Antimicrobial and Ecological Potential of Chlorellaceae and Scenedesmaceae with a Focus on Wastewater Treatment and Industry

Abstract

A complex evaluation of antimicrobial activities of microalgae, including those relevant to wastewater treatment (WWT), in light of the integrated biorefinery concept, is performed. An example of this concept is linking a commercial microalgal system to plants, factories, or farms that emit polluted wastewater (WW). The microalgae would not only metabolize the pollutants—such as nitrogen (N) and phosphorus (P)—from the WW, thus fueling their biomass, but they would exert an antibacterial effect against the pathogenic bacteria there. The biomass then could be harvested and used for biofertilizers, biofuels, and bioplastics and might possibly be utilized as animal feed, antimicrobial and other pharmaceutical agents. A large amount of the research on the antimicrobial activity and WWT potential focuses on the families Chlorellaceae and Scenedesmaceae, which are also some of the most commercially used strains of microalgae. For that reason, they are the species chosen for the current review. Furthermore, the increasing antimicrobial resistance necessitates the search for antibiotic alternatives, and the antibacterial and antifungal activity of Chlorellaceae and Scenedesmaceae is very promising. Microalgae are rich in antibacterial compounds like polyunsaturated fatty acids (PUFAs), polysaccharides, carotenoids, proteins, etc., and for that reason, their extracts possess antimicrobial effects. The in vitro antimicrobial activity of Chlorellaceae and Scenedesmaceae families has varied in a broad range from low to strong activity or no effect. Several strains have fulfilled the criteria for outstanding and high activity, especially C. vulgaris and other Chlorellaceae spp., with an effect equal to or better than the control antibiotics. There were several strains with minimum inhibitory concentrations (MIC) below 80 µg/mL and even 10 and 1.5 µg/mL; some species also had inhibition zones (IZ) over 30 mm, even as high as 48 mm. In vivo results are also promising but scarce, and all this warrants further in vivo and in situ studies—from animal models to clinical and environmental trials. Altogether, important data in the light of the circle economy, the urgent necessity to decrease CO₂ emissions to fight climate change, and to curb the harmful influence of future pandemics are presented. This review paves the way for further utilizing the total potential of a microalgal system.

1. Introduction

The first microalgal antimicrobial agent (chlorellin) was discovered in the 40s [1,2]; however, the antibiotic boom soon occurred, and probably for that reason, that knowledge seems to have been “abandoned” until recently. Then, the rising antimicrobial resistance led to the urgent need for the discovery of new antimicrobial chemotherapeutics. Scientists look for alternative sources for antibiotics other than (semi)synthesis or the classic producers, such as actinomycetes and other microorganisms. Research on the antimicrobial effect of microalgae was renewed and specifically intensified after approximately the year 2010, as a Scopus search shows. It led to promising in vitro and in vivo results. In regard to microalgal secondary metabolites, scholars find the discovery of specific compounds, apart from chlorellin, responsible for the antimicrobial action, to be a relatively emerging field of research [1,2]. It can be speculated that other promising species of terrestrial plants or algae are “rediscovered” today as scientists turn their interest towards ethnobotany and research on important plants treating infections in traditional medicine.

To date, natural products and their derivatives amount to nearly 70% of the FDA-approved antibacterial drugs. The majority (97%) comes from microbes (bacteria and fungi), while algae and higher plant products contribute to just 3% of them [3]. Plant products, in general, can be claimed to include algal products since most of the eukaryotic species that are named with the polyphyletic term (micro)algae are from the kingdom Plantae sensu lato [4,5,6,7].

These 3%, however, do not accurately reflect the potential of algal and higher plant natural products. As Quave (2016) has excellently summarized in her outline, “we have only uncovered the tip of the iceberg in our understanding of the chemical diversity and bioactivity of plant natural products” [3].

The reasons for such low numbers are, on the one hand, that plant natural products were likely being ignored during the golden era of antibiotics, and even today, their development would mean the diversion of resources from the development of conventional antibiotics [8]. On the other hand, there are some inherent difficulties in the development of plant natural products as antimicrobial drugs. Quave lists them as follows: plant extracts are very chemically complex, and the isolation of single antibacterial compounds can be time-consuming and requires a large amount of plant material. Rediscovery of known compounds from unexplored plant species slows the process. It can be challenging to access plant specimens, particularly in an international setting. Synergism between compounds in an extract poses particular challenges, as due to the lack of fully developed scientific technology, it is difficult to study multiple compounds acting simultaneously on multiple biological targets [3].

Particularly regarding microalgae, in addition, proved antimicrobial natural sources, including in ethnobotany, such as eucalyptus, clove, oregano, etc., were likely priorities for research and development.

Similarly, fatty acids, especially polyunsaturated fatty acids (PUFAs), which are abundant in microalgae, have an underappreciated antimicrobial therapeutic potential [9]. For example, increasing their concentration in the animal organism through diet or other means, suppressed infections and also had a prophylactic effect [10]. However, there are other specific deterrents to their application, as outlined in the respective sections of this review.

Based on the outline above, the conclusion that microalgae or fatty acids, perceived as food, have therapeutic potential for treating infections is reached. Chlorella has been known to have an antimicrobial effect for 80 years, but its crude biomass is mostly used as food. Fatty acids have been known for a long time as trivial and ubiquitous components of foods and human physiology and organisms (some longer-chain PUFAs are abundant in fish oil and also precursors of eicosanoids and structural components of mammal nerve tissue). As paradoxical as it sounds, there is accumulating research described below that has begun to unveil their promising therapeutic potential.

Chlorella is a microalgae that is approved by The Food and Drug Administration (FDA) as GRAS (Generally Recognized as Safe) and is consumed as a dietary supplement [11]. Indeed, a GRAS approval does not exclude a possibility for infection treatment use, since undoubtedly antibacterial plant products (even with certified antibacterial use in dentistry) such as clove oil and oregano oil are also GRAS.

This review analyzes the outline of algae process development under the integrated biorefinery concept [12,13]. An advantage of microalgae is that they do not require arable land, can be grown in many substrates with minimal requirements [13] in the frame of system analysis theory [14], and fulfill the idea of integral utilization of algae biomass and their metabolites. The visibility of microalgal technology, in fact, has to pass through conditional optimization of all unit operations (microalgae process development [15,16], photobioreactor (PBR) design and scaling up [17,18], and the downstream processing) for minimal time and with minimal scientific efforts at minimal costs. As a first step of the outline, analysis of the engineering approach of the strain isolation, identification, and its ability to synthesize HVP under given operational conditions [19] and nutrient medium is fundamental. Modern approaches to calculating macro- and micro elements can be considered as a base for an effective first step in the optimization of microalgal kinetics [20]. Its effective modeling, optimization, and design determine all the other steps of upstream process development. The techno-economic analysis should be taken into account under the microalgal biorefinery concept [21]. It is especially important to notice and to know how the production of metabolites relates to the growth of the strains. This is directly linked to the kinetics optimization procedure.

Both marine and freshwater microalgae produce, through various metabolic pathways, a wide variety of chemically active compounds. They act as a chemical defense against herbivores under environmental stress and high competition for space. These molecules have antibacterial, antialgal and antifungal properties, being effective also against bio-fouling [22].

Microalgae are commercial sources of some high-value products (HVP), mainly pigments (carotenoids and phycobiliproteins, which are both food colorants and antioxidants), vitamins, and PUFAs. They are developed in the nutrition and health markets because of their nutraceutical value [2]. For example, carotenoids and vitamin E are antioxidants and may be not approved drugs but are used as food supplements for anti-inflammatory and other effects. Carotenoids are also anticancer compounds [23]. PUFAs are important and fundamental for human metabolism, membrane fluidity, and electron and oxygen transport, and they have antibacterial and anti-inflammatory qualities, anti-carcinogenic effects, and improve cardiovascular diseases [22,24,25,26]. Two kinds of PUFAs are prescription drugs (alone or in combination)—Vascepa^® (Amarin, Dublin, Ireland) and Lovaza^® (Woodward Pharma Services LLC, Wixom, MI, USA)—for lowering triglyceride levels [27]. Marine microalgae are the main primary producers of ω-3 PUFAs, especially the longer chain ones [28].

Microalgae may have resulted in many food supplements and few approved drugs; however, supplements are also used for medical and pharmaceutical applications. They can also be part of therapeutic schemes for chronic diseases, e.g., saline solutions (alkaline citrates), unregistered as drugs, are prescribed for kidney oxalate stones [29].

There are other important medicinal benefits and potential uses of microalgae and their compounds. The mycosporin-like amino acids have ultraviolet light protective effects, the sterols have pharmaceutical applications, and the phycotoxins are cytostatic [23]. Other beneficial biological activities include immunomodulatory, anti-obesity, anti-angiogenic, and neuroprotective [22]. Microalgae are considered one of the most important sources of antimicrobial molecules, including fatty acids, proteins, vitamins, pigments, etc. [22,30,31].

This review reflects the current knowledge of the antimicrobial potential of many Chlorellaceae and Scenedesmaceae species and the features to produce HVP. Another reason for choosing these microalgal families is that, in one report, they generally exhibited more antibacterial activity than, e.g., Dunaliella [32]. The cited studies on the in vitro antimicrobial activity of the two genera can also be viewed as a means to reveal their and their metabolites’ wastewater (WW) decontamination potential. Notably, researchers have to have in mind the complexity of the microalgal system and its relationship with upstream and downstream processes in order to reach the final step of the market realization. The so-called integrated biorefinery concept gives a solid base of cost-effective and robust microalgal-based biotechnology.

2. Why Are Chlorellacea, e.g., C. vulgaris and Scenedesmaceae, among the Most Commercially and Industrially Used Microalgae?

2.1. Overview

Because these taxa are rich sources of HVP, they are appreciated and vastly exploited in biotechnology, although mainly as biomass and extracts from it, for protein-rich food, feed, cosmetics, and bioethanol. Examples of reviews covering that topic are those of Mobin et al. (2017) [33] and Bhalamurugan et al. (2018) [34].

Scientists are divided on whether cyanobacteria such as Spirulina (Arthrospira) and Nostoc are (micro)algae, as they would be nearly the only group of the Bacteria domain in this category. Many credible scientific sources still include them, as the tradition is since (micro)algae is an informal term and do not form a natural group that has descended from a common ancestor. Its eukaryotic members are species from multiple distinct clades, both from the kingdom Plantae sensu lato and not [4,5,6,7]. Cyanobacteria share many characteristics in terms of physiology and ecology, and, especially, many biotechnological applications with the eukaryotic microalgae [35]. For those reasons, they are considered microalgae in this paper.

Over the last 20 years, the biotechnological utilization has focused on four major microalgal species: Spirulina (Arthrospira platensis), Chlorella vulgaris, Dunaliella salina, and Haematococcus lacustris [33]. The most commonly exploited microalgae for the production of bioactive compounds of pharmaceutical interest include Arthrospira, Chlorella, Dunaliella, Haematococcus, and Nostoc [34].

The commercial cultivation of microalgae began in the 1960s in Japan, and Chlorella was the first targeted organism. Chlorella is the most cultivated eukaryotic alga since it is widely used as a feed supplement and health food highly rich in protein. It produces biomass fast—its photosynthetic efficiency can reach 8%, which surpasses that of crops with high efficiency, such as sugar cane [36]. Chlorella and Chlorellaceae are freshwater taxa, although there are exceptions, such as the marine species Chlorella salina.

Moreover, only a few from the approximate 40,000 species of algae have been used by the food industry and Chlorella vulgaris Beijerinck is among them. It is a nutrient-dense superfood containing, as percentages of its dry weight, 18 amino acids, 20 vitamins and minerals, 12–28% carbohydrate, 2–46% lipids, and, notably, it can reach 60% protein. In addition to being directly manufactured into health products like tablets and capsules, it is also used as a supplement to noodles, breads, biscuits, candies, ice cream, bean curd, and other common foods to enhance nutritive and health values [22], as well as coloring matters for food stuffs [33]. An additional reason for this alga to be used as an antimicrobial food supplement may be the presence of flavonoids. The special interest in promoting it in the form of a new medicinal food supplement also comes from its negligible side effects [37]. Until today, Chlorella (together with Spirulina) has been the most commonly sold food-microalgae because both of them can grow very fast. Chlorella is widely used in (health) beverage production [33]. Chlorella spp. e.g., C. vulgaris are also used as animal feed and for extraction of carbohydrates. The increase in global production of Chlorella (2000 t dry weight/year) suggests that in the years to come, microalgae may prove to be a fascinating source of food for humans. Commercial cultures of Chlorella spp. have been produced in PBRs, large circular tanks, and circular or mixed open ponds with paddle wheels. Chlorella’s commercial market value is over 1 billion USD. The production has a compound annual growth rate of 5.3% [34].

A nucleotide-peptide complex, the so-called Chlorella growth factor, is one of the unique properties of the species, allowing the high growth rate of one cell multiplying into four new cells approximately every 20 h. Chlorella is, arguably, the food substance known to be the richest in nucleic acids, and spraying pepper seedlings with Chlorella extract has increased plant height [38,39].

In regard to carotenoids, β-carotene is an antioxidant that works as a vitamin A precursor. Dunaliella spp., e.g., D. salina, are the best source of β-carotene (up to 14% of dry biomass) and sources of production. They are followed by Tetradesmus almeriensis (Scenedesmaceae), which is not industrially used for the purpose yet but is showing promise for production [34]. Scenedesmaceae is a fresh and brackish water family. The carotenoid compounds lutein, zeaxanthin, and canthaxanthin are used for pharmaceutical purposes mainly as antioxidants and for food colorants, e.g., in orange juice and curiously for chicken skin and plumage coloration [33]. The documented for carotenoid production microalgal species are D. salina, C. vulgaris, and Haematococcus pluvialis. Auxenochlorella protothecoides, H. lacustris, the mentioned T. almeriensis, etc., also show promise as sources [40]. When culture conditions are optimal, the biomass of C. vulgaris accumulates a high quantity of secondary carotenoids. This strain is characterized by high volume production of β-carotene [41]. Besides being rich in astaxanthin, the strongest antioxidant found in nature, [42] and lutein, Chlorella is a commercial source of canthaxanthin. All three carotenoids have been in regular use as pigments. For example, they have been added to the feed for salmonid fish, trout, and poultry to enhance the reddish color of the fish or the yellow color of the egg yolk [43]. Astaxanthin is mostly used in aquaculture, while lutein is also highly used as a source for animal nutrition [44]. In the present day, the marigold flower is the only commercial source for lutein [45]. However, because of some drawbacks, alternative sources are searched for, and T. almeriensis is a promising source for lutein production with an approximate content of 4.5 mg/g dry weight [46], followed by Chromochloris zofingiensis (Scenedesmaceae) and A. protothecoides. The cost of 1000 mg of lutein from an algae source is approximately USD 2.5. So far, H. pluvialis is the only microalgal commercial source of astaxanthin. However, similar reasons drive the exploration of alternative algal producers, such as C. zofingiensis [42]. The microalgae T. almeriensis and Nannochloropsis oculata are good candidates to produce zeaxanthin. For example, T. almeriensis yields 0.34 mg/g of zeaxanthin, and the cost of 1000 mg of zeaxanthin from an algae source is USD 10. Scenedesmaceae species could also be used as rich astaxanthin and vitamin sources (C, B1, and B2). The carotenoids β-carotene, lutein, astaxanthin, canthaxanthin, zeaxanthin, violaxanthin, neoxanthin, and antheraxanthin, etc. are among the biomolecular compounds of Chlorella spp., together with chlorophyll-a and b [47,48].

C. vulgaris also has a wound-healing ability which is better in comparison to calcium sodium alginate (brown algae) dressing [41]. Chlorella spp. contain an important compound known as β-1, 3-glucan. It works as a free-radical scavenger, boosting the immune system and reducing blood lipids like cholesterol. It also has antitumor properties and is effective for wound healing, body detoxifying, and treating stomach ulcers. Chlorella extracts have hepatoprotective, blood-sugar-lowering, and hemoglobin-boosting properties. Chlorella spp. are rich in α-carotene, β-carotene, lutein, ascorbic acid, and α-tocopherol, and these compounds can contribute to decreasing the incidence of certain cancers and preventing macular degeneration. They are also rich in chlorophyll, carbohydrates, lipids, and B vitamins (B1, B2, B6, and especially B12).

Chlorella, Dunaliella, and Scenedesmaceae are among the commonly used species of microalgae for farm and aquaculture feed. Scenedesmaceae species can be regarded as a source of monounsaturated, polyunsaturated, and saturated fatty acids in feed [34]; however, their high price is a drawback [25,26].

Various cosmetics frequently contain Spirulina, Chlorella, Dunaliella, and Haematococcus. These microalgae extracts are used to replenish face and body lotions and creams. Additionally, they are a component of shampoos, hair masks, and sun protection creams. The synthesis of skin collagen is reportedly stimulated by the C. vulgaris extract. They also prevent wrinkles from appearing on the skin’s surface and aid in the regeneration of fibers. Protein-enriched Spirulina extract slows down the aging process of the skin [33,34].

Chlorella spp., Scenedesmus spp., and Spirulina fusiformis are among the most commonly used microalgae for bioethanol production. Desmodesmus abundans and Tetradesmus obliquus (Turpin), M.J. Wynne (Scenedesmaceae), and Chlorella sorokiniana are among the microalgae commonly employed in the production of hydrogen [34].

The most common and important for human physiology ω-3 PUFAs are α-linolenic acid (ALA, 18:3, ω-3), eicosapentaenoic acid (EPA 20:5, ω-3), and docosahexaenoic acid (DHA, C22:6, ω-3). EPA acts as a precursor for eicosanoids, such as prostaglandins and leukotrienes. DHA is often among the fatty acids of phosphatidylserine, which, while not only limited to nerve tissues, is the major phospholipid of the cerebral cortex, thus making DHA an important component of the human brain and neuronal phospholipids, retina, etc. [49]. EPA and DHA are the prescription drugs (alone or in combination) Vascepa^TM and Lovaza^TM, used for lowering triglyceride levels [27].

Microalgae, in general, are abundant sources of the three acids. ALA is synthesized in high amounts also in terrestrial higher plants, but in regard to EPA and DHA, few animals (e.g., fishes) can synthesize them with low yield and accumulate them through consumption of microalgae and other microorganisms. The conversion of α-linolenic acid to EPA and DHA occurs at an even more limited rate, with inefficiency, in mammals, including humans. Therefore, ensuring a sufficient level of these acids on a diet that lacks them is very difficult; hence, they must be consumed through the diet, and all vertebrates largely rely on the intake of EPA and DHA from food.

Therefore, it is mainly microalgae that are the primary and original producers of ω-3 PUFAs, especially the longer chain ones, EPA and DHA, and marine microalgae are especially important in that regard [28].

Fish, being higher in the food chain, concentrate EPA from the microalgae they consume, and commercially available EPA is most often derived from fish oil. Nevertheless, the facts that fish oil tends to be one of the most expensive nutritional product on the market, the diminishing fish resources, the need for sustainability, the added risk of contamination such as mercury, the vegetarian preferences of some people, and even the unpleasant flavor and odor for others make microalgae an especially important alternative source of PUFAs [28,50]. Substituting algae for fish oil offers a great chance to develop alternative ω-3 PUFAa for pharmaceutical and nutraceutical uses [22].

PUFAs reach 35–40% of the total lipid content from Chlorella, comprising up to 24% linoleic acid and 27% ALA in C. vulgaris [51]. Some sources claim that chlorophytes (Chlorella, Dunaliella, and Scenedesmaceae spp.) have low and even trace amounts of EPA (up to 3%) and are low and even deficient in DHA [25,26,52], but that is not entirely true. Besides ALA, Chlorella spp. is a ω-3 source also in the form of EPA [53]. However, DHA was found in C. sorokiniana [54]. Auxenochlorella pyrenoidosa and Chloroidium ellipsoideum (Chlorellaceae) are claimed to be among the most common algae employed for the production of DHA-rich algal oil [34]. ALA is the dominant ω-3 fatty acid in T. obliquus, which was found to be deficient in EPA and DHA [55]. However, Scenedesmaceae members have shown high EPA content [56]. Nevertheless, the species rich in DHA are usually dinoflagellates and other marine microorganisms [57], and chlorophytes are not suitable for a single-species diet.

2.2. Production Costs and Economic Robustness of HVP from Chlorellaceae and Scenedesmaceae

World companies first started exploring microalgae for biodiesel and other fuels, but the production costs turned out to be higher than those of petroleum-based fuels. The knowledge about microalgal HVP and nutraceuticals is owed to this fact as it lead to the investment in their intensive research [58].

A detailed techno-economical analysis and a summary of the cost of microalgal low-, middle-, and high-value products, as well as the means of HVP extraction and the optimization for carbon emission reduction, are previously published by Kroumov et al. (2017) [21] and (2021) [59].

Chlorellaceae and Scenedesmaceae are commercially produced mainly as biomass at present. Although academia stresses that HVP from these two families, including antimicrobial ones, are very promising for industrial production [2,60,61,62], single HVP from Chlorellaceae and Scenedesmaceae are currently expected to be isolated and offered mostly on a pilot scale. Apart from the established commercial microalgal HVP production from certain other microalgal species, a number of companies have implemented the biorefinery techniques, enabling cultivating microalgae on a pilot scale and generating diverse products (primarily energy carriers, pigments, and PUFAs) from the same biomass [63].

The exact production costs for specific HVP from a defined species are difficult to calculate. For example, both synthetic (dominating the market) and natural (e.g., microalgal) astaxanthin have been produced for at least three decades. However, which system had the lower cost was claimed to be unknown up until 2011. The analysis showed that the production cost of astaxanthin and microalgae biomass can be as low as $718/kg and $18/kg. This cost might be lower than that of synthetic astaxanthin, and the lack of absolute certainty shows the difficulty in the calculation of production costs [64]. The market demand for natural astaxanthin, the slow growth rate, low biomass production, and ease of contamination with fast-growing microbes ensured the search for an alternative to H. pluvialis sources rich in astaxanthin, such as C. zofingiensis [42].

As mentioned, the expensive costs of harvesting marigold flowers, the commercial lutein source, the land and water use, and the seasonality of the growth [46] drove a comparative analysis. It showed that lutein from marigolds consumed more land and water but less nutrients and power. Therefore, the absolute cost of production is difficult to assess and depends on factors, i.e., microalgal lutein production is suitable for establishments with limited land and water resources and high personnel costs. Therefore, research and development for affordable microalgal commercial lutein production is needed [65].

The health market prefers natural β-carotene because it contains both trans and cis isomers. Although some sources claim that the production of microalgal carotenoids requires less labor and the harvesting process is easier, that production is still very expensive, with significant costs associated with labor, capital, and processing [66]. For those reasons, microalgal β-carotene can be purchased for as high as 700 €/kg, while synthetic forms cannot be purchased for more than half that amount [43].

Microalgal HVP commercialization, in general, undoubtedly has many benefits. As mentioned, algae have higher photosynthetic efficiency compared to terrestrial plants, hence high growth rate and higher yields. They do not require arable land and biomass can be harvested regardless of seasons and geographic area [67]. Microalgal HVPs are usually produced within a biorefinery model, which increases revenue, in essence, an extraction and yield of different co-products, in addition to the ecological benefit of diminishing anthropogenic waste [58,67].

Despite all the listed benefits, and even though microalgal HVP or other ingredients have high demand and price [58,68], their commercialization has not yet reached its peak. This fact is typically ascribed to the exorbitant expenses associated with the extraction and purification procedures currently in use. These high costs, which challenge the profitable commercialization of the compounds, are typically linked to the losses during purification steps and the complexity of the processes that require a lot of organic solvents, have a significant environmental impact or involve more specialized human resources and sophisticated equipment.

For instance, despite microalgae being the only vegetarian source of EPA and DHA, the high cost of fish oil, and the insufficiency of ω-3 products in the market, production costs of PUFAs from microalgal biomass are yet far from competitive, mainly because of the little proportions of PUFAs within the biomass [63].

The listed challenges require efficient and inexpensive extraction and purification processes in order to design new products, opening up new markets [68]. It is necessary to decrease product losses during purification steps [58] and to develop straightforward, efficient processes of hyperaccumulation of the greatest number of HVP [68]. Innovative strategies, such as system analysis theory and computational fluid dynamics [21], are currently being researched and applied; however, hyperaccumulating strain selection, culture condition optimization [67], and minimizing losses due to product degradation [58] are still the holdups to be currently resolved. Economic and environmental studies regarding HVP production from microalgae are needed, as it is a market-driven process. The successful establishment of microalgae products in general, as well as from Chlorellaceae and Scenedesmaceae, depends on all this to ensure a reduction in the cost of production [68].

3. Microalgae-Based Wastewater Treatment (WWT), Potential for Pathogen Removal and Utilization of Biomass

3.1. General Process

As an alternative to removing nutrients and heavy metals from WW and to stop the spread of microorganisms carrying water-borne diseases, microalgal treatment of WW has been studied for more than 50 years. Algal WWT systems are now generally acknowledged to be just as effective as conventional treatment systems [69].

WW-integrated algal biorefinery has recently attracted considerable interest and attention. The state of the art in the field provides evidence that municipal [70,71], agricultural [72], and industrial [73,74] WW can be sources of chemical elements that serve as nutrients for microalgal growth. Microalgae have been rigorously investigated because of their high metabolic flexibility as promising bioremediation agents [75] to be used in tertiary WW treatment for the removal of conventional pollutants, such as nitrogen (N) and phosphorus (P). Chemically- and physically-based technologies to remove excess nitrogen and P from WWs are expensive [76]. In this regard, microalgae are also a sustainable and promising technique for antibiotic removal [76,77]. In addition, microalgae could cooperate with some aerobic bacteria in wastewater, exchanging CO₂ and O₂. This process results in WWT, where the bacteria oxidize the organic compounds.

Furthermore, the ability of microalgae for multi-contaminant removal can be greatly improved using immobilization techniques. Using algal cells in fixed positions, such as biofilms or immobilized cells, leads to better and faster remediation. This accelerated reaction rate is due to an increased cell density and other factors [69].

Chlorella vulgaris has several advantages for WWT. It accumulates nutrients up to 60% of its biomass, and, as noted, they might possibly be processed to HVP. In addition, as Virdi (2019) lists, it grows very quickly, as noted, compared to other microalgal species. It has been found to grow well in WW and can grow dynamically in the presence of essential nutrients that can be readily obtained from any kind of WW. It has the capacity to eliminate organic compounds and heavy metals from its nutrient medium as well. The species is adaptable for WW remediation because it can grow in a variety of environments with different nutrient concentrations, temperatures, and pH levels. Furthermore, all three growth conditions—autotrophic, heterotrophic, and mixotrophic can support the growth of Chlorella spp. [69].

Chlorellaceae, Scenedesmaceae, and Arthrospira species are among the most researched single species for WWT, apart from mixed consortia of microalgae. Virdi (2019) lists several promising studies on WWT and the aforementioned Chlorophyta families [69], and we would like to highlight some more.

Olive mill and industrial WW can be effectively treated by microalgae, specifically Chlorella and Scenedesmaceae [72,78]. For instance, Tao et al. (2017) experimented with cultivating Tetradesmus lagerheimii using biosludge digestate from pulp and paper mills. The findings indicate that, at a biomass concentration of 2.9 g/L, Chlorella and Scenedesmaceae spp. could remove more than 99.99% of NH4⁺-N and more than 96.9% of PO₄⁻³-P. Furthermore, it has been reported that Chlorella sp. can withstand high CO₂ concentrations and convert them into biomass [79]. Wu et al. (2017) showed that Chlorella sp. could grow using raw textile WW [72], and 84% and >60% of the NH4⁺-N removal efficiency and chemical oxygen demand (COD) removal efficiency were achieved with aeration at 0% and 10% dilution rate, respectively. Chlorella sp. cultivated on brewery WW was also found to remove 94% ammonia and 88.5% nitrogen with a decrease in COD [80]. Tetradesmus obliquus has proved to be able to remove ammonia and nitrogen from municipal WW effectively [81]. Heavy metals like Cd²⁺ have the ability to stimulate the production of extracellular sulfated polysaccharide (EPS) and protein by C. vulgaris at concentrations lower than 0.5 mg/L. This can lead to faster interactions between Cd²⁺ and the bacteria in EPS fractions, which in turn improves the ability of C. vulgaris to remove ammonia and phosphate [82].

Special attention has to be given to heavy metal ion removal from WWs and the main mechanisms of ion uptake by the microalgal cells [83]. Since green microalgae are the most widely used species and can adsorb metals, they have a great deal of potential for treating WW contaminated with them [76]. Desmodesmus communis (Scenedesmaceae) was able to reduce nickel and zinc from WWs by 99%, and Chlorella miniata reduced the amount of nickel by 70% [84]. A constraint to the removal process is the presence of organic carbon sources in the growth medium [85,86,87]. Because the WW from the dairy industry is typically high in COD and organic matter, mixotrophic microalgal strains—such as Scenedesmus sp. and Tetraselmis sp.—might be particularly effective at treating these waste streams [88]. While the produced water from the oil and gas industries may be toxic to many microalgal species, some of them, such as Chlorella sp., Dunaliella sp., Scenedesmus sp., etc., may be able to withstand and even thrive in the (pretreated) produced water [89]. EPS from Scenedesmus sp. and C. vulgaris played an important role in the settling of algal biomass via bioflocculation for alga harvesting from WWT for downstream applications [82].

A commercial microalgae system can also be linked to plants, factories, or farms, which emit flue gases, including CO₂. The microalgae would not only metabolize the CO₂, thus fueling their biomass, but this biomass could be used for inoculating WWs to achieve WWT. That process would help the environment to be preserved when using also atmospheric CO₂ [77,90,91]. These two benefits of WW and CO₂-integrated microalgae-based biorefinery are a realization of the clean circle and a profitable economy [91,92,93]. Such a type of microalgal integrated biorefinery concept is given in Figure 1.

3.2. Pathogen Inhibition

Even after the decrease in mortality from infectious diseases, they remain the third leading cause of death, responsible for 14% of the deaths globally in 2019. Among them, diarrheal diseases are second after pneumonia, claiming 1.5 million lives annually, or 2.7% of the deaths in 2019 [94]. According to an estimate by the World Health Organization and other estimates, the mortality of water borne diseases exceeded 3.4 million people in 2001 [95] and between 1.4 to 3.5 million people in 2019 [96,97], remaining one of the leading causes of human morbidity and mortality worldwide [98]. This occurs mainly in low-income countries, mostly due to anthropogenic pollution, and the victims are mainly children under 5 years old. Therefore, improving the global WWT capacity is warranted.

The antibacterial effect of microalgae from environments little affected by anthropogenic influence (natural biotopes) is weaker compared to cultures of microalgae from highly polluted waters (WWs). It is likely that this reflects a defense mechanism in a highly competitive environment of microalgae vs. bacteria [99]. For example, extracts of Dunaliella spp. isolated from clean versus polluted waters were studied by Lustigman (1988). Only species from contaminated water produce a heat-labile, non-proteinous substance that inhibits Escherichia coli [100].

This defense mechanism could be utilized in purifying the WW from pathogenic bacteria including those that have resistance to clinically established antibiotics. This presents the connection between the microalgal antimicrobial effect and WWT, leading to prevention or decreasing the risk of epidemics. This decontamination is a process close to the in vitro conditions of experiments testing the antibacterial activity of microalgae (but in a larger volume, which requires scale-up investigations).

Microalgae have been studied (although to a lesser extent than for conventional pollutants) as promising bioremediation agents in tertiary WW treatment for pathogen removal, such as coliforms [75]. An additional instance demonstrates that there were no direct effects observed when microalgae and Oligella, the predominant risk group 2 (RG₂) pathogen, from piggery WW were co-cultivated. However, the mixotrophic (but not heterotrophic) microalgal cultivation induced a sudden change in the bacterial community, and the RG₂ pathogens were reduced by 63% after the raw piggery WW underwent microalgal treatment. The findings demonstrated that this was an indirect effect of microalgae linked to bacteria—Oligella was indirectly connected with microalgal growth via other bacteria [101].

In fact, C. vulgaris was subjected to one of the first WW pathogen removal research. Interestingly, soon after discovering chlorellin, Pratt et al. (1951) described its practical application during World War II. The authors claimed that it was safe to release open sewage from the military installations into nearby streams due to its high C. vulgaris inoculation. Actually, the coliform count was often lower in effluent from pools heavily inoculated with C. vulgaris and not chlorinated than it was in effluent from chlorinated pools that contained no C. vulgaris. This happened primarily during the summer months when high light intensity and roughly vertical sun rays favored the growth of the algae [102]. More recently, C. vulgaris was able to remove phosphate-phosphorus (PO₄–P) and reduce coliform proliferation and even the antibiotic-resistance genes the sul1 and bla_TEM in the bacteria as well as the intl1 gene. The last is encoding the class 1 integrons integrase, found in pathogenic bacteria of humans and domestic animals and commonly used as a marker for anthropogenic pollution and related to sul1 as it integrates it in the bacterial genome. However, autochthonous microalgal species belonging to the classes Cyanophyceae, Chlorophyceae, and Bacillariophyceae proved to be more efficient [103]. For reasons already described, Virdi (2019) also selected C. vulgaris for his study related to WW remediation and pathogen inhibition of E. coli and Klebsiella pneumoniae. He chose these bacteria because they are frequently discovered in wastewater and notorious for harboring multiple water-borne illnesses, including pneumonia, dysentery, diarrhea, and acute gastroenteritis. Chlorella vulgaris excelled in both tasks, and the efficiency of phycoremediation was improved by immobilizing algal cells in beads of sodium alginate. However, the antimicrobial tests were of an extract in vitro and not in situ or co-cultivation in the WW [69].

The antimicrobial microalgal metabolites can be stored inside the cell (endometabolites) or secreted outside of the cell (exometabolites). Therefore, supernatant and pellet fractions differ in their biological activities [104]. Microalgal exometabolomes remain understudied, and some exometabolites were found to have antialgal effects, suggesting potential roles in allelopathy [105]. Nevertheless, practice shows that antibacterial and antifungal metabolites are more often stored inside than secreted, and the supernatants rarely have antimicrobial activity [106,107]. Another example illustrating this is the fact that the maximal content of free fatty acids (FFAs) in algae culture medium is reported to be in the stationary phase, as it is directly correlated to the process of autolysis when they are released. That could also happen in natural conditions in a water basin. Since FFAs have a general antibacterial effect, their release could secure the resistance and stability of the natural biocenose to the settlement of allochtonic pathogenic microbes. The rate of their elimination could rise with microalgae mass dying and this is one of the mechanisms of self-purification of the water basin. FFAs and other intracellular substances released during microalgal destruction could also play a role in the antibacterial effect of peloid (mud or clay often associated with water basins and used for therapeutic purposes) [32,108]. An example of such effect is the peloid containing extracts of C. vulgaris, T. obliquus, and Desmodesmus magnus var. magnus that had a pronounced antibacterial effect against four opportunistic bacteria—Staphylococcus aureus, E. coli, Klebsiella ozaenae, and Pseudomonas aeruginosa [32]. There is a hypothesis that other microalgal antibacterial compounds are also released after autolysis, or are alternatively induced by the presence of bacteria [109].

3.3. Utilization of Microalgal Biomass

On the other hand, the harvested microalgal biomass could be used for several applications, and this, naturally, would reduce the cost of microalgal cultivation, as there would be little to no requirement for additional nutrients. There is a disagreement between published scientific opinions about these applications because of the obvious safety concerns with microalgae cultivated on WWs (MCW). For example, while some authors state they could not be used for animal feed or the cosmetic industry, others believe that their HVPs are able to enter human nutraceutical and pharmaceutical production, given that they meet the regulatory requirements in terms of toxicology and pathogenic bacteria [69,103,110,111]. If, in the future, MCW is processed into antimicrobial (and other) pharmaceutical agents, another link between the antimicrobial effect of microalgae, WWT with such algae, and antimicrobial MCW would be provided.

The European Union legislation explicitly includes some types of WWs as input materials for obtaining ingredients for fertilizers (EU2019/1009) [112], and the use of MGW is not explicitly prohibited for use in animal feed (EU 68/2013) [113] and cosmetics (1223/2009/EC) [114].

Su et al. (2023) and Al-Jabri et al. (2021) [89] state in their reviews that microalgae cultivated on WWs cannot be used for human applications but only in manners that do not directly affect the human food chain. They could be used as animal feed (except those grown on urban WW), biofertilizers and biostimulants [115], biofuels, bioplastics, bulk chemical production, nutrient recycling, and as ingredients for the cosmetic industry.

Among them, the animal feed application is actively researched. The same authors outline several studies where microalgae cultivated on WWs (MCW) stimulated animal growth performance when fed to fish and pigs [116]. Among them, a consortium of Chlorella sp. and Scenedesmus sp. from anaerobic digested piggery effluent and used for pig feedstock had meal approximately 1.9 value-adding use of ω-3 and ω-6 PUFAs [117]. C. vulgaris and T. obliquus grown on effluent from crabs seafood production and later used for fish diet supplement had high protein and high carotenoid content for attracting colors [118], supporting the view that MCW from aquaculture WW can be used for aquaculture feed [111]. Scenedesmus sp. cultivated in chicken slaughterhouse WW and later fed to fish showed high EPA content [56].

The authors stress that animal-fed utilization is possible only if the end algal product is safe, which would be ensured by processing if needed and strict microbiological control for pathogens [116]. The safety of some MCW, including Chlorella and Scenedesmaceae, in terms of toxicology evaluation and pathogenic bacteria load, suggests no issues for the use of this biomass as an animal feed and has been found to meet regulatory requirements of conventional animal feedstock [119].

In the review of Srimongkol et al., 2022 [103], several studies about species of the two families, alone or combined with other microalgal species, are outlined. They include C. vulgaris, C. zofingiensis, T. obliquus, etc., grown on WWs such as municipal domestic aquaculture, piggery, and the end products of MGW were biodiesel, ethanol, hydrogen, methane, proteins, lipids, and carbohydrates.

If used as fertilizers and biostimulants in the European Union, regulations for permitted levels of heavy metals should be met [116]. In order to produce biodiesel from MCW, the wet biomass needs to be dried, which could be very energy-intensive; therefore, biogas (methane) energy production is more cost- and energy-effective. Bulk chemical production includes alcohol, lactic acid, glycerol, and EPS which could be used as a gelling agent, thickener, stabilizer, etc. [89,116].

Limitations of MCW utilization exist. A success has been demonstrated mostly at a relatively small scale (mainly indoor) while longterm, large-scale demonstrations are limited, thus needed [76,89,116]. One of the major and practical limitations faced in microalgal WWT systems is the harvesting or separation of the algal biomass from the treated water discharge. In fact immobilization of algal cells has been proposed as one of the ways for circumventing that problem [69]. Nevertheless, the potential of microalgae under control conditions for WWT in order to purify WW and, at the same time, to be a source of biomass with economic benefits is extremely promising. Having experience with culturing methods, the researcher may direct the metabolism of microalgae by combining different engineering approaches and complex working conditions where stress factors can be controlled in the proper way, in particular PBR design. This is because the phycoremediation procedure includes physical, chemical, and biological influences for WWT. This field is very challenging and demands efforts in order to explore the antimicrobial potential of microalgae biomass and to identify, isolate, and optimize the production of potential valuable antimicrobial agents, which will dramatically decrease the cost of the overall microalgae technology for phycoremediation of wastewater which in fact represents the concept of integral biorefinery.

4. Antimicrobial Activity of Microalgae, with a Focus on Chlorellaceae and Scenedesmaceae

4.1. Antimicrobial Metabolites from Microalgae

The rising antimicrobial resistance to established antibiotics is an incentive for intensive research of their alternatives and plant or algae natural products provide immense opportunities. The significance of compounds originating from land plants and aquatic organisms is being increasingly acknowledged by the pharmaceutical industries [120]. Apart from oncology and immunoregulation, the therapeutic effects of natural products and drugs derived from them are mainly achieved in infectious diseases, as their targets are major crossroads in signaling pathways in cancer cells or pathogens [121]. Some of the benefits of natural products include their economic advantage and fewer side effects. The high cost and slow rate of the antibiotic drug discovery pipeline processes determine the need for fueling this pipeline with new agents [122]. Natural products often do not have the significant side effects of synthetic drugs, such as hypersensitivity, allergic reactions, and immunosuppression. In addition, substances from the plants used in traditional medicine have a proven effect and compatibility with the living organism [123,124,125]. Microalgae have been used as food from ancient times, such as Arthrospira—by Aztecs and natives of Chad—Nostoc—by ancient Chinese people—and Chlorella in the present time [126].

Another particular example is drugs for amoebic diseases, which have been reported to be mutagenic. It has been suggested that natural products have not only more potential to treat amoebic diseases but are less prone to develop mutagenic effects in the host; the amoebas have a smaller possibility of developing resistance [127]. Although microalgae may not always be as effective as chemical fungicides, they pose fewer environmental risks and fewer food safety problems [22].

Adhoni et al. (2016), after obtaining effects as high as those of gentamycin, called the organic extracts of different algal species, including C. vulgaris, “herbal antibiotics” and were even optimistic that they could be used in place of synthetic or chemically manufactured antibiotics [22]. We hope that, even if found not suitable to replace antibiotics, they will supplement them and acquire more deserved clinical presence. They can be utilized in combination and synergism with clinically approved chemotherapeutics so that the dose of the latter is lowered, leading to fewer adverse effects.

The antibacterial activity within the same species of microalgae can vary due to ecotypes that are tailored to suit distinct environmental conditions [109]. As noted, the antimicrobial metabolites of microalgae can be exometabolites or endometabolites released after the loss of algal integrity. Therefore, supernatant and pellet fractions differ in their biological activities [104]. Nevertheless, practice shows that they are more often stored inside than secreted, and the supernatants rarely have activity [106,107]. The yield of the antimicrobial compounds can be increased by recombinant techniques [30]. As noted, there is a hypothesis that these compounds can also be induced by the presence of bacteria [109].

Because of the new types of compounds discovered in recent years, the identification of the compounds directly responsible for the antimicrobial potential of algae remains a relatively nascent field of research. Furthermore, studies are still in their early stages from a commercial perspective [1,2]. Nevertheless, research has shed light on numerous antimicrobial compounds and their mechanism of action.

4.1.1. Fatty Acids

Generally, most fatty acids have high antibacterial properties. In fact, 10-undecylenic acid (C11:1n-1) is already used clinically to treat fungal infections of the skin and nails. In addition, there are numerous patents utilizing the antimicrobial effect of FFA for medical, agricultural, and other uses, as summarized by Desbois, 2012. Some studies have suggested that FFA may be used to treat systemic infections; however, the patented and most promising uses of FFAs in medicine are where conventional antibiotics are undesirable or forbidden, and especially topical and surface-based, such as preventing microbial colonization of medical equipment, eliminating drug-resistant pathogens from hospital patients’ skin and noses, and treating microbial infections of the skin, wounds, and mucosa. There are also patents for animals, e.g., for prevention and treatment of infections of plants, bees, hooves, mastitis, gastrointestinal infections, or as alternatives to antibiotics for increasing the growth performance of livestock. These substances could be helpful preservatives and disinfectants of food, feed, cosmetics, and medication. Desbois finds that, so far, the full commercial potential of antimicrobial fatty acids has yet to be realized [8]. This is likely because some of them can be unstable or bind nonspecifically to proteins [128], their other biological activities are a complication, their development would divert resources from the development of conventional antibiotics, there may be a perceived lack of patentable intellectual property, etc. However, there are solutions for these problems, and FFAs hold potential for commercial application, especially because they act through different mechanisms to most conventional antibiotics, and the evolution of inducible FFA-resistant bacteria is less problematic and frequent than with conventional antibiotics [8].

The short- and medium-chain saturated fatty acids are most often in the patent literature [128] but that does not exclude the PUFAs and other fatty acids from microalgae to be utilized in the future.

FFA, if extracted to their free form or resulting from autolysis, are among the most potent antibacterial compounds from microalgae, too, and the lipid composition of microalgal extracts influences their antimicrobial activity [1,24,44,129,130,131,132,133,134]. The Chlorophyta (green algae) phylum is known for its ability to synthesize a variety of PUFAs and other lipids. Other lipids, such as glycolipids, can also be antibacterial [135]. The microalgal properties against some specific human pathogens, such as E. coli, P. aeruginosa, S. aureus, Staphylococcus epidermidis, Klebsiella spp., and Bacillus spp., have been attributed to EPA, DHA, palmitoleic, lauric, oleic, lactic, arachidonic, γ-linolenic, stearic, and hexadecatrienoic acids [129]. These pathogens are significant since they are considered to cause water borne and vector diseases, nosocomial infections, pneumonia, etc., therefore causing drastic health impacts [37,99]. It is important to consider that certain compounds, such as cholesterol and vitamin E, can counteract the antimicrobial effects [130], for example, the antiprotozoal effect of PUFAs [10,136].

As FFAs are surfactants, they influence bacterial cell membranes. However, the precise mechanism of action of fatty acids is still not fully elucidated, as they may influence multiple cellular targets. The mechanism is discussed in detail, to the best of our knowledge, by Desbois and Smith, 2010 [128] and later by Chanda et al., 2018 [136]. Even small disruption of membrane integrity leads to electron leakage that may result in cell death. Greater destabilization and membrane damage likely lead to a change of cell surface charge, cell leakage, the entry of harmful components, and the reduction of nutrient uptake. It has been documented that algal FFAs inhibit electron transport systems and cellular respiration. The cell then undergoes lysis, after which peroxidation and auto-oxidation degradation products are formed [128,129,136,137,138,139]. Additionally, FFAs may start peroxidative reactions, impede bacterial fatty acid synthesis (block FabI enzymes) [128,136,138,140,141], and inhibit bacterial enzyme activity [128,136,139]. The antimicrobial mechanisms of PUFAs, including ω-3 ones, may also include the disruption of cell-to-cell communication and ATP production, alteration in membrane hydrophobicity, and increasing membrane poles, which causes cellular leakage [128,136].

Recent structure-function relationship studies suggest that fatty acids’ capacity to impede bacterial growth is dependent on both their chain length and degree of unsaturation [129,142]. It appears that fatty acids containing more than ten carbon atoms cause bacterial protoplasts to lyse. An example of this is EPA and linolenic acid from Ennalax costatus, which demonstrated activity against aquaculture bacteria [130].

4.1.2. Polyunsaturated Fatty Acids (PUFAs)

Polyunsaturated single-cell oils possess antimicrobial and anticancer properties [24]. Among FFAs, the longer-chain polyunsaturated ones, such as EPA and DHA, are especially active antimicrobials and are abundant in microalgae [1,24,44,129,130,131,132,133,134]. EPA and DHA have also demonstrated antibiofilm properties [143,144]. ALA has also shown an antibacterial effect by inhibiting methicillin-resistant S. aureus (MRSA) [47].

It is interesting that EPA, DHA, and other PUFAs are normally found in foods, e.g., fish oil and are components with a physiological role in the organism. Yet, in vivo results show that when increasing their concentration in the organism through consumption, genetic engineering, etc., they suppress infections and also have a prophylactic effect.

In their review, Alhusseiny and Beshbishihave cover the in vivo antimicrobial effect of fish oil or different ω-PUFAs such as DHA and EPA (ω-3), arachidonic and linoleic acid (ω-6), oleic acid (ω-9) against protozoa, such as Plasmodium spp., Trypanosoma, Toxoplasma, etc., in laboratory animals. Increasing these fatty acids in the administered diet or an intra-peritoneal intake route suppressed the infection, decreased parasitemia, and improved the survival of laboratory animals in comparison with the controls (with few exceptions). Specific effects such as decreased cerebral malaria, cardiac parasitism, caecal lesions, and number of Toxoplasma gondii infected cells, and increase in specific antibody levels, etc., were also observed [10].

In addition, increased production of EPA and DHA was achieved by expressing delta-6 desaturase and elongase genes in the liver of transgenic zebrafish. Interestingly, the survival rate of transgenic strains was 70%, while that of wild type was 20% 24 h post Vibrio vulnificus infection. Antimicrobial effects of DHA on Burkholderia cenocepacia using a Galleria mellonella caterpillar in vivo model also showed that ω-3 PUFAs have antimicrobial properties [136]. Transgenic mice that have large amounts of ω-3 PUFAs and low levels of ω-6 PUFAs in their tissues have demonstrated significant reductions in the T. gondii cyst burden in brain tissues [10].

Nevertheless, whether PUFAs exert that suppression of infection in vivo because of direct antimicrobial action, because of their anti-inflammatory effect, or because of both and to what extent is not known. For that reason, PUFAs are also looked into in the next section about immunomodulatory activities. Chanda et al. point out that ω-3-PUFA studies so far typically have focused on ameliorating inflammatory tissue damage rather than getting rid of the etiological agents. Since more evidence of the in vitro and in vivo broad-spectrum nature of fatty acids has accumulated, including direct antimicrobial effect, clinical studies are of utmost importance to substantiate the antimicrobial potential of ω-3 PUFAs, authors conclude [136]. Cheng et al. are of the opinion that EPA and DHA both inhibited bacterial growth directly, as well as enhanced the production of anti-inflammatory cytokines, thereby protecting zebrafish from V. vulnificus infection [145]. More studies aiming to understand the main pathway/s responsible for the activity of PUFAs are needed.

It is true that some drawbacks exist, for example, toxicity, the low oral bioavailability of PUFAs, the fact that vitamin E and cholesterol reverse their antiprotozoal effect, etc., but that could be overcome by technological approaches [10,136].

To date, unfortunately, as Firoozabad and Nasr conclude, the utilization of ω- and specifically ω-3 PUFAs as antimicrobial agents has not been widely appreciated, perhaps due to a lack of understanding of antimicrobial mechanisms, toxicity, and route of administration, but it is worthwhile to consider ω-3 PUFAs in the list of potential antimicrobial agents [9].

Altogether, PUFAs can be considered novel adjuvant drug candidates or alternative therapeutic agents for infections, but future studies are required [9,10,136]. Clinical studies on the effectiveness of ω-3 PUFAs against pathogenic microbes are lacking and, hence, required. Additionally, their benefits are that they have little effect on evolving antimicrobial resistance and are also safe for human use [9]. The incorporation of ω-3 FAs into chewing gums, tooth paste, etc., may provide other routes of treating and preventing oral diseases [136]. In addition, authors encourage dietary intake of PUFAs [10].

4.1.3. Other Compounds

In addition to fatty acids and PUFAs, another important antibacterial class of compounds is polysaccharides (PSs). They are produced and secreted by algae and eukaryotic microalgae, such as Chlorella spp. and Dunaliella spp., at relatively high levels [2,129]. The pharmaceutical and food industries have seen the successful utilization of algal PSs and sulfated PSs. EPS produced by microalgae has acidic properties and may be used as a medicinal agent [146]. The anti-adhesive characteristics of the EPS of certain microalgae against the adherence of microbes may account for the antibacterial action of PSs. Since other carbohydrates on cell surfaces have already been shown to serve as recognition sites for microbes to attach to, PSs may compete with them for this role. The EPS’s capacity to prevent the formation of biofilms may also be connected to its antibacterial action [147] and, therefore, to its anti-adhesive properties. The majority of data point to these compounds’ ability to alter biotic surfaces’ physical characteristics as their mode of action [148]. The distinct cell surfaces of Gram-positive and Gram-negative bacteria may account for the variations in the corresponding inhibitions detected by the EPS [146]. According to Rendueles and Ghigo (2012), PSs may alter the bacterial cell surfaces’ physical characteristics because they are surfactant molecules. PSs derived from microalgae may function similarly to exopolysaccharides from E. coli, which have the ability to prevent bacterial cell adhesins from causing autoaggregation [148,149]. Another mechanism might be the result of PSs binding with substances in the bacterial cell wall and membrane through glycoprotein receptors on the bacterial cell surface. This leads to increased cell membrane permeability, protein leakage, and bacterial DNA binding [137,150]. As sugar polymers, polysaccharides can also function as lectin inhibitors. Lectin is primarily found on the surface of bacterial cells, and it helps bacteria adhere to their hosts by attaching to the glycan substrates on the surface of those cells [151]. By competing with lectins’ sugar-binding domain, PSs prevent pathogen adherence that is dependent on lectin and the creation of biofilms, which lowers the risk of infection. Hydrocolloids’ mechanism of action could be linked to the fact that EPS wound dressings diminish scarring and wound secretions, which in turn lowers bacterial inflammation. [149]. Additionally, sulfated PSs have the ability to prevent infections by preventing their adherence to the gastric surface.

Antibacterial peptides are another class of compounds, although they are mainly cyanopeptides (from cyanobacteria, e.g., Oscillatoriales and Nostocales) [152,153,154]. Cyanopeptides are mainly non-ribosomal and cyclic peptides. Antibacterial microalgal peptides are considerably rarer, e.g., few peptides from Tetraselmis suecica and protein hydrolysates from two Chlorella species. The antibacterial mechanism of action of cyanobacterial and microalgae peptides is generally not known [152]. It has been suggested that Chlorella peptides induced bacterial cell wall destruction and cell growth inhibition [155]. The generally more potent effect against Gram-positive bacteria than against Gram-negative is observed for many compounds and is especially expressed for these peptides. An example is the pepsin hydrolysate and the peptide fractions from C. sorokiniana. This is likely due to hindered penetration of the large oligomeric structure of the peptide by the outer lipopolysaccharide bacterial membrane [156].

Chlorophylls (such as chlorophyll-a derivatives) and carotenoids are some of the major pigments present in microalgae and are not only antioxidants but also effective microbial growth inhibitors, especially β-carotene among the carotenoids [157,158]. There is a hypothesis that most of the antibacterial compounds are synthesized as a result of chlorophyll and FFA oxidation [32,108]. There is also a patented method of using astaxanthin to prevent bacterial infections, such as from Helicobacter pylori [142,159,160].

Phenolic compounds from microalgae are both antibacterial and antifungal. At high concentrations, they can denature cell proteins, which involves the hydrogen bond formed between phenol and protein. Cell walls and cell membranes, which have high protein content, become disrupted by permeability, causing lysis due to leakage [129,130,132]. Phenols, at low concentrations, have been shown to impact the activity of enzymes, particularly those involved in energy production [135]. The antifungal effect may be due to the impact on spore germination [161]. For multicellular parasites, phenols have been suggested to bind to lipids in membranes of the cell and organelles and change their functional, thus killing the cell, a mechanism that could be valid to the eukaryotic microorganisms as well [162].

Other important identified antimicrobials are microalgal amino acids, terpenoids, sterols, saponins, acetogenins, sulfur-containing heterocyclic compounds, and other carbohydrates. Steroids, florotanins, halogenated ketones, and acrylic acid can be used as antimicrobial and anticancer compounds [1,44,129,130,131,132,133,134,139,141]. Steroids destroy the bacterial cell membrane, while saponins lower the surface tension of the cell wall entering the cell easily, then disrupting bacterial metabolism and eventually killing the cells [141]. Other chemical classes such as indoles, terpenoids, acetogenins, and hydrocarbons, such as volatile halogenated hydrocarbons, are accredited to give microalgal extracts not only antibacterial but also anti-protozoal and antiplasmodial effects [2,8,163]. Through the formation of a strong bond polymer, terpenoids have the ability to damage the transmembrane protein Porin in the outer membrane of bacterial cell walls. That would cause a reduction in the permeability of cell walls and, thus, nutritional deficiencies, leading to growth inhibition and even death [141].

Engineering approaches related to the optimization of PBR conditions, as well as gene and metabolic engineering, can be used to boost and maximize the production of antimicrobial or other bioactive compounds.

4.2. Immunomodulatory and Infection Preventing (Prophylactic) Activities

Since microalgae are used as food additives, the antibacterial effect contributes to their nutraceutical properties. Green microalgae have been suggested as a potential substitute for in-feed antibiotics in order to protect the human food chain by preventing disease in livestock and poultry and preserving the microbiological safety of animal products [137]. Pigments in fish are generally from the microalgae they consume. Fish with high levels of carotenoids are more resistant to bacterial and fungal infections [47]. Aquatic animals’ immunity could be strengthened by utilizing the antioxidants found in microalgae, which would solve the issue of antibiotic overuse. It was found that the large yellow croaker Pseudosciaena crocea showed an increase in lysozyme activity, an indicator of fish immunity, with an increase in astaxanthin levels. This suggests that fish immunity is positively correlated with the consumption of microalgal pigments (astaxanthin) [164]. Astaxanthin significantly improves human neutrophil phagocytic and microbicidal capacity [165]. C. vulgaris is a source of (1,3)-β-glucans and by promoting the synthesis and/or expression of interleukins (IL), dectin-1, and toll-like receptors-2 on macrophages and dendritic cells, respectively, those glucans produced antifungal and antibacterial responses in rats and some resistance to mammalian organisms against infections with E. coli. Only C. vulgaris contains homo-polysaccharides of galactose and glucose [149]. Likely as a consequence of this, patients treated with C. vulgaris tablets for non-alcoholic fatty liver disease had significantly lower serum levels of the pro-inflammatory cytokine TNF-α. Using various chemical triggers (such as low oxygen, NaCl, and nutrient starvation), Montero-Lobato et al. (2018) reported that some microalgal species (such as C. zofingiensis, D. salina, A. platensis, etc.) produced more anti-inflammatory molecules (carotenoids, fatty acids, and sulfated polysaccharides) [166]. Studies revealed that macrophages from the mouse peritoneal cavity could be activated to engage in phagocytic activity by extracts of Chlorella stigmatophora polysaccharide. A diet supplemented with C. vulgaris enhanced the natural killer activity of peripheral blood mononuclear cells isolated from the patients receiving treatment and raised the levels of interferon-gamma, IL-1, and IL-12 in the serum [167].

As already elaborated, whether PUFAs exert suppression of infection in vivo because of direct antimicrobial action, because of their anti-inflammatory effect (influencing cytokine and leukotriene production), or both is not known. As Chanda et al. point out, there are a lot of ω-3-PUFA studies that show ameliorating tissue damage caused by activated host response [136]. Alhusseiny and Beshbishihave also state that the protective effect against parasitic infections of ω-PUFAs is due to both anti-microbial properties and anti-inflammatory effect, but stress that they prevent the inflammatory and oxidant reactions harming the human body [10]. Cheng et al. also support the dual action of EPA and DHA [145]. Further studies are warranted. Ω-3-PUFA are potential agents of food preservatives, and their incorporation into products such as chewing gums, drinks, tea, milk, sugar, tooth paste, or dentifrice may provide the antimicrobial requirements for preventing oral diseases [136].

4.3. Chlorella vulgaris and Other Chlorellaceae Strains

The first discovery of the antibacterial effect of microalgae was in the 1940s when “chlorellin”, a mixture of fatty acids from C. vulgaris, was able to inhibit the growth of Gram-positive and Gram-negative bacteria (P. aeruginosa, S. aureus, Streptococcus pyogenes, and Bacillus subtilis) [168]. Although chlorellin was unsuitable for widespread commercial use, it announced the upcoming studies on algal antimicrobial inhibitors [137]. As mentioned, C. vulgaris also has a wound-healing ability better than calcium sodium alginate [41]. This species has the ability to deform E. coli cells, make their surfaces rough, and make depressions in some cell walls [169]. Another study found that E. coli was moderately susceptible to its acetone and ethanolic extracts, while S. aureus had low susceptibility to various solvent extracts of C. vulgaris [170]. The species was reported to reduce dramatically the biofilm of P. aeruginosa without inhibiting its growth and without high bactericidal activity. The effect was focused on the interference of bacterial signaling. Therefore, the extracts inhibited the quorum sensing not due to the reduction of “quorum” but due to the interruption of “sensing” [171]. Plaza et al. (2012) found that almost no differences in the effect of pressurized liquid extracts (PLE) and ultrasound-assisted extracts (UAE) of the species could be observed when changing the extraction temperature. Activity was mainly related to the polarity of the extraction solvent (organic solvent and water) [24].

In vivo activity is more important than in vitro, as an agent may have strong in vitro activity but fail in an in vivo test or show unacceptable side effects. There have been few papers covering the in vivo effect of Chlorella spp. but some of them show very promising results.

Dermatophytes are a highly specialized group of fungi that have adapted to colonize the keratinized tissues of animals. Twenty to twenty-five percent of people worldwide are affected, and the frequency is rising. Impetigo is a bacterial cutaneous infectious disease, and its treatment with antibacterial agents may cause serious problems. C. vulgaris ointment applied twice a day for a total period of one week for impetigo and two weeks for fungal infections was effective, and the histological examinations of the rat’s tissues revealed the fast healing of the skin layers and normal histological patterns of the skin, with minimal inflammatory cellular infiltrate. Positive controls of ciclopirox and nystatin creams were applied as antifungal agents, while mupirocin was applied as an antibacterial agent. These synthetic ointments illustrated partially cured lesions and inflammation (partial healing). The antimicrobial material was defined as a phenolic nitrile [161].

Amoebic dysentery is one of the tropic public health problems. An extract of Chlorella sp. completely eliminated the amoebic cysts from the feces of mice, but there was no information on the amount of extract consumed, only for its concentration. The therapeutic efficiency of the extract was 69%. It reverted to a high extent the pathological changes in the tissues of the large intestine caused by the amoebic trophozoite [162]. Unfortunately, no positive control was applied in order to compare the effect with an established drug.

In a different in vivo study, mice were intragastrically inoculated with H. pylori three times, separated by two days. Mice were given the whole carotenoid extract of Chlorella sp. (100 mg/kg body weight per day, 0.15–0.18 mL in corn oil) orally for two weeks following inoculation. During this time, the stomach and spleen were removed and prepared for additional histopathology analysis. It showed that Chlorella sp. treatment could delay the gastritis process because the denudation of the surface epithelium, hyperplasia of the gastric gland, and swollen lamina propria were observed on day 14 after treatment only. However, the extract did not lead to improvement. It is known that β-carotene accelerates the gastric healing [172]. However, Isochrysis sp. had fewer carotenoids than Chlorella sp. and better activity; therefore, the action regarding these two species was not correlated to carotenoid content [173].

Doses of twice the effective inhibitory concentrations used in the 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. Similarly, the presence of mucin did not affect the polysaccharides in agglutinating H. pylori. Polysaccharides from A. platensis were confirmed to be effective carbohydrate-based antiadhesives in preventing H. pylori from adhering to gastric mucin and colonization without affecting the host [174].

Cepas et al. (2019) investigated the antibiofilm activity of 675 extracts from 225 microalgae, including Chlorophyta and cyanobacteria species, against clinically important bacterial and fungal pathogens [144]. Extracts from the phylum Chlorophyta showed higher minimal biofilm inhibitory concentration values than the other phyla. EPA and DHA were not evident, and the activity was not attributed to any particular compound [143,144].

In regard to the (presumably) active bioactive metabolites of proven antimicrobial C. vulgaris extracts, according to reports in the literature, as mentioned, this species is rich in PUFAs, and they act as antimicrobials [24]. Other antimicrobial compounds are fatty acids in general, carbohydrates, phenols including phytoalexins, phenolic glycosides, saponins, terpenes, unsaturated lactones, cyanogenic glycosides, sulfur-containing heterocyclic compounds, halogenated hydrocarbons, and acrylic acid. An aqueous extract with a concentration of 150 mg/mL was active against all tested organisms but Saccharomyces cerevisiae. The highest antimicrobial activity was observed for the protein fraction which indicated that protein compounds were responsible for the antifungal properties [175]. There was a high content of long-chain fatty acids, e.g., oleic acid, in the intracellular and extracellular extracts, which have been reported to inhibit S. aureus [176]. Phytochemical screening has also revealed the presence of flavonoids, tannins, alkaloids, and cardiac glycosides in various organic solvent extracts [37,170]. Tannins and phlobatannins have already been reported for their antibacterial and antiviral properties [170]. Flavonoids may be an additional reason for this alga to be used as an antimicrobial food supplement [37]. Hexane and chloroform extracts, particularly, have been shown to contain a large group of metabolites, followed by ethyl acetate and methanol extracts. Alkaloids, flavonoids, glycosides, carotenoids, phenols, lignins, saponins, sterols, tannins, reducing sugars, volatile oils, fats, amino acids, and carbohydrates were revealed [22]. The presence of triterpenes, condensed tannins, and saponins in an extract effective in an excision wound model was revealed. These have already been reported in the literature for antimicrobial activities and favor the wound-healing process. A hydrogel had a wound-healing effect by promoting the remodeling of damaged tissue, forming a barrier against and reducing the local bacterial population [11]. Twenty compounds were identified from PLE and UAE extracts; some of them have been described as carotenoid degradation products, such as neophytadiene and derivatives, which were detected in large amounts. This hydrocarbon, from the phytans family, had been identified in other microalgae and had previously demonstrated some antimicrobial activity. Phytol has also been described in different plant essential oils with proved antimicrobial properties [24]. Eight major compounds were found in ethanol and aqueous extracts. Diethyl phthalate and trimethyl(4-tertbutylphenoxy)silane, as the major compounds in the ethanol extract, have been reported to have antibacterial activity [171]. Heptanal, a known biomedical compound, and some other metabolites, such as 2-butanol and 4-methyldocosane, were found in a C. vulgaris extract [169].

Concerning the (presumably) active secondary metabolites of proven antimicrobial extracts of other or unidentified Chlorellaceae species, extracts have been found to contain phenolics, saponins, steroids, and triterpenoids [141]. Minimum inhibitory concentration (MIC) values of the best two extracts from the strain Chlorella SRD3 against Gram-negative bacteria (ethyl acetate and methanol) were tested. The compounds 2-(2-aminoethoxy)-ethanol and 9-octadecyne from the methanol extract had been previously reported to have antibacterial activity [177]. The phytochemical screening of the extract of C. sorokiniana revealed the presence of phytoconstituents, including anthraquinones, cardiac glycosides, flavonoids, saponins, tannins, and terpenoids [178]. Sterols, hydrocarbons, and fatty acid methyl esters are known antimicrobials. For example, polypropylene meshes are coated with a mixture of esterified ω-3 PUFAs to prevent microbial colonization and inflammation [8].

The extracts from A. pyrenoidosa had a broad spectrum of antimicrobial substances against Gram-negative bacteria. The high antimicrobial properties of the methanol extract may be due to the abundance of some lipophilic but polar compounds and corresponded to the highest percentages of the total phenolic and total flavonoid contents [179]. The main constituents detected in the ethanol extract of C. salina were organic acids like the antimicrobial palmitic acid (6%), octadecanoic acid, phytol, and the benzophenone ether octabenzone. When used in aquaculture (food), sea bass in the post-larval stage showed a decrease in the amount of S. aureus, so these algae could be used as a green water technique [131].

An overview of the literature data on the antimicrobial activity of C. vulgaris and different Chlorellaceae strains is given in

Table 1. A review of the research on the antimicrobial activity of Chlorella vulgaris Beijerinck.

Extract or Sample	Test Microorganism	Antibacterial Activity—Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Inhibition Zone (IZ) in mm, etc.	Test Methods	Reference
Ethanol extract	Bacteria isolated from different clinical cases: Enterococcus faecalis, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus lentus, Staphylococcus xylosus, Streptococcus agalactiae, and Streptococcus pyogenes.	The effect was examined. The IZ was the highest (31.6 mm) against S. lentus, while the lowest IZ (20.6 mm) was in the case of S. aureus (both). The chemical composition analysis consisted of terpenes (monoterpenes and sesquiterpenes).	Agar well diffusion method (AWDM) at a concentration of 300 mg/mL	[180]
Aqueous extract from biomass cultured on post-fermentation leachate from a biogas plant and the protein fraction	E. coli, Lactobacillus plantarum, Staphylococcus epidermidis; fungi—Candida albicans, Saccharomyces cerevisiae, Aspergillus niger.	The IZs of the aqueous extract were as follows: A. niger—51 mm; C. albicans—47 mm; E. coli—24 mm; S. aureus—25 mm, S. cerevisiae—0 mm. The MICs were as follows: C. albicans and L. plantarum—37.5–75 mg/mL; E. coli and S. epidermidis—150 mg/mL. The protein fraction had a MICs of 32.5–65 mg/mL against E. coli, 32.5 mg/mL against C. albicans and S. cerevisiae, and 16.25 mg/mL against L. plantarum and S. epidermidis.	AWDM, broth microdilution method (BMD)	[175]
Tris-buffered saline extract from microalgae grown under autotrophic (AE) or mixotrophic (ME) conditions; hydrogel containing the extracts	Three frequently encountered bacteria that cause infections on the skin (Klebsiella pneumoniae, E. faecalis, and E. coli).	The ME had MICs towards K. pneumoniae, E. faecalis, and E. coli 0.781, 50, and 100 mg/mL, respectively. The AE inhibited them at the highest concentration of 100 mg/mL. Wound-healing properties.	BMD, in vivo excision wound model	[11]
Aqueous extract	Aeromonas hydrophila	IZ of 12 mm.	AWDM	[181]
Sodium acetate buffer (SAB), aqueous and chloroform:methanol extracts	Staphylococcus sp. isolates causing goat (19 strains) and bovine (16 strains) mastitis.	Against bovine mastitis, the aqueous was the most efficient, and against goat mastitis, it was the SAB extract with MICs of 50 and 100 μg/mL, respectively.	BMD	[182]
Extracted proteins.	Bacillus cereus, Pseudomonas putida, and S. aureus (highly pathogenic bacteria).	IZs were 14, 13, and 14 mm, respectively. The antibacterial activity was higher than that of the antimicrobial phycocyanin.	Disc diffusion method (DDM)	[183]
Biomass, proteins from it, protein hydrolysates by pepsic digestion	E. coli.	The effect of a protein fraction with 62 kDa was 8.5 and 1.6 times greater than the biomass and its proteins, respectively (a decrease of ca. 34.% in the growth of E. coli).	Optical density of treated bacterial suspension measured at time intervals at 600 nm	[155]
Lyophilized ethanol and aqueous extracts	P. aeruginosa, which has many aquatic pathogenic bacteria, uses a quorum sensing system to establish their biofilm and virulence [47].	The aqueous extract showed inhibition from 60 to 80% on the biofilm, at 500 μg/mL to 2 mg/mL, respectively. The ethanol extract showed inhibition from 60 to 85%, too, but at 62.5 μg/mL to 2 mg/mL, respectively. The extracts, at 2000 and 1000 μg, had a moderate to low or no activity (IZ 10 mm) in comparison to standard (25 mm).	DDM—for antibacterial activity, screening by MTT assay and scanning electron microscopy—for the antibiofilm activity	[171]
Chloroform/methanol/acetone (in ratio of 2/1/1) extract	Streptococcus mutans—involved in dental plaque formation and consequent dental cavities.	IZs at the highest concentrations tested: 13.5 mm (2.5 mg/disc) and 19 mm (7.5 mg/mL). In addition, significant antibiofilm activity was recorded in the concentration of 4 mg/mL.	DDM, AWDM	[184]
Methanol and ethanol extracts	S. aureus, P. aerugenosa, B. cereus, E. coli, Salmonella typhimurium, and C. albicans; fungi—Malassezia pachydermatis, Fusarium graminearum, Fusarium moniliforme, Aspergillus ochraceus, and Penicillium verrucosum.	The IZs of the ethanol extract were 8–10 mm. The methanol extract had no activity against E. coli and P. Aerugenosa, and the IZs against the other strains were between 7–10 mm. The ethanol extract had a high inhibitory effect (9 mm) against M. pachydermatis (8 mm for the methanol extract) and the IZs against F. graminearum, F. moniliforme, A. ochraceus, and P. verrucosum were 7.5–9.5 mm. The methanol extract showed no IZ to the fungi.	AWDM	[185]
Crude extract	Enterobacter, Proteus, E. coli, Klebsiella, S. aureus, Lactobacillus acidophilus, and S. pyogenes (isolated from teeth decay).	IZs 25, 24, 15, 0, 20, 18, and 15 mm, respectively (at 100 µg/mL).	AWDM	[186]
Methanol, ethanol, ethyl acetate, and chloroform extracts	Multi-drug resistant (MDR) pathogens—S. aureus, E. coli, P. aeruginosa, Salmonella sp., and Shigella sp.	The percentage of inhibition was in the range of 66–93%, and the methanol extract was the most effective one.	Disk diffusion method	[187]
Methanol, chloroform, and diethyl ether extracts	P. aeruginosa, E. coli, S. aureus, S. pyogenes grp A (clinical isolate) and Bacillus subtilis.	The diethyl ether extract showed the maximum IZ (28.6 mm) against E. coli, whereas the rest of the extracts were inactive against P. aeruginosa and S. aureus. The MIC of the diethyl ether extract was 0.6 mg/mL against E. coli and S. pyogenes, whereas the MIC of the acetone extract was 1 mg/mL for B. subtilis.	DDM scanning electron microscopy (SEM) was used to examine the morphological changes in E. Coli	[169]
Methanol and aqueous extracts, and the supernatant of the strain CCATM-210	MDR S. aureus	MICs of 0.72, 0.98, and 1 mg/mL, respectively. The interaction turned out to be indifference of the methanol to the aqueous extracts (no synergism or antagonism).	BMD, checkerboard titration method	[178]
Organic and aqueous extracts from a strain from lake Unkal	Gram-positive strains—S. aureus, Streptococcus, Corynebacterium and B. subtilis; Gram-negative strains—K. pneumoniae, Aerobacter aerogenes, and Salmonella Parathypi-B; fungi—A. niger and C. albicans.	The chloroform, ethyl acetate, hexane, and methanol extracts showed the maximum IZ against the Gram-positive strains, K. pneumoniae and A. aerogenes. The highest IZ was 22 mm and the chloroform extract against B. subtilis (the same as gentamycin). The benzene ethanol extracts had an intermediate effect. The petroleum ether extract had the weakest effect—inhibited only S. aureus and A. aerogenes with small IZs of 4 and 8 mm. The extracts showed a marginal effect against E. coli and Salmonella Parathypi-B, and the IZs were 5–8 mm. The poor effect was also observed with the fungal strains A. niger and C. albicans, with a maximum IZ of 6 mm for the aqueous and chloroform extracts.	DDM	[22]
Methanol extract	S. aureus, B. subtilis, Proteus vulgaris, E. coli, and P. aeruginosa.	The IZs against P. vulgaris, E. coli, P. aeruginosa, and B. subtilis were 14.5, 14, 18 and 12 mm, respectively.	AWDM	[38]
Ethanol and water extracts.	S. aureus, S. pyogenes, E. faecalis, P. aeruginosa and E. coli	The ethanol extract at 100–500 mg/mL had mean IZs of 6 to 14 mm. The aqueous extracts only showed minimal activity towards E. coli, with a mean IZ of 7 mm. The MIC values were between 125 and 1000 μg/mL.	AWDM, broth macrodilution assay	[41]
Various solvent extracts	E. coli, Klebsilla sp., Bacillus sp. and Pseudomonas spp.	The acetone extract had IZs 8, 12, 15, and 13 mm, respectively. The IZs of the ethanol extract were 15 (a value better than tetracycline), 17, 13, and 12 mm, respectively. For the chloroform extract, these values were 12, 10, 14, and 11 mm, respectively. The IZs of tetracycline were 13, 18, 16, and 15 mm, respectively.	DDM	[37]
Lipid extracts rich in palmitic and linoleic acid	Propionibacterium acnes	The MIC value was 10 μg/mL.	BMD	[188]
Culture filtrate (intracellular and extracellular extracts) prepared with chloroform, ethanol, methanol, and ethyl acetate	S. aureus and E. coli.	The intracellular extracts had a greater inhibitory effect (IZs 10–19 mm, with the maximum for the ethyl acetate extract on S. aureus) than the extracellular extracts (IZs 0–12 mm).	DDM	[189]
Methanol and acetone extracts	B. subtilis, Bacillus megaterium, S. aureus, Sarcina lutea and K. pneumoniae; fungi—C. albicans, A. niger, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani.	The IZs of the methanol extract against B. subtilis, S. aureus, and K. pneumoniae were 17.5, 17, and 14.5 mm, respectively, with no antifungal activity. The acetone extract inhibited S. lutea, B. subtilis, S. aureus, K. pneumoniae, and F. oxysporum with IZs 30, 20, 18, 16 and 45 mm, respectively. Neither extract inhibited B. megaterium, F. solani, R. solani, A. niger, and C. albicans.	AWDM	[190]
dH2O extracts made with freezing and thawing	S. aureus, E. coli, Klebsiella ozaenae, P. aeruginosa—opportunistic bacteria	A pronounced antagonistic activity on all strains with >90% growth inhibition on K. ozaenae and >82% on P. aeruginosa.	BMD, photometric method	[32]
70% ethanol, 70% methanol, 70% ethyl acetate, and 70% chloroform extracts, antimicrobial material extracted from the algae (AMA)	Methicillin-resistant S. aureus (MRSA) and different isolates of S. aureus.	The methanol extract had the best antibacterial effect on S. aureus, followed by the chloroform, ethyl acetate, and ethanol extracts. The MIC of AMA against the most resistant MRSA isolate was 6.25 mg/mL. The active compound was aromatic in nature.	BMD, MIC determined by agar dilution method	[191]
Methanol extract, pellet, aqueous extracellular supernatant	B. subtilis, E. coli, Pseudomonas fluorescens, C. albicans and Saccharomyces cerevisiae.	No activity.	AWDM	[107]
Organic and aqueous pressurized liquid extracts (PLE) and ultrasound-assisted extracts (UAE) were extracted at different temperatures	E. coli, A. niger, S. aureus and C. albicans	Aqueous extracts (all PLE) had the weakest effect, with MBCs ranging from 11 mg/mL (E. coli) to 16 mg/mL (A. niger). Next were acetone PLE extracts with MBCs in the range of 9.5 mg/mL (C. albicans) to 14.5 mg/mL (A. niger) and the hexane extract (only UAE, MBCs between 9.5 mg/mL against C. albicans and 15 mg/mL against A. niger). Acetone UAE extracts had MBCs ranging from 9 mg/mL (C. albicans) to 14 mg/mL (A. niger). The ethanol extracts were the most effective ones, with the MBCs of the UAE extract 8.5 mg/mL (C. albicans) to 14.5 mg/mL (A. niger) and MBCs of the PLE extract ranging from 7 to 13 mg/mL against the same strains again. MBCs against S. aureus and C. albicans were in the range of 8.5–12.5 mg/mL and 9–12.5 mg/mL, respectively.	BMD	[24]
Chloroform, acetone, ethanol, and aqueous extracts	E. coli, P. vulgaris, S. aureus, P. aeruginosa and B. subtilis	Varying antimicrobial activities at 50–100 μg/mL. IZs at 75 μg/mL for all extracts 11–20 mm, except the aqueous one (0 mm). The ethanol extract had the most pronounced effect, followed by the acetone one, while the rest had less potential. The MICs of the four extracts were at 1.5–25 mg/mL.	AWDM, BMD	[170]
Acetone extract	S. aureus, B. subtilis, K. pnemoniae, and Micrococcus luteus.	IZs 8–15 mm and MIC values 25–40 µg/mL. K. pnemoniae and S. aureus were more susceptible than B. subtilis and M. luteus.	DDM	[192]
Intracellular (food-grade solvent) and extracellular ethanol/water (1:1) extracts	A few of the most relevant foodborne pathogens—S. aureus, E. coli, and Salmonella spp.	Both extracts had low inhibition against E. coli and Salmonella spp. S. aureus was mildly inhibited by the intracellular and weakly inhibited by the extracellular extracts, while P. aeruginosa was affected in the opposite manner.	Mixtures of pathogens and microalgae in the medium were visually assessed for turbidity	[176]
Acetone, methanol, ethanol, and dimethyl sulfoxide (DMSO) extracts	S. aureus, K. pneumoniae, Pseudomonas, Vibrio cholerae, S. pyogenes, and E. coli.	Moderate activities against the tested microorganisms.	DDM	[193]
Ethanol, n-butanol, chloroform, and water extracts	Mycobacterium tuberculosis	No satisfactory results.	Absolute concentration method	[194]
Aqueous and methanol extracts	Pathogens including foodborne: E. coli, P. aeruginosa, Salmonella typhimurium, S. aureus and S. pyogenes.	No effect.	AWDM	[19]
Ethanol extract	E. coli and K. pneumoniae.	The dose of 0.9 μg/mL had IZ of 27 and 18 mm, respectively, similar to the values of streptomycin.	AWDM	[69]
Aqueous, ethanol, and methanol extracts	Different strains of P. fluorescens, P. putida, P. aeruginosa, A. hydrophila, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, Aliivibrio fisheri, Vibrio anguillarum, E. coli, and Edwardsiella tarda.	The ethanol extract showed a minimum IZ of 11 mm against E. coli (strain O111) and a maximum IZ (15–16 mm) against A. hydrophila (strain AH2), V. anguillarum (strain VAN) and E. coli (O1) and was only inactive against one of the P. fluorescens strains. The aqueous extract was also highly active (15 mm) against two A. hydrophila strains. It was inactive against most of the E. coli, some of the Vibrio strains, and E. tarda, and the minimum IZ of 6.3 mm was against V. harveyi.	DDM	[195]
70% methanol extract made into a 14% ointment	Rats cutaneously infected with S. aureus and Microsporum canis, causing impetigo and ringworm, respectively, and C. albicans.	High activity at low concentrations confirmed that the extract would treat and heal impetigo and skin fungal infections without any side effects on skin tissues.	Cutaneous treatment, histological study	[161]

and

Table 2. A review of the research on the antimicrobial activity of different Chlorella spp., including unidentified ones.

Species	Extract or Sample	Test Microorganism	Antibacterial Activity—MIC, MBC, IZ (mm), etc.	Test Methods	Reference
C. zachariaii	Water, hexane, chloroform, ethanol, and methanol extracts	P. aeruginosa, K. pneumoniae, S. aureus, E. coli and Salmonella Typhi	No activity.	AWDM, BMD, agar plate assay for MBC	[196]
C. variabilis	Diethyl ether and methanol extracts	C. albicans, B. subtilis, E. faecalis, S. aureus and P. aeruginosa	The diethyl ether extract had IZs 23, 15, 7.5, 13, and 8 mm, respectively. MICs against all strains were >1000 µg/mL. The methanol extract had IZs of 8 and 8.5 mm against S. aureus and E. faecalis, and both MICs were 282 µg/mL; the MIC against B. subtilis was 563 µg/mL.	DDM, BMD (IZ of >15 mm against at least one pathogen was set as a threshold for MIC determination)	[197]
Chlorella sp. SRD3	Extracts of various solvents	Eight human clinical pathogens—E. faecalis, B. cereus, E. coli, B. megaterium, B. subtilis, S. aureus, P. aeruginosa and Serratia marcescens	Maximum IZ (26 mm) for the ethyl acetate extract against B. megaterium, followed by the methanol extract (23 mm), with effect higher than chloramphenicol (15 mm). The highest IZs for the chloroform and hexane extracts were 15 mm (against E. faecalis and S. marcescens) (the control had an IZ of 17 mm for both). The lowest MIC belonged to the ethyl acetate extract (1.5 μg/mL against E. faecalis), and the range of MIC was 1.5–50 μg/mL. The MICs of the methanol extract were 25–100 μg/mL.	AWDM, BMD	[177]
C. sorokiniana	Proteins	B. cereus, P. putida, and S. aureus—three highly pathogenic bacteria	IZs 15.3, 13.7, and 16 mm, respectively. Better effect than the antibacterial phycocyanin.	Extracted proteins; DDM	[183]
C. sorokiniana	Methanol and acetonic extracts	E. coli and S. aureus	Active against E. coli and S. aureus but not against Streptococcus spp.	DDM	[198]
C. sorokiniana	Protein isolates hydrolyzed by pepsin, bromelain, and thermolysin	E. coli and S. aureus	Only the pepsin hydrolysate and the peptide fractions (<5 and <10 kDa) had an effect and IZs of 13 to 25 mm. The activity of the low molecular weight peptides was stronger than that of the large compounds in the hydrolysates.	AWDM	[156]
Different strains of C. sorokiniana and an unidentified Chlorella sp.	Ethyl acetate and 95% ethanol extracts	S. aureus and E. coli	Best activity for both extracts of C. sorokiniana with IZs 20–22 mm for S. aureus (comparable to ceftazidime) and 18–19 mm for E. coli. The other extracts had lower or no effect.	AWDM (The Oxford cup method variant)	[132]
Edaphochlorella mirabilis (family incertae sedis), C. stigmatophora, different strains of C. sorokiniana, and an unidentified Chlorella sp.	Methanol extract, pellet, aqueous extracellular supernatant	B. subtilis, E. coli, P. fluorescens, C. albicans, and S. cerevisiae.	Only the supernatant of one strain of C. sorokiniana showed 40% inhibition against E. coli (compared to the IZ of 25 mm of chloramphenicol, which was considered to be 100%).	AWDM	[107]
C. sorokiniana, Auxenochlorella pyrenoidosa, Auxenochlorella protothecoides, and Chloroidium ellipsoideum,	Lipid extracts by to the method of Folch et al. with 2:1:0.8 chloroform:methanol:water	Propionibacterium acnes	The MIC values were ≥80 μg/mL for C. sorokiniana and 20 μg/mL for the rest.	BMD	[188]
C. sorokiniana	Aqueous and methanol extracts	Pathogens including foodborne: E. coli, P. aeruginosa, Salmonella typhimurium, S. aureus, and S. pyogenes	No effect	AWDM	[19]
Chlorella spp. from different places in Ecuador	Chlorellin extracted with different solvents	Staphylococcus spp.	The isopropyl alcohol extracts had IZs of 0.19, 0.22, and 0.28 mm. For ethanol-extracted chlorellin, those values were 0.12, 0.17, and 0.19 mm. The zones for positive standards were 5 and 1 mm.	DDM	[199]
Chlorella spp.	Methanol and chloroform extracts	B. subtilis, P. aeruginosa, E. coli, M. luteus, S. aureus Salmonella enterica, Streptococcus iniae, and V. parahaemolyticus.	The methanol extract was active against B. subtilis and P. aeruginosa (IZs 14 mm for both). It had MICs of 0.78 mg/mL against M. luteus and S. aureus and 12.5 mg/mL against S. enterica. The chloroform extract had MICs of 12.5, 6.3, 25, 3, and 12.5 mg/mL against S. aureus, B. subtilis, S. iniae, P. aeruginosa, and V. parahaemolyticus, respectively. E. coli was the only unsusceptible strain.	AWDM, BMD	[200]
Chlorella sp. from Moroccan coastlines	Ethanol extract	E. coli, P. aeruginosa, S. aureus, C. albicans and A. niger	A weak effect only on C. albicans with MIC > 5 mg/mL and did not exhibit any effect against the rest at 5 mg/mL.	BMD	[138]
A. pyrenoidosa	Etheric, hexano–ethanol, and methanol extracts	ATCC strains of S. aureus, B. cereus, B. subtilus, E. coli, Klebsiella sp., P. aeruginosa, S. typhimurium; fungi—C. albicans and Aspergillus brosiliensis	IZs varied from 0 to 48 mm (the methanol extract against E. coli) and often exceeded the IZs of control antibiotics. The methanol extract had a widespread spectrum of strong activity and MICs of <198 μg/mL. MICs of other extracts were 750 to 2250 μg/mL.	AWDM and DDM	[179]
A Chlorella sp. strain from the Aegean Sea	Ethanol extracts	Ten pathogens: E. faecalis, S. aureus, MRSA; E. coli, K. pneumoniae, Shigella flexneri, and V. cholerae; fouling bacteria: Pseudoalteromonas sp., Vibrio furnissii, A. hydrophila, Aeromonas salmonicida, Alcanivorax borkumensis, Alcanivorax sp., Allivibrio salmonicida, Erythrobacter litoralis, Planococcus donghaensis and Pseudomonas mendocina; fungi—Aspergillus fumigatus, C. albicans, and Cryptococcus neoformans	Growth inhibition of 30 and 20% against Pseudoalteromonas sp. and Vibrio furnissii, respectively. Inactive against the rest.	AWDM (1 mg/disc) and BMD	[201]
Chlorella sp.	Methanol extract	E. coli, S. Typhi, B. cereus and S. aureus	Low antibacterial activity. The largest IZs were at 500 µg/mL—12, 9, 10, and 11 mm, respectively, and the zones were only slightly smaller at 10 μg/mL.	DDM	[141]
C. salina	Ethanol extract	S. aureus, E. faecalis, B. cereus, and fish indicator pathogens—E. coli, P. aeruginosa, Vibrio damsela (the strongest pathogen for Dicentrarchus labrax or sea bass larvae), V. fluvialis, and A. hydrophila (a highly pathogenic bacteria to both cultured and wild fish)	Antibacterial effect against E. coli, P. aeruginosa, V. damsela, V. fluvialis, and A. hydrophila with absolute units (AU) 5.4, 2.1, 5.4, 2.8, and 4, respectively. There was no effect against S. aureus, E. faecalis, and B. cereus.	AWDM	[131]
Chlorella sp.	Methanol, ethanol, and chloroform extracts	E. coli, S. aureus, P. aeruginosa, S. Typhi, B. subtillis, K. pneumoniae and C. albicans	The minimal IZ belonged to the chloroform extract against E. coli (12 mm), and the maximal —to the methanol extract against C. albicans (20 mm), which was the most susceptible species. The ethanol and chloroform extract had almost the same activity.	DDM	[202]
Chlorella sp.	Culture medium, lipids, and fatty acids	S. aureus, S. pyogenes, P. aeruginosa, B. cereus, E. coli, Yersinia enterocolitica, S. typhimurium and C. albicans	Fatty acids had 22 and 15 mm IZs towards S. aureus and S. pyogenes, respectively. The culture medium inhibited only C. albicans with an MIC of 12.5 mg/mL. There was no activity towards the rest of the bacteria.	AWDM, BMD	[140]
C. salina and Chloroidium saccharophilum	Ethanol, n-butanol, chloroform, and water extracts	M. tuberculosis	No satisfactory results.	Absolute concentration method	[194]
Chlorella sp.	Total carotenoid extract (55% in dry weight)	E. coli, Salmonella sp., P. aeruginosa, B. cereus, Klebsiella sp.; Mice inoculated with Helicobacter pylori	The highest concentrations tested (50 and 100 mg/mL) inhibited only P. aeruginosa and were probiotic to E. coli and Klebsiella sp. In vivo results showed delayed gastric ailment but no healing.	DDM, in vivo gastritis studies on model mice	[173]
Chlorella sp.	70% ethanol extract	Mice infected with Entamoeba histolytica	The maximum tested concentration of 2 mg/mL for the maximum tested time (20 days) completely eliminated the cysts from the feces.	In vivo oral treatment, histological study	[162]
Chlorella sp.	Polysaccharide extract	H. pylori	An inhibitory effect on the adhesion of H. pylori to gastric mucin. Eighty mg reduced the H. pylori adhering to mucin by about 90%, and the effect was worse than that of Arthrospira. A higher concentration did not increase its effectiveness. No agglutination of H. pylori.	Competitive and blocking inhibition assay, agglutination assay	[174]

, respectively.

4.4. Tetradesmus obliquus and Other Scenedesmaceae Strains

Extracts from Scenedesmaceae spp. have positive results but with varying values of IZs. They have higher antibacterial activity against Gram-positive bacteria than Gram-negative bacteria.

Phosphorus (P) is one of the most important nutritional factors that influence metabolism and cell growth. The optimum P concentration for the antibacterial properties of T. obliquus turned out to be 0.01 g/L P for S. aureus (IZ 26 mm) and 0.007 g/L P for E. coli [203]. MIC values of Desmodesmus subspicatus were more satisfactory using the dimethyl sulfoxide (DMSO) extract, which showed antibacterial activity against most bacteria tested, both Gram-negative and Gram-positive. However, the aqueous extract also had satisfactory activity, and employing an aqueous extract, if effective, is a cost-effective protocol that avoids the usage of toxic solvents [99]. The effect of five strains of Scenedesmus depended on the harvest time, strain-type, and extracting solvent. The majority of the strains had potent antimicrobial activity with MICs less than 1 mg/mL, but the MIC values for the majority of the PE extracts were not determined due to the insufficient yield from the extracting solvent [204].

Eleven compounds were identified in fractions from the diethyl ether crude extract (DEE) of T. obliquus, and most of them have been reported to possess biological activities, e.g., antimicrobial properties against B. subtilis, Enterococcus faecalis, E. coli, S. aureus, P. aeruginosa, K. pneumoniae, Bacillus cereus, Candida albicans, and several other species of mycotoxigenic fungi—Aspergillus niger, Aspergillus flavus, and Aspergillus fumigatus. The main compounds were nonadecane, butylated hydroxytoluene, 9-octadecadienoic acid (Z), and 7,3’,4’-trimethoxyquercetin. There were other compounds that had been reported to have antimicrobial activity when isolated from other plants, such as heptadecane, hexadecane, 2-hexadecenal octasiloxane, etc. [205]. Oleic and linoleic acids have been reported to inhibit S. Aureus, and they were abundantly found in the cells of different T. obliquus strains. The presence of high content of other long-chain fatty acids, such as EPA, may contribute to the antibacterial effect [176]. Nevertheless, there is yet to be a conclusive identification and characterization of specific metabolites responsible for the antibacterial activities of T. obliquus [142].

The most abundant compounds in the methanol extract of Tetradesmus dimorphus included phenols, carboxylic acids such as hexadecatrienoic acid n-4, esters, plasticizer compounds, and terpens, while in the ethanol, N-hexane and diethylether extracts, the most abundant compounds were found to be acyclic diterpene alcohols, plasticizers, carboxylic acids, hydrocarbons like limonene, azulene and eicosane and esters of phthalate and others. Terpenes, carbohydrates, phenols, eicosane, and palmitic acid may be reasons for the antimicrobial effects. Hexadecatrienoic acid n-4 has been found to be active against Gram-positive and Gram-negative bacteria and to be also highly active against MRSA [1]. Methyl esters are known antimicrobials. For example, polypropylene meshes are coated with a mixture of esterified ω-3 PUFAs to prevent microbial colonization and inflammation [8].

Since the crude pigment extract of a Scenedesmus sp. had high antibacterial properties, the authors concluded that the activity was most probably due to chlorophyll [157]. About 30 different fatty acid methyl esters (FAMEs) were identified in Desmodesmus bicellularis among which the methyl esters of DHA (2.3%), EPA (1.1%), linoleic (1.4%), linolenic (6.3%), stearic (0.6%), oleic (1%), and erucic acid (1%) acquire great importance as a result of their potent antimicrobial activity. It was previously established that via inhibiting FabI gene expression, this class of FAMEs inhibits the fatty acid biosynthesis pathway [206].

A summary of the antimicrobial properties of T. obliquus and other Scenedesmaceae spp. is given in

Table 3. A review of the research on the antimicrobial activity of Tetradesmus obliquus (Turpin) M.J. Wynne.

Extract or Sample	Test Microorganism	Antibacterial Activity—MIC, MBC, IZ in mm, etc.	Test Methods	Reference
Various solvent extracts and substances isolated	B. cereus, S. aureus, E. coli, K. pneumoniae, P. aeruginosa, and S. Typhi; nine tested mycotoxigenic fungi—Aspergillus steynii, A. carbonarius, A. ochraceus, Aspergillus flavus, A. parasiticus, Aspergillus westerdijikia, Fusarium verticillioides, Fusarium proliferatum, and Penicillium verrucosum	The aqueous, chloroform, and diethyl ether extracts had antibacterial activity against all tested bacteria, followed by the ethyl acetate extract. The highest inhibition was observed for the diethyl ether crude extract (DEE), recording IZs 12.5–19.5 mm and MICs 0.5–1.2 mg/mL against all tested bacteria (against S. Typhi and P. aeruginosa even 19.5 and 18.5 mm, respectively). IZs were between 9 and 18 mm against the fungi. The highest antifungal effect was shown against A. steynii, followed by A. carbonarius for the DEE with 18 and 16 mm IZs, respectively. DEE had activity against all fungi, followed by the aqueous, methanol, chloroform, ethyl acetate, and hexane extracts, which showed activity against only four fungal species. A. ochraceus, F. verticillioides, and P. verrucosum had resistance against all extracts except the DEE. Fractions F1 and F7 from the DEE had the highest activity against the tested bacteria and fungi.	DDM and tube dilution method	[205]
Methanol extract	S. aureus and E. coli	With optimum phosphorus concentrations, the IZ on S. aureus was 26 mm, and the IZ on E. coli was 23 mm. The IZ of vancomycin was 28 mm.	AWDM	[203]
Ethyl acetate and 95% ethanol extracts	S. aureus and E. coli	The ethyl acetate extract had an IZ of 11 mm against S. aureus and 9 mm against E. coli. The 95% ethanol extract was not active.	AWDM (The Oxford cup method variant)	[132]
dH2O extracts made with freezing and thawing	Four test-strains of opportunistic bacteria—S. aureus, E. coli, K. ozaenae, and P. aeruginosa	A pronounced antagonistic activity was observed against all strains. The growth inhibition on K. ozaenae was 50% and on P. aeruginosa > 82%, respectively.	BMD; photometric method	[32]
Methanol extract, pellet, aqueous extracellular supernatant	B. subtilis, E. coli, P. fluorescens, C. albicans and S. cerevisiae	No activity.	AWDM	[107]
Culture medium, lipids, and fatty acids	S. aureus, B. cereus, S. pyogenes, P. aeruginosa, E. coli, Y. enterocolitica, S. typhimurium and C. albicans	Only S. aureus and S. pyogenes were inhibited with IZs for lipids 16 mm and for fatty acids 18 mm for both of them.	AWDM and BMD	[140]
90% ethanol, 80% methanol, and n-hexane extracts	A. fisheri, Aeromonas fluvialis, B. subtilis subsp. subtilis, E. faecalis, E. coli, P. aeruginosa, Sphaerotilus montanus, Spirillum winogradskyi, S. aureus subsp. aureus; fungi—A. niger and Wallemia sebi	No activity.	DDM and bioluminescence toxicity assay (for A. fisheri only)	[207]
Intracellular (food-grade solvent) and extracellular ethanol/water (1:1, v:v) extracts from strains of T. obliquus.	A few of the most relevant foodborne pathogens—S. aureus, P. aeruginosa, E. coli, and Salmonella sp.	Both extracts from strain B exerted low inhibition against Salmonella sp. and S. aureus and mild inhibition against P. aeruginosa. E. coli was mildly inhibited by the intracellular and weakly by the extracellular extract. Both extracts from strain G had no activity. Strain M2-1 fully inhibited all strains except for Salmonella sp. (low inhibition).	Mixtures of pathogens and microalgae in the medium were visually assessed for turbidity	[176]
Dichloromethane extract	S. aureus, E. coli, S. typhimurium	High MICs at ≥12.5 mg/mL but synergistically potentiated (at 0.01 to 0.05 mg/mL) the activity of penicillin, fluoroquinolones, or oregano essential oil.	BMD, Checkerboard assay	[12]

and

Table 4. A review of the research on the antimicrobial activity of different Scenedesmaceae spp.

Species	Extract or Sample	Test Microorganism	Antibacterial Activity—MIC, MBC, IZ (mm), etc.	Test Methods	Reference
Four Scenedesmus strains from water bodies near abandoned mine sites in northern Canada	Methanol extracts	S. aureus, E. coli, B. subtilis and P. vulgaris	One strain had an IZ of 5 mm against S. aureus and no activity against the rest. No clear association was observed between the metal concentrations, pH, and the antibacterial activity.	Antibacterial activity on agar plate, BMD	[208]
Scenedesmus sp.	Crude pigment extract	S. aureus and Salmonella sp.	Concentrations ranging from 0.35 to 3.48 mg/mL demonstrated high inhibitory activity against S. aureus with IZs from 4.7 up to 17.3 mm. The MIC was as low as 80 µg/mL. On the other hand, there was no inhibitory activity at any concentration of the extract against Salmonella sp.	AWDM, macrobroth dilution method	[157]
Scenedesmus sp. Kiel	Methanol extract, pellet, aqueous extracellular supernatant	B. subtilis, E. coli, P. fluorescens, C. albicans and S. cerevisiae	No activity.	AWDM	[107]
Scenedesmus sp.	Methanol and acetone extracts, 7,3’,4’-trimethoxyquercetin from methanol fractions	B. subtilis, S. aureus, S. lutea, B. megaterium and K. pneumoniae; fungi—C. albicans, A. niger, F. solani, F. oxysporum, Rhizoctonia solani	The methanol extract inhibited only B. subtilis and S. aureus with IZs of 15 and 16 mm, respectively, and the acetone extract inhibited only S. aureus with IZ of 16 mm; both had no activity against the other bacteria. Both extracts inhibited only F. oxysporum with an IZ of 35.5 mm and not the other fungi.	AWDM	[190]
Five strains of Scenedesmus sp. (MACC-411, -422, -493, -720, and -727)	EtOH, dichloromethane (DCM) and petroleum ether (PE) extracts	S. aureus, E. faecalis, E. coli, P. aeruginosa, C. albicans	Most strains had MICs less than 1 mg/mL. Generally, DCM and EtOH extracts had broad spectrum and lower MICs compared to the PE extracts. The three solvent (PE, DCM, and EtOH) extracts of 5-day-old cultures inhibited S. aureus more compared to the 10-day-old ones. Conversely, DCM (all strains) and EtOH (four strains) extracts from the 10-day-old cultures were more active against P. aeruginosa than those from the 5-day-old cultures. Two strains (PE and EtOH extracts) from the 5-day-old cultures were more active against E. coli, while the other three strains (DCM extract) from the 10-day-old cultures were more active. Ten-day-old cultures of three strains and 5-day-old cultures of two strains (one of which was MACC-411 with the highest iridoid content) had higher antifungal activity.	BMD	[204]
Scenedesmus sp.	Acetone, methanol, diethyl ether, and hexane aqueous extracts	Shigella sp., Pseudomonas sp. and Xanthomonas oryzae	The aqueous extract had much greater antibacterial activity compared to the others. Its methanol fractions had the best results. Pseudomonas sp. was the most resistant.		[209]
S. bijuga	Water, hexane, chloroform, ethanol, and methanol extracts	S. aureus, E. coli, P. aeruginosa, K. pneumoniae, and Salmonella Typhi	The chloroform extract had a MIC of 62.5 μg/mL against P. aeruginosa (and 1000 μg/mL MBC) and 16 μg/mL against K. pneumoniae (it had IZ of >11 mm and was subjected to BMD). No effect on S. aureus, E. coli, and S. Typhi.	AWDM, BMD with resazurin, agar plate assay	[196]
Desmodesmus subspicatus	Various solvent extracts	B. subtilis, K. pneumoniae, E. coli, P. mirabilis, E. faecalis and Salmonella enteritidis	The acetone, water, and DMSO extracts inhibited the growth of B. subtilis with IZs 11, 8, and 15 mm, respectively. However, only the DMSO extract inhibited the growth of K. pneumoniae (10 mm) and E. coli (12 mm). The ethanol, methanol, and butanol extracts had no activity. MICs and MBCs of the DMSO extract were 0.72 and 0.85 mg/mL for P. mirabilis, 0.85 and >1.5 for K. pneumoniae, 0.65 and 0.9 mg/mL for B. subtilis, and 1 and >1.5 for E. coli. The water extract had MIC and MBC values 1, and >2 mg/mL for both P. mirabilis and B. subtilis, and the acetone extract had MIC and MBC values of 1.1 and >1.5 mg/mL for B. subtilis. E. faecalis and S. enteritidis were unsusceptible.	DDM and BMD	[99]
Tetradesmus dimorphus	Various solvent extracts	B. subtilis, S. thypimurium, E. coli, MRSA	The IZs of the methanol extract were 13, 10, 10, and 20 mm, respectively; the IZs of the ethyl acetate extracts were 9, 8, 11, and 12 mm, respectively; and of the n-hexane extracts, they were 8, 7, 8 and 8 mm, respectively.	DDM	[210]
T. dimorphus	Methanol extracts	S. aureus, B. subtilis, E. coli and P. vulgaris	No inhibition.	Antibacterial activity on agar plate, BMD	[208]
T. dimorphus	Methanol, ethanol, N-hexane, and diethylether extracts	The pathogens B. subtilis M. luteus, A. hydrophila and E. coli.	Effective inhibition against all strains, especially the diethylether extract with IZs 18–24 mm. The methanol extract effectively inhibited A. hydrophila, unlike the hexane and ethanol extracts.	AWDM	[1]
Tetradesdmus bajacalifornicus	Chloroform, acetone, ethanol, methanol, and aqueous extracts	B. subtilis, E. coli, Salmonella Typhi, and Clostridium perfringens; fungi—C. albicans, A. niger	All extracts had IZs nearly as large as that of fluconazole (21 mm) against C. albicans (19–20 mm). Only the water extract was active against C. perfringens with an IZ of 10 mm, and this extract had an IZ against A. niger of 19 mm, comparable to that of the standard (20 mm). The ethanol extract had an IZ of 18.5 mm against B. subtilis, close to that of the standard (20 mm). The other values ranged between 10 to 18 mm against S. Typhi and E. coli.	AWDM	[211]
Desmodesmus armatus, S. ecornis, and an unidentified Scenedesmus sp.	Ethyl acetate and 95% ethanol extracts	S. aureus, E. coli	The ethanol extract of S. armatus had an IZ of 16 mm against S. aureus (similar to the ethyl acetate extract) and 21 mm against E. coli. The ethanol extract of S. ecornis had an IZ of 22 mm against E. coli. The other extracts had lower or no activity.	AWDM (The Oxford cup method variant)	[132]
Tetradesmus lagerheimii, Ennalax costatus, S. acuminatus, S. obtusus, Pectinodesmus pectinatus, and Tetradesmus wisconsinensis	Methanol extract, pellet, aqueous extracellular supernatant	B. subtilis, E. coli, P. fluorescens, C. albicans and S. cerevisiae	All species (except the pellet of P. pectinatus) inhibited B. subtilis from 20 to 40%, compared with IZ of 25 mm of chloramphenicol, which was considered as 100%.	AWDM	[107]
Desmodesmus magnus var. magnus	dH2O extracts made with freezing and thawing	Opportunistic bacteria—S. aureus, E. coli, K. ozaenae, and P. aeruginosa	A pronounced antagonistic activity against all strains. K. ozaenae had 50% growth inhibition, and P. aeruginosa—> 82% growth inhibition.	BMD; photometric method	[32]
Tetradesmus incrassatulus	Culture medium, lipids, and fatty acids	S. aureus, S. pyogenes, B. cereus, E. coli, P. aeruginosa, Y. enterocolitica, S. typhimurium, and C. albicans	Fatty acids inhibited S. aureus and S. pyogenes with an IZ of 17 mm. The culture medium inhibited only C. albicans with an MIC of 6.25 mg/mL. No effect on the rest.	AWDM and BMD	[140]
Desmodesmus bicellularis	Fatty acid methyl ester (FAME) extracts, lipids and supernatant	MRSA, E. coli and C. albicans	FAME extracts had IZ of 21, 12, and 18 mm, respectively. No or very little IZ was observed in the case of fatty acids (9-octadecenoic acid had the best activity), while the methanol and hexane cell-free supernatant showed no effect at all.	AWDM	[206]
Desmodesmus maximus	Intracellular (food-grade solvent) and extracellular ethanol/water (1:1, v:v) extracts	A few of the most relevant foodborne pathogens—Salmonella sp., P. aeruginosa, E. coli and S. aureus	Low inhibition. The effect was likely derived from the high content of long-chain fatty acids.	Mixtures of pathogens and microalgae in the medium visually assessed for turbidity	[176]
Chromochloris zofingiensis	Ethyl acetate and 95% ethanol extracts	S. aureus and E. coli	Not a significant effect.	AWDM (The Oxford cup method variant)	[132]

, respectively.

4.5. Evaluation of the Antimicrobial Activity of Chlorellaceae and Scenedesmaceae according to Different Criteria

The criteria for assessing antibacterial or antimicrobial activity in vitro are mainly in consideration of the MIC. Eloff, an experienced researcher of antimicrobial activity of natural products, proposed the following criteria for MICs: outstanding activity <20 μg/mL, excellent activity 21–40 μg/mL, very good activity 41–80 μg/mL, good activity 81–160 μg/mL, moderate activity 161–320 μg/mL and weak activity >320 μg/mL. Higher MICs may still be effective in ethnopharmacological studies [212]. Porras et al., 2021 [213] proposed stricter MIC criteria: high activity ≤10 µg/mL and moderate activity 11–100 µg/mL.

There are no universal criteria for IZ. Still, practice shows that, in addition to consulting The European Committee on Antimicrobial Susceptibility Testing (EUCAST) [214], it can be tentatively assumed that zones of inhibition above 20-30 mm are relatively large, therefore the agent that produced it—with relatively strong antimicrobial effect. In addition, the quantity of the extract on the disc has to be considered. When comparing it with the reference antibiotic, we have to remember that extract or fraction is compared to a pure compound.

As Table 1, Table 2, Table 3 and Table 4 show, the antibacterial and antifungal activities of the two genera are in a broad range—from no activity to weak, moderate, strong, or very strong activity. This depends on the algal species, the pathogen, the kind of extract and the criteria used. Often, an extract is weak against a species of bacteria but is highly active against another. For example, a crude pigment extract from a Scenedesmus sp. did not inhibit Salmonella sp. at any concentration, but its MIC was as low as 80 µg/mL on S. aureus [157]. Frequently, one organic solvent produces extracts that are more inhibitory than another. For instance, the ethanol and acetone extracts of C. vulgaris had better antibacterial activity than the chloroform and aqueous ones [170].

The undoubtedly best activity, in terms of MIC, was observed for a strain of Chlorella (SRD3) with a MIC value of only 1.5 µg/mL against E. faecalis (ethyl acetate extract) [177]. Therefore, this strain is with outstanding effect according to the criteria of Eloff and high according to those of Porras et al. Lipid extracts of C. vulgaris, rich in palmitic and linoleic acid, exerted a MIC of 10 µg/mL against Propionibacterium acnes [188]. A chloroform extract of Scenedesmus bijuga also had a very significant effect on K. pneumoniae with a MIC of 16 µg/mL. While both these extracts had an outstanding activity, according to Eloff, the latter had moderate, according to Porras et al., which is valid for all agents described next [196]. C. ellipsoideum, A. protothecoides, and A. pyrenoidosa had a MIC of 20 µg/mL against P. acnes (outstanding effect, according to Eloff) [188]. Other exceptionally good results with C. vulgaris are MIC values as low as 25 to 40 µg/mL (excellent effect according to Eloff), where K. pneumoniae and S. aureus were more susceptible than B. subtilis and Micrococcus luteus [192]. A Scenedesmus sp. showed a MIC of 80 µg/mL (very good activity according to Eloff) against S. aureus [157]. It is obvious that all listed examples fulfill the criteria of [215].

Regarding the IZ, the best results belong to a methanol extract of A. pyrenoidosa with an IZ of 48 mm against E. coli [179]. C. vulgaris showed an exceptionally high antibacterial and antifungal effect because its acetone extract inhibited Sarcina lutea and Fusarium oxysporum with IZs of 30 and 45 mm, respectively. F. oxysporum was also inhibited by a 35.5 mm IZ by a Scenedesmus sp. [190].

Although T. obliquus does not show that significant effect, Table 3 shows that some of its in vitro tests have demonstrated IZs as large as 26–28 mm.

Naturally, that great activity of Chlorella sp. SRD3 predicted correctly that its IZ would be greater than that of chloramphenicol [177]. The IZ of the mentioned A. pyrenoidosa surpassed the IZ of the control antibiotic [179]. Other Chlorellaceae representatives have also shown better results than control antibiotics in an in vitro test. For instance, The IZ of an ethanol extract of C. vulgaris for E. coli surpassed the IZ of tetracycline [37]. Scenedesmaceae spp. have not been found to exceed the activity of antibiotics, but some of their effects are still comparable. Tetradesmus obliquus had IZs nearly as large as that of vancomycin against S. aureus [203]. All tested solvent extracts of Tetradesmus bajacalifornicus had IZs nearly as large as that of fluconazole against C. albicans; the ethanol extract had an IZ comparable to that of the standard against B. subtilis, and interestingly, that was also valid for the aqueous extract against A. niger [211].

5. Conclusions

The biomass of Chlorellaceae and Scenedesmaceae is widely commercialized; however, in order to begin production of specific HVP, efforts ought to be directed toward minimizing product loss as well as the energy and equipment expenses related to the extraction and purification steps. Furthermore, to achieve processes that are both environmentally friendly and economically viable, large-scale downstream processing should be further developed. Research and development for affordable HVP production from the two families, as well as from microalgae in general, is needed.

The antimicrobial activity of the two taxa could be utilized in WWT, diminishing the pathogenic microorganism’s load. Combining the knowledge, scientific, and research experience in the field of microalgal technology under the biorefinery concept, it is worth highlighting the perspectives of the microalgal potential to be an extremely competitive option for WWT and other ecological and industrial applications.

The in vitro antimicrobial activity of Chlorellaceae and Scenedesmaceae is in a broad range. The species could be inactive or have a low, medium, or strong effect. Several strains have fulfilled the criteria of Eloff and Porras et al. for outstanding and prominently high activity, especially those among C. vulgaris and other Chlorellaceae representatives. There were several strains with MICs below 80 µg/mL and even 10 and 1.5 µg/mL. Some strains also had IZs over 30 mm, even as high as 45 and 48 mm. Frequently, the two genera have better or nearly as good as the results of antibiotics against some pathogens.

The reason that the 80-year-old knowledge about the antimicrobial effect of microalgae, e.g., Chlorella, has not led to further development besides a protein-rich food does not accurately reflect their potential and is likely mainly due to the actinomycetes’ antibiotics prioritized for development. FFAs are also nutriments, including the long-chain ω-3 PUFAs that are precursors of eicosanoids and structural components of neural tissue; however, research shows some of them have an infection-suppressing potential when increased in the diet, and others have patents for topical antimicrobial applications. Microalgae, such as Chlorella, have been called “herbal antibiotics” based on vitro studies and the few in vivo studies of Chlorella are very promising. In vitro studies are the solid base for in vivo research and, ideally, the latter, in turn, support clinical trials. We hope that Chlorella and other microalgae will acquire more clinical presence, for example use in synergism with established antibiotics.