ISSN: 0973-7510

E-ISSN: 2581-690X

Review Article | Open Access
Hemlata Sharma1, Jyoti Pal2 and Deepesh Kumar Neelam1
1Department of Microbiology, Faculty of Science, JECRC University, Ramchandrapura Industrial Area, Vidhani, Sitapura Extension, Jaipur – 303 905, Rajasthan, India.
2LNJN National Institute of Criminology and Forensic Sciences, Rohini, Delhi, India – 110 085, India.
J Pure Appl Microbiol. 2021;15(3):1072-1082 | Article Number: 6921
https://doi.org/10.22207/JPAM.15.3.28 | © The Author(s). 2021
Received: 08/03/2021 | Accepted: 08/07/2021 | Published: 17/07/2021
Abstract

Prokaryotic microbial cells especially bacteria are highly emphases for their exopolysaccharides (EPS) production. EPS are the higher molecular weight natural extracellular compounds observe at the surface of the bacterial cells. Nowadays bacterial EPS represent rapidly emerging as new and industrially important biomaterials because it having tremendous physical and chemical properties with novel functionality. Due to its industrial demand as well as research studies the different extraction processes have been discovered to remove the EPS from the microbial biofilm. The novelties of EPS are also based on the microbial habitat conditions such as higher temperature, lower temperature, acidic, alkaliphilic, saline, etc. Based on its chemical structure they can be homopolysaccharide or heteropolysaccharide. EPSs have a wide range of applications in various industries such as food, textile, pharmaceutical, heavy metal recovery, agriculture, etc. So, this review focus on the understanding of the structure, different extraction processes, biosynthesis and genetic engineering of EPS as well as their desirable biotechnological applications.

Keywords

Exopolysaccharides, Biosynthesis, Genetic engineering, Industrial applications

Introduction

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 them8,9. 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 EPS16,17. 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 EPS20,21.

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 exopolymers24,25. 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 biofilms16,29. 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) blocks41,42. 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.

S.No
EPS
Localization
Polymerisation enzyme
Precursors
Micro-organism
Reference
1.
Dextran
Extracellular
Dextransucrase
Saccharose
Leuconostoc sp.
72
2.
Pullulan
Extracellular
UDPG-pyrophosphorylase
UDP–d-glucose
Aureobasidium
Pullulans
73,74
3.
Levan
Extracellular
Levansucrase
      –
Bacilluslicheniformis, Acetobacter sp., Halomonas sp.
 75
4.
Curdlan
Extracellular
Curdlan synthase
UDP–glucose
Rhizobium spp.
76
5.
Cellulose
Extracellular
Cellulose synthase
UDP-d-glucose
Acetobacter
Xylinum
77

 

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 domain47,48. 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 levels50,51. 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 clear53,54. 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 sugar55-57. 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 polymer58,59. 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 pathway60-62 (Table 2).

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

S.No.
EPS
Localization
Polymerisation enzymes
Precursors
Micro-organism
Reference
1.
Succinoglycan
Extracellular
Phosphoglycosyltranferase
UDP-glucose,UDP-galactose
Sinorhizobium meliloti
 78,79
2.
Hyaluronic acid
Extracellular
HA synthase
UDP-glucuronic acid,UDP-N-acetylglucosamine
Psudomonas aeruginosa, Streptococcus sp.
 80,81
3.
Gellan
Extracellular
Gellanlyase
UDP-glucose,TDP-rhamnose,UDP-glucuronic acid
Psudomonas elodea, Sphingomonas paucimobilis
82,83
4.
Alginate
Extracellular
Glycosyl tranferase
GDP–mannuronic
Acid
Pseudomonas sp.
84
5.
Xanthan
Extracellular
Xanthan polymerase
UDP–glucose,
GDP–mannose
and UDP–
glucuronate
Xanthomonas
Campestris
85

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 glucans63,64. 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 complex65,66. 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 perspectives70,71.

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 conditions87,88. 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 biosynthesis91,92.

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

EPS
Name of the  Bacteria
Gene
Reference
Xanthan
Xanthomonas
Campestris
gum D
93
Hyaluronan
Streptococcus zooepidermicus
has A
94
Cellulose
Acetobacter xylinus
bcsA
95
 Levan
Erwinia amylovora
rlsA
96
Gellan
Sphingomonas paucimobilis
pgm G
97
Alginate
Pseudomans aeruginosa
AlgD, AlgC, AlgR, AlgB, AlgZ
98,99,100,101

 

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 niches102-104. 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 shock113,114, 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 food120,121. 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.

CONCLUSION

As described in this review, It is now widely considered that bacterial EPS plays a very important role in various industrial applications. Moreover, EPSs biosynthesis is a complicated process through which various alterations occur and resultant many number of EPSs produces on bacterial cell surface, which have a valuable range of physicochemical properties and highly promising commercial applications. However, the EPS extraction methods from cell surface still required the some novel techniques or tricks that will be easy to handle, time consuming and more effective for understanding of mechanism involved in synthesis and excretion. This study showed the role of EPS in the food, pharmaceutical, heavy metal recovery and agriculture field, but there is still much to learn about their functions in the environment. To understand more about biopolymers synthesis, it will be necessary to explore insight into the some extremophiles from extreme condition these EPSs are highly stable at various physical as well as on chemical parameters more than mesophilic bacterial EPSs. On the other hand genetic engineering is the new tools for changes in the properties of molecules that will be possible by genome annotation and construction of EPS biosynthetic pathways in bacterial cell to understand about how they will incorporate and how they will be affected. The big research gaps still remain that no method exists to extract all microbial polysaccharides but in upcoming scientific studies it could be possibility to explain about EPSs with their specific structure and functions.

Declarations

ACKNOWLEDGMENTS
All listed author(s) are thankful to JECRC University for providing the related support to compile this work.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
None.

ETHICS STATEMENT
This article does not contain any studies with human participants or animals performed by any of the author.

AVAILABILITY OF DATA
All datasets generated or analyzed during this study are included in the manuscript.

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