Exopolysaccharides are the key feature of most of the bacterial sufaces¹. The formation of biofilms takes place through the attachment of bacterial cells to the substratum or cells embedded in a protective extracellular matrix². It is a complex structure of a heterogeneous matrix consists of various molecules³. These natural polymers have emerged as a new alternative to synthetic polymers with marvellous physical characteristics, so they have vast industrial applications. This term was first used by Sutherland in 1972 in his exclusive work on marine bacteria producing EPS⁴. EPS finds in two forms viz. EPS (capsular EPS) and soluble EPS (slime)^5,6. From the last decades, industries are more emphases on natural polymers production and these natural polymers are used by various pharmaceuticals, food and other industries which are developing remarkable interest in polysaccharides produced by microorganisms⁷. The total EPS yield depends upon microorganisms used and cultivation conditions provided to them^8,9. Bacterial EPS’s have been utilized as bio-absorbents, bio-flocculants and heavy metal removal agents¹⁰. The main aim of writing this review is to give an overview of the structure, extraction process, biosynthesis, genetic engineering and applications of microbial EPS.

Structure of EPS
Based on the monomeric unit, EPS’s are classified as homopolysaccharides and heteropolysaccharides¹¹. Homopolysaccharides contain monosaccharides while heteropolysaccharides are composed of more than one type of monosaccharide¹². EPS is classified based on the number and nature of monomers, bonds between them and the type of linkage¹¹. EPS are generally polyanionic due to the presence of uronic acids or ketal-linked pyruvate or inorganic substances like phosphate or sulfate. Some are also neutral macromolecules¹³. A few EPSs may even be polycationic, e.g. polymer obtained from Staphylococcus epidermidis strains¹⁴. However, the physicochemical factors also affect the production of exopolysaccharides which includes pH, temperature, incubation time, and the constituents of culture media (with various organic and inorganic carbon (C) and nitrogen (N) sources)¹⁵.

Extraction of EPS
Various extraction processes have been developed to recover EPS from biofilm of microbes from different environments to identify the contents of EPS, to analyze various properties (chemical, physical and physicochemical) and to observe the functions of EPS^16,17. The various extraction methods include chemical or physical and physical and chemical, and analytical methods. It is estimated that extraction methods are dependent on the water solubility of the EPS separated. The extraction methods of EPS should be cost-effective, eco-friendly and do not damage the structure of EPS¹⁶. The extraction efficiency is calculated as the overall amount of EPS separated from the entire microbial biomass for a particular sample⁵.

Physical method
The physical process mainly involves three extraction techniques i.e. centrifugation, sonication and heating¹⁸. Researchers explored the reason behind variations in extraction efficiency with different physical methods. These variations in results are because during the extraction procedure by heating, the components of EPS might be hydrolyzed, for this particular case, the proteins and polysaccharide content of EPS¹⁸. Another study showed that the heating allowed extracting the capsular EPS to flocs¹⁹. However, several studies suggested that the high EPS extraction yield by the heating methodology could also be thanks to meaningful cell lysis which can lead to high protein content in EPS^20,21.

Chemical method
In the chemical method, different chemicals are used which could break the linkage in the matrix so that EPS can be easily released to the external medium containing water. NaOH treatment can cause the ionization of a great number of charged groups, for instance, carboxylic groups in proteins and polysaccharides. Due to this, a strong repulsion occurs between EPS which enhances its solubility. A lot of polymers can suffer alkaline hydrolysis²². In glycoproteins, disulfide bonds can be broken if exposed to basic environments (pH above 9), which will promote the extraction of these compounds²³. The repulsion and solubility between the compounds of the EPS matrix are resultant of the exchange of divalent cations with mono-valent cations. Resin, EDTA or EGTA are used for the removal of divalent cations. A high concentration of sodium chloride can also be used to carry out cation exchange. This process has been used in Pseudomonas sp. for the extraction of adhesive exopolymers^24,25. The extraction of EPS could be increased by destabilizing biofilm in an enzymatic digestion process²⁶. Furthermore, ethanol from activated sludge has been used to extract lipids²⁷.

Combination of physical and chemical method
Some studies proposed that the chemical extraction method could be of better performance once it is combined with the physical method i.e. defined shear. The shear is often provided by heat, sonication, or stirring under pre-established conditions. The alkaline and heat treatment has been combined to extract capsular EPS from varied microbial species²⁸. On the other hand, shear (stirring) and ion exchange by a Dowex extraction have been used in conjunction to extract EPS from activated sludge and biofilms^16,29. Formaldehyde (CH₂O) and NaCl was applied in combination with ultrasonication, to extract EPS from an anaerobic sludge³⁰. The formaldehyde is added to minimize cell disruption during the extraction process. However, formaldehyde (CH₂O) has the capacity to changes the properties of many EPS components³¹.

Analytical method
Colorimetric analyses
The complex composition of EPS in biofilms is made up of carbohydrates, lipids, humic substances, proteins, nucleic acids, etc. Colorimetric analyses are may be used to quantify the components in EPS³². The measurement of carbohydrate contents performed by two methodologies i.e. the anthrone method or the phenol–sulfuric acid method. The content of protein might be measured by the Lowry technique, the Press-Man technique, or the total N-content technique¹⁶. The m-hydroxydiphenyl sulfuric acid method has been used to measure uronic acid content in EPS³³. To measure the nucleic acid content, three different methodologies could be used, which are the 4,6-diamidino-2-phenylindole (DAPI) fluorescence method¹⁶, the UV absorbance method³⁴, or the diphenylamine method²¹.

Innovative methods
EPS structure, functions and conformation examinations are very difficult to work due to their complex composition. However, recent studies in analytical chemistry develop new techniques such as transmission electron microscopy (TEM)³⁵, scanning electron microscopy (SEM)³⁵, atomic force microscopy (AFM)³⁶, confocal laser scanning microscopy (CLSM)³⁷, fourier transform infrared spectroscopy (FTIR)³⁸, X-rayphotoelectron spectroscopy (XPS)³⁸, nuclear magnetic resonance (NMR)³⁹ and 3-dimensional excitation-emission matrix fluorescence spectroscopy (3D-EEM)⁴⁰ which will help in examining the properties of EPS as well as their nature. The qualitative and quantitative analysis of EPS compositions was reported by using chromatography, mass spectrometry and their combination¹⁶.

Biosynthesis of EPS
Biosynthesis of homopolysaccharides and heteropolysaccharides take place in different-different pathways.

Synthase dependent pathway
The synthesis of homopolysaccharides through synthase, a dependent pathway is quite complicated, but specific enzymes make this process easier, by making modifications in initially synthesized homopolysaccharides such as alginate (Table 1) via the polymerization of GDP-mannuronic acid monomers, the biosynthesis of alginate takes place too. The enzymes involved in alginate biosynthesis are epimerases, lyases and acetylases⁴¹. Diverse alginate epimerases (AlgE1-7) outer of the cell are there that alter the final chemical polymer characteristics through the selective insertion of specific β-D- mannuronic acid (M) and α-L-guluronic acid (G) blocks^41,42. In recent years, the significance of P. aeruginosa was represented as depressingly induces alginate production. The DalgL variant had maximum yields of alginate of equal MW. The higher O-acetylated alginate and lower molecular weight were observed after the overexpression of AlgL. Both are highly focused factors for the pathogenicity of P. aeruginosa⁴³. Therefore, the biosynthesis of alginate functions as a motley-enzyme complex⁴⁴. A pure EPS of biofilm is bacterial cellulose which is a linear glucan β-(1,4)⁴⁵.

Table (1):
List of homopolysaccharides and producing bacteria.

The UDP-activated cytosolic glucose monomers follow the cellulose synthase complex that incorporates a preserved catalytic subunit denominated BcsA. It is related to the GT2 family which is known by performs the polymerization process through upend the mechanism. The structures of the principal subunit and another subunit, BcsA and BcsB both were identified from specific bacteria that was Rhodobacter sphaeroides, show the cell domain of the BcsAB comprised the GT activity and a PILZ domain for interaction with activator c-diGMP⁴⁶. The periplasmic domain is narrowly linked to a flavodoxin-like domain^47,48. The entire cell envelope is permeated by cellulose synthase complex and its productivity is very high⁴⁹. The interesting phenomenon of biosynthesis of cellulose was recently described⁴⁶.

Dextrase/Sucrase dependent pathway
The homopolysaccharide dextran and levan are formed and assembled from the cleavage of sucrose molecules by the action of the extracellular sucrase. After that, the monosaccharide unit is transported to a primer molecule, which may be ramified at distinct levels^50,51. Further, using different primer molecules leads to high oligosaccharide production⁵².

Wzx/Wzy pathway
In this pathway activated sugar nucleotide monomers are transferred by an enzyme called glycosyltransferases (GTs). In this manner, the number of GTs available will determine the sequence of the final polymers. Both the side chain and substituents are incorporated adjacent to the backbone, before the completion of the polymer assembly, but the stage at which this incorporation occurs is not clear^53,54. The Wzx gene encodes the flippase protein which transfers the repeating unit by H+-dependent antiporter mechanism⁵⁵. Different numbers of trans membrane sequences are shown by the structures of the several Wzx proteins, besides lack in similarity. It points out that different types of Wzx protein exist⁵⁶. Evidence has been found to support the preference of the substrate Wzx on kindred O-units further than the first sugar^55-57. As soon are transferred the repeating units towards the periplasm another enzymes, the polymerase will recognize it and helps in the polymerization of repeating units. This procedure is carried out by the polymerase, sometimes accompanied by a co-polymerase, which may be associated with the process of determining the length of the polymer^58,59. The Wzx / Wzy pathway is defined by the involvement of the main protein in the transportation and polymerization of specific repeating units, on which the final structure of EPS depends. Various EPS such as xanthan and succinoglycan are synthesized through this pathway^60-62 (Table 2).

Table (2):
List of heteropolysaccharides and producing bacteria.

ABC transporter pathway
Two synthesis strategies dependent on the ABC transporter have been identified. One of these strategies is combined with the synthesis and export of cytosolic glucans^63,64. The second is the synthesis and export of uncoupled glycans by modifying the non-reducing terminus of the polymer attached to Und-P that ends the chain extension. At this point, the terminator determines the glycan chain length and simultaneously serves as an export signal recognized by the transporter⁶³. A terminal residue linked by the WbdD protein⁶⁵ and the end process depends on the chain size and the stereochemistry of the WbdD-WbdA complex^65,66. To assemble the glycan chain, the domains of GT activity are carried by the WbdA protein. Recently, a protein was described in Raoutella terrigena, that was observed the significant role for polymerization, termination, and quality control within its protein structure⁶⁷. These types of findings show the complexity behind the biosynthesis mechanism. Additionally, specific domain scans could be given within the protein complex. Hence, it was possible to understand an important phase of CPS biosynthesis, whose role is crucial in human pathogens. Recently, in Campylobacter jejuni, the ABC PglK transporter mechanism was described, which is highly dependent on ATP to achieve the transport of lipid-linked oligosaccharide units⁶⁸. The drop-interface-bilayer systems have been novel techniques that have helped to gather the most recent knowledge about the Wza homologs for the export of CPS, allowing to know the complexity of this transport mechanism⁶⁹ and in turn, contributing to the construction of promising perspectives^70,71.

Engineering strategies
The specific operons present on the genome which are encrypted in genes for the biosynthesis of EPS. Thus, the number of open reading frames (ORFs) may differ from one to more than 30 ORFs⁸⁶. EPS is composed of all the genes that are essential for its biosynthesis of polysaccharides units, the turning of the repeating units, as well as the polymerization and the final polymer transport (Table 3). Besides these, there are some specific genes present on the operon which are involved in sugar precursors synthesis, whereas other genes that provide nucleotide sugar are spread all over the chromosomes⁶². It was observed that most of the bacterial genes could encode more than one polysaccharide biosynthesis pathway⁸⁷. The production of EPS depends upon the regulatory effects and cultivation conditions^87,88. The various type of carbon (C) and nitrogen (N) sources can affect the polysaccharide’s expressions in bacteria⁸⁹. It was found that cdi-GMP has an impact on the biosynthesis of EPS, moreover, overexpression resultant as production of novel EPS⁹⁰. The new binding sites could be affecting the EPS biosynthesis^91,92.

Table (3):
List of genes responsible for EPS production.

Applications of EPS
Agriculture
EPS has a wide range of applications in agricultural fields and is known to have the ability to increase productivity. EPS’s secreted by microorganisms play a crucial role during soil development¹⁰². It is also capable of entrapping nutrients and provides protection to microbes against unfavorable environmental conditions by forming niches^102-104. EPS also plays an important role in protecting a crop against desiccation and predation by other organisms¹⁰⁵. EPS also protects the seedlings from drought. The ability to freeze water, technically called ice nucleation activity (INA), is widely used in biotechnology, for example for the production of energy-saving artificial snow and ice. Additionally, in industries such as food processing, it has been used during ice-cream production and freezes concentration efficiently avoiding loss of flavor¹⁰⁶.

Heavy metal degradation
It has been reported in several studies that EPS has a high affinity for heavy metals present in wastewater¹⁰⁷. The binding affinity of EPS towards heavy metals depends upon the composition and binding sites present in EPS¹⁰⁸. EPS is associated with the surface so that it protects micro-organisms from heavy metal toxicity¹⁰⁹. It has been reported that the adsorption capacity of most of the heavy metals such as copper (Cu), lead (Pb), cadmium (Cd), and zinc (Zn), etc. depends on the components of EPS, which shows that the main reason for the adsorption performance of EPS is the protein¹¹⁰.

The major issues of heavy metal, contamination have been seen in agricultural soils because heavy metals can easily enter into the food chain and possess serious health hazards to the humans as well as ecosystem¹¹¹. Toxic heavy metals include cadmium, lead, copper, zinc and manganese¹¹².

Biomedical applications
EPS’s have a wide range of applications in the biomedical sector. It is used as a plasma volume expander for controlling wound shock^113,114, as an antiacid stomach protector in capsules and as a stabilizing agent in pharmaceutical suspensions and emulsions¹¹⁵. It is also used during eye surgery, in wound healing, used in cosmetics, in the treatment of osteoarthritis¹¹⁶, as a drug-controlled release carrier¹¹⁷ and also used in skin repair¹¹⁸.

Food applications
The need of today’s hour is the healthier food without compromising with the safety of food¹¹⁹. There are a lot of microbes producing EPS for eg. lactic acid bacteria mainly produce EPS which improves the quality, texture and safety of various food products and also inhibits the growth of disease-causing organisms in food^120,121. The EPS in the food industry has been used as an emulsifier, stabilizer and thickener. It is also used in the packaging of food products. Mostly xanthan, gellan and cellulose which are secreted by bacteria other than lactic acid bacteria, are predominantly used in the food industry¹²².