N-3 polyunsaturated fatty acids (n-3 PUFAs), and especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential compounds for human health. They have been proven to act positively on a panel of diseases and have interesting anti-oxidative, anti-inflammatory or anti-cancer properties. For these reasons, they are receiving more and more attention in recent years, especially future food or feed development. EPA and DHA come mainly from marine sources like fish or seaweed. Unfortunately, due to global warming, these compounds are becoming scarce for humans because of overfishing and stock reduction. Although increasing in recent years, aquaculture appears insufficient to meet the increasing requirements of these healthy molecules for humans. One alternative resides in the cultivation of microalgae, the initial producers of EPA and DHA. They are also rich in biochemicals with interesting properties.
- microalgae
- n-3 PUFA
- EPA
- DHA
1. Algae, a Source of Bioactive Compounds
The term “algae” describes a diversity of micro and macro aquatic organisms able to survive and proliferate by photosynthesis [1]. Macroalgae (multicellular) and microalgae (unicellular) develop in both freshwater and marine environments [1]. Microalgae are planktonic or benthic algae floating in the water, while macroalgae are benthic and sedentary [2][3]. They include either eukaryotic species or prokaryotic cyanobacteria that are devoid of a membrane-bound nucleus and are classified between bacteria and plants [1]. The most abundant microalgal phyla are blue-green algae (
Cyanophyceae
Chlorophyceae
Bacillariophyceae
Chrysophyceae (including golden algae) [1][4].
2, and nutrients that allow them to be very effective in converting energy to biomass [3][5]. They are also easy to produce and can develop under various trophic regimes. Like terrestrial plants, most microalgae are autotrophic organisms efficiently using light to grow [6]. However, some species, for example, those belonging to the phylum Dinoflagellata, can use organic molecules to grow in addition to the inorganic ones and are called mixotrophic [7]. Heterotrophic microalgae, such as the Dinoflagellate
Crypthecodinium cohnii
Schizochytrium. Even if they cannot be considered algae as they lack plastid and chlorophyll, these organisms are exclusively heterotrophic [8][9]. Microalgae are also very diverse, and more than 30,000 species have been described so far [10][11]. The ease with which they can be cultured explains in part why they have been receiving increasing interest in recent years. Microalgae chemical composition has been widely studied in the literature, with proportions varying widely between the different species and culture conditions used [1][12]. They have been recognized as a reliable source of bioactive compounds such as proteins, lipids, carotenoids, hydrocarbons and vitamins [3][6]. The levels of these compounds of interest in microalgae are similar or even higher than they are in plants and animals [5][6]. The diversity of the compounds associated with the critical number of species available makes it possible to select and choose specific strains and molecules for various applications. These molecules also exhibit very interesting properties such as antioxidative, anti-microbial, anti-inflammatory, coloring, texturing, stabilizing, or even emulsifying capacities, which explains why they are so widely valued in the food and feed industry, in the cosmetics and medical sectors, and for bioenergy, biodiesel or even aquaculture [3][6][12].
In recent years, particular attention has been given to microalgae lipids. Indeed, they can represent up to 74% of microalgae’s total biochemical content according to species [12]. These molecules are built with fatty acids with 12 to 24 atoms of carbon and include the polyunsaturated fatty acids of the n-3 or n-6 families (n-3 PUFAs and n-6 PUFAs, respectively) [1]. N-3 PUFAs, also called omega-3 fatty acids, are characterized by their health benefits for both animals and humans. Their properties are expected to be of central interest in developing novel ingredients or compounds for various sectors, including the feed and food industry, with high commercial value. So far, microalgae are still poorly explored and valued as a natural source for a healthy diet [13]. Among the critical number of species known to date, only a few are commercially produced due to strict food safety regulations, namely in Europe, and because their cultivation in industrial quantities is only a few decades old [10][11]. This translates into a high potential for the production and commercialization of these organisms in the coming years.
2. Increasing Demand for n-3 Polyunsaturated Fatty Acids
A significant increase in fish and marine product consumption was registered between 1961 and 2016, with an average annual growth of 3.2% according to the FAO (2018) [14]. However, these trends are being challenged by supply issues [15]. Supply of n-3 PUFA comes mainly from the ocean and the vast majority (almost 90%) from capture fisheries [15]. Thus, access to fish and seafood determines to what extend the supply can meet PUFA demand. Global fisheries have reached a plateau following stock reduction (90 million tons per annum) [15][14]. Areas that were previously unexploited or underexploited in the 1950s are now undergoing an unprecedented expansion in fishing by industrial fleets [16]. Exploitation in the Southwestern Atlantic, the Indian Ocean, and the Western Central and Southwest Pacific has peaked [16]. These demanding industrial fisheries are responsible for removing top predators and thus the release of predatory pressure on lower trophic levels [16]. Pauly et al. (1998) [17] observed a decline in mean trophic levels. They proposed the concept of “fishing down marine food webs” which illustrates how the intensive capture pressure exercised on top predators leads to a change of target for lower trophic levels. Indeed, the diminution of top-down predatory pressure on smaller fish leads to a subsequent increase in their biomass, thus promoting new intensive captures by fisheries [16]. Overfishing is also responsible for stabilizing fish harvesting in the last few decades (33.1% of stocks were overfished in 2018) [14]. Most fisheries are at or beyond their sustainable levels of exploitation [15]. Technological improvement in fishing gear keeps increasing catching efficiency [18]. The fishing power of fleets and their range of action and versatility are dramatically affecting small pelagic fish [16].
A significant increase in fish and marine product consumption was registered between 1961 and 2016, with an average annual growth of 3.2% according to the FAO (2018) [69]. However, these trends are being challenged by supply issues [40]. Supply of n-3 PUFA comes mainly from the ocean and the vast majority (almost 90%) from capture fisheries [40]. Thus, access to fish and seafood determines to what extend the supply can meet PUFA demand. Global fisheries have reached a plateau following stock reduction (90 million tons per annum) [40,69]. Areas that were previously unexploited or underexploited in the 1950s are now undergoing an unprecedented expansion in fishing by industrial fleets [70]. Exploitation in the Southwestern Atlantic, the Indian Ocean, and the Western Central and Southwest Pacific has peaked [70]. These demanding industrial fisheries are responsible for removing top predators and thus the release of predatory pressure on lower trophic levels [70]. Pauly et al. (1998) [71] observed a decline in mean trophic levels. They proposed the concept of “fishing down marine food webs” which illustrates how the intensive capture pressure exercised on top predators leads to a change of target for lower trophic levels. Indeed, the diminution of top-down predatory pressure on smaller fish leads to a subsequent increase in their biomass, thus promoting new intensive captures by fisheries [70]. Overfishing is also responsible for stabilizing fish harvesting in the last few decades (33.1% of stocks were overfished in 2018) [69]. Most fisheries are at or beyond their sustainable levels of exploitation [40]. Technological improvement in fishing gear keeps increasing catching efficiency [72]. The fishing power of fleets and their range of action and versatility are dramatically affecting small pelagic fish [70].However, these small trophic levels, including phytoplankton are also affected by climate change [19]. Global warming is suspected to impact polyunsaturated fatty acids production by phytoplankton, thus reducing the availability of these essential compounds for higher trophic levels [20]. Temperature is expected to have effects on the quantity and quality of fatty acids in phytoplankton [20]. At the bottom of marine food webs, phytoplankton can adapt to changing temperatures by modifying their membrane characteristics [21]. Indeed, lipids and especially fatty acids are critical elements of cell membranes and can be modulated in response to environmental changes to maintain the desired level of fluidity [19][22][23]. The unsaturation of PUFA enhances the fatty acid’s ability to fold and increase flexibility [20]. Generally, n-3 PUFAs decrease with increasing temperature, while n-6 PUFAs have antagonist dynamics with rising temperature [20]. The increase in water temperature may then induce major shifts in global PUFA production by phytoplankton and their first consumers, microzooplankton, which could then have reverberating effects on higher terrestrial and aquatic trophic levels [24][20].
However, these small trophic levels, including phytoplankton are also affected by climate change [73]. Global warming is suspected to impact polyunsaturated fatty acids production by phytoplankton, thus reducing the availability of these essential compounds for higher trophic levels [74]. Temperature is expected to have effects on the quantity and quality of fatty acids in phytoplankton [74]. At the bottom of marine food webs, phytoplankton can adapt to changing temperatures by modifying their membrane characteristics [75]. Indeed, lipids and especially fatty acids are critical elements of cell membranes and can be modulated in response to environmental changes to maintain the desired level of fluidity [73,76,77]. The unsaturation of PUFA enhances the fatty acid’s ability to fold and increase flexibility [74]. Generally, n-3 PUFAs decrease with increasing temperature, while n-6 PUFAs have antagonist dynamics with rising temperature [74]. The increase in water temperature may then induce major shifts in global PUFA production by phytoplankton and their first consumers, microzooplankton, which could then have reverberating effects on higher terrestrial and aquatic trophic levels [18,74].Consequently, considering the constant growth of the global population and the resulting demand for these compounds, aquaculture now appears to be one of the solutions for meeting future seafood needs and filling the gap between supply and demand [15][25]. World aquaculture’s contribution to world fish production increased to 46% in 2016–2018 compared to only 26% in 2000 [26]. It is the first source of fish and ensures a continuing rise in fish supply for human consumption [14]. Global aquaculture production in 2018 consisted of 82.1 million tons of aquatic animals, 32.4 million tons of aquatic algae, and 26,000 tons of ornamental seashells and pearls [26]. Unfortunately, aquaculture remains mostly dependent on other fisheries’ products such as fish meal or fish oils (22 million tons in 2018) [25][26]. Consequently, finite and limited fisheries of small pelagic species like anchovy or sardine are more harvested, exacerbating pressure on fish stocks and significantly increasing sales prices [26]. In the longer term, aquaculture thus appears not to be economically sustainable, especially since climate change already impacts the natural production of these oils.
Consequently, considering the constant growth of the global population and the resulting demand for these compounds, aquaculture now appears to be one of the solutions for meeting future seafood needs and filling the gap between supply and demand [40,78]. World aquaculture’s contribution to world fish production increased to 46% in 2016–2018 compared to only 26% in 2000 [79]. It is the first source of fish and ensures a continuing rise in fish supply for human consumption [69]. Global aquaculture production in 2018 consisted of 82.1 million tons of aquatic animals, 32.4 million tons of aquatic algae, and 26,000 tons of ornamental seashells and pearls [79]. Unfortunately, aquaculture remains mostly dependent on other fisheries’ products such as fish meal or fish oils (22 million tons in 2018) [78,79]. Consequently, finite and limited fisheries of small pelagic species like anchovy or sardine are more harvested, exacerbating pressure on fish stocks and significantly increasing sales prices [79]. In the longer term, aquaculture thus appears not to be economically sustainable, especially since climate change already impacts the natural production of these oils.The new task is to find alternative n-3 PUFA sources and more sustainable fish oils and meal origins able to supplement aquaculture production and capture fisheries. One of such alternatives is the use of lower trophic level species like zooplankton, krill or copepods. This option is challenging in terms of harvesting and raises environmental questions on higher trophic levels. Top predators like whales and penguins are directly relying on these groups to survive. By collecting the prey this alternative might impact the predators, and then not be sustainable in the longer term [18]. Utilization of higher plants as a substitute for aquaculture feed is conceivable [18][27], however since they are not able to synthesize 20:5n-3 and 22:6n-3 due to the absence of dedicated enzymes [25][28], they would only supply precursors (like 18:2n-6 or 18:3n-3) and be difficult to promote for n-3 long-chain PUFAs (LC-PUFA) production by aquacultured fish. Consequently, the production of EPA and DHA by higher plants is currently attempted to implement genes of organisms naturally producing these compounds, i.e., microalgae, yeast and/or bacteria [25].
The new task is to find alternative n-3 PUFA sources and more sustainable fish oils and meal origins able to supplement aquaculture production and capture fisheries. One of such alternatives is the use of lower trophic level species like zooplankton, krill or copepods. This option is challenging in terms of harvesting and raises environmental questions on higher trophic levels. Top predators like whales and penguins are directly relying on these groups to survive. By collecting the prey this alternative might impact the predators, and then not be sustainable in the longer term [72]. Utilization of higher plants as a substitute for aquaculture feed is conceivable [72,80], however since they are not able to synthesize 20:5n-3 and 22:6n-3 due to the absence of dedicated enzymes [78,81], they would only supply precursors (like 18:2n-6 or 18:3n-3) and be difficult to promote for n-3 long-chain PUFAs (LC-PUFA) production by aquacultured fish. Consequently, the production of EPA and DHA by higher plants is currently attempted to implement genes of organisms naturally producing these compounds, i.e., microalgae, yeast and/or bacteria [78].Developed in the mid-1970s for use on common crops, genetic engineering has been adapted in recent years, thanks to improvement in techniques, more specific DNA-markers and the use of genomics [29]. Genetic modifications have already introduced the n-3 PUFA biosynthesis trait to oleaginous plants. For example, this genetic engineering has been tested on
Developed in the mid-1970s for use on common crops, genetic engineering has been adapted in recent years, thanks to improvement in techniques, more specific DNA-markers and the use of genomics [82]. Genetic modifications have already introduced the n-3 PUFA biosynthesis trait to oleaginous plants. For example, this genetic engineering has been tested onNicotiana tabacum [30],
[83],Arabidopsis thaliana [31],
[84],Brassica juncea [32],
[85],Cannabis sativa
, andCamelina sativa [33][34]. At first, these transgenic plants could not match the level of EPA and DHA of microalgae or the requirement for farmed fish [25], especially in DHA [28]. However, recent studies have shown more promising results, proving, for example, the efficiency of
[86,87]. At first, these transgenic plants could not match the level of EPA and DHA of microalgae or the requirement for farmed fish [78], especially in DHA [81]. However, recent studies have shown more promising results, proving, for example, the efficiency ofCamelina crops to substitute both fish and vegetable oil without any negative effects on fish development or health [35][34][36]. They are real alternatives to less sustainable aquafeeds satisfying the demand for essential fatty acids as well as increasing 20:5n-3 and 22:6n-3 content for the human diet [15][36]. However, these genetically modified plants are often negatively regarded by consumers as artificial and unnatural and are perceived to carry risks for health and the environment [37]. Industries have to work on their transparency and communication strategies in order to increase the acceptance of such products on the market.
crops to substitute both fish and vegetable oil without any negative effects on fish development or health [38,87,88]. They are real alternatives to less sustainable aquafeeds satisfying the demand for essential fatty acids as well as increasing 20:5n-3 and 22:6n-3 content for the human diet [40,88]. However, these genetically modified plants are often negatively regarded by consumers as artificial and unnatural and are perceived to carry risks for health and the environment [89]. Industries have to work on their transparency and communication strategies in order to increase the acceptance of such products on the market.Another encouraging solution is the direct production of microalgae rich in these two PUFAs. Algal biomasses are currently used and formulated for farmed fish feed and human consumption and are commercialized by different industries [15]. These aspects will be developed later on in this review. However, even if the fatty acid composition of the different microalgae species is widely studied [22][38][39][40][41][42][43], more knowledge is needed to elucidate their synthesis pathways, as they will be particularly useful in developing new sustainable products for aquafeed as well as for human food.
Another encouraging solution is the direct production of microalgae rich in these two PUFAs. Algal biomasses are currently used and formulated for farmed fish feed and human consumption and are commercialized by different industries [40]. These aspects will be developed later on in this review. However, even if the fatty acid composition of the different microalgae species is widely studied [76,90,91,92,93,94,95], more knowledge is needed to elucidate their synthesis pathways, as they will be particularly useful in developing new sustainable products for aquafeed as well as for human food.3. Microalgae as n-3 Polyunsaturated Fatty Acids Producers
As already stated above, humans or top predators cannot synthesize de novo n-3 PUFAs or produce them from their precursors in large enough quantities. They thus have to obtain them from their diet [44][45]. It is well accepted that the main sources of LC-PUFAs are microalgae. Their average lipid content varies from between 1 and 40% according to the species and growth conditions [5]. Fatty acids that cannot be synthesized by all organisms are considered essential fatty acids [46]. This is the case for linoleic acid, α-linolenic acid or even longer PUFAs such as EPA, DHA or ARA [45].
As already stated above, humans or top predators cannot synthesize de novo n-3 PUFAs or produce them from their precursors in large enough quantities. They thus have to obtain them from their diet [96,97]. It is well accepted that the main sources of LC-PUFAs are microalgae. Their average lipid content varies from between 1 and 40% according to the species and growth conditions [5]. Fatty acids that cannot be synthesized by all organisms are considered essential fatty acids [98]. This is the case for linoleic acid, α-linolenic acid or even longer PUFAs such as EPA, DHA or ARA [97].The percentage of 20:5n-3 and 22:6n-3 varies with microalgae species and depends on the productivity rate and the growth conditions imposed [44]. Microalgae richer in EPA and DHA consequently present a higher nutritional value for consumers than microalgae with lower n-3 PUFA levels [44].
The percentage of 20:5n-3 and 22:6n-3 varies with microalgae species and depends on the productivity rate and the growth conditions imposed [96]. Microalgae richer in EPA and DHA consequently present a higher nutritional value for consumers than microalgae with lower n-3 PUFA levels [96].DHA-producers are known to be Dinophytes, Haptophytes, some Cryptophytes, Thraustochytrids, or Euglenoids [40][41][45][47][48]. Dinophytes can produce high amounts of 22:6n-3 with up to 40% of the total fatty acids in some taxa [47][49][50]. In Haptophytes, DHA production can reach 30% of total fatty acids [51]. Thraustochytrids remain some of the most essential 22:6n-3 producers and synthesize around 60% of total fatty acids in the form of triacylglycerides (TAG) in the genus
DHA-producers are known to be Dinophytes, Haptophytes, some Cryptophytes, Thraustochytrids, or Euglenoids [92,93,97,99,100]. Dinophytes can produce high amounts of 22:6n-3 with up to 40% of the total fatty acids in some taxa [99,101,102]. In Haptophytes, DHA production can reach 30% of total fatty acids [103]. Thraustochytrids remain some of the most essential 22:6n-3 producers and synthesize around 60% of total fatty acids in the form of triacylglycerides (TAG) in the genusAurantiochytrium
and [100,104]. EPA-synthesizing taxa are Diatoms, such asPhaeodactylum tricornutum
, Eustigmatophyte such asNannochloropsis sp. and some Haptophytes [45][53][54][55][56][57][58][59]. Diatoms can synthesize around 20% of EPA and a low proportion of DHA [57]. The Eustigmatophyte,
sp. and some Haptophytes [97,105,106,107,108,109,110,111]. Diatoms can synthesize around 20% of EPA and a low proportion of DHA [109]. The Eustigmatophyte,Nannochloropsis oculata, produces between 15 and 30% of 20:5n-3 [58].
, produces between 15 and 30% of 20:5n-3 [110].For other taxa such as Cyanobacteria or Chlorophytes, long chain n-3 PUFA production can be assumed negligible [44], and only certain levels of PUFAs such as ALA and stearidonic acid (SDA—18:4n-3) can be found and play a subsiding role in consumer development, namely
For other taxa such as Cyanobacteria or Chlorophytes, long chain n-3 PUFA production can be assumed negligible [96], and only certain levels of PUFAs such as ALA and stearidonic acid (SDA—18:4n-3) can be found and play a subsiding role in consumer development, namelyChlorella vulgaris [45].
[97].In addition to inter-species variability, LC-PUFA production can also be modulated by environmental parameters and varies with phytoplankton growth. Factors such as temperature, growth rate, irradiance, salinity, and nutrient availability can control phytoplankton PUFA production. Indeed, as stated earlier in this review, microalgae can modify their membrane fluidity in response to temperature variations, thus adapting their fatty acid composition [22][23]. Anterior works studied the impact of nitrogen and nutrient availability and showed that microalgae tended to accumulate neutral lipids when nutrients became scarce [60][61]. Microalgae sensitivity varies with taxa and can also have a tremendous impact on this lipid accumulation intensity following environmental stress [60]. In contrast, phytoplankton’s exponential growth phase associated with replete nutrient conditions would be more likely linked to higher production of polar lipids used to build cell membranes [61]. Taipale et al. [45] studied the influence of the growth stage on the production of n-3 and n-6 PUFAs in 16 species belonging to the six main groups of phytoplankton (Cryptophytes, Dinophytes, Chrysophytes, Diatoms, Chlorophytes and Cyanobacteria). They showed that PUFA content can vary greatly within phytoplanktonic groups to variation in cell size. Both increasing temperature and concentration of nitrogen were related to the production of SDA, EPA, DHA and docosapentaenoic acid (DPA-6—22:5n-6) in Cryptophytes, Chrysophytes, and Dinophytes, and the PUFA production of some taxa was more favored during the stationary phase (Cryptophytes and Chrysophytes), while others would be during exponential phase (such as fast-growing diatoms and dinophytes) [45].
In addition to inter-species variability, LC-PUFA production can also be modulated by environmental parameters and varies with phytoplankton growth. Factors such as temperature, growth rate, irradiance, salinity, and nutrient availability can control phytoplankton PUFA production. Indeed, as stated earlier in this review, microalgae can modify their membrane fluidity in response to temperature variations, thus adapting their fatty acid composition [76,77]. Anterior works studied the impact of nitrogen and nutrient availability and showed that microalgae tended to accumulate neutral lipids when nutrients became scarce [112,113]. Microalgae sensitivity varies with taxa and can also have a tremendous impact on this lipid accumulation intensity following environmental stress [112]. In contrast, phytoplankton’s exponential growth phase associated with replete nutrient conditions would be more likely linked to higher production of polar lipids used to build cell membranes [113]. Taipale et al. [97] studied the influence of the growth stage on the production of n-3 and n-6 PUFAs in 16 species belonging to the six main groups of phytoplankton (Cryptophytes, Dinophytes, Chrysophytes, Diatoms, Chlorophytes and Cyanobacteria). They showed that PUFA content can vary greatly within phytoplanktonic groups to variation in cell size. Both increasing temperature and concentration of nitrogen were related to the production of SDA, EPA, DHA and docosapentaenoic acid (DPA-6—22:5n-6) in Cryptophytes, Chrysophytes, and Dinophytes, and the PUFA production of some taxa was more favored during the stationary phase (Cryptophytes and Chrysophytes), while others would be during exponential phase (such as fast-growing diatoms and dinophytes) [97]. Despite the extensive knowledge of the diverse species able to produce these two compounds, the synthesis pathways leading to the production of EPA and DHA are still under investigation. They would be of vital interest in improving microalgae cultures and enhancing the n-3 PUFA yields for industries, especially for healthy food and feed preparations.