Health benefits of microalgae and their microbiomes

Summary Microalgae comprise a phylogenetically very diverse group of photosynthetic unicellular pro‐ and eukaryotic organisms growing in marine and other aquatic environments. While they are well explored for the generation of biofuels, their potential as a source of antimicrobial and prebiotic substances have recently received increasing interest. Within this framework, microalgae may offer solutions to the societal challenge we face, concerning the lack of antibiotics treating the growing level of antimicrobial resistant bacteria and fungi in clinical settings. While the vast majority of microalgae and their associated microbiota remain unstudied, they may be a fascinating and rewarding source for novel and more sustainable antimicrobials and alternative molecules and compounds. In this review, we present an overview of the current knowledge on health benefits of microalgae and their associated microbiota. Finally, we describe remaining issues and limitation, and suggest several promising research potentials that should be given attention.


Introduction and background
Microalgae and their associated microbiota grow and survive in all climate zones and many species are well adapted to extreme temperatures and pH values. Since microalgae are photosynthetic active organisms, which can be grown under a wide variety of conditions, they are highly attractive for the biotechnological production of a wide range of different chemical compounds. They are particularly well known for their use in the production of advanced biofuels (e.g. drop-in biofuels and fourthgeneration biofuels) and to some extend for the production of bioplastics (Chisti, 2007;Mata et al., 2010;Hempel et al., 2011;Rahman and Miller, 2017;Khan et al., 2018;Onen Cinar et al., 2020;Keasling et al., 2021).
Recently, it has become clear that algae and their microbiota harbour a large and diverse set of genes for the biosynthesis of molecules that supress bacterial pathogens (Table 1) (Krohn-Molt et al., 2013. One of the example are sterols with anti-inflammatory capacity, like diacylglycerols, triacylglycerols and phytosterols (Ostlund et al., 2003;Bilbao et al., 2016;Randhir 0  1  0  1  4  0  0  0  6  17  68  21  15   Alkylhydroperoxidase 0  0  0  1  13  0  0  1  69  72  276  138  64  Deferrochelatase/  peroxidase   0  0  4  0  1  0  0  0  5  11  41  26   Peroxiredoxin  9  11  12  8  54  9  11  6  54  78  314  139  74  Chlorophyll a  biosynthesis   5  4  4  4    Key features of antibacterial, antiviral, antioxidant activity and anti-inflammatory and anti-cancer properties as well as immune promoters and immunomodulatory activity of microalgae communities' genomes and metagenomes using IMG function search including IMG ID and total size of bp. Data shown in total number of hits for possible antibacterial activity, antiviral activity, antioxidant activity, anti-inflammatory and anti-cancer properties, and immune promoters and immunomodulatory activity. et al., 2020). In this respect, they bear a great potential to make major contributions to important societal needs linked to the treatment of infections due to human, animal and plant pathogenic microorganisms (Fig. 1). The appearance of untreatable antibiotic resistant microorganisms in clinical settings is a major concern to human health (O'Neill, 2016;WHO, 2019). Thus, there is a need to develop novel antimicrobials that are distinct in their mode of action from those already known and on the market. The awareness in the scientific community about the largely unexplored potential in microalgae has led to increased interest during the last decade, as evidenced by an exponential increase in the number of publications and patents on the subject of microalgae and health. However, compared to the large number of microalgal species, our knowledge remains sparse, and further research requires a focused and more systematic approach to better explore this promising resource with a special emphasis on human, animal and plant health and well-being.
Here, we summarize current knowledge on the benefits of microalgae in health management. We further point out current limitations hindering their exploitation and address technologies that could provide a basis for a more systematic exploitation of their potential.

Antibacterial activity
Quorum sensing and quorum quenching as drives for antibiofilm strategies Quorum sensing (QS) and Quorum Quenching (QQ) play an important role in the expression of virulence factors and antimicrobial resistance, and are involved in the formation of bacterial biofilms (Ahlgren et al., 2011;Waters and Goldberg, 2019). The latter are of major concern in clinical and industrial settings, as they are very difficult to control and treat and cause severe problems in patients and industries. Both QQ and various mechanisms of QS interference have been outlined and discussed as possible strategies to prevent and treat microbial biofilm formation (Singh et al., 2000;Ahlgren et al., 2011;Fetzner, 2015;Waters and Goldberg, 2019). Microalgae and their associated bacterial microbiota may be valuable tools to further verify this concept (Table 1). Microalgae microbiomes offer both QQ enzymes and a broad variety of QS molecules that have been shown to interfere with pathogens (Ghanei-Motlagh et al., 2021a, 2021b. In this framework, the screening of 19 strains of microalgae, reported that one microbial community of Chlorella saccharophila and one of Chlorella vulgaris both degraded N-acyl homoserine lactones (AHLs), resulting in the inhibition of violacein production in the reporter strain (Natrah et al., 2011). When using a E. coli (JB523)-strain sensitive to N-(3-oxohexanoyl)-L-homoserine lactone, the C. saccharophila associated microbiome was found to significantly suppress bacterial QS and further to inhibit AHL-regulated bioluminescence in the pathogen Vibrio harveyi (Natrah et al., 2011). AHL degradation can occur indirectly, such as in cultures of Tetraselmis suecica and Chaetoceros muelleri that were associated with AHLdegrading bacteria belonging to the genera Bacillus and Pseudomonas (Pande et al., 2015). These bacterial isolates were found to degrade AHL molecules, and Bacillus sp. was reported to suppress the quorum sensing system of Vibrio campbelli and thus protected the larvae of the giant river prawn (Macrobrachium rosenbergii) from infection, improving survival from 42 to 67% during an infection challenge.
Another route of interference with quorum sensing systems is through the secretion of molecular mimics. In general, in Gram-negative bacteria, many important changes in gene expression and behaviour are regulated in a population density-dependent fashion by N-acyl homoserine lactone (AHL) signal molecules. Plants are able to secrete substances, which mimic bacterial N-acyl homoserine lactones. These mechanisms affect population density-dependent behaviours in associated bacteria. For example, ethyl acetate, extracted AHL mimics from Chlamydomonas reinhardii, was found to affect the expression of 34 proteins, 25 of which were also affected by AHL (Teplitski et al., 2004).

Phycobiliproteins have antimicrobial effects
Multiple compounds from microalgae and their affiliated microbiota have been reported to have antimicrobial properties, sometimes as a secondary benefit distinct from their primary function. This is the case for phycobiliproteins, a family of water-soluble lightharvesting pigments, that play a central role in the photosynthesis of cyanobacteria and the red algae Rhodophyta. Phycobiliproteins are divided into four groups: phycocyanin, phycoerythrin, phycoerythrocyanin and allophycocyanin, of which phycocyanin is the most common in the environment (Li et al., 2019, Pagels et al., 2019. Overall, in our bioinformatic analyses, it was found that publicly available genomes of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169 (IMG ID 2507525016) contain phycoerythrobilin, phycocyanobilin, phycoviolobilin and phycourobilin biosynthesis genes (Table 1). For example, screening of 19 microalgae supernatants containing the whole microbial community identified multiple active phycobiliproteins, including phycocyanin and phycoerythrin, which exhibited significant antifungal property and growth inhibition of both Gram-positive and -negative bacteria (Najdenski et al., 2013). A phycobiliprotein extract from Arthrospira platensis was found to have antifungal effect against the plant pathogen Botrytis cinerea when applied at doses from 0.3 to 4.8 mg ml À1 , both reducing the fungal growth as well as protecting tomato fruits from infection (Righini et al., 2020). Interestingly, environmental conditions have been found to affect the production of phycobiliproteins, both in terms of quantity and of the distribution of phycobiliprotein produced, with factors such as the pH, quality of light and nutrient source all having a significant effect (Pagels et al., 2019). Khattar et al. (2015) reported optimized culture conditions for Anabaena fertilissima that involved a slightly alkaline pH as well as supplement of nitrite and illumination with blue light, resulting in a 1.6-fold increase in the total production of phycobiliproteins (from 383 to 627 lg mg À1 ; P < 0.05) and a 4.5-fold in the production of phycoerythrin (from slightly more than 100 to almost 500 lg mg À1 ; P < 0.05). Overall, as every microalgae will require its own optimization to achieve its maximum potential, it is one of the main issues for practical application of these products.

Fatty acids play a key role as antimicrobials
Fatty acids, including those from microalgae, have strong antimicrobial effects. Notably, extracts from cyanobacteria have been shown to be inhibitory against Streptococcus pyogenes and Staphylococcus aureus, while fatty acids from the cyanobacterium Synechocystis sp. were inhibitory against Bacillus cereus, Escherichia coli and the yeast Candida albicans (Najdenski et al., 2013). Ruffel et al. tested 29 different types of purified fatty acids from different species of microalgae using disk-diffusion assay. The results show, that 3 of 29 fatty acids were inhibitory against E. coli, while 15 were inhibitory against S. aureus (Ruffell et al., 2016). The effective dose ranged from 250 to 2000 lg per disk and polyunsaturated fatty acids (PUFA) were found to be more significantly more likely to display antimicrobial activity compared to monounsaturated or saturated acids (11 of 13, compared to two of seven and two of nine respectively).
Testing of extracts from Chlorococcum strain HS-IO1 and Dunaliella primolecta showed that a-linolenic acid from these algae had antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) (Ohta et al., 1995). Similarly, the fatty acid fraction of the acidophilic Coccomyxa onubensis has been shown to have antimicrobial activity against multiple Gram-positive and -negative bacterial pathogens. Although these authors did not test the individual fatty acids involved in this activity, the most common fatty acids in the extracts included palmitic acid and oleic acid alongside the PUFA linoleic acid and linolenic acid (Navarro et al., 2017). In Chlorella spp., a mixture of fatty acids, termed 'chlorellin' is known to have antimicrobial properties, and, for example, ethanol and isopropanol-extracts from Chlorella spp. were shown to have inhibitory capacity equivalent to ampicillin and oxacillin against Staphylococcus spp., although this author did not investigate the effect of the individual fatty acids (Acurio et al., 2018). Taken together, these results suggest that various microalgal fatty acids can exert antimicrobial activity, although PUFA appeared more likely to do so. Unfortunately, drawing firm conclusions is hindered by the fact that many authors treat all fatty acids together rather than attempt to separate the various species of fatty acids to test them separately.
Contrary to the percentage of lipids in microalgal cells which is considered roughly comparable between microalgal species, fatty acids are highly variable both in terms of their relative concentration and the repartition of the various species of fatty acids (Hu, 2013). Several environmental factors have been reported to influence the fatty acid profile of phytoplankton, for example, higher temperatures are associated with an increased in the proportion of saturated fatty acids, whereas decreased light levels were associated with an increased in polyunsaturated fatty acids (plausibly due to an increase in the presence of thylakoids to improve photosynthetic activity) (Guedes et al., 2010;Li et al., 2011). Interestingly, comparison of the fatty acid profiles of diatoms and dinoflagellates by Peltomaa et al. (2019) suggested that fatty acid contents were higher in freshwater species than marine ones. However, genetic and phylogeny appear to be the main factor dictating the fatty acid profile of microalgae. For example, screening of 1145 species, representing six major groups of both marine and freshwater phytoplankton species, showed that phylogeny was the main factor influencing the fatty acid profile of microalgae (Galloway and Winder, 2015) accounting for about 36 to 44% of the total variation in fatty acid profiles. Similar results were reported by J onasd ottir (2019) based on the screening of the fatty acid profiles of 160 species representing seven phyla of marine phytoplankton.
For example, Cavonius et al. (2014) reported that fatty acid represented 12% of the dry mass of Tetraselmis galbana, but only 4-5% in Phaeodactylum tricornutum. Similarly, screening of 2076 strains of microalgae by Lang et al. (2011) showed that fatty acids were particularly low in Chlorophyta and Streptophyta. Comparison of the fatty acid profiles of six commonly cultivated strains of microalgae showed that C. vulgaris had a comparatively high concentration of 16:2 fatty acid. In addition, both C. vulgaris and Tetradesmus obliquus had comparatively high levels of 18:1 fatty acids as well as 18:3, linolenic acid (Chac on- Lee and Gonz alez-Mariño, 2010). This is in accordance with the reports of Cepas et al. (2021) who, after screening the fatty acid profiles of several strains of cyanobacteria also reported that C. vulgaris and T. obliquus were particularly rich in linolenic acid.
Other microalgal molecules with antibacterial effects EPS from several algal cultures were inhibitory against multiple bacterial and fungal isolates when tested using both the agar diffusion and the minimal inhibitory concentration (MIC) (Najdenski et al., 2013). Of these, the most effective were EPS from Gloeocapsa sp. with MIC values ranging from 0.125 for S. aureus to 1.0 mg ml À1 for S. pyogenes (Najdenski et al., 2013). Similarly, crude extracts from H. pluvialis demonstrated inhibitory effects against several bacterial pathogens using disk-diffusion assays, resulting in inhibition zone between 6.1 and 10.2 mm (Rather et al., 2021).
Extraction of A. platensis compounds using different solvents revealed that methanolic extracts had the highest antimicrobial activity against bacterial pathogens. Whereby, MIC of were 128 and 256 lg ml À1 against S. aureus and E. coli, respectively, although the compounds involved were not further characterized (Kaushik and Chauhan, 2008). Extraction of a variety of antimicrobial compounds from Cosmarium sp. showed that all had some potential as antimicrobials, although the methanol, hexane and aqueous extracts were not effective against the Gram-positive bacteria tested (Challouf et al., 2012).
Other microalgae cultures including their microbiomes have been found to display antimicrobial properties, but without the responsible compounds being further characterized. This is the case for eight freshwater microalgae (belonging to the genera Oscillatoria, Lyngbya, Oedogonium and Spirogyra) whose ethanolic and methanolic fractions, tested at concentrations ranging from 0.16 to 0.66 mg ml À1 using the disk-diffusion method, demonstrated some inhibitory properties against some Gram-negative and one Gram-positive pathogenic bacteria with zone of inhibitions ranging from 7 to 12 mm, for O. sancta extracted using ethanol at a dose of 0.35 mg ml À1 and S. decimina extracted using a methanol solvent and applied at 0.20 mg ml À1 respectively (Prakash et al., 2011).
For example, methanolic extracts from the cyanobacterium A. platensis at 100 ng ml À1 have clear inhibitory effects on the biofilm formation of several bacteria, including pathogens. Biofilm formation of Vibrio parahaemolyticus was inhibited by 90%, of Vibrio alginolyticus by 88%; of Aeromonas hydrophila by 74%; and by 61 to 84%, in S. aureus (LewisOscar et al., 2017).
In addition, for the green alga Chlamydomonas reinhardtii, Vishwakarma and Sirisha (2020) reported that extracted sulfated polysaccharides displayed activity against the biofilms of Salmonella enterica and V. harveyi, distorting the biofilms and reducing their formation by about 50% when applied at concentrations of 0.5 mg ml À1 against S. enterica and 8 mg ml À1 against V. harveyi. Ghaidaa et al. (2020) reported similar findings with C. reinhardtii reducing the formation of biofilms of several bacterial species by about 50%. Several human pathogens were also more susceptible to these compounds since S. aureus biofilms were reduced by up to 90%.

Antiviral activity
In cyanobacteria, a variety of antiviral and antimicrobial molecules has been described over the years, as recently reviewed by Mazur-Marzec et al. (2021) and Khalifa et al. (2021), including cyanovirin N, isolated from Nostoc ellipsosporum, which is known to interfere with human immunodeficiency virus (HIV's) binding onto CD+ T-cells. The antiviral compound cyanovirin-N binds the viral spike protein gp120 that is required for HIV interactions with receptors on the host cells and has been shown to have antiviral activity against HIV (Dey et al., 2000;Singh et al., 2005). Cyanovirin-N has also demonstrated inhibitory action on other enveloped viruses such as herpes virus and measles virus as well as feline immunodeficiency virus (FIV) at concentrations as low as 10 nM (Dey et al., 2000). More recently, in silico docking simulations have suggested that cyanovirin-N could form stable covalent bonds with the homotrimeric transmembrane spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (Lokhande et al., 2020).
Among these, lectins are ubiquitous proteins, whose carbohydrate domains have been shown to interact with the surface of cells and viruses. For example, a lectin recently isolated from the cyanobacterium Oscillatoria acuminate was found to be inhibitory against herpes simplex virus type-1 at doses at low as 20 ng ml À1 with highest inhibition at a dose of 2 mg ml À1 (Saad et al., 2020). Because pre-treating the cells prior to viral infection was not found to be protective, it is most likely that this lectin interacts and interferes with viral receptors. Figure 2 shows as an example the structures of cyanovirin N and oscillatorial lectin.
A further interesting fact is that many microalgae communities are able to produce polysaccharides, which are well known to exert a broad spectrum of biological activities, especially antiviral properties (Chaisuwan et al. 2021). These polysaccharides were found among numerous algal microbiomes, including S. quadricauda (IMG ID 3300005759), C. saccharophila (IMG ID 3300008885), C. sorokiniana (IMG ID 3300042370) and M. crux-melitensis (IMG ID 3300008886) ( Table 1).

Antioxidant activity
It is well established that multiple compounds from microalgae have antioxidant activity. The best known algal antioxidant is the keto-carotenoid pigment astaxanthin, in particular extracted from the green algae Haematococcus pluvialis (Plaza et al., 2009, Fig. 2). Astaxanthin can neutralize singlet oxygen and scavenge free radicals, resulting in a powerful antioxidant effect, approximately 10 times stronger than b-carotene and 100 times greater than that of a-tocopherol (Shimidzu et al., 1996;Lorenz and Cysewski, 2000). H. pluvialis exists under different morphotypes, influenced by environmental conditions, including high temperature, intense light, in particular UV-light, alongside other stressors such as salinity, drought or nutrient scarcity. Under the effect of these environmental stressors, it switches from a flagellated free-living cell to a coccoid cyst called an aplanospore (Lim et al., 2018;Molino et al., 2018). This is accompanied by a degeneration of the cell's chloroplasts, hence the name 'red phase' given to this phase of the life cycle, a thickening of the outer cell wall and secretion of two layers of extracellular matrix as well as the accumulation of large quantities of astaxanthin in oil droplets within the aplanospore (Wayama et al., 2013). During this process, the relative volume of astaxanthin changes from 0.2% of the total free-living cell to 52% in the aplanospores.
The antioxidant capacity of astaxanthin has recently been shown in juvenile Asian tiger shrimp (Penaeus monodon). Supplementation of 80 mg astaxanthin per kg of diet was associated with a significant increase in the total antioxidant status and superoxide dismutase (SOD) in the haemolymph of the shrimp (Pan et al., 2003). This was connected to an improved recovery following exposure to various stressors and a decrease in both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values in the haemolymph, suggesting a hepatoprotective effect of astaxanthin. Comparable results were reported after feeding diet supplemented with 8 g kg À1 of astaxanthin to yellow catfish (Pelteobagrus fulvidraco), which resulted in an increase in SOD and HSP70 activity, and a reduction in both ALT and AST, as well as an increased survival following stress and infectious challenge with Proteus mirabilis (Liu et al., 2016).
Another example of compounds with antioxidant activity is malondialdehyde (MDA, Fig. 2). Feeding of lambs with A. platensis (incorporated at 0.1 g kg À1 of food) resulted in a decrease of the animals´serum concentrations of MDA (from 99 to 16 nmol ml À1 in the animals receiving the control and supplemented feed respectively). At the same time, an increase in both vitamin A (from 690 to 710 ng ml À1 ) and glutathione (from 90 to 140 ng ml À1 ) was recorded in the animals' sera (EL-Sabagh et al., 2014). The same authors further reported reduction in the sera's alanine and aspartate aminotransferase, which is consistent with reduced oxidative stress to the liver. Diet supplemented with A. platensis (at doses ranging from 25 to 100 g kg À1 of feed) fed to the fish O. mykiss was correlated with an increase in the serum antioxidant activity (at doses ranging from 50 to 100 g kg À1 of feed) alongside an increased expression of the superoxide dismutase and catalase genes in the liver of the fish, when given at doses of 75 or 100 g kg À1 of feed (Teimouri et al., 2019). Feeding of the fruit fly Drosophila melanogaster with Chlorella sorokiniana (incorporated at doses of 2 or 4 mg ml À1 ) resulted in an increased expression of SOD1, a superoxide dismutase encoding gene, as well as resistance against H 2 O 2 -induced oxidative stress (Qiu et al., 2020). Phycobiliproteins have also demonstrated potent antioxidant capacities and promoted the elimination of reactive oxygen species and increasing the concentration of antioxidative enzymes (Li et al., 2019). Future analyses could be more investigated for bioinformatical analysis of algae and their microbiomes. So far, data sets published at IMG/MER revealed genes coding for SOD, catalases and rhodanese-related sulfurtransferases (Table 1). Furthermore, the studying of algal genomes (C. variabilis NC64A and C. subellipsoidea C-169) demonstrated the presence of genes coding for the biosynthesis of known antioxidants, such as chlorophyll, carotenoid, lutein and astaxanthin. These genomes are available under the accession number IMG ID 2507525016 (Table 1).

Anti-inflammatory and anti-cancer properties
Sterols represent a subgroup of steroid molecules and are widespread in the cell membranes of eukaryotic organisms. Figure 2 shows the molecular structures of saringosterol and phytosterols. Various sterols have been linked to diverse health benefits, including antiinflammatory and anti-cancer properties. Phytosterols from H. pluvialis have been shown to have cytotoxic effects on human IMR-32 neuroblastoma cells, with a dose of 100 or 200 lM inhibiting neuronal activity by about 60% (Bilbao et al., 2016). Sanjeewa et al. (2016) have also reported anti-inflammatory effect of the hexanoic fraction of extracts from Nannochloropsis oculata, suppressing nitric oxide production in LPS activated macrophages when applied at doses of 6.25, 12.5 or 25 lg ml À1 . This fraction also showed anti-proliferative and pro-apoptotic effects, when applied at doses of 25 lg ml À1 , in several human cancer cell lines.
Sterols are mostly employed in cardiovascular health, because of their ability to hinder cholesterol adsorption in the intestine (Ostlund et al., 2003). The saringosterol from the kelp species Lessonia nigrescens has been shown to have antimicrobial activity on Mycobacterium tuberculosis with MIC values equivalent to rifampin at 0.25 lg ml À1 (W€ achter et al., 2001). Interestingly, it has been estimated that the current source of phytosterols will be unable to meet demands by 2030 (Randhir et al., 2020). The presence of a high variety of sterols is well established in eukaryotic algae and, more controversially, also in cyanobacteria (Volkman, 2003), although the subject has received very little considerations and much remains to be investigated in this field (Randhir et al., 2020).

Immune promoters and immunomodulatory activity
A large number of plants and microorganisms are known to possess an immunostimulatory activity (Riccio and Lauritano, 2020), although the mechanisms through which microalgae cultures exert this immunostimulatory effect, often remain to be clarified. However, microalgal products have been known to induce the expression of various immune genes. Moreover, Xu et al. (2014) have reported an increase in digestive enzyme and an improvement in the growth performance of gibel carp (Carassius auratus gibelio) fed dried powder of Chlorella sp. (incorporated at doses as low as 4 g kg À1 of food). Similarly, Adel et al. (2016) reported a significant increase in protease activity as well as the population of lactic acid bacteria in the intestine of sturgeons (Huso huso) fed A. platensis at dose of 50 or 100 g kg À1 of food. These results suggested that an improvement in the digestive health of the fish, possibly linked to a prebiotic effect of the algae, may have contributed to the improved immune parameters reported in the studies. For example, polysaccharides from Chlorella vulgaris have been shown to promote the transcription of nitric oxide, prostaglandin E2, TNF-a, IL-6 and IL-10, as well as promote cell proliferation in the murine macrophage cell line RAW264.7 (Tabarsa et al., 2015). Feeding of Nile tilapia (Oreochromis niloticus) with feed supplemented with 50 mg kg À1 of either b-Carotene or phycocyanin-supplemented feed resulted in a significant elevation of the activity of multiple blood immune parameters (phagocytic and lysozyme activity, immunoglobulin M levels), while expression of the genes coding for the interferon gamma and interleukin 1b was upregulated (Hassaan et al., 2021). Addition of dry powder from C. vulgaris to the diet of the Koi carp Cyprinus carpio at doses ranging from 50 to 100 g kg À1 of feed resulted in the proliferation of red and white blood cells, while inclusion of the algae at doses ranging from 20 to 100 g kg À1 of feed increased lysozyme activity (Khani et al., 2017).
Reports on supplementing feed for rainbow trout (Oncorhynchus mykiss) with b-carotene rich extracts from the marine phytoplankton Dunaliella salina at doses of 100 to 200 mg kg À1 resulted in an increase in the phagocytic rate and the serum complement and lysozyme activity in the fish (Amar et al., 2004). In shrimp, it has been reported that feed supplementation with 3 g kg À1 of Arthrospira platensis resulted in an improvement of the phagocytic activity of haemocytes from the banana shrimp Penaeus merguiensis, as well as resistance to infection by Vibrio harveyi (Lee et al., 2003). In mammals, diet supplementation with extracts from A. platensis was found to increase the levels of IgG1 in the serum and IgA in the intestine, alongside the antibody produced in the supernatants of lymphoid cell cultures from the spleens and mesenteric lymph nodes of A. platensis-fed mice (Hayashi et al., 1998). Interestingly, the effect was class-specific as IgE levels were unaffected by this feed supplement. Comparable results were obtained by dogs where supplementation of the diet with 0.2% of spray-dried A. platensis resulted in an increase in the levels of serum antibodies and faecal IgA with the following vaccination with a commercial anti-rabies vaccines (Satyaraj et al., 2021). Investigations using Dunaliella tertiolecta found that extracts and purified sterols (at concentrations of 0.4 mg ml À1 and 0.8 mg ml À1 diluted 1 in 3) from this microalgae had anti-inflammatory effects in sheep, reducing proliferation of peripheral blood mononuclear cells as well as the production of interleukin-6, which was the opposite to what Tabarsa et al. (2015) reported in C. vulgaris, while promoting secretion of IL-10 (Caroprese et al., 2012). Administration of 50 mL a day of warm-water extracts from A. platensis to sheep was reported to induce secretion interleukin 12 subunit beta (IL12 p40) by peripheral blood mononuclear cells as well as the secretion of interferon-gamma and the cytotoxic activity of activated NK cells (Hirahashi et al., 2002). However, diet supplemented with increasing doses of A. platensis (ranging from 5 to 20 g kg À1 ) resulted a dose-dependent increase in harvested macrophages, with a higher percentage of macrophages phagocytosing sheep red blood cells (SRBC) and a higher average number of SRBC in each macrophage (Al-Batshan et al., 2001). The authors of this study also reported a significant increase in nitricoxide production in macrophage stimulated with bacterial LPS.

Prebiotic activity
The indigenous microbiota of microalgae represent an early and important barrier to infection. Local bacteria can inhibit bacterial infections either directly through the secretion of antimicrobial or antiviral compounds, or by competing with them for nutrient and attachment sites, a phenomenon known as competitive exclusion (Irianto and Austin, 2002;Ghanei-Motlagh et al., 2021a, 2021b. Consequently, research has been performed on the possibility to defend against infection either through the direct ingestion of beneficial microorganisms (probiotic treatments) or the ingestion of substances that promote the growth of beneficial bacteria (prebiotic treatments). Several microalgae have been shown to have prebiotic activity, for example, C. vulgaris and A. platensis are known to increase the viability and survival of multiple beneficial bacteria such as lactobacilli and bifidobacteria when incorporated at doses ranging from 0.25 to 1.00% (Beheshtipour et al., 2012). Moreover, co-culture with C. vulgaris or Nannochloropsis oculata has been shown to improve the antimicrobial activity of Sulfitobacter spp. or Roseobacter sp., respectively, against Vibrio anguillarum Eguchi, 2011, 2012). More recently, it has been reported that supplementation of dogs' diets with 2 g kg À1 spray-dried A. platensis improved the stability of the gut microbiota in dogs during periods of physical exercises (Satyaraj et al., 2021). It has been suggested that some algae have the opposite effect, such as sequestering valuable nutrients and reducing their availability to bacteria.

Discussion and limitations
Compound production and toxic compounds The production of compounds from microalgae microbiomes is complicated by the fact that their production is often strongly influenced by the culture conditions of the algae and that these conditions are not always known for all algal strains (Abu-Ghannam and Rajauria, 2013;Fatma, 2009). This is further complicated by different culture conditions that may affect various beneficial factors in different ways. For example, cultivation in Zarrouk medium improved the production of b-carotene and the antioxidant properties of several strains of Arthrospira spp. (several dozen times for some strains) while a medium deprived of some mineral ingredients, RM6, allowed for an improved production of phycobiliproteins (Tarko et al., 2012). A strong seasonal effect has also been reported, although this may simply be a side-effect of changes in light and temperature conditions (Abu-Ghannam and Rajauria, 2013). Changes in light intensity to intensities inducing light stress have been shown to increase production of triacylglycerol by 250% and sterols by 1200% in H. pluvialis (Bilbao et al., 2016).
Optimal culture conditions for the algae will often be different from the conditions for the optimal production of the compounds of interest, as is the case for the production of carotene or antimicrobial fatty acids (Ruffell et al., 2016;Molino et al., 2018;Kaha et al., 2021). As one could expect, protective secondary metabolites are often produced in response to stressors which will impair algal growth (Little et al., 2021). However, there is no universal rule correlating harsher culture conditions with the accumulation of beneficial compounds. For example, Ru et al. (2020) have reported that poor growth conditions lead to an increase in the starch content of C. vulgaris (Ru et al., 2020). Even within the same species, there can be considerable differences between strains, and it will be necessary to confirm that the strain does produce the compound of interest in high quantities under the expected culture conditions (Tarko et al., 2012).
Several microalgae are known to secrete phycotoxins. In particular dinoflagellates are known as a major source of toxins in the marine environment (Wang, 2008). These toxins, including the alkaloid saxitoxin that is considered the most toxic among them, have been associated with neurotoxicity. Dinoflagellates toxins are normally present at relatively low levels in the environment, although this level increases during algal blooms involving these species and have been correlated with mortality events in aquatic life, for example, Alexandrium tamarense has been associated with mass mortalities in fish, birds and aquatic mammals in the Saint Lawrence Estuary in Canada (Starr et al., 2017). These toxins are known to bioaccumulate along the food chain with predatory carnivorous fish harbouring higher levels of the toxins, and diseases in humans are often associated with the consumption of contaminated seafood. In the case of ciguatera, caused by ciguatoxin and maitotoxin produced by microalgae of the genus Gambierdiscus, cases are more generally associated with the consumption of contaminated reef-dwelling fish. The presence of these toxins is likely a major reason why dinoflagellates have received less attention as a source of health products (Friedman et al., 2017).
Cyanobacteria are known to secrete a large number of various toxins with hepatotoxic effects as well as known neurotoxins such as kalkitoxin and saxitoxin, and more than 100 species of cyanobacteria have been shown to secrete toxins (Singh et al., 2005;Sacilotto Detoni et al., 2016;Zerrifi et al., 2021). Extracts from the marine cyanobacterium Trichodesmium erythraeum from microalgal blooms on the Brazilian coastline were found to contain microcystins, cylindrospermopsins and saxitoxins, and showed toxic antimitotic activity against larvae of the green sea urchin (Lytechinus variegatus) but not against mice (Proenc ßa et al., 2009). Saxitoxins from T. erythraeum were also connected to mortality events in farmed pearl oysters (Negri et al., 2004). Some of these cytotoxic activities may prove beneficial, for example, in the development and antitumor therapeutants; however, they will make commercial adoption of microalgae more complex (Parra-Riofr ıo et al., 2020).

Sustainabilityeffect of environmental conditions and costs
From a sustainability perspective, microalgae production allows to capture CO 2 with higher average bioenergetic yield on sunlight than higher plants: 10% vs. 5% respectively (Williams and Laurens, 2010). Such higher yield allows to reduce the use of land for cultivation. While closed photobioreactors enable higher productivity and finer control of growth conditions than open pond systems, they also display higher energy consumptions (Brentner et al., 2011;Valdovinos-Garc ıa et al., 2021). Recent Life Cycle Assessments (LCA) studies of microalgae cultivation in closed reactors (P erez-l opez et al., 2014a;2014b;Porcelli et al., 2020) showed that in pilot scale astaxanthin production from Haematococcus pluvialis and in the production of Tetraselmis suecica and Phaeodactylum tricornutum, the main contributor to most environmental impact categories were by far the electricity consumption during the cultivation stage. In these cultivation systems, electricity is mainly consumed for mixing and pumping large water volumes and for lighting the reactor when necessary. Regarding outdoor cultivation, the need to thermoregulate the system is one of the main drivers for electricity consumption (P erez-L opez et al., 2017;Smetana et al., 2017;Schade and Meier, 2019;Duran Quintero et al., 2021). A common insight from these studies is that the environmental performance of cultivating a specific strain of microalgae is highly dependent on the location, cultivation period and suitable thermal range of the strain (Duran Quintero et al., 2021).
Microalgae are becoming increasingly interesting for the extraction of high value compounds, rather than as feedstock for the refining of low value products such as biofuels. Concerning this extraction, P erez-l opez et al. (2014a, 2014b) showed that the extraction stage (methanol and KOH solutions) for the production of PUFAs, a-tocopherol, chlorophyll, b-carotenoid and polyphenols by Tetraselmis suecica was the second most important contributor to most environmental impact categories, but remained far behind the cultivation stage. Supercritical CO 2 fluid extraction was used for astaxanthin extraction P erez-l opez et al. (2014a, 2014b) and accounted for less than 10% of the considered impact categories. Overall, even if the extraction method depends on the targeted bioactive compound, cultivation will remain the main environmental hotspot for new microalgal strains. Crucial to limit the environmental impacts of a microalgal production is the valorization of coproducts within an integrated biorefinery approach (Da Silva et al., 2014;'t Lam et al., 2018). Microalgae residual biomass can serve as substrate for biogas production via anaerobic digestion and the residual digestate can substitute the production of fertilizers (Collet et al., 2011;P erez-l opez et al., 2014a, 2014b. Depending on the nutrient profile of the biomass, it could also be used to substitute animal feed (Draganovic, 2013). Due to the high diversity of the assumptions, parameters and production technologies, for strains that have not been cultivated yet, it is worth noting that their behaviour, optimal growth conditions and productivity in given reactors and locations are currently difficult to anticipate and therefore highly uncertain (Mata et al., 2010;Barra et al., 2014).
The potential need to induce bioactive molecule production by specific cultivation conditions, such as high-intensity lighting for astaxanthin production from Haematococcus pluvialis, could greatly affect the final environmental impacts (P erez-l opez et al., 2014a;2014b;Onorato and R€ osch, 2020). Ultimately, the overall sustainability of producing microalgae-based bioactive molecules depends on the final efficiency and quality of the produced compounds. Finding synergistic products would therefore be of great interest. Indeed, the compound's capacity to substitute alternatives, tackle key issues such as fish farming health management and the needed doses will highly affect its environmental performance (Liu et al., 2016;Lieke et al., 2020).

Opportunities for future research
Currently, the main knowledge gap is linked to the correspondingly low numbers of algal species studied, as only a small percentage of species have been investigated compared to the large number of microalgal species that exist (Connelly, 2014), as illustrated by the number of times the same genus names are repeated in the present review. This is especially true when taking into account the strain differences previously mentioned. Therefore, one of the most urgent tasks is to increase our knowledge by investigating new microalgal species, and focusing on other than well-known genera (Yarnold et al., 2019).
Optimal culture conditions are not always known for all algal strains, and neither are the optimal conditions for the production of the compounds of interest. This means that additional research is needed for each individual strain to optimize the farming protocol for both biomass and the production of the compounds of interest (Ruffell et al., 2016).
Another approach is to identify or create mutants that overexpress the compounds of interest. Genetic engineering of microalgae is shown to be possible, although it is more technically difficult than for other organisms (Qin et al., 2012;Spicer and Purton, 2017;Charoonnart et al., 2018).
Consequently, alternative and potentially more promising pathways could be integrated in multi-omics approaches to stimulate production within microalgae and their microbiota, and transfer and expression of biosynthetic pathways in suitable heterologous host species and strains. (Meta)genomics, (meta)transcriptomics, (meta)proteomics and MS-based metabolomics approaches can thereby help unravelling biosynthetic pathway activity constraints in microalgae and their microbiomes and identifying beneficial chemical compounds, that is, elicitors, that can be used to stimulate the production of compounds of interest (Maghembe et al., 2020). Such approaches in combination with advanced bioinformatics assessment of (meta)genomic contents, for example, using tools like antiSMASH and MiBIG (Kautsar et al., 2020;Blin et al., 2021) can also help identifying the boundaries if biosynthetic gene clusters (BGCs) that encode the biosynthetic machinery for such compounds (Table 1). Combining this knowledge with advanced long-insert cloning technologies and transfer to a panel of heterologous expression hosts can yield production of the compound of interest in a system that is better accessible to genetic modification for the purpose of optimizing production and further compound engineering (Nah et al., 2017;Ke and Yoshikuni, 2020). Such techniques may also be helpful in reducing the presence of detrimental compounds, for example, toxic compounds, produced by these algae.

Conclusions
Microalgae in combination with their associated microbiota are very promising as health management tools: not only have several species shown potential, harbouring antimicrobial, immune-stimulating and antioxidant substances but also because of their variety and the relatively small numbers that have been investigated, it is likely that many useful compounds remain to be discovered. In this context, much research remains to be performed to identify new compounds. In addition, we have to clarify their mechanisms of action and make their application practical, by optimizing production methods and reducing their costs of production as well as multi-omics approaches.