Exopolysaccharides (EPSs) are natural biopolymers synthesized by microorganisms and are secreted for various respective functions, such as defense, biofilm formation, pathogenicity, structure, adhesion, etc. [1]. EPSs are long-chain biomolecules with molecular weights ranging from 10 to 30 kD, and are produced during the late exponential and or stationary phase of microbial growth. EPSs are produced in response to environmental stress conditions, such as pH and temperature, and exposure to heavy metals or inhibitors, etc. ^[2][3][2,3]. Biochemically, EPSs are carbohydrate polymers composed of glucose, galactose, and rhamnose, accompanied by non-carbohydrate moieties such as proteins, enzymes, nucleic acid, etc. The exact composition may vary with microorganisms and growth conditions.

Based on the biochemical structure and composition, EPSs can be categorized into homo-exopolysaccharides (HOEPSs) and hetero-exopolysaccharides (HEEPSs) [4]. HOEPSs are composed of one type of monosaccharide, such as α-D-glucans, β-D-glucans, fructans, and polygalactans, interlinked with α-1-6, α-1-3, β-1-2, β-1-3, β-2-6, and β-2-1 linkage among the subunits, depending upon the monomeric units. Dextran is one of the well-known examples of HOEPSs, which is made of glucose interlinked with α-1-6 glucoside linkage. In contrast, HEEPSs are comprised of different sugar monomers, along with their respective derivatives and non-carbohydrate moieties. Succinoglycan is one of HEEPSs present in bacterial biofilm. It is comprised of saccharide-based oligomers in which sugar molecules are derivatized with acyl, pyruvate, and succinic acid ^[4][5][6][4,5,6]. Both classes are further recognized as subgroups based on the dominant sugar residues [4]. In recent years, EPSs have gained wide attention for their cost-effective production and diverse applications as pharma and healthcare products, cosmeceutical, nutraceutical, functional food, and biocontrol agents in agriculture [7], oil recovery in petroleum industries [8], heavy metal removal [9], drug delivery, and tissue regeneration and repair [10]. Microbial exopolysaccharides have applications in various sectors, as depicted in Figure 1.

Exopolysaccharides are produced by diverse microorganisms, including yeasts, fungi, and bacteria, utilizing various raw materials (Figure 2). Besides the native physiological role, microbial EPSs also have diverse applications in many industries. Several efforts have been made by various researchers for the production of EPSs (Table 1). In comparison to yeasts and other fungi, probiotic bacteria are commonly used due to non-pathogenicity and categorization of generally regarded as safe (GRAS) ^[11][25]. It has been found that carbon source has a direct relation with EPS production and composition. A study on 20 strains of Lactobacillus paracasei revealed that with a change in carbon source, not only does EPS yield, but it also influences the monosaccharide composition. The yield of EPS was increased by 115% with optimized carbon sources, including fructose, glucose, galactose, lactose, mannose, and trehalose ^[12][26]. Among different carbon sources, including sucrose, maltose, lactose, glycerol, and sorbitol, maximum EPS production has been achieved with maltose by Candida guilliermondii and Candida famata (0.505 and 0.321, respectively) ^[13][27]. Among 156 lactic acid bacteria isolated from healthy young children’s feces, the maximum EPS production, i.e., 59.99 g/L, was reported from Weissella confusa VP30 after 48 h in growth media containing 10% sucrose ^[14][28].

The use of commercial-grade sugars/substrates has a direct impact on product cost, and is one of the factors responsible for high production costs. Hence low-cost waste materials, such as lignocellulosic residues and wastewater, are preferred as raw materials for EPS production. The carbohydrate and organic fractions present in the waste can be used by microorganisms. It opens up the opportunity to reduce product costs, along with waste management. Da Silva et al. ^[15][29] have compared coconut shells, cocoa husks, and sucrose for xanthan gum production by Xanthomonas campestris pv. campestris IBSBF 1866 and 1867. The study revealed that xanthan gum yields were higher, i.e., 4.48 g/L and 3.89 g/L, in the case of cocoa husk by Xanthomonas strains 1866 and 1867, respectively, but the apparent viscosity was higher than sucrose, i.e., 181.88 mPas over cocoa husk, with a viscosity of 112.06 mPas.

Choi et al. ^[16][30] used spent media wastewater (originated from kimchi fermentation) for EPS production using Leuconostoc mesenteroides WiKim32. Under optimal conditions, the maximum EPS productivity was 7.7–9.0 g/L, with a conversion of 38.6–45.1%. The EPSs were nontoxic and exhibited thermal tolerance and antioxidant activity. Pan et al. ^[17][31] optimized dextran production from Leuconostoc pseudomesenteroides XG5 using an L9 (33) orthogonal test. Under optimal conditions (sucrose 100 g/L, pH 7.0, 25 °C, at 100 rpm for 36 h), the maximum dextran yield of 26.02 g/L and 40.07 g/L were recorded at a laboratory scale and fed-batch fermentation, respectively. The EPS was also exhibiting water-holding capacity and antioxidant activity. It reduced the chewiness and hardness of yogurt, but the resilience increased during the 14 days of storage. Product cost is one of the main obstacles in commercialization, hence process economics is one of the major factors that must be assessed. Integration of multiple processes might improve process economics, as well as environmental adaptability. Sphingobium yanoikuyae was evaluated for the coproduction of EPS and polyhydroxyalkanoates (PHAs) using lignocellulosic hydrolysate. Hydroxymethyl furfural (HMF), one of the hydrolysis byproducts, improved the consumption of glucose and xylose during fermentation. The optimum C/N ratio of 5 resulted in the maximum EPS production of 3.24 ± 0.05 g/L, however, a further increase in the C/N ratio (30) favored PHB accumulation (38.7 ± 0.08% w/w) ^[18][32]. Biomass hydrolysate is usually accompanied by phenolics, furfurals, and HMF, which also act as fermentation inhibitors and hinder microbial action. Some of the methods, including activated carbon-based adsorption and membrane filtration, have been proposed for hydrolysate detoxification ^[19][20][33,34]. Removal of inhibitors might improve microbial action and product yield. Bhatia et al. ^[18][32] have compared the potential of various raw and detoxified hydrolysates for EPS production. In comparison to raw hydrolysate, detoxified biomass hydrolysate showed increased EPS production and maximum EPS production was reported with detoxified pine biomass hydrolysate, i.e., 2.83 ± 0.03 g/L. Table 1 summarizes the recent efforts for EPS production using various microbes (yeasts, bacteria, and fungi), utilizing various pure carbon and organic waste materials.

Preparation of composites for healthcare applications needs high purity, therefore the downstream processing becomes an inseparable part of processing after fermentation. After production recovery, the identification of structural and chemical characteristics is necessary for further applications. The characterization of EPSs is quantitative, as well as qualitative. EPSs are mainly comprised of carbohydrates conjugated with other biomolecules, hence the basic characterization techniques employed are a colorimetric estimation and use spectrophotometry ^[31][45]. For carbohydrate estimation ‘Dinitrosalicylic Acid Reagent’ is one of the common methods which quantify the reducing sugars ^[32][46]. Similarly, for protein, Bradford’s dye-binding method ^[33][47] and Lowry’s method ^[34][48] are used. Besides basic characterization with colorimetric methods, Fourier transform infrared spectroscopy (FTIR) is employed to detect the available functional groups and structural functionalities of EPSs. For the detailed structures of EPSs, nuclear magnetic resonance (NMR) and mass spectra are used. In NMR, the sample is dissolved in deuterated solvents for quantification with respect to internal standards ^[35][36][49,50]. The mass spectrum of EPS provides a monosaccharide composition. For analysis, EPSs are hydrolyzed with acid hydrolysis, followed by silylation derivatization. The derivatives are detected and identified by gas chromatography-mass spectrometry ^[35][49]. The biological potential of EPS has followed the general procedure for antimicrobial, antioxidant, anti-inflammatory and other activities ^[31][45].