Antimicrobial Activity of Arthrospira (Former Spirulina) and Dunaliella Related to Recognized Antimicrobial Bioactive Compounds

With the increasing rate of the antimicrobial resistance phenomenon, natural products gain our attention as potential drug candidates. Apart from being used as nutraceuticals and for biotechnological purposes, microalgae and phytoplankton have well-recognized antimicrobial compounds and proved anti-infectious potential. In this review, we comprehensively outline the antimicrobial activity of one genus of cyanobacteria (Arthrospira, formerly Spirulina) and of eukaryotic microalgae (Dunaliella). Both, especially Arthrospira, are mostly used as nutraceuticals and as a source of antioxidants for health supplements, cancer therapy and cosmetics. Their diverse bioactive compounds provide other bioactivities and potential for various medical applications. Their antibacterial and antifungal activity vary in a broad range and are strain specific. There are strains of Arthrospira platensis with very potent activity and minimum inhibitory concentrations (MICs) as low as 2–15 µg/mL against bacterial fish pathogens including Bacillus and Vibrio spp. Arthrospira sp. has demonstrated an inhibition zone (IZ) of 50 mm against Staphylococcus aureus. Remarkable is the substantial amount of in vivo studies of Arthrospira showing it to be very promising for preventing vibriosis in shrimp and Helicobacter pylori infection and for wound healing. The innovative laser irradiation of the chlorophyll it releases can cause photodynamic destruction of bacteria. Dunaliella salina has exhibited MIC values lower than 300 µg/mL and an IZ value of 25.4 mm on different bacteria, while Dunaliella tertiolecta has demonstrated MIC values of 25 and 50 μg/mL against some Staphylococcus spp. These values fulfill the criteria for significant antimicrobial activity and sometimes are comparable or exceed the activity of the control antibiotics. The bioactive compounds which are responsible for that action are fatty acids including PUFAs, polysaccharides, glycosides, peptides, neophytadiene, etc. Cyanobacteria, such as Arthrospira, also particularly have antimicrobial flavonoids, terpenes, alkaloids, saponins, quinones and some unique-to-them compounds, such as phycobiliproteins, polyhydroxybutyrate, the peptide microcystin, etc. These metabolites can be optimized by using stress factors in a two-step process of fermentation in closed photobioreactors (PBRs).


Introduction
Almost every report on the antimicrobial effect of natural products notes that the dangerously rising antimicrobial resistance (AMR) of microbe pathogens necessitates and increases the search for novel sources of antibiotics and other antimicrobials, other than from traditional sources, such as Streptomyces.
It is interesting that microalgae and cyanobacteria, such as spirulina, that we associate with food emerge with high antimicrobial activity and contain important antimicrobial molecules [1].This fact may raise concerns, specifically how Generally Recognized as Safe (GRAS) nutraceuticals could hold anti-infectious therapeutic potential, especially given that the first microalgal antibacterial agent (chlorellin) was discovered in the 1940s [2,3].
Although it has been known for a long time, it has not yet been utilized clinically.However, we must not forget that it is very likely that the antibiotic boom that followed caused chlorellin to be ignored.Pure antimicrobial compounds with low concentrations in GRAS organisms may be isolated and concentrated.The identification of other compounds with direct antimicrobial activity from algae and cyanobacteria is still a relatively young and slow field of research, and new kinds of compounds have been reported in recent years [2,4,5] and research and innovations for commercial application is still in a very early phase [3].In addition, plants and algae extracts have their specific technological obstructing points for drug development, such as chemical complexity, rediscovery of known compounds, etc., as summarized by Quave (2016) [6].
For those reasons, the fact that terrestrial plant and algal products contribute to just 3% of the nearly 70% natural products and derivatives that are approved by the Food and Drug Administration (FDA) as antibacterial drugs does not accurately reflect their potential [6].
Bactericidal antibiotics and antimicrobials are especially prone to elicit resistance [7], and established antimicrobial agents may cause other serious medical problems, such as destroying normal gut and skin flora and producing gastrointestinal disorders and irritations, dermatitis or serious hypersensitivity problems.Thus, the test of new microbial infection-fighting natural compounds is especially urgent [8].
Natural extracts and compounds also have their well-described benefits for drug development including chirality, the chemical diversity-mentioned also as a drawback-as well as chemical synergy.For instance, complex extracts of Artemisia annua.were reported to have antiplasmodial activity that is 6 to 18-fold greater than what was expected based on artemisinin content alone [9][10][11].
In addition, we would like to highlight some other benefits here.Natural products often may not have strong direct bactericidal activity but favor homeostasis towards the host, by enhancing the immunity, etc., thus inhibiting the pathogen [6].Terrestrial plants and algae used in ethnobotany and traditional medicine have a proven effect and compatibility with the living organism [12][13][14].Spirulina (present scientific name of the species is Arthrospira spp.) has been used as an endurance-booster food from ancient times by Aztecs and natives of Chad, the cyanobacterium Nostoc was used by ancient Chinese and Chlorella is consumed in the present time [15].
Another benefit of extract synergy is overcoming resistance, and we would like to give the example of Quave (2016) [6] again with artemisinin.It was discovered and crystalized in the early 1970s in China from A. annua [16].Regretfully, it is becoming more and more problematic that resistance to artemisinin monotherapy appeared in a short period of time and is globally spreading [17].However, A. annua is a traditional Chinese medicinal plant named Qinghao, and it is a therapy known to have been in use for millennia and has not yielded resistance [16].Whole plant therapy was even effective at overcoming artemisinin resistance in an animal model [9][10][11].These studies supported the idea that the synergetic action of multiple compounds in this species can overcome resistance noted in monotherapy, apart from other factors, such as the widespread use of the drug due to global distribution.This idea might apply in the future to the creation of novel antimicrobial formulations from natural products intended to prevent the development of resistance [6].
Regarding the taxonomy of microalgae, there is not yet a full consensus among academia whether cyanobacteria such as Arthrospira and Nostoc are (micro)algae, as they would be nearly the only group of the prokaryotic Bacteria domain in this category.Many credible scientific sources still include them, as the tradition is, since (micro)algae is an informal polyphyletic term, meaning it does not form a natural group that has descended from a common ancestor.Its eukaryotic members are species from multiple distinct clades and both from the kingdom Plantae sensu lato and not [18][19][20][21].Cyanobacteria and eukaryotic microalgae share many traits in terms of physiology, ecology and, particularly, biotechnological applications [22].We will collectively refer to them as phytoplankton.
The medicinal potential of microalgae in brief includes their ability to synthesize anti-inflammatory antioxidants, such as carotenoids, vitamin E [23] and polyunsaturated fatty acids (PUFAs).PUFAs are vital for human metabolism and cardiovascular health, two of them with longer chains even being prescription drugs for lowering triglyceride levels [1,[24][25][26][27].In fact, marine microalgae are the main primary producers of those longer chain ω-3 PUFAs [28].As for the only approved drug derived from a unique to microalgae (cyanobacteria) compound, it is the anticancer drug dolastatin [29].
Microalgae also produce sterols with pharmaceutical application and cytostatic phycotoxins [23] and also have immunomodulatory, anti-obesity and other effects [1].Microalgae are reported to be one of the most important producers of antimicrobial molecules, including PUFAs and other fatty acids, proteins, vitamins, pigments, etc. [1].
Spirulina is the consumed biomass of filamentous nutraceutical cyanobacteria and may consist of Arthrospira platensis Gomont (synonym Arthrospira fusiformis or formerly Spirulina platensis) or Arthrospira maxima.It is approved by the FDA as GRAS and sold as a food supplement, largely because of the 65% protein in the dry mass.A. platensis inhabits tropical lakes with alkaline waters (pH 11) and a high concentration of salt and bicarbonates.While these circumstances hinder the development of other microorganisms, they permit the cultivation of spirulina in open reactors.A. platensis and A. maxima inhabit highly alkaline lakes in Africa and Mexico, while other Arthrospira spp., besides brackish water, can be practically found everywhere-seawater, freshwater, soil, marshes and thermal springs [30,31].The protein of spirulina contains all the essential amino acids, and A. platensis contains 10% w/w carbohydrates and 7% w/w lipids, of which 1.5-2% are PUFAs [32].Arthrospira is a substantial source for complementary and alternative medicine and has a significant potential in conventional medicine because of its proven in vitro and in vivo bioactivities [30,31,33].It has immunostimulant, antioxidant, anti-inflammatory, antimicrobial, antineoplastic and a broad spectrum of antiviral activity [30].Spirulina is effective against certain allergies and rhinitis and has antidiabetic, anti-obesity and metalloprotective hepatoprotective properties.It is also reported to influence neurodegenerative disorders, anemia, cardiovascular diseases, hyperglycemia and hyperlipidemia.These activities are attributed, individually or synergistically, to Arthrospira's bioactive compounds.In fact, cyanobacteria such as Arthrospira, Anabaena, Nostoc and Oscillatoria are claimed to synthesize a great variety of secondary metabolites comparable to the metabolite diversity of actinomycetes [34].Arthrospira is rich in fatty acids, including essential omega-3 or omega-6 PUFAs, β-carotene, α-tocopherol (vitamin E), phycocyanin, phenol compounds, caffeic and chlorogenic acid, dietary minerals and vitamins and a sulphated polysaccharide with antiviral property (calcium spirulan) [30,31,33].
Dunaliella salina (Dunal) Teodoresco is a halophile green microalga.It is especially found in hypersaline environments, such as salt lakes and salt evaporation ponds, where it contributes to their pink color.It has high concentrations of glycerol to counter the salinity by osmotic pressure and high concentrations of β-carotene.Dunaliella has two flagella and a single cup-shaped chloroplast containing the β-carotene, which makes it appear orange-red.It is nutrient rich, hence used for dietary supplements [35].Dunaliella spp.can also be found in marine and freshwater habitats [36].
Although products from the two genera have been so far developed only into food supplements but not into approved drugs, we must not underestimate the former.Dietary supplements can also be part of clinical therapeutic schemes for chronic diseases, e.g., kidney stones.
The topic of bioactive and nutraceutical metabolites is also relevant to the circumstances under which the organisms, in our case, microalgae or phytoplankton, of interest optimally synthesize secondary metabolites.Since uncontaminated microalgal biomass can be obtained from bioreactors and not from the field, authors have to highlight the main strategy of boosted production during controlled fermentation conditions.Theory in the field is well established, and the two-step process of maximization of secondary metabolites is well studied and proven.The first step consists of maximum growth rate by optimal conditions, and the second step applies stress factors, which induce and increase some secondary metabolites.For example, carotenoids, fatty acids and sulphated polysaccharides have been reported to increase after various chemical triggers, such as low oxygen and salt and nutrient starvation in A. platensis, D. salina, Chlorella zofingiensis, etc. [37].All this is related to the trend of cultivating Arthrospira in bioreactors instead of open ponds, which provides an increase in biomass due to the lack of water evaporation and a higher quality of the product (open ponds still have the benefit that control of temperature and light is not necessary) [30].Figure 1 presents a schematic diagram of production and isolation of antimicrobial metabolites from phytoplankton.More details in regard to the stress factors can be found in the corresponding section.
metabolites is well studied and proven.The first step consists of maximum grow by optimal conditions, and the second step applies stress factors, which induce a crease some secondary metabolites.For example, carotenoids, fatty acids and sulp polysaccharides have been reported to increase after various chemical triggers, s low oxygen and salt and nutrient starvation in A. platensis, D. salina, Chlorella zofin etc. [37].All this is related to the trend of cultivating Arthrospira in bioreactors ins open ponds, which provides an increase in biomass due to the lack of water evapo and a higher quality of the product (open ponds still have the benefit that control o perature and light is not necessary) [30].Figure 1 presents a schematic diagram of p tion and isolation of antimicrobial metabolites from phytoplankton.More details in to the stress factors can be found in the corresponding section.
Reviews covering some of the reports on the antimicrobial activity of Arthrosp Dunaliella, in addition to other phytoplankton species, are those of Falaise et al., 201 and Senhorinho et al., 2015 [39].This review aims to comprehensively describe an lyze the reported in vitro and in vivo antimicrobial activity (antibacterial and antif of the genera Arthrospira and Dunaliella as well as to provide some focus on their bio compounds and the strategies for their enhancement in bioreactors.Reviews covering some of the reports on the antimicrobial activity of Arthrospira and Dunaliella, in addition to other phytoplankton species, are those of Falaise et al., 2016 [38], and Senhorinho et al., 2015 [39].This review aims to comprehensively describe and analyze the reported in vitro and in vivo antimicrobial activity (antibacterial and antifungal) of the genera Arthrospira and Dunaliella as well as to provide some focus on their bioactive compounds and the strategies for their enhancement in bioreactors.

Arthrospira and Dunaliella Are among the Most Commercially and Industrially Used Cyanobacteria and Microalgae in Biotechnology
Arthrospira and Dunaliella are among the most exploited phytoplankton species in biotechnology.As the reviews of Mobin et al., 2017 [36], and Bhalamurugan et al., 2018 [40], outline, the reason is that they are rich sources of high-value compounds.The large amount of obtained biomass that is already used for biotechnology purposes would favor the production/extraction of antimicrobial high-value products.Overall, this strategy would result in diminishing the cost of the microalgal technology.
Over the last 20 years, four major phytoplankton species-Arthrospira, Chlorella vulgaris, D. salina and Haematococcus pluvialis-have been utilized in biotechnology [36,41].The most commonly exploited microalgae and cyanobacteria for the production of bioactive compounds with pharmaceutical purposes include Arthrospira, Chlorella, Dunaliella, Haematococcus and Nostoc [40].
To date, Arthrospira (together with Chlorella) has been the most commonly sold phytoplankton species for food because of its robust growth.In fact, only a few from the approximate 40,000 species of algae and cyanobacteria have been used by the food industry, and Dunaliella spp., as well as A. maxima and A. platensis (still frequently and popularly named spirulina), are among them [42].They are used as a supplement to enhance the nutritional and health benefits of breads, candies, ice cream and other common foods, pastry and sweets.They are directly manufactured into health products like tablets and capsules and also used as food coloring [1].Arthrospira spp.are very valuable, as they are rich in proteins, carbohydrates and vitamins.Their proteins and vitamins, especially B12, are used for antioxidants and immune enhancers.The genus is rich also in essential amino acids, minerals, essential PUFAs, etc. Due to this, it holds second place in (health) beverage production.Proteins of the nutrient-rich D. salina are also used in the baking industry [36,43].
However, Arthrospira and chlorophytes, such as Dunaliella and Chlorella, are not suitable as a single-species diet, as they are not rich sources of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [25,26,44].Microalgae may be the main source of the physiologically important ω-3 PUFAs EPA and DHA in fish oil; however, this role is attributed mainly to dinoflagellates [45].
According to a report by The Food and Agriculture Organization, the global production of Arthrospira sp. was 56,000 t in 2019 [41].The increase in global production of species, such as Arthrospira (3000 t dry weight/y), D. salina (1200 t dry weight/y) and, e.g., Chlorella (2000 t dry weight/y), indicates a positive trend of human consumption, which will likely rise even more in the years to come.Microalgae and cyanobacteria production has a compound annual growth rate of 5.32% [40].
Dunaliella and Arthrospira (together with Chlorella and Scenedesmus) are also among the commonly used species of microalgae for animal and aquaculture feed, especially because they can grow in highly saline and alkaline medium [43].
Phycobiliproteins are photosynthetic pigment-protein complexes found within cyanobacteria, in addition to chlorophyll.Their major sources are A. platensis, other Arthrospira spp.and Amphanizomenon floaaquae.Examples of these proteins include phycocyanin and phycoerythrin.In fact, the biomass from A. platensis and Arthrospira spp. is claimed to be used mainly for the extraction of phycocyanin.All phycobiliproteins are used in the pharmaceutical industry, as they are antioxidant, anti-inflammatory, neuroprotective and hepatoprotective agents and are utilized in photodynamic therapies of various solid cancers and leukemias.Next, they are used in clinical laboratories and immunology research due to their effective molecular absorption, high fluorescence ability for marker production and photostability.Last, but not least, they are used commercially as natural food dyes and in cosmetics in perfumes and eye makeup [36,[46][47][48].
Dunaliella salina is the best source of β-carotene (up to 14% of dry biomass), followed by Scenedesmus almeriensis, and the sole source of naturally derived β-carotene.This carotenoid is an antioxidant and a vitamin A precursor.Another commonly used microalgae for production of β-carotene is Dunaliella bardawil (also a GRAS algae).The amount of βcarotene it produces is 1.65 pg/cell with an approximate market value of 0.6 US$ per 1000 mg of β-carotene [49].D. salina is responsible for most of the primary production in hypersaline environments worldwide and also a source of glycerol [35].
The carotenoids lutein, zeaxanthin and canthaxanthin have pharmaceutical value mainly as antioxidants for health supplements and in the food industry as natural colorants (interestingly, for, e.g., chicken skin).Dunaliella salina is among the most utilized microalgal producers together with some Chlorella and other species.Arthrospira is also rich in zeaxanthin [50].
Other pharmaceutical benefits of the two genera include sulphated polysaccharides of Arthrospira, which are widely used as an antiviral agent and tablets, being marketed since 1975 in Japan [36].Humans have eaten Arthrospira to lower arterial pressure, induce the growth of intestinal Lactobacillus and reduce hyperlipidemia [48].
Dunaliella and Arthrospira, in addition to Haematococcus and Chlorella, are commonly found in a variety of cosmetics.Lotions and creams for the face and body are replenished with these microalgae extracts.They are also an ingredient in sun protection creams, hair masks and shampoos.They also help in the regeneration of fibers and stop wrinkles from developing on the surface of the skin.Arthrospira extract enhanced with proteins slows down the aging process of the skin [48,51,52].
A. platensis, as well as Chlorella and Scenedesmus spp., and are among the most commonly used phytoplankton species for bioethanol production [40].Other industrial uses of the two genera include wastewater treatment.Many microalgal species may find the produced water from the oil and gas industries toxic, but Dunaliella spp.(as well as Chlorella and Scenedesmus spp., again) may be able to survive and even flourish in the (pretreated) produced water [53].

Antimicrobial Metabolites from Microalgae and Cyanobacteria
It is well known that free fatty acids (FFAs) have a general antibacterial effect [54].There are reports that the maximal content of FFAs in algae culture medium is in the stationary phase and is directly correlated to the process of autolysis.That could be due to their release after cell lysis and could also happen in natural conditions in the aquatic environment.These compounds could secure the resistance of the natural biotope to settlement of allochtonic microbes and stability of the structure of the biocenose.Antibacterial compounds are also synthesized as a result of chlorophyll and FFA oxidation.A protein-chlorophyll complex of D. maritima inhibits the growth of the bacteria Pseudomonas saccharophila.This complex could also be released after cell lysis, and that could also happen in the water basin.The velocity of elimination of foreign microorganisms could rise at microalgal mass dying, and this is one of the mechanisms of self-purification of water bodies.The antibacterial effect of peloid (mud or clay often associated with water basins and used for therapeutic purposes) could be largely due to the intracellular compounds released during microalgal destruction [55,56].
Fatty acids, especially PUFAs, are one of the most abundant and highly antibacterial compounds in microalgae and have an underappreciated antimicrobial therapeutic potential [57].The double bonds of unsaturated fatty acids are claimed to contribute to higher antimicrobial activity in comparison with the saturated fatty acids [58].Their mechanism of action is mostly related to their surfactant nature, leading to cell membrane damage and cell and electron leakage.They also inhibit electron transport systems, ATP production and other bacterial enzymes and induce peroxidative reactions [59][60][61][62][63][64][65][66].EPA and DHA have infection-suppressing properties in vivo [67]; however, as noted, Arthrospira and Dunaliella are not rich sources of them.Nevertheless, A. platensis is rich in the antibacterial γ-linolenic acid (36% of the total PUFAs) [32].However, this acid has a high activity only against Gram-positive bacteria but not against Gram-negative ones [58].
Other lipids, as well as polysaccharides and other carbohydrates, can also have antimicrobial properties [3,60,68,69].Dunaliella spp., as well as other eukaryotic microalgae, such as Chlorella spp., produce and secrete relatively large amounts of antibacterial polysaccharides (PSs) [3,60].The pigments chlorophylls and carotenoids, especially β-carotene, are also effective microbial growth inhibitors [70,71].PSs, e.g., sulphated exoPSs, are also surfactants and have anti-adhesive properties against microbes.That may be due to their competing with carbohydrates (glycans) on host cell surfaces, which are the recognition sites for bacteria to attach to using their lectins on the bacterial surfaces [72,73].PSs binding with bacterial glycoprotein receptors leads to increased cell permeability and bacterial DNA binding [59,74].The biofilm-preventing effect is related to prevention of bacterial autoaggregation, which is also lectin-dependent [75,76].
Other major antimicrobial (antibacterial, antifungal and antiviral) compounds of Arthrospira are phycocyanin, phycocyanobilin and allophycocyanin, and they have certain anticancer effects as well [69,77].The taxon possesses also flavonoids and other phenolic compounds and terpenes, which are proven antimicrobials [69,78].Phenolic compounds at high concentrations can denature proteins in bacterial cell walls and membranes through hydrogen bond formation, thus damaging them and causing lysis due to leakage [60,79,80].At low concentrations, they likely influence enzymes, especially those involved in energy production [68].The antifungal effect may be due to binding and damaging lipids in cell membranes and organelles and the impact on spore germination [81,82].Terpenoids are capable of damaging the porins in the outer membrane of bacterial cell walls through the formation of a strong bond polymer, thus impeding the flux of nutrients [66]. A. plantentis also has alkaloids, saponins and quinones.Alkaloids can interfere with components of peptidoglycan in bacterial cells wall, thus damaging the wall and causing cell death.Saponins are detergents which can decrease the voltage between the bacterial cell wall and permeabilize the membrane.They reduce the surface tension of the cell wall, thus penetrating the cell, disrupting cell metabolism, and ultimately causing the bacteria to die.Quinone compounds can bind to cell proteins, rendering them dysfunctional, thus disrupting cell metabolism [66,79].
Phytoplankton is an emerging source of antibacterial peptides.For example, all cyanobacteria, including Arthrospira are reported to be able to synthesize the antimicrobial toxins microcystins, which are cyclic heptapeptides.They are produced in large quantities during algal blooms and are a major threat to drinking and irrigation in terms of toxicity.While this does raise safety issues associated with commercial spirulina products, the potential of microcystin as an antibacterial agent can be explored [79], similarly to how they are explored as cancer drugs [86].Antibacterial peptides are mainly cyanopeptides, meaning they are mostly in cyanobacteria, such as Oscillatoriales and Nostocales [69,87].Apart from microcystin, few antibacterial peptides have been isolated from A. platensis [88,89].The mechanism of action of cyanobacterial and microalgal peptides is not generally known [69].It is interesting that Arthrospira spp.are one of the marine microbial species that contain intracellular polyhydroxybutyrate (PHB) polymer.There are many reports demonstrating that the PHB particles are stored as carbon and energy reserves under severe stress conditions.PHB has exerted a strong inhibitory and anti-adhesive effect against Vibrio species pathogenic to shrimp and Nile tilapia fish [90].Not only can it be used as an effective antimicrobial agent, but it could present opportunities to be used as a packaging polymer [91].
Arthrospira spp.also has antimicrobial glycosides [78] and gallic and caffeic acids, which generally have an antibacterial effect [91].Other specific antibacterial compounds from phytoplankton are listed in the work of Rojas et al., 2020 [69].An illustration of most of the known mechanisms of antimicrobial action of microalgal and cyanobacterial metabolites is presented in Figure 2.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 8 of 40 used as an effective antimicrobial agent, but it could present opportunities to be used as a packaging polymer [91].Arthrospira spp.also has antimicrobial glycosides [78] and gallic and caffeic acids, which generally have an antibacterial effect [91].Other specific antibacterial compounds from phytoplankton are listed in the work of Rojas et al., 2020 [69].An illustration of most of the known mechanisms of antimicrobial action of microalgal and cyanobacterial metabolites is presented in Figure 2.

The Two-Step Process
Theory in the field is well established, and the two-step process of maximization of secondary metabolites is well studied and proven.
The first step considers the working condition under which the microalgal growth is kept close to the maximum growth rate by applying well-balanced and rich medium where no limitation of growth by nutrients may occur.Hence, microalgal cells' growth in

Maximizing the Synthesis of Bioactive Metabolites including Antimicrobial Compounds by Phytoplankton Microorganisms, Involving Stress Factors 4.1. The Two-Step Process
Theory in the field is well established, and the two-step process of maximization of secondary metabolites is well studied and proven.
The first step considers the working condition under which the microalgal growth is kept close to the maximum growth rate by applying well-balanced and rich medium where no limitation of growth by nutrients may occur.Hence, microalgal cells' growth in non-limiting conditions in the logarithmic phase up to the stationary phase is supplied by keeping state parameters (CO 2 content, pH, T, pressure, light intensity, mixing conditions, changing organic source of carbon, etc.) in optimal values.When the culture reaches the stationary phase, usually secondary metabolites are synthesized under stress factors, which directs metabolism of microalgae to their overproduction.Most common manipulation of microalgal metabolism is under stress conditions, such as nutrient starvation, high salinity, high temperature, light stress and supplementation of organic carbon source or by using several stress factors together.The results can be production of high-value compounds (for example, lectins) expressing anticancer, antiviral, antibacterial and anti-oxidant activities.Especially important to notice is that in open ponds, biomass concentration is low and production of secondary metabolites is low as well.Hence, application of closed photobioreactors (PBRs) is advanced and provides many benefits, such as high-density culture, controlled process conditions and precise manipulation of stress factors directing the metabolism of microalgae to over production of bioactive compounds.Studying PBRs performance as a complex system is fundamental for overall process development [92,93].

Stress Factors Such as Salinity, Nutrient Deficiency, Temperature and Light Increase Carotenoids and Other Metabolites in Dunaliella
Of special interest is the synthesis of carotenoids [94,95].The main microalgal carotenoids are astaxanthin, β-carotene and lutein.Many microalgal species are known to accumulate these carotenoids; however, the main species that are extensively studied are Dunaliella salina, Haematococcus pluvialis, C. zofingiensis and Chlorella vulgaris due to their capability of commercial production in large scale cultures.Secondary carotenoids synthesis is affected by the variation of the cultivation settings, and their accumulation is induced by the exposure of the cells to several stress factors [96].Carotenogenesis is enhanced by reactive oxygen species (ROS), which are generated by stress conditions like high light intensity, salt stress or high temperature [97].
D. salina contains β-carotene typically about 0.5-1% of dry weight, as many other green microalgae [98].However, as noted, under stress cultivation conditions, β-carotene is accumulated within lipid globules in the chloroplast to more than 12% of the algal dry weight, which is the highest content of β-carotene of any known source, making D. salina the best source of β-carotene [98].The β-carotene accumulation and the rate of synthesis depend on certain environmental parameters [99].As in the case of astaxanthin, β-carotene accumulation in Dunaliella sp. is also primary triggered when the cell division rate is either slowed down or arrested due to the effect of the stressing factors [100].Nutrient starvation in cultures of D. salina is well investigated, and it is well known that under nitrogen, phosphorus and sulfur starvation, the cells have elevated content of β-carotene [101].Between sulfur, nitrogen and phosphorus starvation, the highest increase of β-carotene accumulation was obtained with phosphorus starvation [102].However, in the large and commercial-scale production of β-carotene by D. salina, the technique used is high-salinity stress and nitrogen deficiency [103].In nitrogen starved cultures of D. salina, the β-carotene content increased up to 2.7% of ash free dry weight from about a content of 0.75% in nitrogen replete cultures [100].It was observed that in nitrogen-starved cells, total fatty acid content did not increase, but only an increase of specific fatty acids occurred [100].In contrast to lipids, D. salina under nitrogen starvation accumulated carbohydrates over 55% of dry weight, while its total protein content along with lipids decreased [104].
D. salina is able to grow under a very wide salinity values from 0.5 to as high as 35% v/w NaCl [105].The majority of the Dunaliella species have optimal growth in cultural medium containing 1-2 M NaCl.Salinity tolerance is positively linked with temperature of the liquid phase.For example, at 15 • C, the cells grow in about 1 M, and at over 30 • C, the culture adapts to over 3 M [106].Certainly, increasing the salinity in the culture has to follow a particular strategy in order to achieve optimal results [107].On the other hand, salinity stress is responsible for over-production of carbohydrates in D. salina.If the medium contained 5.5 M salt, the culture reportedly synthesized 2.5-fold more carbohydrates [108].
Decreasing the culture temperature from 30 • C to 10 • C resulted in a 2-fold increase of β-carotene value [109].It has to be noticed, that cultivation in low temperature increased the 9-cis-β-carotene and α-carotene value of the cells [110].Nutrient deficiency, for example, nitrogen starvation, resulted in an increase of carbohydrate content up to 40% [104].
Microalgal culturing under light illumination stress conditions is a challenging method for β-carotene over-synthesis.The effect of light changes has a positive influence of production of β-carotene [111].In cultures of D. salina, transferring the light conditions from 100 to 1000 µmol photons m −2 s −1 resulted in a significant increase in β-carotene content, where the maximum at 3.1% of dry weight was achieved [112].However, combined light stress and nitrogen starvation conditions seem to result in more accumulation of β-carotene [100,112].Cellular β-carotene accumulation due to light stress results in accumulation of specific fatty acid species (C16:0 and C18:1) and especially of C18:1, while total fatty acid content is not significantly affected [112].The degree of light intensity affects also the isomers of β-carotene; low light intensity favors the synthesis of the 9-cis isomer, while high light favors the all trans β-carotene.It has to be noted that light illumination in a particular wavelength influences the β-carotene synthesis, for instance, blue light [113].It is interesting that, recently, application of closed PBRs led to very promising results with some other microalgal species (eustigmatophytes) regarding β-carotene accumulation [114,115].Many frontiers for optimization of secondary metabolites over-production are achieved by using mixoptrophic and heterotrophic growth of microalgae.Strategies for such approaches can be found in the special literature about the mode of microalgae cultivation.

Optimization of Metabolites from Arthrospira spp.
The influence of light wavelength on A. platensis metabolism is studied in detail by Sohani et al., 2023 [116].They investigated cell behavior under blue, red, yellow and white light illumination.
Especially important once again to be noted is that manipulation of environmental factors as stress ones is based on a robust theory about methodology and established principles of optimization of cell cultivation.An excellent example can be given by citing the work of Chentir et al., 2018 [117].In their work, the established methods of successful culturing are applied in order to achieve intracellular and extracellular valuable compounds.Using statistical methods and the effect of applying stress factors such as light intensity, NaCl, nitrogen and phosphorus, they succeed in maximizing intracellular and extracellular substance produced by Arthrospira sp.Certainly, the working conditions were realized in a two-phase cultivation process.The carbohydrate content was maximized up to 26.6%.Using this approach, the maximum content of lipids (15.6%) was achieved.
The conclusion from this work is fundamental and demonstrates that manipulation and combination of the multiple stress factors together with application of advanced culturing techniques such as the multi-step culturing process is the only winning strategy which saves time and resources for research.The strategy can be applied for any microalgal species by taking into account specific requirements of its metabolism and is thought to be a promising tool to produce biomass enriched in various high-value compounds.

Immunomodulatory Activity
Microalgae, cyanobacteria and their compounds have great ability to fight and prevent infections indirectly through their immunostimulating properties.Carotenoids and vitamins have immunological activity, e.g., the relationship between vitamin E supplementation and membrane lipid protection from oxidative damage has been the subject of numerous studies.This is largely due to the fact that carotenoids and many vitamins are antioxidants having general immunostimulant activity [118].As noted, the proteins, B12 and other vitamins from Arthrospira are used for antioxidants and immune enhancers [36].Arthrospira platensis is rich in β-carotene, essential fatty acids, vitamin E, B12 and other vitamins and minerals, which makes it a great antioxidant and immune enhancer.In fact, not all immunological compounds from Arthrospira are elucidated, but the genus, as well as some microalgae, has proven immunological activity due to the activation of the innate immune system by increased interferon production and natural killer (NK) cytotoxicity in humans [118,119].For instance, oral administration of hot water extract of spirulina significantly increased the production of interferon-γ in NK cells [120].
As noted, PUFAs have anti-inflammatory action.They, together with phytosterols, carotenoids and vitamins are the most explored phytoplankton compounds for their effects in the regulation of immunological responses.Dunaliella tertiolecta and D. salina are rich in phytosterols, giving the species an immunomodulatory potential [118].Indeed, D. tertiolecta and D. bardawil have been reported to have anti-inflammatory properties.D. salina activates natural killers and macrophages in mice and interleukin (IL)-6 in human peripheral blood mononuclear cells (PBMCs) [119].A mixture of phytosterols from D. tertiolecta inhibits the proliferation of PBMCs and cytokine production in a sheep model of inflammation [118,119].
As we noted, some anti-inflammatory metabolites, such as carotenoids, fatty acids and sulphated polysaccharides, have also been reported to increase after various stress chemical triggers in A. platensis, D. salina, C. zofingiensis, etc. [37].
The two genera assessed in the current review have a clear non-specific immune response enhancing effect in shrimp and fish.Thus, they have vibriosis preventing and Aeromonas infection preventing effect.While it is certain that it is largely due to their immune stimulating effect and immune prophylactic [121] action, it is also very likely that their antibacterial activity has a role [85].Artemia (brine shrimp) fed with a daily supplementation with two strains of D. tertiolecta were fully protected against vibriosis after challenge with Vibrio sp. in terms of survival and viable parameters.The results were better than when using probiotics and dead bacteria.It is not known whether this effect is due to the immune stimulating effect or the synthesis of antibacterial compounds [122].Carp that were fed with A. platensis had an enhanced response of phagocytosis, superoxide anion production and IL-1β and tumor necrosis factor (TNF)-α genes expression in kidney phagocytic cells [123].Phagocytes from channel catfish fed also with A. plantesis showed enhanced phagocytic activity to zymosan and increased chemotaxis to Edwardsiella ictaluri exoantigen [124].
Dry powder and hot water extract of A. platensis increased resistance against Vibrio alginolyticus in the white shrimp Litopenaeus vannamei due to increased phagocytic and lysozyme activities for pathogen clearance [121,125,126].Superoxide dismutase was induced and activated, and this is an essential antioxidant enzyme playing a vital role in the immune system of shrimp in scavenging superoxide anion that damages the host tissues [121,125].All shrimp that survived vibriosis in the study of Al-Ghanayem (2023) developed resistance to Vibrio pathogens, which is explained by the fact that antioxidant enzyme levels of shrimps had increased after treatment with A. platensis extract and antioxidant enzymes increase immunity patterns of the host.According to this study, Arthrospira bioactive compounds have the potential to function as both inexpensive and environmentally friendly immune stimulants as well as an effective preventive measure against Vibrio bacterial infections [121].

In Vivo Antimicrobial Activity
It is noteworthy that a substantial amount of in vivo research (Table 1) has been dedicated to Arthrospira in comparison to other phytoplankton, such as Chlorella, or generally the former Chlorella genus, Scenedesmus, Dunaliella, etc.
An example of such research is about impetigo.This is a highly infectious skin bacterial disease, most common among pre-school children, and is mostly caused by Staphylococcus aureus.Gas chromatography-mass spectrometry (GC-MS) analysis of both crude cell biomass and its methanol (MeOH) extract from A. platensis detected a high percentage of linoleic and palmitic acid and the presence of squalane in the MeOH extract, which could act in synergy, resulting in impetigo infection suppression [8].
Arthrospira platensis had an in vivo antimicrobial effect on the bacterial groups investigated in the rabbit cecum but only in combination with thyme.The qPCR detected significantly lower bacterial load in the samples, while the classical microbiological methods did not measure a substantial effect on the composition of the cecal microbiota.Samples were collected on the 14th, 28th and 48th day of the supplementation (a 48-day growing period).The number of Escherichia coli, total anaerobic and strictly anaerobic bacteria decreased by age, regardless of the diet type.There was a drawback: A. platensis plus thyme had a negative effect on cellulose and on crude protein digestibility, impairing the rabbit diet's digestible protein content [127].The authors suggested testing the effects of A. platensis and/or thyme on health status under poorer sanitary conditions [128].
Vibriosis is a common lethal bacterial infection in shrimp in hatcheries and farms.Vibrio species amount for more than 80% of the bacterial population in seawater and are opportunistic pathogens infecting shrimps under environmental stress [121,129].V. alginolyticus-infected shrimp are more susceptible to other pathogens and ultimately dying.Vibrio harveyi is a luminescent bacterium that causes luminous vibriosis and is among the most common pathogens that increase the mortality in aquaculture shrimp species, such as the white shrimp L. vannamei [130].Vibrio parahaemolyticus is also a major pathogenic strain for L. vannamei and causes acute hepato-pancreatic necrosis disease, a.k.a.early mortality syndrome, which is a relatively new disease that emerged in Southeast Asia in ca.2010 and may cause 100% mortality within 20 days of infection [131,132].The AMR in Vibrio spp.towards antibiotics in aquaculture and antibiotic residues left in water are also pressing problems [133].
The in vivo study of Al-Ghanayem (2023) utilized tests, one of which included separate suspensions of the three Vibrio species mixed with a MeOH A. platensis extract intramuscularly injected in an abdominal segment of 12 white shrimp.The positive control inoculated shrimps were severely infected within 72 h and died within 120 h, except the V. harveyi-infected shrimp, which survived slightly longer.Vibrio alginolyticus was the most virulent.A challenge test included shrimps fed with 2500 µg/g extract of A. platensis and injected with suspensions of the three Vibrio species separately into the ventral sinus of the cephalothorax.The survival rate was recorded every 24 h for 168 h.The two assays clearly showed increased survival rate and reduced shrimp mortality and demonstrated that A. platensis considerably influenced pathogen multiplication regulation.Apart from the immunostimulant action, which contributes to overcoming environmental stress, the direct antibacterial activity could also contribute to the results.The conclusion is that A. platensis could effectively prevent Vibrio infection and considerably protected L. vannamei against vibriosis [121].
Doses of 1 mg, 10 mg or 25 mg A. platensis per fish were administered orally to carp by intubation for 3 days.After Aeromonas hydrophila challenge, fish were sacrificed and the livers and kidneys were removed, pooled, homogenized and diluted.Suspensions were plated on ager and the number of colonies was counted after 12 h.The results showed that the numbers of bacteria in the liver and kidney in the control group peaked at 8 and 1 h, after the challenge, respectively, and then decreased on their own.However, A. platensis feed aided that process.The kidneys of the treated groups had lower bacterial numbers than the control only at the early hours (1-4), while the liver of the treated groups had lower bacterial numbers at hours 4-12 [123].
Wound assays have a considerable part in the in vivo research regarding Arthrospira.An example of these reports used 30 rats and a clinical in vivo wound procedure with contamination with Pseudomonas aeruginosa and S. aureus for 24 h before treatment.The signs of inflammation were more obvious with P. aeruginosa.The negative control (without treatment) showed partial healing and shrinkage in the wound area but also formation of scar tissue after seven days.Treatment with the aqueous extract or with standard antibiotics (azithromycin for S. aureus and ceftazidime for P. aeruginosa) showed comparable results.In comparison with the untreated group, there was a complete or accelerated healing, sometimes without remnant scar tissue.Wound shrinkage and a process of re-epithelialization were observed.All this is a reflection of a good antibacterial activity, as inhibition of bacteria in the wound site is, authors say, probably what led to cell proliferation and regeneration.However, the antibacterial activity is not the only cause of healing, which might be attributed also to stimulation of angiogenesis, collagen formation and deposition, and epithelial cell proliferation.Flavonoids, alkaloid, terpenes, glycosides and saponins but not tannins were found in the extract, and some of them are not only antimicrobials but antioxidants.Since antioxidants are also reported to have a significant role in the wound healing process by protecting tissues from oxidative damage, antibacterial and antioxidant compounds might act synergistically in the wound healing process [78].
Arthrospira platensis cream of a MeOH extract proved to be better that a nystatin cream for murine wounds infected with Candida albicans.The wound healing recorded for nystatin was 50-90% from days 4 to 13 after treatment.However, A. platensis cream caused wound healing of 55-95% for the same time interval.At the 13th day, the wound's redness was at its lowest and hair growth was moderate; by the end of the 17-day study period, all inflammation had disappeared and there was significant hair growth.The histopathological analysis of the untreated wounds showed several missing layers of the epidermis, rounded, short, elongated cells, hypha swelling and keratinized fiber that were loose and disordered in the stratum corneum.The epidermis also had a chronic inflammatory cellular infiltration mainly of lymphocytes and plasma cells.The nystatin-treated wounds still had abnormal epidermis as the keratinized fibers of stratum corneum were also disrupted and had separation of the epidermis.The dermis also had edema with some inflammatory lymphocyte infiltrate.Mice treated with A. platensis showed a finding similar to the healthy skin, with no hypha swelling, no discernible toxic consequences compared to the control, normal looking skin tissue and epidermis, normal dermis with minimal inflammatory cellular infiltration and with keratinized fibers of the stratum corneum consistently and regularly arranged and condensed without any disruption.The high antioxidant activity of the MeOH extract could also contribute to the wound healing effect.Alkaloids, phytol, fatty acids hydrocarbons, phenolics and phthalates were found in the extract [134].
Chlorophyll is a natural photosensitizer, and when irradiated with a laser, it generates reactive oxygen species (ROS), thus causing photodynamic destruction of bacteria, for example, in an infected area.This interesting and innovative phenomenon was developed by Li et al., 2021, [135] for A. platensis coated with a formulated natural chitosan polymer.The hydrogel promotes the adhesion to wounds and the cyanobacteria release chlorophyll, which is irradiated with a 650 nm laser.However, the main function of the photosynthetic cyanobacteria was the production and local delivery of oxygen to alleviate acute and chronic tissue hypoxia in wounds.This combined action enhances wound healing.With increasing time of laser irradiation, the ROS level increased, demonstrating the laser-induced ROS participated in the antibacterial process.Arthrospia platensis with a 650 nm laser could significantly inhibit E. coli and S. aureus growth in vitro, with over 80% antibacterial efficiency, compared to the control.In vivo, comparing with the untreated control, the groups of chitosan, SP gel and SP gel + laser showed enhanced wound healing, which was slowest for the chitosan-treated mice.Laser irradiation could significantly enhance the effect and prolong the activation time of A. platensis gel in the wound healing process and could satisfy the oxygen requirement for wound healing, leading to best neovascularization.The authors named the formulation a living hydrogel, which relies on both photosynthetic ability and photodynamic effect to inhibit infection, alleviate hypoxia and accelerate wound closure and is promising for clinical translation [135].
The same mechanism was developed in another study; however, A. platensis in the chitosan gel was loaded with the plant alkaloid berberine, a quorum sensing inhibitor and antibacterial agent.All in vitro and in vivo antibacterial results, i.e., in wound healing, were the synergetic effect of laser-irradiated A. platensis and berberine, therefore not a subject of this study [136].
In an animal study feeding mice with phytoplankton agents and Helicobacter pylori inoculation, the short-term treatment was repeated for two consecutive days.To study the effect of a longer period, other mice were fed either with polysaccharides or 35 mg of A. platensis powder 3 times per week for 4 weeks before infection with H. pylori.All mice were sacrificed two weeks after the last treatment, and the stomach of each animal was removed for further analysis.Helicobacter pylori that were present in it were isolated, identified and enumerated.Treatment of mice with the polysaccharides only up to 2 h before inoculation with H. pylori significantly reduced the mean bacterial load in their stomach by more than 50%.Arthrospira platensis powder had a positive but not statistically significant effect.When the mice were fed with polysaccharides and powder for 4 weeks before infection, reduction in H. pylori load was significantly and more obviously reduced by 94% and 87%, respectively.Doses of twice the effective inhibitory concentrations used in an in vitro inhibition study did not kill H. pylori and AGS gastric epithelial cells.A polysaccharide agglutination assay showed that allowing porcine gastric mucin to interact with the polysaccharides before the addition of H. pylori did not prevent the agglutination of H. pylori by them.Similarly, the presence of mucin did not impede the polysaccharides in agglutinating H. pylori.Polysaccharides from A. platensis were confirmed to be effective carbohydrate-based antiadhesives in preventing H. pylori adhering to gastric mucin and colonization without affecting the host [137].
Cold atmospheric pressure plasma (CAPP) technology is currently considered an effective and cost-effective new technology to sterilize food and pharmaceutical matrices in a few minutes.However, more research is required in relation to food-plasma interaction and food macromolecules stability/functionality under the new CAPP technology.Pina-Pérez et al., 2022, [138] conducted an in vivo assessment of this technology on the bioactive activity of Arthrospira powder using Caenorhabditis elegans as an animal model.This nematode has been used very frequently as a model of infection by pathogenic microorganisms, whereas survival of infected worms, either exposed to a tested antimicrobial agent or not, is assessed.CAPP did not have negative effects on the nematode, and both CAPP-treated and untreated A. platensis improved C. elegans lifespan.On the topic of the current review, no antimicrobial effect, measured as an increase in C. elegans lifespan, was detected in worms infected with Salmonella enterica serovar Typhimurium when CAPP-treated or untreated Arthrospira was added to the media.Approximately half the worms of all groups were dead at ca. 12 days [138].

In Vitro Antimicrobial Activity
In vitro reports on the antimicrobial properties of Arthrospira are the most numerous in the current review (Tables 2 and 3).
Non-polar extracts from the species were found to have higher antimicrobial activity than polar ones in one report [68].Atomic force microscopy (AFM) showed that one hour of exposure to an acetone-soluble fraction from A. platensis cells caused considerable damage to cell walls of A. hydrophila bacteria-pores, holes and grooves had formed.After two hours of exposure, the cells had become permeabilized and collapsed due to fragmentation of the cell wall [139].
Many aquatic pathogenic bacteria use quorum sensing system to establish their virulence [140], and Vibrio spp.are one of them.Co-culturing of A. platensis and different Vibrio spp.leads to positive results-high inhibition of bacterial growth.This antibacterial activity was not influenced by the presence or absence of light as well as for other phytoplankton species.One potential course of action might involve interfering with quorum sensing.Previous studies have demonstrated the high sensitivity of Vibrio to these mechanisms.The findings from that study could provide an explanation for the beneficial effects of adding phytoplankton to fish larvae generally [85].
De Souza et al., 2011, [141] found out that the dry mass of Aspergillus flavus was on average 4× lower in the presence of phenolic compounds from A. platensis than in the control.They analyzed the indicators of A. flavus biomass development (glucosamine, ergosterol and protein content) and concluded that glucosamine content is the most suitable to investigate the effect of phenols on the fungal biomass development because its production was linear along the time in the control experiment and was the most affected in the culture medium.The membrane component ergosterol was hardly affected, and fungal biomass protein content had an unstable pattern of production over time [141].
Biofilms are held together and covered by a matrix made up of strong biopolymers called extracellular polymeric substance (EPS).A decrease in EPS will cause the biofilm structure to weaken, making the bacteria living there more susceptible to antimicrobial agents and simpler to remove.Bacterial cell surface hydrophobicity (CSH) plays a crucial role in the initial bonding of the bacterial cell for the first few seconds in the biofilm.Weakening of CSH would reduce the initial attachment and leads to decreased bacterial population in the biofilm.Therefore, according to LewisOscar et al., 2017, [7] an ideal antibiofilm strategy has to control these factors (CSH and EPS).Arthrospira platensis extract achieved biofilm inhibition and significant reduction in CSH in some bacterial species.In addition, the biofilm inhibitory concentration did not exert antibacterial activity and was not bactericidal.This makes the cyanobacterium a potential candidate for antipathogenic and antibiofilm agents, less prone to induce antimicrobial resistance [7].
As noted, the PHB from Arthrospira spp.could be used as an antimicrobial therapeutic and for the production of packaging polymers [90,91].The PHB from Spirulina sp.LEB 18 is biodegradable and has similar mechanical and thermal properties to polymers of petrochemical origin.PHB nanofibers were prepared by injecting polymer solution of PHB through capillaries with a diameter of 0.45-0.8mm.That solution contained phenolics extracted from the biomass of the same species, namely gallic (72%) and caffeic acids (28%), which generally have an antibacterial effect.This material (35% PHB/1% phenolic compounds), albeit not highly active against E. coli and S. aureus, showed retaining of the phenolics' activity after the process of electrospinning.This new nanomaterial combined a biodegradable packaging function with the antibacterial activity that prevents the multiplying of microorganisms and ensures the quality and preservation of food [91].
The active ingredients from A. platensis with proved antimicrobial activity were found to contain some antimicrobial compounds.Naturally, high content of long-chain fatty acids [142], e.g., in non-polar extracts [68], other fatty acid compounds [143], γ-linolenic acid and the synergetic effect of lauric and palmitoleic acid [144,145] have been considered responsible for the antibacterial action.Other compounds included proteins, carbohydrates, flavonoids, phenols, steroids [146,147], alkaloids, saponins, phenols, quinones (from an ethanol (EtOH) extract) [79], terpenes (monoterpenes and sesquiterpenes) [146,148] and glycosides [147].A positive correlation between the antimicrobial effect and the phenolic content in the respective extracts has been found in a study, and the more active extracts, the non-polar ones, contained lipid-soluble phenolics [68].In another study, the phenolic compounds did not turn out to be the main component of the most active samples [88].
The peptides with <3 KDa molecular weight from the species inhibited E. coli and S. aureus.As previous studies had shown that only peptides with <6 KDa molecular weight can pass through gastro-intestinal epithelial cells [149], the isolated peptides should be capable of being developed as oral drug candidates.In another study, the antibacterial substance was indicated to be an aliphatic compound with different active groups (-OH, =C=O, =CH 2 and -CH 3 ) [150].MICs of ASF = 1.9-15 µg/mL, MBCs (7.8-250 µg/mL) and highest IZs = 31-41 mm.E. tarda was the most susceptible pathogen to ASF AIF-moderate effect Phycocyanin IZ on A. hydrophila = 16 mm AFM of A. hydrophila showed cell wall decay AWDM BMD Atomic force microscopy (AFM) [139] Live cells in a co-culture Six Vibrio bacterial strains-V.parahaemolyticus, V. anguillarum, V. splendidus, V. scophthalmi, V. alginolyticus and V. lentus Strong inhibition of bacterial growth ca.1000 times after 96-120 h co-culturing V. alginolyticus and V. anguillarum: most resistant slower decrease in bacterial count Co-culturing of microalgae or cyanobacteria with bacteria-OD measurement  EtOH, MeOH and aqueous extracts Fish and shellfish pathogens-P.putida, P. aeruginosa, P. fluorescens, A. hydrophila, V. alginolyticus, V. anguillarum, V. fluvialis, V. parahaemolyticus, V. harveyi, V. fisheri, E. tarda and animal isolates of E. coli  7. Antimicrobial Activity of Dunaliella spp.
Substantial in vitro research, as well as some in vivo studies, has been conducted in regard to D. salina and other Dunaliella spp.(Tables 4 and 5).
To the best of our knowledge, two in vivo studies have been carried out studying D. salina.Research was carried out onto plant pathogens, in addition to in vitro tests.A hexane extract from the algal species had an MIC of only 0.3 mg/mL against B. subtilis and 3 mg/mL against the other bacteria.The other two extracts had MICs of 3 mg/mL on B. subtilis and >3 mg/mL on the other bacteria.Pseudomonas syringae was the most resistant in the DDM, with inhibition zones (IZs) 8-12 mm (24 mm for ciprofloxacin), while B. subtilis was the most sensitive, again with IZs of 20 and 21 mm of the hexane and EtOH extracts, respectively (32 mm for the control).The mean IZ of the extracts on Pectobacterium carotovorum was half that of ciprofloxacin (20 mm).The hexane extract showed the highest amount of carotene, so it was selected for in vivo analyses.The leaves and fruits of tomato plants and zucchini fruits were artificially wounded, and treated groups were sprayed with or immersed in a hexane extract.Then, the respective bacterial suspension was (spray)inoculated.The tomato plants that were treated showed a reduction in bacterial speck spot disease of 66% in incidence and 77% in severity.Similarly, treated tomato and zucchini fruits showed less signs of soft rot, with disease incidences of 5% and 13% compared to 91% and 100%, respectively, for the positive control [170].
Another in vivo research studied the effect of chitosan nanoparticles loaded with hexane: ethyl acetate (HEAE-CNPs) and methanol (ME-CNPs) extracts on wound healing.As noted, antioxidants such as carotenoids contribute to wound healing, as they are scavengers of ROS and anti-inflammatory agents, e.g., NF-κβ signaling pathways inhibitors.The hexane extract contained 19 mg/g β-carotene and 16 mg/g zeaxanthin, while the MeOH extract contained only traces of zeaxanthin.That would explain the better effect of the hexane extract.Both agents increased vascular endothelial growth factor, angiogenesis, collagen skin contents and tissue remodeling and decreased (TNF)-α.Therefore, both HEAE-CNPs and ME-CNPs exerted wound healing and regeneration not only due to the antibacterial effect but also to the antioxidant and anti-inflammatory activity [171].
Another in vivo research deployed intragastrical inoculation of mice with H. pylori three times at two-day intervals.Two weeks after inoculation, mice were treated orally with the total carotenoid extract of a Dunaliella sp.(100 mg/kg body weight per day, 0.15-0.18mL in corn oil) for two weeks, during which stomach and spleen were removed and prepared for further histopathology analysis.The histological changes of the infection started with the denudation of the surface epithelium that affected the gastric pit, followed by gastric gland hyperplasia and lamina propria swelling.After 14 days of treatment, the Dunaliella sp.treatment group showed no hyperplasia gastric gland, reduced swelling of lamina propria and normal gastric pit condition.Unlike Chlorella sp., Dunaliella sp. did not delay the gastric ailment but promoted the healing process during gastritis and restored the normal histology of the stomach relatively rapidly, e.g., faster than Isochrysis sp.It is known that β-carotene reduces the inducible nitric oxide synthases and cyclooxygenase-2 expression and suppresses the ROS inflammation and inflammatory signaling during gastritis, thus accelerating the gastric healing [172].In addition, a high intake of carotenoids such as astaxanthin has been suggested to prevent gastritis by lowering colonization levels of H. pylori.The total carotenoid present in dry weight of Dunaliella sp. was especially high (99%).Since Dunaliella sp.treatment healed the stomach in 14 days, authors supported the view that carotenoids, which combine antibacterial and antioxidant effect, may be a new strategy for treating gastritis-causing H. pylori [173].
Previous studies have found oleic, alpha-linoleic and palmitic acids to be the major components in the EtOH or hexanic extract of D. salina and to be mainly responsible for its in vitro antimicrobial activity.The EtOH extract also contained the highest amount of EtOH-extractable PUFAs (77%), of which linolenic acid was 45% [58].There is a hypothesis that free fatty acids and phytol are derived from galactolipid and chlorophyll catabolism in that microalgae (but likely not limited to D. salina).Together with the products related to carotenoid degradation, they act as antimicrobials but can likely be extracted only by a sufficiently aggressive extraction protocol.Probably for that reason, EtOAc and MeOH extracts from a hexane extract lacked antimicrobial activity against E. coli and S. aureus in the study of Iglesias et al., 2019 [174].Another study concluded that not only the several fatty acids but also the polysaccharide-rich fraction and the compounds cyclocitral, neophytadiene and phytol explained the antimicrobial properties of D. salina [3,175].Pressurized extracts from the same species also contained β-cyclocitral, α and β-ionone, neophytadiene, phytol and hexadecanoic acid.They are compounds with documented antimicrobial activity and were present in higher amounts in the most active antibacterial extracts.The major compound in the pressurized extracts was identified as 9,12,15-octadecatrienoic acid methyl ester, and all extracts yielded fifteen different volatile compounds as a whole, as well as several fatty acids (mainly palmitic, α-linolenic and oleic acids) that could also have been responsible for the antimicrobial activity were identified in the extracts [83].
Other unique compounds which are potential antimicrobial agents, such as 3,3,5trimethylheptane and n-hexadecane, have also been revealed [175].Maadane et al., 2017, found that the antimicrobial effect of D. salina and other Dunaliella species correlated with the content of fatty acids, which were main compounds of the biomass, but also with carotenoids and polyphenols.The authors suggested additive or synergetic action for them [61,140].
Biofouling or biological fouling is defined as the accumulation of microorganisms, plants, filamentous algae or small invertebrate animals on ships or other surfaces where it is not wanted, such in as submarine hulls and devices such as water inlets, pipework, ponds and rivers, where it degrades the item's main purpose [176].To date, the testing of the anti-fouling properties of D. salina and other species from the genus turned out to be negative [177].
According to previous studies, among extract from different microalgal species, only the EtOH extracts from Dunaliella sp.contained a significant quantity of carotenoids; therefore, they could also be responsible for the antibacterial effect [61].The antimicrobial activity of Dunaliella primolecta is likely due to pheophorbide α and β-like compounds [3], while that of Dunaliella tertiolecta-to polyphenols, notably gentisic acid, (+) catechin and (−) epicatechin [178].
The same species (D. tertiolecta) had no clear anti-Vibrio activity when used in vivo as green-water cultures in Vibrio-challenged L. vannamei cultures.That study is interesting, however, because no differences in mortality of juvenile L. vannamei were observed between different groups of treatments and the control, suggesting that the pathogenicity of V. campbellii could be related to the growth stage of shrimps [84].Full protection against vibriosis in terms of survival and viable parameters.This effect could be due to the immune-stimulating effect or antibacterial compounds In vivo challenge test after daily feeding [122]

Evaluation of the In Vitro Antimicrobial Activity of Arthrospira and Dunaliella According to Different Criteria
We assessed the antimicrobial activity of the two genera according to different criteria.Different standards apply when evaluating antimicrobial or antibacterial activity in vitro.Today, we still use Eloff's criteria, which state that an extract or fraction has significant antibacterial activity if its MIC against a particular microorganism is equal to or lower than 0.1 mg/mL [182].Plant extracts with MIC values up to 0.5 mg/mL are regarded as strong inhibitors, those between 0.6 and 1.5 mg/mL as moderate inhibitors and those above 1.6 mg/mL as weak inhibitors, according to Aligiannis et al., 2001 [183].
Based on the diameter of the IZ, antimicrobial activity can be divided into four classes, according to Greenwood (1995).When an IZ is greater than 20 mm, the antimicrobial effect is strong; at 16-20 mm, it is medium; at 11-15 mm, it is weak; at <10 mm, it is nonexistent [184].Other authors claim that an IZ with a diameter of more than 15 mm exhibits strong inhibitory activity, a diameter of 9-14 mm exhibits moderate inhibitory activity and a diameter of less than 8 mm exhibits weak inhibitory activity [185,186].
The antimicrobial activity of A. platensis is strain-specific and also varies with the extraction solvent and procedure.The antimicrobial properties vary from no or weak activity through moderate to strong and excellent according to all criteria and sometimes are comparable or exceed the activity of the control antibiotics.
The most potent ingredient, arguably, is an acetone soluble fraction from a strain with MICs as low as 1.9-15 µg/mL and IZs as high as 31-41 mm against E. tarda and other bacterial fish pathogens including Bacillus and Vibrio spp.[139].The sodium acetate and aqueous extracts of A. platensis had MIC values of 25 µg/mL against Staphylococcus sp.[151].The MeOH and hexano-EtOH extracts of another strain inhibited B. subtilis and B. cereus with IZs of 43 and 41 mm, respectively.The latter had an IZ of 28 mm on E. coli, and together with the IZ of the acetone extract of 24 mm, they exceeded the effect of gentamycin (23 mm) [152].
Other Arthrospira species generally had weak to moderate and rarely strong effect, except for A. maxima, whose aqueous extract inhibited S. aureus with more than 50 mm IZ, while the MeOH extract showed IZs of 37-45 mm against E. coli, P. vulgaris, S. aureus and B. subtilis [168].
Purified compounds generally have higher activity than extracts; therefore, the strong activity of purified phycocyanin, which had MIC values against E. coli, K. pneumoniae and P. aeruginosa of 100, 75 and 50 µg/mL, respectively, was expected [159].Also anticipated was the significant effect of a purified unidentified compound with MICs in the range of 30-85 µg/mL against C. albicans, B. subtilis, S. aureus, E. coli and P. aeruginosa [144].
The activity of D. salina is also strain-specific and varies from no effect to strong inhibition.The CHCl 3 /MeOH/acetone extract had an IZ of 25.4 mm against caries-causing S. mutans [179].An EtOH extract had an IZ of 23 mm against Y. enterocolitica and an MBC of 0.63 mg/mL against fish bacterial pathogens.A DCM extract from the same strain had an MBC value of 0.32 mg/mL against B. subtilis.MICs are at lower concentrations than MBCs, meaning this strain fulfills the criteria of Aligiannis and most likely of Eloff for significant activity [58].Hexane and EtOH extracts against B. subtilis had IZs of 20 and 21 mm, respectively and an MIC of 0.3 mg/mL for the hexane extract [170].Other Dunaliella species generally had weak or no effect, except for D. tertiolecta, with MICs as low as 25, 50 and 100 µg/mL for the aqueous and sodium acetate extract against some Staphylococcus spp.[151].

Conclusions
In addition to their already exploited roles for nutraceuticals, and in biotechnology, the genera Arthrospira and Dunaliella have different bioactivities and potential in medical applications due to their high diversity of bioactive compounds.One such activity is the antimicrobial action (antibacterial and antifungal), which varies in a broad range for the two genera and is strain-specific.There are strains of A. platensis with very potent activity with MIC values as low as 1.9-15 µg/mL, and Arthrospira sp. with an IZ of 50 mm.Noteworthy are the substantial amounts of in vivo studies of Arthrospira which show that the cyanobacterium is very promising for preventing vibriosis in shrimp and Helicobacter pylori infection and for wound healing.The innovative laser irradiation of the chlorophyll it releases can cause photodynamic destruction of bacteria.D. salina exhibits MIC values lower than 300 µg/mL and an IZ value of 25.4 mm, while D. tertiolecta has demonstrated MIC values of 25 and 50 µg/mL.These values fulfill all criteria for significant antimicrobial activity and sometimes are comparable or exceed the activity of the control antibiotics.The antimicrobial bioactive compounds which are responsible for that action are fatty acids including PUFAs, polysaccharides, glycosides, peptides, neophytadiene, etc. Cyanobacteria, such as Arthrospira, also particularly have antimicrobial flavonoids, terpenes, alkaloids, saponins, quinones and some unique-to-them compounds, such as phycobiliproteins, polyhydroxybutyrate, the peptide microcystin, etc.All these substances are subject to maximization by stress factors in a two-step process during controlled fermentation conditions.
The current state of the art shows that the antimicrobial metabolites from Arthrospira and Dunaliella hold potential, but the market products are still limited, which is valid for other microalgal species too.More research is warranted so that they can reach practical application.

Figure 1 .
Figure 1.Production and isolation of antimicrobial metabolites from microalgae and cyanob and possible applications-flow diagram of the process.

Figure 1 .
Figure 1.Production and isolation of antimicrobial metabolites from microalgae and cyanobacteria and possible applications-flow diagram of the process.

Figure 2 .
Figure 2. Examples of mechanisms of action of antimicrobial microalgal and cyanobacterial metabolites.Legend: PUFAs-polyunsaturated fatty acids, FFA-free fatty acid.

Figure 2 .
Figure 2. Examples of mechanisms of action of antimicrobial microalgal and cyanobacterial metabolites.Legend: PUFAs-polyunsaturated fatty acids, FFA-free fatty acid.

Table 1 .
Research which included studying the in vivo antimicrobial activity of Arthrospira platensis Gomont.

Table 2 .
Research on the in vitro antimicrobial activity of Arthrospira platensis.

Table 3 .
A review of the in vitro research on the antimicrobial activity of Arthrospira.spp., including unidentified ones.

Table 4 .
A review of the research on the antimicrobial activity of Dunaliella salina (Dunal) Teodoresco.

Table 5 .
Cont.Salmonella sp., P. aeruginosa, B. cereus, Klebsiella sp.; Mice inoculated with H. pylori E. coli and Salmonella sp.: no activity up to 100 mg/mL; P. aeruginosa: probiotic activity at 100 mg/mL only; B. cereus: inhibition at 6.25 mg/mL; Klebsiella sp.: inhibition at 25 mg/mL.From three microalgal species, only Dunaliella sp.healed the stomach in 14 days, highest ability to promote gastric healing due to antioxidant and antimicrobial effect 13% growth inhibition (the aqueous extract at 78-313 µg/mL.Exact MIC not determined due to variation in the inhibition at different concentrations.No clear anti-Vibrio activity for the MeOH extract and in the in vivo test when the alga was used as green-water cultures