Extracellular polymeric substances (EPS) are natural polymers of high molecular weight secreted by microorganisms into their environment. EPS establish the functional and structural integrity of biofilms, and are considered the fundamental component that determines the physicochemical properties of a biofilm. EPS in the matrix of biofilms provides compositional support and protection of microbial communities from the harsh environments. Components of EPS can be of different classes of polysaccharides, lipids, nucleic acids, proteins, lipopolysaccharides, and minerals.

Components EPS are mostly composed of polysaccharides (exopolysaccharides) and proteins, but include other macromolecules such as DNA, lipids and humic substances. EPS are the construction material of bacterial settlements and either remain attached to the cell's outer surface, or are secreted into its growth medium. These compounds are important in biofilm formation and cells' attachment to surfaces. EPS constitute 50% to 90% of a biofilm's total organic matter.

Exopolysaccharides

Exopolysaccharides (also sometimes abbreviated EPS; EPS sugars thereafter) are the sugar-based parts of EPS. Microorganisms synthesize a wide spectrum of multifunctional polysaccharides including intracellular polysaccharides, structural polysaccharides and extracellular polysaccharides or exopolysaccharides. Exopolysaccharides generally consist of monosaccharides and some non-carbohydrate substituents (such as acetate, pyruvate, succinate, and phosphate).

Exopolysaccharides are secreted from microorganisms including microalgae into the surrounding environment during their growth or propagation. They can either be loosely attached to the cell wall or excreted into the environment. Many microalgae, especially a variety of red algae and cyanobacteria, are producers of structurally diverse exopolysaccharides. Additionally, exopolysaccharides are involved in cell-to-cell interactions, adhesion, and biofilm formation.

Exopolysaccharides are widely used in the food industry as thickeners and gelling additives, which improve food quality and texture. Currently, exopolysaccharides have received much attention for their antibacterial, anti-oxidative, and anticancer properties, which lead to the development of promising pharmaceutical candidates. Since exopolysaccharides are released into the culture medium, they can be easily recovered and purified. Different strategies used for the economical extraction and other downstream processing were discussed in a chapter of the referenced book.

The minerals, results of biomineralization processes regulated by the environment or bacteria, are also essential components of the exopolysaccharides. They provide structural integrity to biofilm matrix and act as a scaffold to protect bacterial cells from shear forces and antimicrobial chemicals. The minerals in EPS were found to contribute to morphogenesis of bacteria and the structural integrity of the matrix. For example, in Bacillus subtilis, Mycobacterium smegmatis, and Pseudomonas aeruginosa biofilms, calcite () contributes to the integrity of the matrix. The minerals also associate with medical conditions. In the biofilms of Proteus mirabilis, Proteus vulgaris, and Providencia rettgeri, the minerals calcium and magnesium cause catheter encrustation.

Constituents A 2013 review described sulfated polysaccharides synthesized by 120 marine microalgae, most of which are EPS. These heteropolymers consist mainly of galactose, glucose, and xylose in different proportions except those from Gyrodinium impudicum, which are homopolymers.Raposo M.F., de Morais R.M., de Morais A.M.B. Bioactivity and applications of sulphated polysaccharides from marine microalgae. Mar. Drugs. 2013;11:233–252. doi: 10.3390/md11010233. Most EPS from cyanobacteria are also complex anionic heteropolymers containing six to ten different monosaccharides, one or more uronic acids, and various functional substituents such as methyl, acetate, pyruvate, sulfate groups, and proteins.De Philippis R., Sili C., Paperi R., Vincenzini M. Exopolysaccharide-producing cyanobacteria and their possible exploitation: A review. J. Appl. Phycol. 2001;13:293–299. doi: 10.1023/A:1017590425924. For instance, the EPS from Arthrospira platensis are heteropolymer with protein (55%) moieties and a complex polysaccharide composition, containing seven neutral sugars: glucose, rhamnose, fructose, galactose, xylose, arabinose, and mannose, as well as two uronic acids, galacturonic acid and glucuronic acid.Trabelsi L., M'sakni N.H., Ben Ouada H., Bacha H., Roudesli S. Partial characterization of extracellular polysaccharides produced by cyanobacterium Arthrospira platensis. Biotechnol. Bioprocess Eng. 2009;14:27–31. doi: 10.1007/s12257-008-0102-8.

Dunaliella salina is a unicellular green alga of outstanding halotolerance.Chen X.J., Wu M.J., Jiang Y., Yang Y., Yan Y.B. Dunaliella salina Hsp90 is halotolerant. Int. J. Biol. Macromol. 2015;75:418–425. doi: 10.1016/j.ijbiomac.2015.01.057. Salt stress induces the secretion of extracellular polymeric substances from D. salina. It is speculated that the release of complex mixtures of macromolecular polyelectrolytes with high polysaccharide content contributes to the survival strategy of D. salina in varying salt concentrations. Four monosaccharides (galactose, glucose, xylose, and fructose) were detected in the hydrolysate of EPS from D. salina under salt stress.Mishra A., Jha B. Isolation and characterization of extracellular polymeric substances from micro-algae Dunaliella salina under salt stress. Bioresour. Technol. 2009;100:3382–3386. doi: 10.1016/j.biortech.2009.02.006.Mishra A., Kavita K., Jha B. Characterization of extracellular polymeric substances produced by micro-algae Dunaliella salina. Carbohydr. Polym. 2011;83:852–857. doi: 10.1016/j.carbpol.2010.08.067. In contrast, the water-soluble polysaccharides released by Chlorella pyrenoidosa contain galactose, arabinose, mannose, ribose, xylose, fucose, and rhamnose; their release depends on the cell photosynthetic activity and reproductive state.Maksimova I.V., Bratkovskaya L.B., Plekhanov S.E. Extracellular carbohydrates and polysaccharides of the alga Chlorella pyrenoidosa chick S-39. Biol. Bull. 2004;31:175–181. doi: 10.1023/B:BIBU.0000022474.43555.ec.

Strategies for EPS yield-increase Although the EPS from microalgae have many potential applications, their low yield is one of the major limitations for scale-up in industry. The type and amount of EPS obtained from a certain microalgae-culture depends on several factors, such as culture system design, nutritional and culture conditions, as well as the recovery and purification process. Therefore, the configuration and optimization of production systems are critical for the further development of applications.

Examples of successful increase of EPS yield include

an optimized medium (for Chlamydomonas reinhardtii), an examination of the nutritional conditions including higher salinity and nitrogen concentration (for Botryococcus braunii), the addition of sulfate and magnesium salts in the culture medium (P. cruentum), a co-culturing of Chlorella and Spirulina with the Basidiomycete Trametes versicolor, and a novel mutagenesis tool (atmospheric and room temperature plasma, ARTP), leading to an increase of EPS production of up to 34% (volumetric yield of 1.02 g/L).

It was suggested that co-cultures of microalgae and other microorganisms can be used more universally as a technology to increase the production of EPS, since microorganisms may respond to the interaction partners by secreting EPS as a strategy during unfavorable conditions.

List of Exopolysaccharides (EPS)

acetan (Acetobacter xylinum) alginate (Azotobacter vinelandii, Pseudomonas spp.) cellulose (Acetobacter xylinum) chitosan (Mucorales spp.) curdlan (Alcaligenes faecalis var. myxogenes) cyclosophorans (Agrobacterium spp., Rhizobium spp. and Xanthomonas spp.) dextran (Leuconostoc mesenteroides, Leuconostoc dextranicum and Lactobacillus hilgardii) emulsan (Acinetobacter calcoaceticus) galactoglucopolysaccharides (Achromobacter spp., Agrobacterium radiobacter, Pseudomonas marginalis, Rhizobium spp. and Zooglea spp.) galactosaminogalactan (Aspergillus spp.) gellan (Aureomonas elodea and Sphingomonas paucimobilis) glucuronan (Sinorhizobium meliloti) N-acetylglucosamine (Staphylococcus epidermidis) N-acetyl-heparosan (Escherichia coli) hyaluronic acid (Streptococcus equi) indican (Beijerinckia indica) kefiran (Lactobacillus hilgardii) lentinan (Lentinus elodes) levan (Alcaligenes viscosus, Zymomonas mobilis, Bacillus subtilis) pullulan (Aureobasidium pullulans) scleroglucan (Sclerotium rolfsii, Sclerotium delfinii and Sclerotium glucanicum) schizophyllan (Schizophyllum commune) stewartan (Pantoea stewartii subsp. stewartii) succinoglycan (Alcaligenes faecalis var. myxogenes, Sinorhizobium meliloti) xanthan (Xanthomonas campestris) welan (Alcaligenes spp.)

Exoenzymes Exoenzymes are enzymes secreted by microorganisms, such as bacteria and fungi, to function outside their cells. These enzymes are crucial for breaking down large molecules in the environment into smaller ones that the microorganisms can absorb (transport into their cells) and use for growth and energy.

Several studies have demonstrated that the activity of extracellular enzymes in aquatic microbial ecology is of algal origin. These exoenzymes released from microalgae include alkaline phosphatases, chitinases, β-d-glucosidases, proteases etc. and can influence the growth of microorganisms, chemical signaling, and biogeochemical cycling in ecosystems. The study of these exoenzymes may help to optimize the nutrient supplement strategy in aquaculture. Nevertheless, only a few of the enzymes were isolated and purified. Selected prominent enzyme classes are highlighted in the cited literature.

Extracellular proteases The green microalgae Chlamydomonas coccoides and Dunaliella sp. and chlorella sphaerkii (a unicellular marine chlorophyte) were found to produce extracellular proteases. The diatom Chaetoceros didymus releases substantial amounts of proteases into the medium, this production is induced by the presence of the lytic bacterium Kordia algicida and is connected to the resistance of this alga against the effects of this bacterium. Some proteases are of functional importance in viral life cycles, thus being attractive targets for drug development.

Phycoerythrin-like proteins Phycobiliproteins are water soluble light-capturing proteins, produced by cyanobacteria, and several algae. These pigments have been explored as fluorescent tags, food coloring agents, cosmetics, and immunological diagnostic agents. Most of these pigments are synthesized and accumulated intracellularly. As an exception, the cyanobacteria Oscillatoria and Scytonema sp. release an extracellular phycoerythrin-like 250 kDa protein. This pigment inhibits the growth of the green algae Chlorella fusca and Chlamydomonas and can be potentially used as an algicide.

Extracellular phenoloxidases