Microalgal Cell Biofactory—Therapeutic, Nutraceutical and Functional Food Applications

Microalgae are multifaceted photosynthetic microorganisms with emerging business potential. They are present ubiquitously in terrestrial and aquatic environments with rich species diversity and are capable of producing significant biomass. Traditionally, microalgal biomass is being used as food and feed in many countries around the globe. The production of microalgal-based bioactive compounds at an industrial scale through biotechnological interventions is gaining interest more recently. The present review provides a detailed overview of the key algal metabolites, which plays a crucial role in nutraceutical, functional foods, and animal/aquaculture feed industries. Bioactive compounds of microalgae known to exhibit antioxidant, antimicrobial, antitumor, and immunomodulatory effects were comprehensively reviewed. The potential microalgal species and biological extracts against human pathogens were also discussed. Further, current technologies involved in upstream and downstream bioprocessing including cultivation, harvesting, and cell disruption were documented. Establishing microalgae as an alternative supplement would complement the sustainable and environmental requirements in the framework of human health and well-being.


Introduction
Prognoses speculate the world population to reach almost 9.5 billion by 2050 and the food produced must be increased two-fold to meet the global necessities [1]. Escalating agricultural area, crop rotation, and yield increase could meet the rising demands for food; nevertheless, these practices deepen the existing environmental problems i.e., deforestation, soil degradation, and loss of biodiversity [2][3][4]. In this perspective, microalgae are emerging as a sustainable feedstock and alternative food source [5]. The primordial theory suggests that life began in a water body (ocean/ponds) and within the earth's biome, algal strains are characterized by high biodiversity [6]. Microalgae present in freshwater and marine environments are unicellular, microscopic, and photosynthetic microorganisms. Rapid proliferation rate, high photosynthetic activity, CO 2 sequestration, biomass production, and ability to grow on wastewater makes algae an amenable microbe for the production of value-added biochemicals [7][8][9]. Globally, 7000 tons of dry algal biomass is produced with a market value of 3800 to 5500 million USD per annum [10]. Utilizing microalgal biomass for developing pharmaceuticals, nutraceuticals, and functional foods for health benefits is rapidly soaring. For example, Chlorella and Arthospira species (2000 and 5000 tons/year) are dominant dietary supplements with a global production of USD 40 million/year [11]. The worldwide Spirulina market is valued at USD 629.6 million, and it is anticipated to reach a CAGR of 9.4% by 2025 [12]. Bioactive compounds synthesis has spread all over the world including countries with higher temperatures as well as longer-sunlightduration countries (Southern Asia, Middle East, Central, and South America), which allows for more cost-effective cultivation of microalgae [13]. This communication will to be USD 898.7 million by 2025 [22]. Humans are unable to synthesize lipids themselves endogenously, and external administration in diet is crucial for maintaining hemostasis [40]. PUFA reduces the prevalence of several chronic diseases and has proven to have several health benefits, i.e., the central nervous system and human vision reinforced by FDA (Food and Drug Administration) and FAO (Food and Agricultural Organization) [11]. The balanced diet ratio of polyunsaturated fatty acids to saturated fatty acids recommended by the World Health Organization (WHO) and the FDA should be above 0.4 and prescribed intake should be 0.2-0.3 g DHA and EPA per day for a healthy individual [41]. Dihomo-γlinolenic acid (DGLA), a trace amount long chain PUFA is important in developing infant formulations [36]. Chaetoceros calcitrans, Crythecodinium cohnii, Isochrysis galbana, Monodus subterraneus, Nannochloropsis sp., Pavlova salina, Phaeodactylum tricornutum, and Porphyridium cruentum are some of the PUFAs producing microalgal species [42,43].

Carbohydrates
Carbohydrates are major intracellular components acquired from photosynthesis and can reach up to 50% of microalgae dry weight. Carbohydrates are synthesized in the form of reducing sugars like glucose, lactose, sucrose, fructose, and polysaccharides [44]. Polysaccharides have attracted widespread attention from bioengineering and pharmaceutical companies as they are natural, biodegradable, non-toxic, and biocompatible. These provide health benefits by boosting the immune system, blocking tumorigenesis, and were first exploited for their rheological property as thickening or gelling agents [11]. β-1,3 glucan, an agile immunostimulator from Chlorella, alleviates hydroxyl radicals, atherosclerosis, gastric ulcers, cholesterol, constipation, and hypercholesterolemia [45]. Polysaccharides, namely immulina and immurella isolated from Spirulina platensis and Chlorella pyrenoidosa, exhibited high anticancer activity when compared to fungal polysaccharides such as schizoplyllan, lentinan, and krestin [46]. Animals fed with polysaccharide-rich algae exhibited lower cholesterol levels in serum with the subsequent reduction in insulin and glucose [47]. Sulfate ester-containing algal polysaccharides are classified as sulfated polysaccharides and are mainly produced by Chlorella vulgaris and Scenedesmus quadricauda. These polysaccharides consist of 3-linked β-D-galactose and 4-linkedά-D-galactose as alternate remnants in their backbone [48]. Sulfated polysaccharides produced from Porphyridium aerugineum act as a coating material on the surface of sanitary items to prevent infections from epidemic diseases like COVID-19 [49].

Proteins
Proteins are building blocks and essential macro-nutrients responsible for the overall growth of human body. Compared to animal-based proteins (42-52 m 2 for chicken, 47-64 m 2 for pork, and 144-258 m 2 for beef production), microalgae proteins (<2.5 m 2 per kg of protein) require minimal land for cultivation [50]. Algal proteins are used as a dietary supplement (tablets, powder, and paste), and the amino acid composition is equivalent to high-quality protein foods such as β-lactoglobulin, tofu, mushrooms, soybean, and egg white [51]. Chlorella sp., Dunaliella sp., and Arthrospira sp. produce top-notch proteins and amino acid profiles according to FAO/WHO guidelines and are legally marketed as nutraceuticals or fortified foods to prevent health disorders and abnormalities in cells. Spirulina contains 50-70% protein (dry weight) and essential amino acids, such as isoleucine, leucine, and valine, which exhibit high digestibility (83-90%). A spoonful of Spirulina (7 g), of which dried biomass contains almost 4 g of protein, is also known as a "superfood" by the World Health Organization [17]. Hainan Simai Pharmaceutical Co. Ltd., Haikou in China annually produces around 3 × 10 3 tons of Spirulina biomass. Cyanotech (Hawaii, USA) under the name Spirulina Pacifica produces and markets products (http://www.cyanotech.com, accessed on 2 September 2009). Microalgas Macronutricao factory in Brazil produces Spirulina lozenges for dietary nourishments (http://www.olson.com.br, accessed on 14 July 2001). Phycobiliprotein are hydrophilic protein-pigment complexes present only in cyanobacteria. These are used as fluorescent labels in antibodies and receptors, flow-cytometry, immunohistochemistry, immunoblotting experiments, and microscopy or fluorescence diagnosis [52]. Triton Health and Nutrition (USA) and Algenics (France) are the first manufacturers to use microalgae in high-throughput recombinant protein expressions. Chlamydomonas reinhardtii and Chlamydomonas elipsoidea contain recombinant proteins viz. vascular endothelial growth factor (VEGF), erythropoietin, Human IgG αPA83, hGAD65, immunotoxin αCD22-PE40, Plasmodium surface proteins (AMS1, MSP1, VP28, Pfs25, Pfs28, CtxB-Pfs25 and Pfs 48/45), Anti-CD-22-gelonin, lsc αHSV glycoprotein D, CSFV-E2, hTRAIL, and Anti-PA 83 anthrax IgG1, which possess therapeutical applications in anemia treatment, wound healing, anti-malarial vaccines, and as an antibody against anthrax, Herpes simplex virus, human papillomavirus, and foot-and-mouth disease [10,53,54]. Besides these, it functions as an immunotoxin against B-cell lymphoma and has increased resistance to UV-induced stress [55].

Vitamins
Microalgae are the cradle homes of vitamins that can be used as ingredients or food supplements to nourish the human body, revitalize cells, detoxify, as well as activate the immune system [56]. Vitamin A (β-carotene), vitamin C, E, and B i.e., thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), folic acid (B9), and cyanocobalamin (B12) are commonly produced vitamins. Vitamin B12 and provitamin A (β-carotene) are rich in Spirulina, which upon consuming, improves Lactobacillus in the intestine and makes it possible to digest vitamin B1 and other vitamins from food more efficiently [57]. Lutein and Zeaxanthin synthesized from Spirulina maximaare rich in vitamin A and act as protective factors from harmful light sources and age-related macular degeneration (AMD) [41]. Anabaena cylindrica, a cyanobacterium, produces a high concentration of vitamin K1 (200 µg g −1 ), which plays a significant role in the inhibition of chronic ailments [58]. Microalgae containing vitamin D 2 and D 3 together with provitamin D 3 acts as a chemopreventive agent and exerts antiproliferative and immunomodulatory activity on tumor-growing cells [59]. Vitamin E synthesized in microalgal species such as Chaetoceros calcitrans, Dunaliella tertiolecta, Nannochloropsis sp., Porphyridium sp., and Tetraselmis suecica improves endothelial function, vascular health, and inhibits prostate cancer cell growth [60].

Sterols
Phytosterols are structurally similar and functionally analogous to cholesterol with an additional alkyl group in the side chain of the sterol nucleus. They are used as additives in many food products and characterized by the presence of different phytosterols such as stigmasterol, sitosterol, and brassicasterol [20]. Sterols are essential constituents of cell membranes and play a key role in stabilizing phospholipid bilayers and in signal transduction as hormone precursors. These are crucial components of a healthy diet for reducing blood cholesterol levels in hyper and normocholesterolemic people and inhibiting colon cancer development [61]. Methanolic extracts of Chlorella vulgaris exhibited anticancer and anti-inflammation properties due to the presence of 2 delta (5, 7)-sterols (ergosterol and 7-dehydroporiferasterol peroxide), 7-oxo-delta(5)-sterol (7-oxocholesterol), and 5,8 alphaepidioxy-delta(6)-sterol [62]. Pavlova lutheria, Nanochloropsis sp. (BR2), and Tetraselmis sp. (M8) are good phytosterol producers, especially Pavlova lutheria, with 5186 mg/100 g. The minimal intake of 2 to 3 g day −1 promotes the smooth functioning of the heart, blood vessels, and minimizes lipoprotein [63].

Therapeutical Function of Microalgae
Microalgae and their extracts are an enormous and under-explored source of biologically active compound (Table 2).

Antimicrobial Activity
Microalgae are a valuable source of antibiotics and show potent antimicrobial activity because of their ability to produce carbohydrates, proteins, lipids, halogenated hydrocarbons, phytosterols, monocyclic aromatic compounds, phenols, and polyketide products [46]. Lipid structure and other compounds such asά-and β-ionone, β-cyclocitral, neophytadiene, and phytol are attributed to the antimicrobial action of microalgae extracts. Microalgal derived composites are used as adjuvants in fortified foods and fodder formulations to reinstate existing synthetic compounds of microbial origin.

Antibacterial Activity
The growing resistance of pathogenic bacteria for antibiotics with consequences on human health is of great concern. These bacteria contaminate foods and, in turn, infect humans causing several foodborne diseases like diarrhea [95]. Pratt was the pioneer who isolated chlorellin, a fatty acid mixture from Chlorella, which exhibited antibacterial activity against several Gram positive and Gram negative bacteria [96]. 'Parsiguine,' an antimicrobial compound isolated from Fischerella ambigua, showed a minimum inhibitory concentration (MIC) of 40 mg/mL and 20 mg/mL against S. epidermidis PTCC 1114 and C. krusei ATCC 44507 [97]. The extracts from algae paved a path to develop new drugs against bacterial infections, foodborne, and human pathogens. Microalgae antibacterial activity functions against many human pathogens such as Salmonella, Escherichia coli, Campylobacter, Pseudomonas, Shigella, and Staphylococcus, which can be attributed to the presence of monounsaturated and polyunsaturated fatty acids. These fatty acids possessing antibacterial activity can also impede bacterial fatty acid biosynthesis [98]. Algal fatty acids interfere with bacterial growth and cause harmful effects to the microorganisms, i.e., cell leakage, membrane damage, reduced nutrient uptake, and inhibit cellular respiration. Fatty acids with more than ten carbon atom induce bacterial protoplast lysis [99]. Gram (−) bacteria are more resistant than Gram (+) bacteria due to their complex membrane permeability and impenetrable cell wall. Methanol, benzene, petroleum ether, and hexane are the solvents used to extract antibacterial compounds from microalgae [100].

Antiviral Activity
Viruses are the smallest infectious agents for leading death rates globally. Preventing the invasion or synthesis of viral components is ambiguous as they have a deleterious impact on the host system. Although several antiviral drugs originated from plant secondary metabolites, mammals, and synthetic chemicals, drug-resistant mutations are continually occurring. Microalgae have gained renowned recognition as potential sources of antiviral agents in conjunction with the promotion of blue technology [108]. In antiviral compounds, microalgae predominate and cyanobacteria are a potential source of antiviral agents on an industrial scale [109]. Polysaccharides isolated from G. cartilagenium were the first antiviral compounds to safeguard egg embryos from mumps and flu viruses [110]. Herpes simplex virus (HSV1/HSV2) is a prevailing contagious disease, and around 30% of the global population is affected by genital and oral herpes. Several studies documented that marine polysaccharides present a wide antiviral spectrum against enveloped viruses. Spirulan, a sulfated polysaccharide isolated from Spirulina platensis, demonstrated good antiviral activity against human immunodeficiency virus (HIV1) and herpes simplex virus (HSV1) [111].
Acyclovir ® , an antiviral compound isolated from Dunaliella sp., also deactivates the viral infections of HSV and HIV-1. Higher sulfate content induces higher antiviral activity, and the inhibitory effect arises by blocking the receptor sites of viral glycoprotein [74]. Polysaccharides (PS) isolated from A. platensis, R. reticulate, Porphyridium sp., G. impudicum, and C. polykrikoides are the prominent microalga exhibiting antiviral properties against innumerable viruses like Human alphaherpesvirus 3 (HHV-3), Herpes simplex virus (HSV1/HSV2), Human beta herpesvirus, murine leukemia virus (MuLV), Flu-A viruses, Hepatitis B virus (HBV), Encephalomyocarditis virus, Flu-A and B, viral hemorrhagic septicemia virus (VHSV), measles, African swine fever virus (ASFV), vaccinia virus (VACV), mumps, vesicular stomatitis virus (VSV), and respiratory syncytial virus (RSV-A& B) [101,[112][113][114][115]. Cyanovirin, a potential protein moiety produced by Nostoc species, showed promising effects in curing human influenza A and HIV [82]. The Algevir technology expressed an antigenic protein called ZK from Schizochytrium sp. in the Zika virus (ZIKV) glycoprotein envelope and displayed substantial humoral responses to those caused by subcutaneous immunization at greater magnitudes upon oral administration [81]. Extracted from Gyrodinium impudicum, sulfated polysaccharide p-KG03 demonstrated strong efficacy against the encephalomyocarditis RNA virus (EMCV) relative to current medicines [80]. Polysaccharide (TK V3) and exopolysaccharide from A. platensis and P. purpureum were shown to be successful against Vaccinia and Ectromelia orthopoxvirus with IC 50 values (0.78 and 0.65 mg/mL) when examined with HEp-2 and Vero C1008 cells lines [116]. Naviculan isolated from diatom N. directa and A1/A2 from C. polykrikoides showed strong antiviral activity against contagious diseases like HIV1, HSV1, and type A flu viruses [117]. Carrageenan lozenges are used for treating patients with sore throat and carrageenan pills are particularly potent against HRV1a HRV8, influenzavirus A H1N1n, Coxsackievirus A10, and human coronavirus (hCoV) OC43 [72]. Similar studies were carried out by Graf et al. for treating rhinitis and sinusitis caused by human rhinovirus 1a and human coronavirus OC43 [118]. The viral hemorrhagic septicemia virus (VHSV) affects more than 50 species of commercially important freshwater and marine fish, including salmonid fish. Nevertheless, fewer studies were conducted on the antiviral properties of microalgae.

Antifungal Activity
Fungal contagions were mostly due to intensive chemotherapy regimens that depress the patient's immune system, organ transplantation, and spread of prevalent diseases [119]. Screening of microalgae for antifungal compounds began far beyond antibacterial activity. Aspergillosis induced by Aspergillus sp. and candidemia caused by Candida sp. has been increasing exuberantly since ancient periods [120,121]. The formulation of new drugs and antifungal activity varies widely depending on microalgal species. Marine microalgae (e.g., Chaetoceros sp.) have more capability in developing novel antifungal compounds compared to fresh habitats [102]. Several antifungal compounds such as polysaccharides, organic solvent extracts, polyethers, and lipid fractions isolated from green (H. pluvialis, Chlorella vulgaris and Scenedesmus sp.) and red microalgae (Porphyridium sp. and R. reticulata), Bacillariophytes, and dinoflagellates show activity against microorganisms such as A. fumigatus, A. niger, Penicillium sp., Saccharomycessp., Candida sp., Microsporum sp., and E. floccosum [122,123]. Gambieric acid, a potent antifungal agent with polycyclic ether isolated from Gambierdiscus toxicus, acts against molds and dermatophytes while the diatom Thalassiothrix frauenfeldii was involved against active yeasts [124]. Amphidinol, a potent antifungal agent isolated from Amphidinium klebsii, targets fungi membrane, mainly the ergosterol biosynthetic pathway [125]. Pigments produced by H. karadagensis showed antifungal activity against C. maritima, D. salina, and Lulworthia sp. responsible for biological fouling [126]. Organic extracts from C. lauderi, G. toxicus, and C. vulgaris inhibit Aspergillus fumigatus, a toxin-producing fungus that contaminates seafood of marine bivalves [127].

Anticancer Activity
Cancer is the single most serious disorder causing irregular cell growth that has the ability to infiltrate other body parts. A lump, irregular bleeding, excessive coughing, weight loss, and a decrease in bowel movements are potential symptoms. Human beings are affected by over 100 types of diseases, the bulk of which are due to hereditary abnormalities, i.e., environmental and lifestyle causes (90-95%), and the remaining 5-10% is due to inherited genetics [128]. Chemotherapy is the most promising anticancer approach aimed at reducing mortality by delaying the carcinogenesis process. Several enzymatic and non-enzymatic antioxidants from natural sources suppress tumorgenesis by inhibiting tumor cell protease activity, fostering cell division, triggering cell death, and immunomodulations [83]. Chinery et al. reported that the combined effect of antineoplastic agents and antioxidants helps in remission of colon cancer [129]. Cyanobacteria are an abundant source of natural foods, and their medicinal value was recognized at the beginning of the 15th century when Nostoc strains were used to treat malignant tumors, arthritis, and fistula. Anabaena and Nostoc sp. are important cyanobacteria that produce over 120 natural products with potent activity against several cancer-causing agents [130]. Chlorophyllin, a derivative of chlorophyll, acts against HCT1 16 human colon cancer cells because of its high effectiveness as a chemo-preventive agent [64]. Aqueous extract of Nostoc muscorum showed the highest anticancer activity in humans against Ehrlich cell (87.25%) and hepatocellular carcinoma (89.4%) [90]. This is attributed to the high content of phycobiliproteins, polysaccharides, and polyphenols, which induced programmed cell death of tumor cells. Bioactive compounds dragonamide A-E and dragomabin isolated from cyanobacterium Lyngbya sp. exhibited in vivo activity against leishmaniasis. Moore's group isolated twenty-six cryptophycin from Nostoc sp. GSV 224, of which cryptophycin 52 was reported to be successful in lab trials for treating ovarian carcinoma and alveoli cancers [131]. C-phycocyanin, an aqueous extract of red algae, exhibits antiproliferation inducing apoptosis [132]. β-(1, 3)-glucan, an active immunostimulator from C. vulgaris, is used as a potential antitumor agent [133]. Sulfated polysaccharides isolated from P. cruentum exhibited intense antitumor activity in rhodent peritoneum by hindering cancer cell progression and increasing the spleen and thymus indexes as well as boosting the immune system [134]. Exopolysaccharides of Porphyridium sp. retarded neoplasia, and its biomass in rats prevented the growth of colon cancer [135]. Fewer reports supported that gastric cancer can be treated using microalgae-derived peptides [136]. Vitamin B12, isolated from marine algae, is used as an encapsulating agent for anti-cancer drugs and plays a vital role in histone methylation, DNA synthesis, and the regeneration of blood cells. Cobalmin and folate extracted from diatomous microalgae with high proportions lower the risk of breast cancer [86].

Anti-Inflammatory
Inflammation is a cell or tissue's biological reaction to harmful factors, such as pathogenic agents, pollutants, weakened cell, and irritants, as well as a defensive response containing skin cells, blood cells and molecular mediators [137]. Inflammation normally occurs in heat, pain, swelling, and loss of control at the site of infection, but diabetes, obesity, renal, cardiovascular, and neurodegenerative disorders may be caused by chronic inflammation [138]. Oxidative stress is the predominant factor responsible for several inflammatory responses inducing mitochondrial dysfunction and beta-amyloid aggregation [139]. Besides, ROS generated in brain tissue also causes neurodegeneration, cellular death, and memory loss by modulating synaptic and non-synaptic communication between neurons [140]. Oral uptake/consumption of anti-inflammatory compounds revitalizes body immunity and aids in the healing process. Microalgae produce several antiinflammatory metabolites such as carotenoids, PUFAs, phycobiliproteins, carbohydrates, and sulfurized polysaccharides that suppress chronic inflammation and exert a protective function when consumed as food or used as pharmaceutical supplements [141,142]. Because of the metabolites mentioned above, microalgae are being considered in regenera-tive medicine for developing tissue scaffolds, especially in patients with skin burns [143]. The PUFAs (ω3 and ω6 fatty acids), especially arachidonic, docosahexaenoic, and eicosapentaenoic acids, are mainly involved in treating rheumatoid arthritis and dermatitis. Anti-inflammatory effects of DHA are prominent in developing breast milk, human fetus, and act against colon and breast cancer [144]. DHA and EPA produced from Schizochytrium species are widely used for nourishment in women in maternity and cardiac patients. DHA intake as a subsidiary food modulated fibrillar oligomers in transgenic APP/PS1 rat brains by blocking βeta-amyloid aggregation [77]. Docosapentaenoic acid (DPA) extracted from Schizochytrium sp. inhibits cytokines (IL-1 and TNF-α) in peripheral blood mononuclear cells (PMBC) of humans [145]. Astaxanthin produced from Haematococcus pluvialis had more remarkable inhibitory effects on lipopolysaccharide-induced inflammation and anti-inflammatory action than prednisolone [146]. ASX could prevent lipopolysaccharideinduced acute respiratory distress syndrome (ARDS) and sepsis by suppressing the signaling pathway of MPAK/NF-KB and decreasing pro-inflammatory cytokine level [65]. It also showed protective effects against lung injury, repressed alveolar wall swelling, myeloperoxidase activity, and attenuation of number of pulmonary alveoli in lung tissues [32]. Research conducted by Chang et al. reported that astaxanthin defends nerve cells against Aβ25-35-induced apoptosis by inhibiting radical oxidative destruction, Bax expression, nuclear transcription factor-kB (NFkB), and P-38 MAP kinase dephosphorylation (mitogen-activated protein) [66]. In a clinical trial performed on healthy young women, a daily dosage of two milligrams of astaxanthin for two months lowered inflammatory cytokines, C-reactive protein, and also decreased ROS production by the transcription factors regulating nuclear factor (NF-κB) and activator protein (AP-1) [67]. Algal metabolites produce various pro-inflammatory mediators, and its mechanism includes enzyme modulation (prostaglandin-endoperoxide synthase, phospholipase A2, and NO synthase), modulating cellular processes, and intervening with nuclear factor κB (NF-κB) and the mitogen-activated protein kinase pathways [147,148]. Inhibitory effects and reduced production of nitric oxide synthase and prostaglandins in phagocytic cells were shown by Fucoidan, a sulfated polysaccharide isolated from brown algae [149]. Among the pigments, fucoxanthin isolated from Bacillariophyceae is capable of eliciting metastasis in humans, and phycobiliproteins from cyanobacteria can inhibit histamine release [150]. Spirulan isolated from A. platensis showed similar properties to that of glycosaminoglycans (GAG), present in animals with bioactivities involved in human physiological and pathological responses [151]. Besides oxidative stress, exopolysaccharides from Porphyridiumcruentum enhanced immunostimulating activity in vitro and inhibited membrane peroxidation [134]. Sulfated polysaccharides possessing anti-inflammatory properties can be used in dermal treatments by blocking the polymorphonuclear leukocytes stimulations. Sulfolipids isolated from Scenedesmus rubescens, Scenedesmus acuminatus, and Phaeodactylum tricornutum are effective in inhibiting alpha-glucosidase, glutaminyl-peptide cyclotransferase, and telomerase activities [73]. In addition, natural ASX, with its proven anti-inflammatory and anti-oxidant activity backed by multiple preclinical and human trials and with its extraordinary safety profile, can be one of the most promising candidates to be tried against COVID-19 [32].

Other Activities
Hypertension, including diabetes, kidney failure, stroke, and coronary diseases, is one of the leading risk factors for many chronic illnesses. Blood pressure in the human body is regulated by the angiotensin I conversion enzyme (ACE) found in the renin-angiotensinaldosterone system [154]. Captopril, lisinopril, and enalapril are commercially available synthetic ACE inhibitors that are used for treating hypertension. This showed numerous side effects such as coughing, dizziness, fatigue, headaches, and angioedema [155]. Widely used microalgae to produce ACE-inhibitory peptides are Chlorella vulgaris, Nannochloropsis oculata, C. ellipsiodea, and Arthrospira platensis. Dermochlorella DG, a product derived from Chlorella vulgaris and Arthrospira proteins, activates collagen synthesis, reduces striae distensae, vascular lesions, and also acts as skin resurfacing agent [11]. A purified peptide sequence isolated from C. vulgaris (Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe) and C. ellipsoidea (Val-Glu-Gly-Tyr) showed IC 50 values of 29.6 µM and 128.4 µM when administered orally [156]. Similarly, the peptide sequence (Gly-Met-Asn-Asn-Leu-Thr-Pro and Leu-Glu-Gln) from N. oculata exhibited ACE-inhibitory activity of 123 µM and 173 µM, respectively [157]. These peptide sequences bind to C-and N-terminal active sites of ACE and block Angiotensin II production and prevent organ damage. Nanofibrils extracted from A. platensis could be used as an extracellular matrix for treating spinal cord injuries by culturing stem cells [158]. Spirulan isolated from A. platensis exhibits anti-thrombogenic properties by interfering with the blood coagulation-fibrinolytic system [159]. Aeruginosins and depsipeptides isolated from Microcystis aeruginosa and other microalgaeshow inhibitory effects against plasmin, thrombin, trypsin, and chymotrypsin [41]. Spriulina extract (250 mg) plus Zinc (2 mg) twice daily helps with the detoxification of heavy metals like Arsenic [160].

Functional Food
Microalgae are considered as "GRAS" (Generally Regarded as Safe) [165] and marketed as functional foods that benefit health beyond the basic role of nutrition. Nostoc sp. was used in China for food (over 2000 years ago), and later Chlorella sp. and Spirulina sp. were consumed as healthy foods in Japan, Taiwan, and Mexico (39). Microalgae are commercially produced in the Asia-Pacific region with capacities ranging from 3 to 500 tons/year, and the market value for human consumption is 100 €/kg [166]. Currently, microalgae are available in markets as health foods, and their products are mixed with pastes, snacks, candy, gums, noodles, wine, beverages, and breakfast cereals. Auxenochlorella protothecoides, Chlorella vulgaris, Chlorella pyrenoidesa, Dunaliella salina, Dunaliella tertiolecta, and Spirulina platensis are some of the microalgae species widely used in human foods because of their high protein and nutritive value [167]. Besides, Scenedesmus sp. is also used as a nutritional food source in desserts, fruit puddings, ravioli, noodles, and soups, but their commercial production is limited [168]. In fact, the omega-3 acids in fish come from the microalgae consumed at the bottom of the food pyramid and are gradually passed up to the fish at the top [39]. Schizochytrium sp is cultivated by OmegaTech (USA) as an alternative to fish oil to produce DHA-rich oils, namely 'DHA Gold' [75]. In infant-grade milk in Europe, purified PUFAs are added, and chickens are supplemented with microalgae to produce omega-eggs [75]. In USA, EPA + DHA-rich algal oil capsules (Pure One TM ) are marketed as food supplements [169].

Feed-Animal and Aquaculture
Microalgae are a treasure trove when it comes to nourishment, since they act efficiently as animal and aquaculture hatchery dietary supplements. The global microalgae production for aqua farming reached 1 kiloton (molluscs −62%; crustaceans −21%; fishes −16%) and 4.5 kilotons for ruminants and plants [170]. Moreover, microalgae is cost-effective and eco-friendly when compared to other feeds [171]. The protein content (510-710 g/kg) and essential amino acids in some microalgal species is greater than normal eggs (132 g/kg) and Glycine max (370 g/kg). Spirulina platensis exhibit a protein digestibility rate of 87-97% [172]. Microalgae-based eggs are marketed as vegan eggs by a UK company with similar nutrition to that of an egg (www.mygenefood.com, accessed on 15 March 2021). As microalgae feed is thick and recalcitrant owing to cell wall with cellulose and polysaccharides, CAZymes are used as a flavor enhancer, which helps in food assimilation [173]. Microalgae species such as Nannochloropsis, Chlorella, Dunaliella, Haematococcus, and Schizochytrium with sizes of 25 µM or less are used as feed for zooplankton [174,175]. Shrimps feed entirely on microalgae and zooplankton during their metamorphosis. An algal diet rich in DHA and arachidonic acid (ARA) improved hemocyte count, larvae growth, and antioxidant and bactericidal properties. It also protects against white spot syndrome virus [176]. Fish like sea bream, turbot, seabass, striped mullet, flounders, halibut, Atlantic cod, lobster, and Senegalese solely depend on microalgae as live prey/feed in hatcheries [177]. Haematococcus pluvialis, a rich source of astaxanthin, provides pinkening, a nutrient profile, as well as increasing the selling price of the fishes. Utilizing H. pluvialis as feed for farmed salmonid has been recently recommended by the Food and Drug Administration [1]. Carassius auratus (ornamental goldfish), when fed with microalgae, showed enhanced pigmentation. Nannochloropsis sp. is used in finfish hatcheries while Pavlova sp. is used for oyster, broodstock, clams, and mussels to enhance DPA/EPA levels [178]. Chlamydomonas reinhardtii, genetically engineered microalgae, produce bovine lactoferricin, increased PUFA levels, fatty acids, and anti-p57 antibodies [179]. Around 30% of total algal biomass is marketed as feed or utilized in food formulations [85].
Chlorella sp., Spirulina sp., Arthrospira sp., Porphyridium sp., and Isochrysis sp., are important microalgae species used in animal husbandry [173]. Canthaxanthin containing microalgae is associated with an improved content of vitamin E in the liver and is also used as a poultry dietary supplement with antioxidant, anti-inflammatory, and neuroprotective properties [41]. Holstein cows fed with microalgae produced enhanced milk yield (34 kg day −1 ), plasma metabolites, DAH, and linolenic acid [180]. Chlorella pyrenoidosa and Spirulina platensis contained essential nutrients like lactobiose, protein, and saturated fatty acids in the paunch of ruminants [181]. Sheep and Damascus goats, when fed with microalgae, showed high strength, increased protein, and lactobiose content with the same pH of the alimentary canal. Pregnant ewes supplemented with Spirulina gave birth to ewes 4 times larger than that of normal ones [182,183]. Poultry birds (chickens, ducks, turkeys, and quail) supplemented with microalgae reported being productive concerning body weight, yield, and reduced cholesterol [184]. Feeding chickens with H. pluvilais, N. gaditana, and Spirulina resulted in higher muscle pigmentation, antioxidant properties in the liver, and carotenoid pigmentation in egg yolk [185,186]. The average daily benefit and DHA levels in loin and subcutaneous meat are higher in pigs. Defatted algal biomass of Nannochloropsis sp., on feeding to anemic pigs, elevated blood hemoglobin, hematocrit, and growth [187]. Drosophila melanogaster, when fed with Chlorella vulgaris and Senedesmus obliqus, showed increased total weight, longer travel distance, and higher serotonin levels [188].

Microalgae Cultivation
Microalgae are versatile microorganisms that can grow under phototrophic, heterotrophic, and mixotrophic growth conditions and possess the ability to adapt to different environmental conditions by altering cellular mechanisms [189]. Microalgae are grown in a wide variety of indoor and outdoor cultivation systems, such as vertical column photobioreactors, raceway pools, tubular closed photobioreactors, lagoons, bubble columns, pilot tanks, earthen pots, basins, and concrete tanks ( Figure 1) [13,20,190]. The cultivation system should have a short path length, adequate microalgae culture for proper mixing, and full light dispersion [191]. Microalgae growth and product formation do not occur simultaneously. To overcome this challenge, microalgae are grown under a two-stage cultivation system, where microalgae are grown initially under nutrient-rich conditions to achieve maximum biomass productivity, followed by nutrient-deprived conditions for cellular morphogenesis and stimulation of value-added products [192,193]. Proteins, carbohydrates, and lipids are synthesized during the growth phase, while amelioration of these products occurs under nutrient-deprived and stress conditions [8]. Light [194], temperature [195], pH [196], salinity [197], trace metals [198], Ultraviolet Radiation [199], and dark-light photoperiods are the important key factors essential for enhanced biomass production and targeted biomolecules [52]. Chlorella vulgaris grown under nitrogen-rich conditions achieved a maximum lipid productivity of 71.1 mg L −1 d −1 [200]. Chlorococcum, a cosmopolitan microalga tolerant to extreme pH and temperature, produces astaxanthin, canthaxanthin, lutein, β-carotene, adonixanthin, and cis-isomers of ketocarotenoid [201]. H. Plulvalis accumulates 95% of total carotenoids and increased fatty acid biosynthesis due to improvements in environmental conditions (light strength, temperature, salinity, and nutrient limitation) [202]. Although reduced amounts of nutrient have shown propitious results, utilizing wastewater as a nutrient source has been considered promising and an alternative to economic cultivation [203,204]. Nitrates and phosphates can serve as essential nutrients while acetate can be utilized as organic carbon for algal cultivation [205,206]. Apart from external stress conditions, researchers have now targeted genetic engineering and manipulation of metabolic pathways that redirect cellular functions in the synthesis of compounds of interest, enlarging the competences of microalgae. Homologous recombination, RNA silencing, selection marker, biolistic transformation, artificial hybrids, somatic variants, and mutant's development are communal tools used in genetic engineering for pharmaceutical and nutraceutical products of monetary value [5,20]. To date, over 40 different microalgae species have been successful in genetic manipulation including the green algae Chlorellavulgaris, Haematococcus pluvialis, Dunaliellasalina, Chlamydomonas reinhardtii, and the Bacillariophyceae Phaeodactlyum tricornutum and Thalassiosira pseudonana [207]. Today, microalgae represent a viable source for recovery of high value-added products and have drawn attention for their use in health products in addition to biofuels.

Harvesting and Downstream Process of Microalgae
The downstream method entails lower-cost exploitation of high-value microalgae metabolites with high product recovery. The composition and productivity of microalgae mainly depends on cultivation mode, nutrient composition, and harvesting time [208]. The most commonly used harvesting techniques for performance, operating economy, and technological feasibility are membrane filtration, centrifugation, microfiltration, ultrafiltration, gravity sedimentation, electrolytic phase, electrophoresis, and magnetic separation [209]. Harvesting of microalgae is challenging as these are microscopic (3-30 µm), with low sedimentation rates and negative surface charges, which cause repulsion between the cells. Gravitational sedimentation and filtration are widely employed to harvest relatively large (>70 µm) microalgae Spirulina and Coelastrum [210]. However, microalgae with dimensions (<30 µm), such as Scenedesmus, Dunaliella, and Chlorella, often blind the filter, and frequent replacement results in high operational costs [211]. The preferable harvesting method is chosen according to the attributes of the microalgae (size, species, and cell density) and the final product. Conjoining chemical or biological coagulation/flocculation harvesting methods will improve process productivity and minimize operational costs [212]. The microalgae cell wall is a complex and tri-layered structure typically composed of polysaccharides (cellulose, pectin, and xylan), proteins (glycoproteins), and sugars, making them highly recalcitrant to degradation [213]. The cell wall has high mechanical strength and chemical resistance due to the presence of covalent, hydrogen, and van der Waals forces [214]. The exposure of microalgae cells to harsh shear levels, high temperatures, and pressure may change the integrity, loss of functionality, and subsequent release of the compound. For example, microalgal cell wall tensile strength is up to 9.5 MPa, which is three times that of Daucus carota [215]. It is important to use mild pre-treatment procedures to promote the recovery of the delicate intracellular compounds from the microscopic cell. Microalgae cells can be pre-treated/disrupted individually or in amalgamations, i.e., (1) mechanical/physical, (2) chemical, and (3) enzymatic/biological pre-treatment method (Figure 1).

Mechanical/Physical Pre-Treatment
Mechanical/physical cell disruption involves the breakdown of the cell wall to expedite the entry of solvents and solubilize intracellular complexes. Pre-treatment of microalgae cells can be carried out using bead milling, steam explosion, ultrasonication, and microwave-assisted extraction. Steam explosion is a cost-effective and efficient cell disruption method. Lorente et al., reported that N. gaditana, C. sorokiniana, and P. tricornutum achieved the highest lipid extraction yields using steam explosion [216]. Glass beads, zirconia-silica, zirconium oxide, or titanium carbide with 0.5 mm diameter is effective in microalgae disruption for the release of intracellular compounds. Bead milling of C. vulgaris releases water-soluble proteins with yields ranging between 32% to 42% and energy consumption below 2.5 kWhkg −1 dry weight [217]. Bead milling perturbation of H. pluvialis improves the recovery of astaxanthin by up to 98% during extraction at 400 rpm for 5 min [218]. Ultrasound-assisted extraction (UAE) is an efficient, economical, and rapid extraction method for the disruption of cell walls [219]. It works with low amounts of solvents and low temperatures to preserve thermolabile compounds and structural properties of the by-product. Polyphenols, antioxidants, and phycobiliproteins are the main compounds extracted from microalgae using UAE. In the destruction of cell membranes and the extraction of separate intracellular compounds, the energy strength of 0.4 kWh L −1 gained worldwide interest [220]. The UAE process obtained a yield of 15.6% phycocyanin in 20 min in Spirulina platensis, while the extraction of Soxhlet took 4 h to extract 11.1% [221]. Microwave-assisted extraction (MAE) yields high-value biological compounds by dissipating electromagnetic waves in the irradiated medium [222]. MAE has been used for the pretreatment of a variety of microalgae classes including Chlorophyceae, Bacillariophyceae, Eustigmatophyceae, and Phaeophyceae [223]. MAE is widely employed to extract bioactive compounds such as alkaline galactans, carrageenans, polysaccharide, and agar from Chlorella, Haematococcus, Bacillariophyceae, and seaweeds with high mechanical resistance and silica frustules [224,225].

Chemical Pre-Treatment
Chemical pre-treatments are easy with less energy inputs and high scalability but the major drawback is chemical toxicity and product recovery. Chemical solvents, salts, bases, ozone, and ionic liquids are widely used to extract bioactive compounds from algal biomass [226]. For their use in pharmaceuticals/nutraceuticals, the US Food and Drug Administration (FDA) categorized solvents into different groups and proposed allowed daily exposure (PDE) limits for each solvent. Chloroform PDE, for example, is equivalent to 0.6 mgday −1 , whereas hexane can be used up to 2.9 mgday −1 [227]. Chemical treatment of Thraustochytrids biomass for astaxanthin extraction showed higher yields with inorganic acids compared to organic ones [228]. Ionic liquids are liquid-state molten organic salts composed of an asymmetric cation and inorganic anion. Owing to their synthetic stability, solubility, conductivity, polarity, and hydrophobicity in the range of cationic or anionic constituents, these are known as designer solvents [22]. Ionic liquids break down complex networks of lignin, cellulose, and hemicellulose due to their high capacity to accept hydrogen bonds [229]. Imidazolium-and cholinium-based ILs are widely investigated for their potential to extract bioactive compounds from Synechocystis sp., Scenedesmus sp., C. vulgaris, and Spirulina [230].
Supercritical fluid extraction (SFE) is a promising technology for green extraction, focused on the use of solvents above their critical temperatures and pressures. SFE is popularly used for the recovery of lipids from microalgae [226]. Different solvents such as ethanol, methanol, hexane, butane, and sulfur hexafluoride are used for the abstraction of biological composites using CO 2 as a supercritical fluid. SCF-CO 2 easily infiltrates the microalgae cell wall due to its high permeability and diffusivity that solubilizes intracellular compounds [231]. Sanzo et al., stated that astaxanthin and lutein yield was enhanced to 98.6% and 52% by varying the pressure from 10 to 50 MPa at 50 • C and CO 2 flow rate of 14.48 gmin −1 in H. pluvialis [35]. Increased extraction time, temperature, and pressure increased the recovery of 79.6% DHA from Nannochloropsis sp. [232]. A similar phenomenon was observed by Molino et al., with the isolation of carotenoids from D. salina [233]. Pressurized Fluid Extraction (PFE) is a technique that uses high temperature (below critical point) and pressure during the extraction process to retain the solvent in a liquid state [234]. Water is used as extraction solvent and an alternative to organic solvents in Pressurized Liquid Extraction (PLE) because it has the same similar dielectric constant (ethanol or methanol) and solvent characteristics to that of organic solvents. Carotenoids are widely extracted compounds through PLE method. Ethanol/acetone/water has shown promising results as solvents in the PLE method for the extraction of different kinds of carotenoids from Phormidium sp., Phaeodactylum tricornutum, Chlorococcum sp., Neochloris oleoabundans, Scenedesmus sp., and C. vulgaris [235,236].

Enzymatic/Biological Pre-Treatment
Enzymatic disruption is a non-destructive pre-treatment method to facilitate the access of microalgae cellular components. It is a cost-intensive process, is highly effective, and operates under mild environmental conditions with less energy demand [237]. During lysis, enzymes act selectively, lyse, and degrade specific chemical linkage without destroying particle existence or any non-specific reaction in the solution. For cell lysis, lytic enzymes such as glycosidases, glucanases, cellulose, pectinase, and lipases have been used extensively [214]. Tang et al., reported that the utilization of carbohydrases disrupts the cell wall and enhances protein yield from rice bran [238]. Papain hydrolyzed from N. incerta demonstrated higher antioxidant activity relative to alcalase, neutrase, alpha-chymotrypsin, pepsin, pronase-E, and trypsin [239]. Recent studies demonstrated that the simultaneous use of enzymes facilitates the extraction of intracellular compounds. For example, cellulase hydrolyzes the cellulosic structure while lysozyme degrades resilient polymers present on the microalgae envelope [240]. The combination of chemical or mechanical treatment with enzymatic cell disruption may minimize the expense of the enzymatic procedure, the use of solvents, energy consumption, and improve the efficiency of intracellular material recovery. Emerging cell destruction developments that are still under development are explosive decompression, microfluidization, pulsed arc technology, and disruption using cationic polymer-coated membranes.

Conclusions and Future Perspective
Bioactive metabolites of microalgal origin have gained the biotechnological spotlight to decelerate the rate of malnourishment in developing nations. Even today, 1 out of 10 is undernourished due to malnutrition. Bioactive compounds obtained from microalgae, such as polyunsaturated fatty acids, carotenoids, phycobiliproteins, polysaccharides, antioxidants, sterols, and other secondary metabolites, have significant potential through a sustainable approach to meet increasing global demands. Microalgae metabolites act as the repository of natural antibiotics benefitting animal/aquaculture and human health by replacing antimicrobial compounds of synthetic origin. Microalgae are more nutritious, productive, and could be the next alternative for traditional sources of animal and aquatic feed with enriched eggs, meat, and milk. Microalgae-derived bioactive metabolites are renowned for antioxidant and broad-spectrum anti-inflammatory and antibacterial properties with impressive safety profiles, which help in the reduction and prevention of diseases associated with oxidative stress. The application of bioactive compounds proved to be beneficial and much more effective when compared to present traditional treatments and therapies. These metabolites act widely against emerging infectious diseases, antibiotic resistant bacteria, viral infections (epidemic and pandemic), and can prevent the delay or onset of chronic and carcinogenic diseases. Although numbers of products isolated from microalgae are commercially available for human health and nutrition, the social acceptance and awareness of health benefits of microalgae are still lacking. When eaten entirely, the chemical constituents of microalgae can exert synergistic results. In order to exploit the pharmacological and biological properties of algal-derived materials, numerous in vivo clinical studies are also required. Microalgae cultivation in different wastewater streams using genetically modified techniques explores their biotechnological potential to favor bio-based process. Sequential or co-extraction of multiple bioactive compounds with low energy consumption and high yields needs to be prioritized. Nonetheless, more studies are needed to turn the current technology into green technology for the exploitation of microalgae in bioengineering and fulfill the requirements compared to plants.