NON-TRADITIONAL PRODUCERS OF MICROBIAL EXOPOLYSACCHARIDES

Microbial exopolysaccharides (EPS) are high molecular hydrocarbonic exogenous products of microbial metabolism [1–3]. They are widely used in industry (food production, chemistry, oil production, etc.) due to their ability to gel, emulsify, flocculate, form suspensions and to change rheological parameters of aqueous systems [3–5]. Most of currently known microbial EPS have similar functional properties that determine their practical significance [2, 4]. Thus, it is not surprising that only a few of many isolated, described and studied polysaccharides of microbial origin (xanthan, gellan, alginate, dextran) are produced industrially [1, 4]. A polysaccharide must now have unique properties to enter the free niches of rapidly developing fields like medicine, pharmacy, cosmetics, and nature conservation. Since late ХХ century, scientists actively study microorganisms living in habitats previously overlooked in the search for bioactive compounds-producing microorganisms (permafrost, hot springs, oceanic depths, salt marshes, etc.). Quite possibly, they survive in such places due to specific adaptive mechanisms and synthesis of protective compounds [5], including EPS with new properties. Such organisms are known as extremophiles, or microorganisms isolated from extreme habitats [6, 7]. We argue that the terms “extremophile” and “extreme” are not quite applicable, since microbiology considers “extreme” conditions in which only specialized microorganisms survive and many other taxa perish. Therefore this review refers to them simply as “nontraditional”. To date, a number of reviews have been published about synthesis of EPS by nontraditional producers [6–17]. However, the reviewers mostly paid attention to habitat description, physico-chemical properties and environmental significance of the synthesized polysaccharides and almost ignored the possibility of practical applications [13]. In addition, the reviews were devoted to a specific group of microorganisms (thermophilic [15], halophylic bacteriae [14, 16] and archaea [18], cryophilic yeast [19], sea microbes [9, 10, 17], and microorganisms isolated from hydrothermal vents [8]). Only a few papers reviewed several unusual producers at once [6, 7, 12]. The listed studies were published in 2010–2012 and include mostly summaries of specifics of EPS biosynthesis and their physico-chemical properties. A recent paper [11] discusses practical applications of several polysaccharides, synthesized by bacteriae isolated from hydrothermal sources. UDC 579.841: 577.114 https://doi.org/10.15407/biotech11.04.005

Most of currently known microbial EPS have similar functional properties that determine their practical significance [2,4]. Thus, it is not surprising that only a few of many isolated, described and studied polysaccharides of microbial origin (xanthan, gellan, alginate, dextran) are produced industrially [1,4].
A polysaccharide must now have unique properties to enter the free niches of rapidly developing fields like medicine, pharmacy, cosmetics, and nature conservation.
Since late ХХ century, scientists actively study microorganisms living in habitats previously overlooked in the search for bioactive compounds-producing microorganisms (permafrost, hot springs, oceanic depths, salt marshes, etc.). Quite possibly, they survive in such places due to specific adaptive mechanisms and synthesis of protective compounds [5], including EPS with new properties.
Such organisms are known as extremophiles, or microorganisms isolated from extreme habitats [6,7]. We argue that the terms "extremophile" and "extreme" are not quite applicable, since microbiology considers "extreme" conditions in which only specialized microorganisms survive and many other taxa perish. Therefore this review refers to them simply as "nontraditional".
This review aimed to summarize the available information on EPS synthesis by non-traditional producers (thermo-, cryo, halophilic microorganisms and bacteriae isolated from deep-sea hydrothermal vents), and properties of polysaccharides that support their potential practical application in medicine, pharmaceutical, food industries and nature conservation. Thermophiles The studies of thermophilic microorganisms started approximately in 1967 [20]. The paper briefly summarized the available knowledge about the microorganisms. In those days, attention was mostly paid to their environmental niche and the mechanisms enabling their survival at high temperatures.
One of those adaptive mechanisms is synthesis of microbial EPS. It should be noted that, unlike industrial mesophilic producers, using thermophils for the preparation of polysaccharides has a number of technological advantages, in particular, at elevated temperatures, the viscosity of the culture fluid and the possibility of the process infection are reduced, as well as mass exchange processes increase, etc. [21][22][23][24][25].
The main disadvantage of those archaea as well as almost all other thermophilic producers of EPS is the low concentration of the target product (Table 1). This can be caused by low concentrations of the carbon and energy source (2-9 g/l) in the cultivation medium. Special attention was paid to the polysaccharide effect on physiology. Thus (Table 1). Notably, the first reports of EPS synthesis by thermophilic bacteriae also included representatives of these families. Thus, Manca et al. [35] in 1996 reported isolation of extremely thermophilic bacteriae Geobacillus thermoantarcticus, which at 65 C synthesized up to 400 mg/l sulfated EPS from soil near the crater of Melbourne volcano (Antarctica).
Besides representatives of Bacillaceae and Paenibacillaceae, synthesis of polysaccharides is known for hyperthermophilic bacteriae of the genus Thermotoga (optimal temperatures 80-85 C) [27] and thermophiles of the genus Thermus (optimal temperature 60 C) [38].
Thermophilic obligate methanotroph Methylococcus thermophilus 111п synthesizes up to 5 g/l EPS [2] and thus is a much better choice. Those amounts were achieved after a complex investigation of pH, temperature, diluted oxygen concentration, gaseous methane to oxygen ratio conditions, and the pre-treatment of the inoculum. The exogenous addition of 0.5 g/l aspartic acid (obtained by transferring amino group to oxaloacetic acid) to the culture medium of strain 111п was followed by an almost two-fold increase in the polysaccharide biosynthesis rate [2].
Lin et al. [38] isolated from the biofilm of Thermus aquaticus YT-1 a polysaccharide that heightened immune response. That EPS was observed to act as an agonist of TLR2 receptor and helped induce synthesis of cytokines IL-6, TNF-, and nitrogen monoxide (NO) by murine macrophages and human monocytes. That immunoregulatory activity supposedly was caused by galactofuranose in its structure [38].
Recently Spanò et al. [40] found that EPS of B. licheniformis T14 at 400 μg/ml inhibited biofilm formation by multiresistant strains Escherichia coli 463, Klebsiella pneumoniae 2659, Pseudomonas aeruginosa 445 and Staphylococcus aureus 210 by 74, 56, 54 and 60%, respectively. The researchers suggested that due to the emulsifying properties of the polysaccharide it is able to impact the hydrophobicity of bacterial cells and so prevent their primary adhesion to surfaces [40].
A summary of EPS biosynthesis by thermophilic and thermotolerant microbes is given in Table 1. Currently, the microbes are not considered promising due to low EPS synthesis ability. Meanwhile such polysaccharides have properties important for medicine and pharmacy (antiviral, immunomodulating, anticytostatic, etc.), which can stimulate work on intensifying their synthesis.
52 hours of culture [45]. Further research [51] of EPS of strain HYD657 established that they efficiently protect keratinocytes from inflammation agents. The protective effect was also found towards Langerhans cells, which are sensitive to the ultraviolet and play an important role in the system of human skin immune protection. Nowadays, cosmetic preparation Abyssine ® was developed based on the polysaccharide (deepsane). It is recommended for soothing and protection against irritation of sensitive skin [52].
Notably, the polysaccharide of strain HYD657 has an unusual component, a residue of 3-O-(1-carboxyethyl)-D-glucuronic acid [45]. Currently, the compound was also found in EPS of the strain Alteromonas sp. HYD1644, isolated from the epidermis of the polychaete Alvinella caudata [46], and in drought-resistant cyanobacteriae Nostoc commune DRH-1 [53]. Helm et al. [53] suggested that this and other uronic acids with carboxyethyl moieties play a key part in providing survival in unfavorable conditions. For example, such functional groups can help EPS attach to adjacent chains of the polymer, organic (biofilms) or inorganic surfaces, etc.
The strain Vibrio diabolicus HE800 Т was isolated from polychaete Alvinella pompejana. The strain produces a polysaccharide similar to hyaluronic acid [49]. The EPS is made up equally from glucuronic acid and hexosamines (N-acetylglucosamine and N-acetylgalactosamine) [54]. Treating damaged skullcap skin of Wistar rats with the EPS made the wound close sooner, while the trabecular and cortical anatomic structure of the defect fully recovered [55]. Zanchetta et al. [55,56] suppose that the effect is caused by the ability of EPS to form extracellular matrix that helps direct adhesion of osteoblasts and pericytes, generally protect the damaged site while it heals, and to bind calcium.
Senni et al. [57] suggested that glycosoamino glycan polysaccharide of strain НE800 Т is a promising agent for various derivatives (heparan sulfate, chondroitin sulfate, etc.). Such depolymerization of native polysaccharide to molecular mass of 22 kDa with further deacetylation and sulfation (sulfate content 34%) resulted in a polymer similar to heparan sulfate. Those derivatives were observed to stimulate proliferation of dermal and gingival fibroblasts and inhibit secretion of matrix metalloproteinases [57].
The EPS of Alteromonas infernus GY785 after sulfation (sulfate content 40%) and controlled depolymerization by free radicals to molecular mass of 24 kDa substantially raised APTT (activated partial thromboplastine time) [58,59]. The anticoagulant activity of the polysaccharide was on the level of calcium pentosan polysulfate though 2.5-6.5 times lower compared to heparin [58]. Notably, due to the low sulfate content in the native polysaccharide (5.5-10%) it did not have anticoagulant activity [58].
Recently the effect of depolymerized EPS of strains V. diabolicus HE800 Т and A. infernus GY785 on the complement system was studies [60]. The low molecular (2.9 kDa) derivative of the polysaccharide of strain HE800 Т to a large extent activated the system (60% activation at 50 μg EPS), while the depolymerized (molecular mass 23 kDa) and sulfated (sulfate content 37-42%) EPS of strain GY785, conversely, caused its significant inhibition (78% inhibition at 10 μg EPS). Due to those properties, the polysaccharides are promising for treating diseases caused by deregulation of immune system and over activation of the complement system. Therefore, EPS of bacteriae isolated from hydrothermal vents can become widely accepted into medical, pharmaceutical and cosmetic industries due to anticoagulant, protectant, immunomodulatory and regene rative activities. Notably, such microorganisms can synthesize up to 11 g/l of the product, and some polysaccharides from hydrothermaldwelling bacteriae are already mass-produced. For example, EPS of A. macleodii subsp. fijiensis biovar deepsane HYD657 is used for cosmetics (Abyssine ® ).
Data on EPS of bacteriae isolated from hydroterms are summarized in Table 2.

Psychrophiles
Cold environments are found from deep seas to snow-laden mountaintops, from Arctic to Antarctica. Temperature of almost 75-80% of the Earth surface is below 5 C [60][61][62]. Cold habitats are characterized by frequent sharp changes in temperature (cycles of freezing and thawing, etc.), UVradiation, nutrient concentration [63,64]. Oceanic and sea waters also have pressure and salinity oscillations [21]. Evidently, microorganisms would not survive in such conditions without relevant adaptive mechanisms [62,65,66].
EPS play a large role in it. Exopolymers, including polysaccharides, take part in Fastens bone fusion [49,[54][55][56][57] aggregation, adhesion to surfaces and other microorganisms, biofilm formation, nutrient storage, etc. in marine bacterial communities [66][67][68]. Often aggregates of salty drops remain unfrozen after the sea water freezes, and the microbes are trapped in salt canals [63,66]. Then, EPS are cryoprotectants and protectants from high salinity [62,65,66]. The majority of microorganisms, able to survive at low temperature, are yeasts and bacteriae [8]. Notably, phylogenetic research also registers a lot of representatives of Archaea [61], although they have not been cultured.
Fungi. EPS synthesis by fungi at relatively low temperatures is a novel approach. The first report of polysaccharide production by cryotolerant mycelial fungi appeared only at the beginning of ХХІ century. In 2002, Selbmann et al. [69] established the ability of Phoma herbarum CCFEE 5080 cultured on medium containing sorbitol (60 g/l) to produce 13.4 g/l 7412 kDa glucan. Due to cryoprotectant properties of the polysaccharide, strain CCFEE 5080 is able to grow at 0-5 C (optimal temperature 28 C) [70].
Another glucan-producing fungus is strain Thelebolus sp. IITKGP-BT12 [68]. Unlike the strain CCFEE 5080, at 18 C it synthesizes only 1.94 g/l EPS. Experiments have shown that the glucan has significant antiproliferative effect on cells of skin cancer in B16-F0 mice. IC 50 (the concentration at which maximal inhibition occurred) of the EPS was 275.4 μg/ml. The polysaccharide had almost no effect on normal fibroblasts of the L929 line (at the concentration of 187.5-1500 μg/ml cytotoxicity was almost absent) [67].
Research of economically valuable properties of EPS of yeasts from the Livingstone Island confirmed their possible use in cosmetics, food industry [73,75,76] and medicine [78]. EPS of strain Cryptococcus laurentii AL 100 exhibited high emulgent activity, significantly enhanced by other polysaccharides (xanthan, guar gum, cellulose, etc.) [73].
EPS of cryotolerant fungi can be used as emulgents and thickeners in food and cosmetic practices at low temperatures. They are promising for medicine and pharmacy due to antitumor and anticytostatic activities.
Bacteriae. Reports of EPS synthesis by cryophilic and cryotolerant bacteriae started shortly after the first study about polysaccharides of cryotolerant fungi [69].
Polysaccharides of cryotolerant bacteriae isolated from free ice and marine aggregates in the Antarctic ocean, with in situ temperature of 4 C were described in 2005 [78]. Six of the studied isolates belonged to the genus Pseudoalteromonas, three to the genera Shewanella, Polaribacter, and Flavobacterium. A strain САМ030 Т represented the family Flavobacteriaceae, later it became a new taxon Olleya marilimosa [79]. Most cryophilic bacterial producers isolated after 2005 belong to the genera Pseudoalteromonas, Polaribacter and Flavobacterium (Table 3).
By their monosaccharide content, the polysaccharides of cryophilic bacteriae are similar to EPS of marine bacteriae ( Table 2).
Lowering the growth temperature from 20 to 10, or to -2 C caused an almost 30fold rise in EPS-producing ability of strain Pseudoalteromonas sp. САМ025 (up to 99.9 and 97.2 mg EPS/g biomass, respectively), and a changed monosaccharide ratio [80].
Cryoprotectant properties of EPS of Pseudoaltermonas sp. SM20310 were studied in [63]. At 30 mg/ml EPS the number of living cells of strain SM20310 and E. coli DH5 was 7 to 18 times as high as in the control group (without EPS) after three cycles of freezing-thawing. Other researchers [68] report that adding the polysaccharide of cryotolerant bacteriae Flavobacterium sp. ASB 3-3 at 50 mg/ml led to a four times increase in the number of living cells of strains ASB 3-3 and E. coli DH5 after two cycles of freezing-thawing compared to the cultures without EPS.
Cryotolerant bacteriae Pseudoalteromonas elyakovii ArcPo 15 isolated from Chukchi Sea were observed to synthesize 1.7 MDa EPS with high cryoprotectant activity [81]. Adding the EPS (0.5%) to a suspension of E. coli DH5 resulted in 94.2% survival of the cells after five cycles of freezing-thawing. Adding 20% glycerin resulted in 54.1% survival of the cells.
Due to the cryoprotectant ability of bacterial EPS we suggest using them as alternative cryoprotectant agents for longterm storage of suspended cultures [82,83].
According to Carrión et al. at 10% EPS of Pseudomonas sp. ID1, survival of E. coli ATCC 10536 after freezing and storing for seven days at -20 and -80 C was 36 and 64%, respectively [82]. Cell survival decreased at lower EPS concentrations. After similar freezing of EPS-synthesizing strain ID1, the cell survival rates were 75 and 94%, respectively. Another study [84] showed that EPS of cryophilic Colwellia psychrerythraea 34H are a better cryoprotectant agent for freezing cells at -80 C than 10% glycerin solution.
Notably, cryoprotectant properties of polysaccharides are not limited to merely the protection of microbial cells. Sun et al. [84] reported that, survival rate of human dermal fibroblasts after 20 hours at 4 C reached 76.1% with 500 μg/mg EPS of Polaribacter sp. SM1127, while without the polysaccharide it was only 44.2%.
In the native environment, other physicochemical factors besides temperature can induce EPS synthesis, such as pressure and salinity [63,83]. For example, culturing C. psychrerythraea 34H at high hydrostatic pressure (up to 400 atm) resulted in EPS content increasing 4.5-7.5 times.
After optimization of the culture medium [88] in the fed-batch culture [85], the concentration of EPS of strain Z. profunda SM-A87 increased to 17 g/l, which is 1.93 times higher compared to the initial.
Recently Sathiyanarayanan et al. [68,86] isolated cryotolerant Flavobacterium sp. ASB 3-3 and Pseudomonas sp. PAMC 28620 (AS-06/29) from the soil of Svalbard Arctic glacier fore-field. The optimal carbon and energy source for those bacteriae, unlike other microbial sources of EPS (Table 3) is glycerin. At the medium with 30 g/l of this substrate, the bacteriae produced 7.25 g/l EPS with flocculant and emulgent properties.
Unlike thermophilic and thermotolerant sources ( Table 1 and Table 2), cryophilic and cryotolerant microorganisms synthesize more EPS (up to 17 g/l; Table 3), and their polysaccharides have cryoprotectant, emulsifying properties, retain moisture and adsorb heavy metals. That, consequently, makes the polysaccharides potentially attractive for various fields from food industry (foodstuffs storage) and cosmetics (production of protective cosmetics) to environmentfriendly technology (purification of waste waters).

Halophiles
Halophiles are organisms able to survive in briny habitats, whose development requires salt. The salt in question is generally NaCl, while many researchers in their experiments on halophilic cultures use sea salt which contains not only NaCl but also comparatively small amounts of other salts of two-and monovalent metals [89].  [80] As to salinity, halophiles can be halotolerant (upper salinity limit 15%), weak (NaCl content of 2-5%), moderate (5-5%) and extreme halophiles (20-30%) [16].
In 1988, Antón et al. [96] established that extremely halophilic archaea Haloferax mediterranei ATCC 33500 cultured on a medium with 1% glucose and 25% sea salt produced 3 g/l of sulfated high molecular polysaccharide. Viscosity of EPS solutions was stable in wide ranges of pH, temperature and salinity. Hence EPS of strain ATCC 33500 can be utilized in increasing oil production from wells with high salt content. Later, researchers established the structure of repeating sequences of EPS strain ATCC 33500 [98] and other EPSsynthesizing archaea, in particular Haloferax gibbonsii ATCC 33959 [97] and Haloferax denitrificans ATCC 35960 [99].
At the end of the twentieth century, for new producers of polyhydroxyalkanoates and EPS, Nicolaus et al. [94] isolated three obligate halophilic strains Т5, Т6 and Т7, which synthesized 35-370 mg/l EPS, from the salt works of Tunisia. The isolates belonged to the genus Haloarcula. Among halophilic EPS-synthesizing archaea is strain Halobacterium volcanii 1539, which produces 300 mg/l sulfated polysaccharide [100].
There have been no new studies on EPS synthesis by halophilic archaea after that, until a recent  [85,87] report of EPS-synthesizing archaea Haloterrigena turkmenica DSM-5511, isolated from briny soil (Turkmenistan) [101]. The polysaccharide has high emulsifying (emulsification index of sunflower and olive oils are 62.2 and 59.6%, respectively) and antioxidant activity (68.2% neutralization of DPPH· at 10 mg/ml EPS). The EPS also better than hyaluronic acid and sodium alginate retained moisture. Similar properties were found in certain polysaccharides of cryophilic bacteriae [85,86] (Table 3). However, the level of target product is too low (at least now) in strain H. turkmenica DSM-5511 to consider it a marketable EPS source.
Soon, wide-scale screening of possibly halophilic producers isolated from solar salterns in Morocco was published [92]. Thirty two isolates of the genus Halomonas were selected for a more detailed analysis out of more than 500 isolates. Only four of them accumulated over 2 g/l polysaccharide, and the highest amount (2.8 g/l) was produced by strain S-30. According to phylogenetic analysis, the strain and isolates S-7, S-31 Т and S-36 were combined into a new species Halomonas maura [115]. Further optimization of the cultivation medium (reducing sea salt concentration, instead adding 2.5% NaCl and 0.05% MgCl 2 ·6H 2 O) increased EPS production of strain S-30 to 3.8 g/l [103].
Strain Halomonas xianhensis SUR308, isolated from soil of a solar saltern (India) [90,91], on a medium with glucose (1%) and NaCl (10%) produced 2.56 g/l EPS [91]. Further increase of glucose content to 3% and decrease of NaCl to 2.5% was followed by increased EPS production to 7.87 g/l [90]. The polysaccharide was not toxic for Huh7 human hepatocytes. Also, the polymer had high antioxidant activity: the level of neutralization of DPPH· was 72% at 1 mg/ml EPS 72% [91].
Poli et al. [95] reported isolating a moderately halophilic bacteria Halomonas sp. AAD6 Т from Turkish salterns. Later it was identified as the typical strain of a new species Halomonas smyrnensis [113]. It produced levan (a fructose homopolysaccharide). Adding 50 мМ boric acid, 0.8 mg/l thiamine and trace quantities of salts of Mn, Zn, Fe and Cu to the culture medium resulted in a five times increase in levan concentration (up to 8.84 g/l) compared with the initial medium [116].
Further studies aimed to lower the production cost of the target product by using various molasses instead of sucrose in the EPS biosynthesis medium [105]. EPS concentration reached 7.56 g/l (12.4 g/l after 210 hours of cultivation) in culture medium with beet pre-treated with calcium phosphate, sulfate acid and activated carbon. In culture medium with likewise pre-treated starch molasses (a side product of manufacturing dextrose from starchy materials) it was 4.38 g/l. Using starch molasses as a substrate resulted in levan with high emulgent activity [117]. Levan of strain AAD6 Т was shown to be useful in targeted delivery of drugs, in particular, of antibiotic vancomicyn [118]. It also increased LD 50 of avarol from 0.18 ppm to 10 ppm [95]. Anticoagulant activity of artificially sulfated derivatives of that EPS was studied in [119].
Ruiz-Ruiz et al. [110] studied antitumor properties of polysaccharides of halophilic bacteriae Halomonas stenophila В100 and N12 T . Artificially sulfated EPS (sEPS) of strains В100 and N12 T (sulfate content 23 and 17%, respectively) efficiently decreased proliferation of Т-cells of acute lymphoblast leukemia line Jurkat (500 μg/ml sEPS of strain В100 resulted in 100% inhibition of cell proliferation). Only sEPS of strain В100 induced apoptosis of tumour cells (lines CEM, MOLT-4, HPB-ALL, etc.), while healthy Т-cells resisted the apoptosis induction [111]. Authors considered that antitumor effect to directly depend on the concentration of sulfates. It was suggested that sulfates change the charge of polymer molecule to negative and affect its structure, increasing the interaction between EPS and the target cell surface [110].
Thus, studies of EPS from non-traditional sources (cryophilic fungi and bacteriae, haloand thermophilic archaea and bacteriae, including those from deep-sea hydrothermal vents) is a novel field which began to develop rapidly at the end of the twentieth century. Many of those isolated microorganisms produce polysaccharides. The physiological effect, physico-chemical properties and possibilities of industrial application of those EPS are studied. Those substances due to their immunomodulating, antiviral, anticoagulant, antitumor, antioxidant activities can be widely employed, in medicine and pharmacy, etc.
Meanwhile the practical implementation of polysaccharides is limited by the low efficiency of production. Non-traditional sources produce EPS in much lower concentrations than the traditional ones. In our opinion, solving this problem is only a question of time, because various approaches to metabolic and gene engineering for microbial synthesis intensification are already developed [88,112,[120][121][122].
EPS biosynthesis by non-traditional sources currently requires expensive carbohydrate materials (glucose, fructose, sucrose, and maltose) (Tables 1-3, 6). At the same time, many new studies aim to substitute carbohydrate substrates with cheap industrial wastes (whey, crude glycerin, oil-containing wastes, and agricultural wastes) in culturing traditional producers of polysaccharides. Those approaches to microbial polysaccharide production are reviewed in [123]. We demonstrated that it is possible to obtain microbial EPS ethapolan using fried vegetable oil [124] and its mixture with molasses [125].