Iron is involved in controlling microalgal growth, as shown in the diatom Cyclotella meneghiniana, while its requirement is nitrogen source dependent [187,188]. Manganese is needed in large amounts for cells growing under low light conditions, as it is involved in photosynthesis [189]. Zinc is present in proteins involved in DNA transcription and in alkaline phosphatase; it is also used as a co-factor for carbonic anhydrase, a critical enzyme that transports and fixes CO 2 [190]. Copper is also needed for photosynthesis, being required in cytochrome oxidase and plastocyanin, even though it can be substituted in some species by iron when cytochrome c 6 is present [191]. Nickel is a co-factor of urease, mainly required when urea is present as nitrogen source [192]. Other micronutrients, such as selenium, cadmium and mercury, can be added to the medium, albeit carefully because of their toxic effects. Moreover, three vitamins-vitamin B 12 (cyanocobalamin), thiamine, and biotin-are also essential for microalgal growth [193].
Peculiar functional groups of microalgae concerning resource uptake are diazotrophs and mixotrophs (Table 2). Diazotrophs, primarily cyanophytes like the species Trichodesmium, fix atmospheric nitrogen and are able to grow without external sources of nitrogen. Beside a potential advantage of such strategy for biotechnological mass cultivation, the low metabolic rate efficiency of these species limits their applications in biotechnology [135]. Mixotrophs instead are able to perform both photosynthesis and heterotrophic grazing on particles or assimilate dissolved organic carbon (osmotrophy) [136][137][138]. Cells can therefore grow under inorganic nutrient depletion and this feature might favour the allocation of intracellular carbon into proteins [194]. This functional group can account for up to 20% of primary production, explaining plankton blooms under extreme conditions [195].
However, growth rate is generally low [196] and is strongly dependent on temperature, together with the efficiency of grazing activity. It has been recently shown that heterotrophic algae, such as Scenedesmus spp., can be used for fatty acid production [197]. Mixotroph species might be potentially used in the framework of the first kind of microalgae application, for pollutants bioremediation (Table 1).

Functional Diversity: Functional Traits versus Environmental Variables
Microalgae can also be characterized by their adaptive traits to optical, physical or hydrological variables. Since production is strictly related to the amount of energy supplied to the photosynthetic apparatus, light is the main variable driving photosynthetic capacity, and therefore growth. In aquatic ecosystems, light intensity is the most variable parameter, changing over different time and spatial scales and ranging from limiting to excessive. Light limitation influences nutrient uptake [198][199][200][201][202][203] and degree saturation of lipid [204,205]. Excessive light, which is damaging for cells, results in photoinhibition [206], in reduction of maximum quantum yield, and CO 2 uptake [207][208][209]. Microalgae have developed protective mechanisms, to prevent or limit irreversible photoinhibition, in order to cope with the formation of reactive oxygen species (ROS) by synthesizing certain carotenoids or other molecules with antioxidant activities [66]. The first response to high light is the activation of photoprotective xanthophyll cycle [23], leading to the synthesis of zeaxanthin (in green algae) or diatoxanthin (in many chlorophyll c-containing algae). The role of these photoprotective xanthophylls, in countering peroxidative damage, has been reported in diatoms [210]. In Haematococcus pluvialis high light conditions increase the astaxanthin content three-fold as compared to low light [211,212].
Two functional groups, discriminated by light responses, have been proposed: high light and shade-acclimated species [139]. The high light group is characterized by low photosynthetic pigment content relative to the shade-acclimated species. More recently, Dimier et al. [24] discriminated three functional groups based on their photoregulation capacity: high light adapted, low light adapted and variable light adapted. The latter group, composed by coastal microalgae, is characterized by efficient and fast development of photoprotective processes such as xanthophyll cycle and non-photochemical quenching [24].
Recently, it has been shown that the growth capacity of the toxic Ostreopsis ovata (dinophyte) is strongly dependent on the effects of both temperature and light [21]. Temperature influences the growth of photoautotrophs through its control of enzymatic kinetics [213,214] and changes in cellular membrane composition [215]. Generally in microalgae, an increase in temperature leads to an enhancement in growth [216][217][218][219], as well as in carbon content [218][219][220][221][222]. Indeed, it has also been shown by Toseland and collaborators [142] that temperature strongly affects phytoplankton metabolism in the field as nutrients and light do. Microalgal responses to temperature also vary between groups (Table 2) [88,216,223,224]. The optimal temperature for growth capacity is usually below 10 °C for polar species [225,226], around 10-25 °C for temperate ones [227,228], close and beyond 25 °C for tropical species [88] and between 25 and 35 °C for desert-inhabiting microalgae [229].
Another relevant variable is salinity ( Table 2). Osmoregulation is achieved by uptake of ions [230,231], the synthesis of osmotic active compounds or the expulsion of water [76,230,[232][233][234][235]. Biochemical changes occur in cells exposed to high salinity, including an increase in ash content [236,237] and protein synthesis inhibition [238]. Estuarine species are tolerant to a wide range of salinities, though fewer species are able to grow in high salinity conditions. The success of these halophytic species is related to enhanced synthesis of osmotic stress regulation molecules such as the zeaxanthin in cyanophytes [239] or glycerol in the green alga Dunaliella salina [240]. The latter also accumulates β-carotene under high salinity stress [176,241,242] while Haematococcus pluvialis accumulates astaxanthin [175]. These species are already used in biotechnology to produce stress-response molecules, useful in cosmetics or nutrition [144].
In a culture batch, it is also important to monitor pH since it can vary in relation to photosynthesis. Moreover, pH variations can induce: (i) the differential availability of inorganic carbon sources (CO 2 /HCO 3 − /CO 3 2− ) [243][244][245]; (ii) the co-precipitation of phosphate with calcium, magnesium and carbonate at high carbonate concentrations; (iii) the low solubility and availability of trace metals at high pH [246]; (iv) intracellular pH regulation; and (v) changes in the uptake of essential nutrients, such as nitrate and phosphate [22,247,248]. The diatom Phaeodactylum tricornutum presents high plasticity to pH variation being able to grow at pH > 10, making it one of the most common contaminants in poorly buffered cultures [249,250]. This property could be advantageous in outdoor mass cultivation.

Enhancing Production and Growth: "Photosynthetic Regulation Biotechnology" and Genetic Modifications
One of the main challenges for increasing biotechnological applications of microalgae is the enhancement of their growth and yield. The growth rate of a microalgal population is the result of the balance between actively dividing and death cells. This -performance index‖ mainly depends on both cells' acclimation to the culture conditions and the energetic cost of internal processes regulation. The latter depends on how ecophysiological requirements match the environmental conditions and on physiological plasticity of the cultivated strains. Two of the most promising routes to progress, in enhancing biomass or interesting molecules production, are the -Photosynthetic Regulation Biotechnology‖ ( Figure 1) and the genetic engineering of strains.

Light Control and the "Photosynthetic Regulation Biotechnology"
Light is the most relevant trigger for photosynthesis even though it is the most variable parameter at sea over different temporal and spatial scales (see previous section). Many recent studies, using physiological, biochemical or molecular approaches, have shown how light intensity, variability and spectral composition affect the physiological state and growth of cells [24,25,[251][252][253]. By consequence, light manipulation might be a way for modifying microalgal productive performance [254,255], and therefore a relevant issue for biotechnological purposes (see the review by Murchie and collaborators [256]), in the field of -photosynthetic regulation biotechnology‖.
Photosynthesis is a unique process that converts light energy into biochemical energy, through light and dark reactions. In response to light intensity variations, cells modulate two stages of the photosynthetic process by modifying the photosynthetic apparatus structure and pigments, as well as the enzymes involved in carbon fixation [257]. Recent studies have shown that these two steps do not respond in the same way to light changes, showing an uncoupling of their regulative processes that depend on the ecophysiological properties of cells, and this also affects the growth rate maintenance [25]. Photosynthetic properties and growth can be poorly coupled, as observed in a diatom and a prasinophyte subjected to fluctuating irradiance [258], reinforcing the observations on the variability of the relationship between photosynthetic organic production and cell growth [259]. This variability can be attributed to many factors, such as variations in respiration, changes in dissolved organic losses and metabolite storage, as for instance polysaccharides [258]. The discrepancy between growth and production is less severe in small cells with smaller intracellular energy storage [25,126,251]. The term -light‖ includes different key variables (Figure 1): instantaneous photon flux density (PFD), daily light dose intensity, photoperiod, light distribution, frequency of PFD variability and spectral characteristics [20,260]. The integrated daily light dose depends on the photoperiod and light distribution. The daily light dose experienced by cells under sinusoidal light distribution compared to quadratic shape is around 1.9 times less, when provided using the same 12:12 h light-dark photoperiod and maximal light value [164]. Light is generally provided following a quadratic distribution, i.e., with an -on/off‖ switch system. Recent studies have shown that applying a sinusoidal light regime allows cells to activate a gradual and efficient photoregulation on the contrary of a quadratic distribution [24,251]. It has been observed that the slow increase of light at dawn allows cells to efficiently perform photoregulation and prepares the photosynthetic apparatus to cope with the high light midday peak [24,251]. By contrast, a constant excessive light induces considerable damage, increasing the biochemical costs associated with defence, protection and recovery, and decreasing the energy fuelled towards growth. Reciprocally, when constant light is limiting for optimal photosynthesis, cell performance will be reduced.
The photoperiod, i.e., the succession of illuminated and dark periods during the day, influences microalgal growth and photosynthetic rate [20,261]. It is known that biomass synthesis, in terms of produced carbon, may be higher under continuous light than alternating light-dark phases [203,262]. However, cells do not appear healthy under continuous light [263]; the light-dark succession allows cells to recover and uncouple many biological processes, such as photosynthesis, from cell division [20,264]. Moreover, photosystem repairing and relaxing occur during the dark period; it is also known that during dark phytoplankton cells uptake and assimilate NH 4 [265,266].
The very fast frequency of light variability (scale of milliseconds) positively influences photosynthetic efficiency, due to the re-oxidation of the electron transporters of the photosynthetic apparatus during dark phases [261]. Again, the frequency of the light variability, on an hour scale, is highly relevant. The shade acclimation state of cells is strengthened by the enhancement of the light fluctuations experienced, driving production of different carotenoids and antioxidants [251]. Light fluctuation dynamics also strongly affect the growth capacity of cells [251,258,259,267].
The spectral radiation of light does influence growth, photoacclimation state, and cell biochemistry [268,269]. Blue light affects many physiological processes in algae, such as photo-morphogenesis [270], chloroplast movement [271], cell division and photosynthetic acclimation in diatoms [252,253,270,272]. The spectral composition of light plays a key role in the ability of diatoms to finely balance light harvesting and photoprotective capacities [273]. Indeed, red radiation mixed to blue is necessary for activating the photoprotective pathway, such as the synthesis of xanthophyll pigments with an antioxidant role [274,275]. The spectral properties of microalgal absorption are the base for designing new models of photobioreactors and improving microalgal growth [276].

Genetic Transformations
Until now, biotechnological production from microalgae, such as food additives, cosmetics, animal feed additives, pigments, polysaccharides and fatty acids, is done in a non-transgenic way. However, genetic engineering is already being applied to microalgal research field, and selectable marker genes, promoters, reporter genes, transformation techniques, together with other genetic tools are available for various species. At present, about 20 species are accessible to genetic transformation [27], and large-scale sequencing projects are in progress for several microalgae species [277]. Sequences are available at the NCBI organelle database [278] and at the Organelle Genome Database [279].
The complete genome for about 30 cyanobacteria have already been sequenced; genome sequences of the cyanophyte Synechococcus [280], the green alga Micromonas [281] and Ostreococcus [128] are also available. For a more exhaustive list, please refer to the website from the Roscoff Culture Collection [282,283]. The following species' genomes (nuclear, mitochondrial and plastidial): Alexandrium tamarense, Amphidinium operculatum, Aureococcus anophagefferens, Chlorella vulgaris, Cyanophora paradoxa, and Dunaliella salina are being sequenced, and 37 microalgae transcriptomic projects are currently taking place [8,284].
In the last few years, about 20 new microalgal species have been genetically modified with success: nine green microalgae, five diatoms, three cyanobacteria, two dinophytes, two red microalgae and one euglenoid have been successfully transformed; most of these were achieved by nuclear and stable transformation [27]. Stable transformation of four diatom species (Phaeodactylum tricornutum, Navicula saprophila, Cylindrotecha fusiformis and Cyclotella cryptica) has also been reported, and 62 promoters have already been tested for microalgae transformations [8]. The use of engineered nucleases to genetically reprogram diatoms, with the aim of producing bio-fuels, has been successfully demonstrated [285]. By targeting specific sequences within diatoms' genomes, nucleases can be used to accurately insert, correct, or inactivate specific genes. With the whole genome sequencing of several diatom species, such as Thalassiosira pseudonana and Phaeodactylum tricornutum, a new era of post-genomics research has begun, with opportunities to improve fundamental understanding of the diatoms biology, and to build a molecular foundation for new industrial applications. Stable mutants of some species of microalgae have been obtained in the last years such as Phaeodactylum tricornutum, expressing a heterologous functional glucose transporter, Dunaliella salina (zea1) overproducing zeaxanthin, and cyanobacteria-synthetizing mosquito larvacides [7,27,80,[286][287][288][289]. Euglena gracilis has been transformed at the chloroplast level [290], while Thalassiosira weissflogii has been only transiently transformed [287]. Some groups are more difficult to be transformed by electroporation or conjugation [291], as for instance diatoms with their silica wall, compared to species from other groups such as Spirulina, Anabaena, or Synechocystis. Recently Synechocystis sp PCC6803 was deeply analysed, through several growth-coupled knockouts, to identify its main metabolic properties in the aim of biotechnological applications [292].
One of the main goals in biotechnological genetic engineering is their use as -bioreactors‖ in producing vaccines [293][294][295], specific proteins [296], or bioenergetic molecules [297]. Some therapeutic proteins have been successfully produced using microalgae, mainly the Chlorophyta Chlamydomonas reinhardtii, for which suitable transgenic tools and genomic data are available (for all three genomes: nuclear, chloroplastic and mitochondrial). The chloroplast of C. reinhardtii has been used to produce a range of recombinant proteins, including reporters such as glucuronidase (GUS), luciferase (LUC), green fluorescent protein (GFP), industrial enzymes, vaccines and therapeutic enzymes [298]. To date [8], 18 biopharmaceutical proteins have been expressed in C. reinhardtii and one in Chlorella ellipsoidea [298,299]. Diatoms have not been employed for expression of any biopharmaceutical proteins, but Hempel and collaborators [300] have recently reported the first stable expression of a full-length human antibody and the respective antigen in P. tricornutum.
In the bio-fuel production field, the Heterokonta Nannochloropsis represents a new model with potential for further development into an integrated photons-to-fuel production platform [301]. Several isolates of Nannochloropsis spp. produce large quantities of triacylglycerols, related to over-representation of genes involved in lipid biosynthesis together with rapid growth capacity, and industrial-scale cultivation [301]. The genome of the high PUFA-content species Nannochloropsis oceanica has been recently sequenced by Pan and collaborators [302]. Sequence similarity-based investigation identified new elongase-and desaturase-encoding genes involved in the biosynthesis of PUFAs, which provide a genetic basis for its rich eicosapentaenoic acid (EPA) content, making this species suitable for the genetic engineering of a triacylglycerols pathway.
To conclude, we can assert that genetic transformation could be a valid tool to -revolutionize‖ blue biotechnology. However, cautious measures have to be taken for ecosystem bio-safety and to monitor transgenic microorganisms in nature.

Conclusions
The functional biodiversity of microalgae has to be explored thoroughly, and mass culturing conditions presently used have to be revisited in order to optimize the fitness of cultivated species and decrease production costs. Indoor culturing systems would be preferable to outdoor systems for the reasons already discussed on the controlled vs. uncontrolled environmental parameters. We also suggest use of local microalgal species and seawater from which the species have been isolated, i.e., cultivation next to aquatic ecosystems, in the frame of what we can call the -Km-0 mass cultivation‖ strategy. This strategy might reduce the cost of cultivation, allow for the use of freshly isolated species and strains, and potentially also provide high flexibility in species choice that share common ecophysiological requirements. This could also solve the potential loss of growth efficiency due to long-term cultivation maintenance.
Furthermore, we recommend the use of ammonium, as nitrogen source, to increase biomass production, since it is assimilated faster than nitrate, and this allows an increase in the production efficiency (see above). We suggest providing light with an intra-diel light-dark cycle, with a sinusoidal shape instead of quadratic distribution, for the reasons discussed in the previous sections. Turbulence, reproducing coastal ecosystems, can be applied to coastal species cultivation, through a light variation frequency program. We propose the use of illumination systems that allow regulation of the photon flux density and the spectral composition to better manage photosynthetic productivity. For many microalgal groups (e.g., diatoms or chlorophytes), green radiation may be excluded from the light spectrum since it is not harvested, in order to increase the growth yield and reduce production costs [276]. Since the effect of light variations on physiology and growth of autotrophs is relevant, we suggest that genetic engineering should mainly target the photophysiological response system (e.g., [252,253,303]), entering the -photosynthetic regulation biotechnology.‖ The photophysiological pathway transformation of microalgae could be useful but with a high probability of obtaining a productive strain with a slow growth.
Diatoms that include many coastal species might be the ideal model to cultivate with biotechnological aims, for many reasons, such as the huge biodiversity of this group and many biological peculiarities (see previous). Many diatom species grow on benthic substrates, at least during one step of their life cycle; therefore, their cultivation for biotechnological purposes needs a deeper communication between ecophysiological and process engineering researchers. Moreover, they are able to reproduce sexually [134], allowing this group frequent gene recombination processes and present high physiological flexibility [274]. Due to this relevant feature, we could easily manipulate their photophysiological responses and biochemical pathways to increase the production of targeted molecules by varying the culture conditions.