**5. Applications**

*Microalgae - From Physiology to Application*

to operate and economical for scale-up [30].

was more effective than the other methods.

water for 48 h, and finally, the extracts were lyophilized.

microalgal cell [9].

pretreatment is effective qualitatively and quantitatively, and the technology is simple

Zhao's research team [30] investigated three methods of conventional solvent extraction (CSE), fluidized bed extraction (FBE), and ultrasonic-assisted extraction (UAE) to obtain an effective extraction method of carbohydrates/glucose. The CSE employed lyophilized microalgae extracted with distilled water and agitation in a vortex. For FBE, the *Chlorella* sp. culture was harvested and washed with distilled water and then diluted using distilled water and added into a fluidized reactor with air aeration. To UAE, the algal cells were harvested and washed with distilled water, diluted, and taken to the ultrasonic processor. The ultrasonic-assisted extraction

Information in the literature related to the amount of cell wall microalgae polysaccharides is scarce. Usually, the quantification of polysaccharides in microalgal is made by analyzing the total carbohydrate, thus including storage polysaccharides (SPS) and cell wall-related polysaccharides, which exhibit different functions in the

In the extraction of CWPS, the pellets were suspended in MOPS buffer, and the cells were disrupted using UHPH after cold ethanol was added to the suspensions and for pellet recovery. Lipids were removed by addition of hexane/isopropanol to the pellet, mixed and centrifuged to remove the upper solvent layer. Afterward, SPS and protein were enzymatically removed using endo-β-1,3-glucanase or a combination of α-amylase/amyloglucosidase and *Subtilisin* A protease, respectively. The mixtures were incubated and after addition of cold ethanol they were centrifuged. The pellet was finally washed in acetone, vacuum filtered, and dried overnight at 40°C, and this residue was considered as CWPS. Monosaccharide and uronic acid composition of CWPS and EPMS were hydrolyzed according to De Ruiter et al. [36]

After sample preparation, carbohydrate analysis is a very complex field. Usually, after microalgal acid hydrolysis, the total carbohydrate content of the hydrolysate can be determined using colorimetric procedures like the phenol-sulfuric acid [37–39] or anthrone-based [40–42]. These methods are available, giving excellent and robust results with low effort in a very short time. Nevertheless, detailed information about the monosaccharide composition cannot be generated [29]. Qualitative investigations can be performed using TLC methods with silica-based separation materials making the separation of most monosaccharides possible. However, quantification with the TLC methods is not possible [29], and for quantification of monosaccharides, analytical methods such as high performance liquid chromatography (HPLC) are often used. The HPLC equipped with a refractive index detector (RID) [32] and HPLC combined with pulsed amperometric

using methanolysis combined with trifluoroacetic acid (TFA) hydrolysis.

According to Bernaerts et al. [9], the insight into the composition of cell wall-related polysaccharides, such as the monosaccharide profile or the degree of sulfation, is not only desired in terms of process optimization but also as a potential for several biotechnological. Thereby, the authors investigated to apply a universal procedure for extraction of the total cell wall-related polysaccharides, including cell wall polysaccharides (CWPS) and extracellular polymeric substances (EPMS), of 10 commercially available microalgae species followed by a characterization of the monosaccharide profile, uronic acid content, and sulfate [9]. Initially, the procedure consisted of dry biomass suspended in saline solution incubated for 16 h at 25°C, followed by a two-step centrifugation. Afterward, the supernatant was submitted for extraction of EPMS and the residual biomass (pellet) for extraction of CWPS. Ethanol was added to the supernatant precipitating EPMS; the solution was vacuum filtered, and the insoluble residue was dialyzed against demineralized

**162**

Microalgae have several types of polysaccharides in their composition, such as phycocolloids, agar, alginate, carrageenan, fucoidan, ulvana, and cellulose, among others. phycocolloids can be formed by different monomers such as glucose, galactose, mannuronic acid, guluronic acid, mannitol, and laminarin. These carbohydrates can be inserted into functional beverages and food products such as functional bread, ready to serve soups, functional snack foods and a variety of sauces, creams, bakery products, and additional food products [43, 44].

Due to the high carbohydrate content, poultry and aquaculture feed is one of the main study targets for the use of microalgae biomass. In 2007, around 30% of the world's current algae production was sold for animal feed application [8]. Microalgae are also a suitable alternative for growing fish, larvae, and zooplankton. *Chlorella* is one of the main examples of microalgae that can play a key role in food and feed due to the properties of its biomass, which can simultaneously provide high concentrations of carbohydrates, vitamins, and proteins [45]. Besides *Chlorella*, other species used in aquaculture can be highlighted: *Tetraselmis*, *Isochrysis*, *Pavlova*, *Phaeodactylum*, *Chaetoceros*, *Nannochloropsis*, *Skeletonema*, and *Thalassiosira*. *Spirulina* and *Chlorella* microalgae can be applied in the feeding of cats, dogs, aquarium fish, ornamental birds, horses, birds, cows, and breeding bulls. The most common genera of larval microalgae include *Chaetoceros*, *Thalassiosira*, *Tetraselmis*, *Isochrysis*, and *Nannochloropsis* [46].

1,3-*β*-glucan is an important carbohydrate present in microalgae composition due to its applications in the food industry as a thickener, and health applications, especially in the protection against infections and also to inhibit cancer cell growth in vivo [44, 47]. According to [48], the global β-glucan market was valued at USD 307.8 million in 2016, and it is predicted that in 2022, the global carbohydrate market could reach up to USD 476.5 million, which indicates the huge potential for development in many different types of applications.

According to Koller et al. [49], sulfated polysaccharides produced by microalgae can be applied in therapies against bacterial infections. Carrageenan polysaccharide, also known as food additive E407, can be used in pharmaceutical applications. Marine carbohydrates have been widely used in the cosmetics industries due to their chemical and physical properties. Brown algal fucoidans/alginates, green algal ulcers, and red algal carrageenans/agar are used as gelling, thickening, and stabilizing agents. In addition, marine carbohydrates have potential skin benefits, and biological activities are linked to their structure as determined by molecular weights or the presence of sulfate groups and other sugars [50].

Red algae, such as *Chondrus* sp., *Gigartina* sp. *Eucheuma* sp., *Hypnea* sp., and *Furcellaran* sp., are widely used for the production of carrageenan. This compound can be used in food and pharmaceutical industries for applications in fruit gel, fruit juices, sweets, and jellies, among others. Another carbohydrate group

molecule is fucoidan, which is associated with brown algal cell wall components (*Phaeophyceae*). Among the bioactivities derived from this molecule, the anticoagulant, antitumor, antivirus, and antioxidant properties stand out, making it attractive for pharmaceutical applications [51].

Besides these applications, the remaining biomass of microalgae presents carbohydrate-rich molecules, which have been widely used in the production of bioplastics, agar, sugars, and other high-added value chemicals. However, despite being a growing area, the biorefinery stage must be studied in order to extend its applicability on an industrial scale [51]. According to Mihranyan [52], the rheological behavior of cellulose found in *Cladophora* algae is similar to micro fibrillated cellulose. Because this cellulose is very robust and not susceptible to chemical reactions, the properties of cellulose found in these algae provide excellent rheological properties making this material interesting in food, pharmaceuticals, paints, dressings, and biodegradable plastic applications.

The high carbohydrate content and low-ash values make microalgae more suitable for conversion to biofuels [43]. The production of bioethanol from microalgae gained importance due to their high biomass productivity, diversity, variable chemical composition, and high photosynthetic rates of these organisms [53]. Due to the large amount of carbohydrates/polysaccharides and cellulose walls, these microorganisms become favorable for the production of this biofuel [54, 55]. In many countries, ethanol is produced on a large scale from crops containing sugars and starches in its composition through fermentation. The biomass is ground, and the starch is converted into sugars by different methods. Polysaccharide starch is also accessible as a storage material for various algal species and can be anaerobically converted into bioethanol [49].
