Ocean represents an unusual diversity of life. The largest proportion of microbial diversity has been found in the oceanic and terrestrial subsurface respectively. Marine habitats are inhabited by several microbial populations adapted to these ecosystems. Among these populations, bacteria are one of the important and dominant inhabitants of such environments. Marine bacteria themselves or their products such as enzymes, exopolymers, pigments, antimicrobial compounds and biosurfactants represent a wide range of applications in food, textile and pharmaceutical industries as well as in many environmental processes. This review aims to collect the majority of papers containing structural characterization focusing on relationships with polysaccharide activities or properties. A better understinding of the structure-property relationships of marine bacteria extrapolysaccharide (EPS) is important to evaluate their ecological roles and for exploring their possible biotechnological and industrial applications.
Keywords: extremophile, marine bacteria, EPS, chemical characterization, capsular polysaccharide, purification, structure-activity relationship
About 71% of the Earth’s surface is covered with oceans having an average depth of 3.8 km and an average pressure of 38 MPa (van Eldik and Hubbard, 1996). Temperatures at deep-sea surfaces and the upper surfaces of the sea are different. The different conditions that prevail in the marine ecosystems are responsible for the existence of various extreme habitats, such as salt lakes, marine salterns, deep-sea, volcanic and hydrothermal marine areas as well as in the sea ice in Polar Regions, where organisms grow in such extreme conditions and flourish (Casillo et al., 2018). The marine organisms have developed unusual metabolic processes and defensive mechanisms for their survival under such extreme conditions, which might have resulted in the ability to produce novel bioactive compounds in comparison to other natural habitats (Chi and Fang, 2005). Marine microorganisms produce many organic substances and one such product is exopolysaccharide (EPS) (Abreu and Taga, 2016). There has been a growing interest in the isolation and identification of new marine microorganisms capable of producing polysaccharides. These polymers participate in the maintenance of marine environments by contributing to several processes like sedimentation, particle formation, cycling of dissolved metals and dissolved organic carbon (Verdugo, 2012).
Although microbial cells require up to 70% of the total energy for EPS production, but once formed it benefits the microbes in multiple ways. EPS helps the organisms to grow and survive under adverse environmental conditions (Poli et al., 2010). Apart from these, EPS play a vital role in nutrient uptake, aggregation, adhesion to surfaces, and biofilms formation (Dave et al., 2016; Shukla and Dave, 2018). EPS possess active and ionisable functional groups and non-carbohydrate substituents like amine, sulfhydryl, carboxyl, hydroxyl, phosphate, and sulphate groups that are responsible for the negative charge of the polymer. Due to this property, various heavy metals can bind to EPS by ion exchange, complexation, and entrapment like mechanisms (Gupta and Diwan, 2017). Loaec et al., (1998) and Wuertz et al., (2000) have described the role of EPS producing heavy metal resistant isolates from deep-sea hydrothermal vents and purified EPS for metals and toxic substances binding ability.
Nowadays the focus is to isolate novel marine microorganisms for the production of EPS with diverse properties. Different genera of marine bacteria such as Alteromonas, Bacillus, Cobetia, Colwellia, Geobacillus, Halomonas, Hyphomonas, Idiomarina, Pseudoalteromonas, Pseudomonas, Polaribacter, Rhodococcus, Shewanella, Vibrio, Exiguobacterium, Kocuria, Pontibacter, Planococcus, Marinobacter have been reported as EPS producers (Le Costaouec et al., 2012; Kumar et al., 2004; Lelchat et al., 2015; Carillo et al.,2015; Arena et al., 2009; Bouchotroch et al., 2000; Arias et al., 2003; Quintero et al., 2001; Martínez-Cánovas et al., 2004; Saravanan and Jayachandran, 2008; Wu et al., 2016a, b; Sun et al., 2015; Urai et al., 2006; Vinogradov et al., 2005; Bramhachari and Dubey, 2006; Upadhyay et al., 2016).
The increasing commercial importance of marine microbial EPS has stimulated the efforts in the development of rapid and efficient techniques for their recovery and purification. The presence of microbial cells, medium ingredients in the fermentation broth, as well as its high viscosity, often causes problems in the recovery of EPS (Yang et al., 1998; Kumar et al., 2007). The steps involved in EPS recovery and purification are the removal of microbial cells and protein followed by precipitation, dialysis and lyophilisation of EPS (Smith and Pace, 1982; Laroche and Michaud, 2010; Castillo et al., 2015). Elucidations of chemical compositions and structures of EPS are necessary to establish their structure-function relationship. But the precise characterization of the EPS is a challenge to researchers because of its structural complexity (Chowdhury et al., 2011). Although the chief components of EPS are carbohydrates, it is difficult to derive their unique monomer linkage patterns and biochemical properties (Jiao et al., 2010). Thus acid hydrolysis, Fourier-transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC), methylation analysis, gas chromatography with mass spectroscopy (GC–MS), and 1H- and 13C-NMR (one and two dimensions) have been used for chemical characterisation of EPS (Liang and Wang, 2015). The development of purification techniques and sophisticated analytical approaches permit researchers to provide an insight into primary structures and conformation of polysaccharides, which are important to acquire information about complete polymeric structures.
Aim of this review is to collect the majority of papers containing structural characterization focusing on relationships with polysaccharide activities or properties. A better understanding of the structure property relationships of marine bacteria EPSs is important to evaluate their ecological roles and for exploring their possible biotechnological and industrial applications.
PURIFICATION AND CHARACTERIZATION METHODOLOGIES
Visualization of polysaccharide material during the microbial growth on agar plates can be reached through colorimetric methods. It is well known that some monosaccharides are able to interact with dyes and this phenomenon can be used to quickly and easily identify the presence of EPS (Rühmann et al., 2015). Among these dyes, Alcian Blue is sensitive to the presence of negative charges of the polymer (Decho, 1990; Deming and Young, 2017), while Aniline Blue (Ma and Yin, 2011), Congo Red (Darwish and Asfour, 2013) and Calcofluor White (Leigh et al., 1985) are able to detect glucan polysaccharides. The electrophoresis is the optimal technique for screening the presence of ionic polysaccharides as well as for following the purification during the various steps. Electrophoresis on polyacrylamide gels (SDS- or DOC-PAGE) of carbohydrates containing molecules is familiar to people working on polysaccharides structural elucidation (Laemmli, 1970). Indeed, lipopolysaccharides (LPSs), proteoglycans and charged polysaccharides can be analysed with this technique. Two different staining methods can be used, the silver staining (Tsai and Frasch, 1982) and the Alcian Blue (Krueger and Schwartz, 1987); Thornton et al., 2007). The last one needs the presence of negative charges to be effective. Exopolysaccharides can be also identified by bacteria phenotypes, both in liquid and solid media. Usually, a “mucoid” strain indicates that the microorganism is able to produce exopolymeric substances but only the determination of carbohydrates content can ensure the presence of exopolysaccharides (Tallgren et al., 1999; Ortega-Morales et al., 2007). A number of colorimetric assays has been developed for detecting carbohydrates in a sample. Most of them rely on the action of concentrated (or near concentrated) sulphuric acid, causing the hydrolysis of all glycosidic linkages with a resulting dehydration of the monosaccharides, producing derivatives of furfural. These products react with several reagents, such as phenol (Dubois et al., 1956), m-hydroxy-diphenyl (Blumenkrantz et al., 1973) and carbazole (Bitter and Muir, 1962), to give coloured compounds. EPSs can be extracted by a plethora of physicochemical methods. All of them are able to use external forces to ensure the complete detachment of the polymer from the cells or the complete recovery from the medium. Centrifugation, filtration, dialysis, precipitation, alkaline extraction, metals complexation by EDTA or crown ether and ultrasonication are among the most used methods. It is important to underline that care should be taken when using such methods, in order to avoid cells lysis. However, the extent of cells lysis during these procedures is difficult to evaluate (Sheng et al., 2010).
Extraction from Cell-bound Polysaccharides: Capsular Polysaccharides (CPSs)
Exopolysaccharide isolation is different depending on the polymer localization. Capsular polysaccharides, visualized by Transmission Electron Microscopy (Stukalov et al., 2008) and/or light microscopy (Beveridge et al., 2007), are mainly recovered from the cells. Indeed, they are strictly associated to the membrane and form ionic interactions with the molecules included in the membrane. The first step consists in the separation of cells from growth medium by centrifugation. During this operation, some CPS molecules could pass into the growth medium, distributing the polymers into the two phases. It turns out that the polymer yield calculation is quite complex. Then, the isolation of capsular polysaccharides proceeds with a solvent extraction of the cells, usually by saline solution (Reddy et al., 1992) or by the hot phenol/water method (Carillo et al., 2015; Vinogradov et al., 2005). Since with the last procedure LPSs are extracted from the cells together with the CPSs, an acetic acid hydrolysis is demanding to remove the glycolipid portion (lipid A) from the polysaccharide sample. After extraction and taking into consideration the sample characteristics, anionic exchange chromatography and/or gel filtration are usually performed.
Extraction from Growth Medium: Medium-released Polysaccharides (MRPs)
MRP-EPSs can be obtained from the growth medium, after centrifugation or filtration of the cells. Then, the supernatant is treated with various chemical substances in order to obtain a precipitate. The majority of the reports indicate alcohol precipitation as the main method for obtaining polysaccharidic material, either by ethanol or 2-propanol. In addition, acetone (Kwon et al., 1994) or detergent salts such as the cetylpyridinium chloride (Kim et al., 2016), can be used. However, since it is hard to directly obtain a purified polysaccharide, the chromatographic step is determining.
Extraction of EPS from Biofilm
Extraction of polysaccharides from biofilm is much more difficult, due to the necessity to firstly break down the interactions between the EPSs and the complex matrix, mainly constituted by proteins and nucleic acids, where they are embedded (Lembre et al., 2012). EPSs can be extracted from biofilm by EDTA (Oliveira et al., 1999; Boualam et al., 2002; Liu and Fang, 2002), NaOH (Boualam et al., 2002), NaCl (Lin et al., 2011) and formaldehyde (Liu and Fang, 2002). EPSs can also be extracted by centrifugation, as reported for Pseudomonas aeruginosa (Ma et al., 2007). After extraction and precipitation by ethanol, chromatographies are requested to obtain a pure sample of EPS.
Methods for Structural Characterization
To solve the complete primary structure of an oligo-/polysaccharidic chain the following questions must be addressed: (i) the monosaccharide composition, with their absolute configuration; (ii) the anomeric configurations of the monosaccharides; (iii) the ring size of each monosaccharide; (iv) the glycosylation sites; (v) the sequence of the monosaccharides; (vi) the presence and the positions of the appended groups.
Current analytical methods to characterize a polysaccharidic chain exploit both chemical and spectroscopic methods. Glycosyl analysis is usually performed to establish the type and the relative amount of the monosaccharides constituting the investigated polymer. Chemical derivatization of monosaccharides is necessary to perform such analysis by Gas Chromatography Mass Spectrometry (GC-MS). Therefore, a portion of the polysaccharide is usually subjected to a complete degradation with acids, with two different procedures, owing to the different stability of some monosaccharides. The sample can be either hydrolysed in water and treated with dry acid methanol in a solvolysis reaction (methanolysis). After neutralization and concentration, the sugars are converted into their alditol acetates (AA) (Abazia et al., 2003) or acetylated methyl glycosides (MGA), respectively (Fresno et al., 2007). The components are separated into the GC column and identified by their relative retention times and Electron Ionization-Mass Spectrometry (EI-MS) fragmentation patterns (Lönngren et al., 1974). For the determination of absolute D or L configurations, the solvolysis is performed with an optical chiral pure alcohol, such as 2-(+)-butanol or 2-(+)-octanol (Leontein et al., 1978). The diastereoisomeric butylor octyl-glycosides obtained, can be acetylated and analysed by GC-MS and the configuration can be deduced by comparison with authentic standards. The determination of the ring size and the glycosylation sites of the monosaccharides is obtained through the analysis of the partially methylated alditol acetates. These are achieved by a complete methylation of the polysaccharide in alkaline conditions, followed by the acid hydrolysis, the reduction with a deuterated reagent of anomeric positions and acetylation (Ciucanu et al., 1982). The observed fragments in MS spectra of GC-MS chromatograms are diagnostic for the specific substitution pattern of methyl and acetyl groups. Spectroscopic methods include Infrared Spectroscopy (IR) and Nuclear Magnetic Resonance (NMR). FT-IR can furnish several information about polysaccharide structures, such as the type of sugar rings, the presence of amino sugars, uronic acids and sulphate groups. Nevertheless, infrared spectroscopy is not suitable for an in-depth structural analysis. Instead, Nuclear Magnetic Resonance provides the most useful tool in the field of structural determination of polysaccharides. Anomeric resonances in both 1H and 13C spectra are usually displayed in a different region with respect to the carbinolic signals, thus helping in determining not only the alpha or beta anomeric configuration of each single residue but also in establishing, in a quite confident way, the number and their relative proportions (Perlin and Casu, 1982). Finally, the observation of long-range scalar couplings between anomeric signals and proton/carbon resonances of attachment points in modern 2-D techniques (HMBC), together with the measurement of nuclear Over hauser effects (NOE) between protons two consecutive residues, give information about the sequence. Mono and two-dimensional spectra of both 1H and 13C nuclei provide essential and definitive information on the primary structure of a carbohydrate chain. The detailed use of NMR in carbohydrates structural elucidation have been extensively reviewed in dedicated papers (Agrawal, 1992; Duus et al., 2000; Bubb, 2003; Larsen and Engelsen, 2015).
All these methods allow the determination of the primary structure of EPSs, that is only the first step to understand the relationship between the polysaccharidic structure and its function and/or activity. Therefore, secondary structure definition, that is the conformation of the polymer in a solution and in some cases, supramolecular structures characterization, are mandatory to define the shape of the macromolecules in solution. Among the methods used for secondary structure determination, NMR NOEs measurement coupled with Molecular Mechanics (MM) and Molecular Dynamics (MD) simulations (Allen et al., 1999), Circular Dichroism (Stevens, 1997) and Small-Angle Neutron Scattering (SANS) (Masuelli and Renard, 2017), are the most used.
STRUCTURE OF EXOPOLYSACCHARIDES (EPSS) FROM MARINE BACTERIA
In the following sections, the structural features of EPSs produced by marine bacteria will be reported. Since many bacteria are ubiquitous, in this review we have considered only marine strains.
The Gram-negative genus Alteromonas, belonging to the Alteromonadaceae family, was established by Baumann and co-workers (Baumann et al., 1972) for marine Gram-negative heterotrophic bacteria requiring sodium to grow (López-Pérez and Rodriguez-Valera, 2014). Several species belong to this genus and A. macleodii was designated as the type species (Baumann et al., 1972). Numerous rod-shaped bacteria, which produced EPS, were isolated from alvinellids: Alteromonas macleodii sub. fijiensis biovar deepsane HYD 657, Alteromonas HYD-1545 and Alteromonas sp. strain 1644. Alteromonas macleodii subsp. fijiensis biovar deepsane HYD 657 (Cambon-Bonavita et al., 2002; Le Costaouëc et al., 2012) produces a high molecular weight exopolysaccharide, named deepsane. This EPS was found to contain different type of sugars carrying sulphate, lactate and pyruvate substituents (Table 1).
Deepsane is currently used in cosmetics due to its protective effect on keratinocytes. The exopolysaccharide produced by Alteromonas HYD-1545 was composed of uronic acids and neutral sugars, of which, one galactose unit is substituted by a pyruvic group. This polysaccharide showed a good heavy-metal-binding capability, providing in this way protection to the polychete worm (Vincent et al., 1994). The deep-sea bacterium Alteromonas macleodii subsp. fijiensis strain ST716 produced an EPS with a good intrinsic viscosity similar to the xanthan’s (Raguenes et al., 1996). The polymer was constituted by a branched hexasaccharide repetitive unit containing Glc, GlcA, Gal and GalA and terminating with a 4,6-O-(1-carboxyethylidene)-Manp (Table 1) (Rougeaux et al., 1998). Alteromonas sp. JL2810 was isolated from the surface seawater of the South China (Zhang et al., 2015). The exopolysaccharide produced was purified from the culture medium by adding cold ethanol, followed by an anion-exchange chromatography. NMR analysis of the pure EPS showed a trisaccharide repeating unit containing rhamnose, mannose and galacturonic acid (Table 1). JL2810 EPS showed high capacity to absorb heavy metals (Cu2+, Ni2+ and Cr6+) (Zhang et al., 2017). Raguènés et al. (1997) reported EPS structural features produced by Alteromonas infernus strain GY785. This bacterium, isolated near an active hydrothermal vent, produces two different polysaccharides: a water-soluble EPS (EPS1) and a not-soluble EPS (EPS 2) embedded in a gelatinous matrix containing also bacterial cells. Chemical analysis, performed only for EPS1, revealed an acidic polysaccharide containing galactose, glucose, galacturonic and glucuronic acids (Table 1) (Raguénès et al., 1997). An in-depth characterization of the polymer was achieved by studying oligosaccharide fractions obtained from an acidic hydrolysis. The polysaccharide structure consists of a nonasaccharide repeating unit decorated with sulphate groups (Roger et al., 2004). The high content of uronic acids correlated to the metal binding ability and it makes the polymer a good candidate in the waste-water treatment and metal recovery. Alteromonas hispanica F32, a moderate halophilic bacterium isolated from hypersaline habitat in Spain, produces an exopolysaccharide when grown in MY medium supplemented with 7.5% (w/v) of sea-salts. The molecular weight reported was 1.9- 107 Da, the highest reported for this type of microorganism. Chemical analysis revealed the presence of Glc, Man, Rha and Xyl as monosaccharide components, together with sulphate and phosphate groups (Table 1). A. hispanica EPS showed to be a more efficient emulsifier respect to the commercial surfactants (Mata et al., 2008).
Bacillus and Geobacillus
The family of Bacillaceae comprises the rod-shaped bacteria which form endospores. They are mostly saprophytes, having a wide diversity of physiological characteristics. Their ability to form hardy spores enables them to be widely distributed in nature, from Arctic environments to hot springs and desert sands and from fresh water to salt or marine sediments. Very few reports are available on EPSs production from Bacillus and Geobacillus genera. Usually, genus Geobacillus comprises a group of Gram-positive thermophilic bacteria that can grow over a range of 45–75°C. Geobacillus strain 4004 isolated from geothermal sea sand in Italy (Ischia Island) and able to grow at 60°C as optimal temperature, released an exopolysaccharide in the culture medium. The EPS fraction, recovered by cold-ethanol precipitation, was further purified and three different EPS fractions (named EPS 1–3) were obtained (Table 1). EPS 1 and EPS 2 contained Man, Glc and Gal; EPS 3 showed the presence of Gal, Man, GlcN and Ara. A preliminary structural characterization was carried out only for EPS 3: the 1H and 13C NMR spectra revealed that this polymer essentially consists of two sugars having a gluco- and galacto- configurations and three with a manno- configuration. In addition, the molecular weight for EPS was found to be about 1- 106 Da (Schiano Moriello et al., 2003). Geobacillus tepidamans strain V264 was isolated from Velingrad hot spring (Bulgaria) at 79° C and pH 7.8. The microorganism synthesized an exopolysaccharide showing a glucan-like structure, with a molecular weight higher than 1- 106 Da (Table 1). The bio-polymer isolated from this bacterium was found to be anti-cytotoxic in brine shrimps (Kambourova et al., 2009). Arena et al. (Arena et al., 2009) reported the immunomodulatory and antiviral effects of an EPS produced by a strain of Geobacillus thermodenitrificans, isolated from a shallow marine vent of Vulcano island, in Italy. The EPS showed a molecular weight of 400 KDa and displayed mannose and glucose as main monosaccharidic components (Table 1) (Arena et al., 2009). Bacillus licheniformis strain B3-15 is a halophilic and thermotolerant bacterium, isolated from the shallow marine hot springs at Vulcano island (Italy). It is reported to produce an exocellular polysaccharide when grown on kerosene as carbon source (Maugeri et al., 2002). The EPS obtained after ethanol precipitation, was desalted on Sephadex G-50 and further purified on a DEAE-Sepharose CL-6B. The most abundant fraction gave an EPS essentially constituted by mannose (Table 1) (Maugeri et al., 2002), with the ability to enhance the metal immobilisation (e.g. cadmium immobilisation). In addition, antiviral and immunomodulatory effects of EPS were evaluated: data suggested that EPS treatment impaired HSV-2 replication in human peripheral blood mononuclear cells (PBMC) (Arena et al., 2006). Still from Porto Levante (Vulcano, Italy), another thermophilic Bacillus strain B3-72 was found to be able to produce two exocellular polysaccharides (EPS1 and EPS2) (Table 1). EPS1 and EPS2 structures were based on mannose and glucose in a relative ratio of 0.3:1 and 1:0.2, respectively (Nicolaus et al., 2000). The haloalkalophilic bacterium Bacillus sp. I-450, recovered from mudflats (Korea), displayed interesting flocculating activity. The polysaccharide consisted of neutral sugars such as glucose, galactose and fructose (Table 1). The polymer, observed by SEM microscopy, showed a porous surface, where the water molecules are entrapped. In addition, the small pores structure may also be responsible of the polymer compactness, of the gel stability when subjected to external forces and of the maintenance of the texture properties during storage (Mao et al., 2001; Kumar et al., 2004).
The genus Colwellia comprises heterotrophic and halophilic Gamma- proteo-bacteria. It contains 12 psychrophilic species (Deming et al., 1988; Kim et al., 2013) and among them Colwellia psychrerythraea 34H (Cp34H) is the one best characterized (Huston et al., 2000; Methé et al., 2005; Nunn et al., 2015). Cp34H was isolated from enriched Arctic marine sediments at 1°C and it is considered a model organism for cold-adaptation mechanisms (Boetius et al., 2015). This strain, when grown in marine broth at 4°C, is able to produce cryoprotectant carbohydrate polymers, an EPS (Marx et al., 2009; Casillo et al., 2017) and a CPS (Carillo et al., 2015). In addition, it has been reported that these polymers can be useful for overcoming threshold requirements for dissolved organic carbon in cold environments (Methé et al., 2005). The repeating unit of both CPS and EPS have been completely delucidated and both contain amino sugars and uronic acids (Table 1). Another common feature is the presence of an amino acidic decoration, consisting of a threonine and an alanine in CPS and EPS, respectively. The shape of these two polymers in aqueous solution can explain the ice recrystallization inhibition (IRI) activity, measured for both molecules. When Colwellia is grown at 8°C, it is able to produce a third polysaccharide, which is not endowed with antifreeze activity. This is a polymer containing amino sugars and aminouronic acids and it is not decorated by amino acids (Table 1) (Casillo et al., 2017). It has been hypothesized that the lack of these substituents does not settle the equilibrium between the hydrophobic and the hydrophilic regions necessary to the establishment of the IRI activity.
Halomonas species are moderately halophilic microorganisms that represent one of the major microbial communities populating marine and saline environments (Arahal and Ventosa, 2006). Several Halomonas spp. Have been reported as EPS producers, especially those isolated from high salt concentration habitats (Poli et al., 2009; Martínez-Cánovas et al., 2004). H. eurihalina strain F2-7, previously named Volcaniella eurihalina, is a moderately halophilic bacterium isolated from a solar saltern at Alicante. H. eurihalina produced an exopolysaccharide in several culture media supplemented with hydrocarbons. Chemical analysis suggested as major components rhamnose, glucose and mannose, with a higher content of uronic acids, acetyl and sulphate groups (Table 1) (Bejar et al., 1996). EPS from F2-7 resulted a good emulsifier when growth in hydrocarbons media and showed an interesting application in bioremediation of pollutants (Martínez-Checa et al., 2007). Sulphated polysaccharides are quite uncommon among prokaryotic; nevertheless, another species of Halomonas, named maura, has been reported to produce a sulphated exopolysaccharide, the name of which is mauran. This polymer revealed to be interesting for its potential biotechnological application. Indeed, it showed to share some features with xanthan such as, for example, the ability to form a double helix (Bouchotroch et al., 2001; Arias et al., 2003). In addition, solutions of this polymer showed a thixotropic and a pseudo-plastic behaviour, with viscosity not changing in the presence of high salt concentrations or extreme pH values. The monosaccharides composition (Table 1) reflects the xanthan’s, except for the presence of galactose. Finally, the chelating properties of mauran offers the possibility to use it in bio-remediation. Halomonas sp. OKOH was isolated from the bottom sediment of Algoa Bay of South Africa. The bacterium was reported to produce an exopolysaccharide. The phenol-sulphuric acid method revealed a high carbohydrate content, suggesting the polysaccharidic nature of EPS (Table 1). The polymer showed good bioflocculant activity, enhanced in the presence of glucose and urea, used as carbon and nitrogen source, respectively (Mabinya et al., 2011). The EPS released in the growth medium of Halomonas sp. AAD6 (JCM 15723) has been found to be a levan (Poli et al., 2009). Levans are polymers constituted by β-(2→6)-fructofuranosyl residues (Table 1), with antibacterial (Byun et al., 2014), anti-cancer and anti-oxidant (Abdel-Fattah et al., 2012) activities and with probiotic potential (Abid et al., 2018). Since levan production is very expensive, an industrial production of this product from Halomonas sp. AAD6 has been considered and many efforts have been devoted to obtaining a large-scale production from low-costs substrates (Küçüka et al., 2011). Halomonas alkaliantarctica strain CRSS, isolated from salt sediments near the salt lake in Cape Russell in Antarctica, has been shown to produce different EPSs when grown in different conditions (Poli et al., 2004). After precipitation of the cell-free supernatant with ethanol, the EPS fraction from each growth medium was further investigated. In all conditions, the glycosyl composition indicated the presence of neutral polysaccharides (Table 1). In particular, it was deduced that CRSS produced on complex media a mannan and a xylo-mannan, whereas on minimal medium, it produced a fructo-glucan (Poli et al., 2004).
Members of Hyphomonas genus are Gram-negative bacteria, which are usually colonizer of marine environments. Species of the genus Hyphomonas divide by budding, forming a flagellated cell at the tip of the prosthecum (Abraham and Rohde, 2014). Two marine Hyphomonas strains, MHS-3 and VP-6, are reported to produce exopolysaccharides. Hyphomonas strains MHS-3, isolated from shallow marine sediments in Puget Sound, produces colonies with different morphology, ranging from a slime-producing (MHS-3) to a non-slime-producing (MHS-3 rad) phenotypes (Quintero and Weiner, 1995a). The EPS produced may be involved in the first phase of adhesion process for the formation of the biofilm matrix. The capsule-like exopolysaccharide, produced by MHS-3, is recognized by a GalNAc-specific lectin, suggesting that it contains the amino sugar N-acetylgalactosamine (Table 1). MHS-3 EPS is an acidic polysaccharide, as confirmed by HPAE and IR analyses and may contain N-acetyl-galactosaminuronic acid, due to the binding to the cationic polyferritin (Quintero and Weiner, 1995b). Moreover, it has been reported that MHS-3 EPS has also the ability to sequester gold cations, thus confirming the ionic interactions between the EPS and metal ions (Quintero et al., 2001). Another prosthecatum bacterium, Hyphomonas VP-6, produces two different EPSs, a capsule and holdfast EPS, involved in the first step of biofilm formation (Table 1) (Langille and Weiner, 1998).
Mata et al. (Mata et al., 2008) reported the production of EPSs from two proteobacteria belonging to Alteromonadaceae family, Idiomarina fontislapidosi F23 and Idiomarina ramblicola R22. Idiomarina fontislapidosi F23 was isolated from Fuente de Piedra lagoon in Spain (Martínez-Cánovas et al., 2004), while Idiomarina ramblicola R22 from Spanish rambla, a steep-sided water course, often dry but subject to flash flooding (Martínez-Cánovas et al., 2004). Each bacterium produced two different molecular-mass EPSs, mainly composed of Glc, Man, Xyl and Rha, with traces of sulphate and phosphate groups (Table 1). The EPSs produced very stable emulsions, composed of small and uniform droplets, making them good emulsifier agents (Mata et al., 2008).
The genus Alteromonas, belonging to the family Alteromonadaceae, was determined by Baumann and co-workers (Baumann et al., 1972) to be a genus of marine Gram-negative heterotrophic bacteria. Later, the genus Alteromonas was revised in 1995 to contain only one species, A. macleodii, while the remaining species were reclassified as Pseudoalteromonas (Gauthier et al., 1995; Bosi et al., 2015). Pseudoalteromonas species have been isolated from cold environments (sea-ice, deep sea, Arctic ocean) as well as from marine sea-water. Among these last isolates, there is Pseudoalteromonas strain HYD721, a Gram-negative bacterium recovered from a deep-sea hydrothermal vent. The strain, when grown at 25°C, released a polysaccharide into the growth medium, the structure of which has been completely elucidated (Rougeaux et al., 1999). The polymer displays an octasaccharide repeating unit, decorated with a sulphate group (Table 1). The presence of sulphate groups is quite unusual for Pseudoalteromonas genus (marine microorganisms). Sulphated polysaccharides extracted from seaweeds or animals display a huge number of biological functions (Campo et al., 2009; Toida et al., 2003); nevertheless, no assays have been reported for strain HYD721. Many EPS have found to be involved in metal binding, due to the presence of functional anionic groups, such as carboxyl, phosphoryl, sulfhydryl and hydroxyl groups. These groups can complex both in vivo (Joshi and Juwarkar, 2009) and in vitro (Ha et al., 2010) heavy metals, as in the case of the EPS purified from Pseudoalteromonas sp. strain TG12, named PE12 (Gutierrez et al., 2008). This polymer is a heteropolysaccharide mainly constituted by Glc, GlcN, Xyl and GalA. The sugar analysis revealed also minor components (Table 1). The presence of negative charges of the uronic acids, together with monosaccharides hydroxyl groups has been found to be responsible of the desorption of metals bound to marine sediments (Gutierrez et al., 2008). More interestingly is the emulsifying activity of PE12, able to produce stable emulsions with various food oils even at very low concentration (0.02%). An anti-biofilm activity was revealed for a pool of polysaccharides isolated from Pseudoalteromonas ulvae strain TC14 grown in sessile conditions (Brian-Jaisson et al., 2016). These polymers named LB-EPS and TB-EPS depending on whether they are loosely or tightly bound to the cells and the medium soluble EPS (Sol-EPS), inhibit the biofilm of some marine bacterial strains. Their monosaccharide composition was similar, since all of them showed to contain glucose, while only for LB-EPS and TB-EPS an uronic acids content was revealed (Table 1). After various purification steps, TB-EPS was found to be composed of two polysaccharides, of which one is an-glucan, as suggested by its 1H NMR spectrum, while the other is an heteropolysaccharide. A comparison of P. ulvae polysaccharides production grown in planktonic conditions revealed a higher content of the Sol-EPS.
Pseudoalteromonas ruthenica, isolated from the sea water samples collected from the vicinity of the Bay of Bengal on the coast of India, is able to produce an EPS with interesting properties for potential industrial application (Saravanan and Jayachandran, 2008). Indeed, the polymer solutions indicated a non-Newtonian behaviour, revealing that it is pseudoplastic in nature. GC-MS analysis revealed that the polymer was mainly constituted by mannose, with a low content of uronic acids (Table 1). The last EPS isolated from a non extremophilic Pseudoalteromonas is the one produced by the strain MD12-642. Again, the authors report the capacity of the polymer to confer viscosity to the medium, which suggests potential applications (Roca et al., 2016). The galacturonic acid is the most abundant monosaccharide (44%), followed by glucuronic acid (28%), rhamnose (15%) and glucosamine (14%). Pseudoalteromonas genus is frequently found in cold environments. Among these, there are isolates from sea-ice, Antarctic sea-water, Arctic ocean and deep-sea. The cold-adapted Pseudoalteromonas haloplanktis TAC125, isolated from Antarctic sea water, is a Gram-negative bacterium, the genome of which has been annotated in 2005 (Médigue et al., 2005). It is able to grow even at sub-zero temperatures and it has been considered a good model for the cold adaptation (Médigue et al., 2005). The production of EPS for this microorganism has been reported for three different grown temperatures, namely 4, 15 and 25°C (Corsaro et al., 2004). In all the cases, the medium contained both carbohydrates and proteins and the purification afforded a phosphomannan structure (Table 1), as indicated by NMR spectra and chemical analysis. Some differences were displayed for the EPS structures obtained at the three different temperatures. In particular, a different phosphorylation content was found, being that of EPS recovered at 25°C the highest (4.1%), followed by 4°C (1.4%) and 15°C (1.1%). In addition, the sample obtained at 15°C was less branched, indicating a more linear structure (Corsaro et al., 2004). A mannan structure was also recovered from the medium of Pseudoalteromonas strain SM 20310, an Arctic sea-ice isolate (Corsaro et al., 2004). This microorganism was selected among 110 isolates obtained from samples collected for cold-active enzyme study. It is well known that the organic matter in marine environments is constituted by high molecular weight polymers produced by microorganisms (Yu et al., 2009). These polymers, which in a large amount are polysaccharides, may have different roles, among which is the cryoprotection. The mannan from the strain SM 20310 has been tested for a cryoprotection activity during freeze-thaw cycles of both E. coli and the strain itself cells (Table 1). The results gave indications about a significant improvement of the freeze-thaw survival ratio of both E. coli and strain 20310, thereby suggesting that this EPS may have a universal impact on microorganism cryoprotection (Liu et al., 2013). Moreover, Liu et al. demonstrated that the EPS could enhance the high-salinity tolerance of SM20310, thus facilitating the adaptation of the strain to the sea ice environment. A cryoprotection role was also found for the exopolysaccharides purified from the two species, Pseudoalteromonas arctica KOPRI 21653 (Kim and Yim, 2007) and Pseudoalteromonas elyakovii Arcpo 15 (Kim et al., 2016). The EPS purification method was the same for both strains, even if the biochemical characteristics of the two exopolysaccharides were completely different. Indeed, the glycosyl composition of the arctica polymer revealed only neutral sugar, whereas that of elyakovii species indicated also uronic acids (Table 1). As both neutral and acidic EPS have been found to display cryoprotection to microorganism cells, it is hard to deduce how the glycosyl composition can influence the activity. EPS from marine isolates also play a role in the attachment to surfaces (Decho and Gutierrez, 2017), driving marine particle formation including marine microgels, marine snow and biofilms. Hydrophobic interactions could be responsible of cells aggregation and therefore the sugar composition of the EPS, together with secreted proteins, can influence this phenomenon (Verdugo, 2012). Two Pseudoalteromonas strains, named CAM 025 and CAM 036, were isolated from particles collected in melted Antarctic sea ice and from particles captured by a plankton net towed through the Ocean, respectively. Both the EPSs isolated contain uronic acids, as well as amino sugars, neutral sugars and sulphate groups (Table 1) (Mancuso et al., 2004). The negative charges, due to both carboxylic and sulphate groups, are suggested to be responsible of the “sticky” quality of the EPS in marine environment. In addition, Mancuso-Nichols et al. (Wu et al., 2001) speculated on the possibility that metal ions, such as dissolved iron, interact with organic ligands, thus suggesting a further ecological implications of EPS polymers. A bio-sorption capacity and a flocculation behaviour has been attributed to the EPS isolated from Pseudoalteromonas strain SM 9913 (Qin et al., 2007). This polymer, obtained in large quantity (5.25 g/L) under optimal growth conditions (15°C, 52 h), is also able to bind a wide range of metal cations, suggesting an ecological role in concentrating metal ions to improve the biochemical interactions with the environment (Mancuso Nichols et al., 2007). The glycosyl analysis for this EPS indicated mainly glucose, with arabinose, xylose and a minor content of mannose (Table 1). The complete structure was identified by methylation analysis and by NMR spectra and it was found to have a linear arrangement of ᾳ-(1→6) linkage of glucose residues with a high grade of acetylation. The psychrotroph Pseudoalteromonas sp. MER144 belongs to a pool of 606 isolates of Antarctic origin and it has been selected on the basis of its mucous growth on agar plates (Caruso et al., 2017). To obtain the highest production of the EPS material, many variables were tested, such as different values of carbon source, temperature, pH, NaCl concentration and incubation time. The optimal growth was reached at 4°C, at pH 7, with a 3% of NaCl, using sucrose as carbon source. After purification, the EPS material showed to still contain proteins, together with a polysaccharide containing neutral sugars and uronic acids (Table 1). Caruso et al. (Caruso et al., 2017) demonstrated that in presence of heavy metals Pseudoalteromonas sp. MER 144 is able to increase the production of EPS, suggesting that this could represent an adaptation mechanism to the tested stressed conditions.
Pseudomonas species are widespread in marine ecosystems and therefore many EPSs from this genus have been isolated, characterised and tested for their properties and abilities. One of the first species reported to produce exopolysaccharides involved in adhesion properties of the bacterium is the strain NCMB 2021. Two different polysaccharides have been isolated from this strain, of which polysaccharide A was able to form gel, with the ability to bind multivalent cations (Christensen et al., 1985), in contrast with polysaccharide B which is not able to precipitate in the presence of cations. These properties may be related to the glycosyl composition (Table 1), which is completely different in the two polymers (Table 1). Indeed, the solution of polysaccharide B showed low viscosity, suggesting that it has a very flexible chain (random coil). Another EPS involved in the adhesion properties of bacterial cells is that from Pseudomonas sp. S9. Under laboratory conditions, the cells were grown in static and, after 1 to 5 h of starvation, they were detected to be surrounded by an exopolymer material (Wrangstadh et al., 1986). In this case, the cells did not show adhesion to hydrophobic surfaces. With extended starvation, the cells appeared to gradually lose the extracellular layer of polysaccharides. When the cells were grown under agitated conditions, the adhesion characteristic grew between 1 and 4 h of starvation, followed by a slow decrease in attachment with time. In this case, no exopolymer was observed around the cells. Three neutral sugars were revealed for the purified exopolysaccharide (Table 1), even if no relationships with adhesion properties of the cells were suggested. Sulphated exopolysaccharides are usually isolated from algae and, due to their similarity with glycosaminoglycans, very often display biological activity, anticoagulant, antiviral and immuno-inflammatory activities that might find relevance in nutraceutical/functional food, cosmetic/cosmeceutical and pharmaceutical applications (Jiao et al., 2011). An anti-cancer sulphated polysaccharide has been isolated and characterised from Pseudomonas sp. WAK1. The structure, obtained by methylation analysis and both mono- and two-dimensional NMR experiments, indicated a backbone of two sulphated galactoses, of which one is branched at 6 positions with a glucose unit (Table 1) (Matsuda et al., 2013). This polymer, showing a cytotoxic effect towards human cancer cell lines MT-4, could provide an example of bacterial exopolysaccharide with suitable functions for obtaining new drugs. Among the most interesting bioactive polysaccharides isolated from Pseudomonas species there is the recently reported polymer from P. stutzeri 273, which showed anti-biofilm, anti-biofouling and antioxidant activities (Table 1) (Wu et al., 2016). All these properties indicated that it could be find applications in challenging bacterial biofilm-associated infection, food-processing contamination and marine biofouling. The glycosyl analysis indicated only neutral and amino sugars, even if the presence of carboxylic acids was suggested by the IR spectrum (Wu et al., 2016 ). Therefore, an in-depth analysis of the primary and secondary structures of this polysaccharide would be interesting in order to advance knowledge of antibiofilm activity mechanism. A cryoprotectant polysaccharide, containing both neutral monosaccharides and uronic acids, has been isolated and purified from the Antarctic cold-adapted Pseudomonas sp ID1 cells (Table 1). Interestingly, the chemical analysis revealed also the presence of amino acids, which could be part of the EPS (Carrión et al., 2015), a feature revealed also for Colwellia psychrerythraea 34H CPS and EPS (Carillo et al., 2015; Casillo et al., 2017). In addition, the polysaccharide was revealed to possess the ability to form a long-term stable emulsion against different food and cosmetic oils, such as olive, sunflower and corn oils and cetiol V oil, respectively (Carrión et al., 2015).
Rhodococcus sp. 33, a marine bacterium of genus Rhodococcus, was isolated from Port Botany (Sidney), in a contaminated site near a chemical plant (Luz et al., 1997). This bacterium is able to tolerate and degrade highs levels of benzene. Aizawa et al. (Aizawa et al., 2005) reported a different benzene-tolerance for rough and mucoid Rhodococcus sp. 33 cells. They demonstrated that the spontaneous mutants rough-type, producing very low amount of EPS, were more sensitive to benzene, resulting in an absent or reduced growth in the presence of the pollutant. In a different way, wild-type colonies that produced EPS appeared mucoid and they were resistant to benzene. These data suggested the direct involvement of exopolysaccharides in the protection against this pollutant (Aizawa et al., 2005). The EPS, purified through an enzymatic digestion and gel filtration chromatography, was analysed by way of chemical and spectroscopic experiments. The polymer consists of a tetrasaccaridic repeating unit containing Glc, Gal, GlcA and Man substituted by a pyruvic acid (Table 1). Authors demonstrated that the pyruvic residue and the uronic carboxyl group were responsible for the protecting activity, since the de-pyruvylated and carboxyl-reduced EPS that tested for benzene sensitivity showed no activity (Urai et al., 2006). Rhodococcus erythropolis PR4 was isolated from Pacific Ocean, in Japan. As reported for other strains (Iwabuchi et al., 2002; Urai et al., 2002; Urai et al., 2004; Urai et al., 2006), R. erythropolis produces a FACEPS, a fatty acid containing exopolysaccharide. R. erythropolis was grown on IB agar plates at 25°C; the EPS purified through an ion exchange chromatography, showed two peaks, named FR1 and FR2, displaying different monosaccharide composition and emulsifying activity. EPS FR1 contained Glc, GlcN, Man and GlcA and did not show any emulsifying activity. Differently, EPS FR2 showed good activity, probably related to the different chemical structure. Indeed, it displays a tetrasaccharidic repeating unit containing Gal, Glc, Man and GlcA and a pyruvic acid substituting the mannose residue (Table 1). Furthermore, only FR2 EPS contained stearic and palmitic acids. These data allowed the conclusion that EPS FR2 named PR4 was the FACEPS, while FR1 was assigned as a mucoidan (Table 1) (Urai et al., 2007).
The genus Shewanella comprises species largely distributed in marine environments, typically isolated from the deep-sea (DeLong et al., 1997; Nogi et al., 2017). They can be both mesophiles and psychrophiles. Shewanella genus belongs to Gammaproteobacteria and about 60 species have been isolated and characterised. Among these, there are S. onedeinsis MR-4 ( Murray et al., 2001) and S. colwelliana (Weiner et al., 1998; Weiner et al., 1985). Both species produce capsular polysaccharides. In particular, S. oneidensis polymer has been fully characterised, where neutral monosaccharides, amino sugars and uronic acids are included (Table 1) (Vinogradov et al., 2005). The repeating unit has been identified on the basis of chemical analysis and NMR experiments. Instead, S. colwelliana capsule has been characterised by cross-reaction with six monoclonal anti-bodies (Sledjeski and Weiner, 1993), indicating a specific and identical epitope recognition. Unfortunately, no structure has been identified in this case, only glycosyl analysis and pyruvate content have been reported (Table 1) (Sledjeski and Weiner, 1993).
The genus Vibrio consists of more than 100 species that occur naturally in marine, estuarine and freshwater systems worldwide. They occupy habitats ranging from the deep sea to shallow aquatic environments (Reen et al., 2006) but are also associated with a wide variety of molluscan and animals, including humans, for some of which are pathogens (Romalde et al., 2014). Many EPS associated to Vibrio bacteria have been reported, of which some are acidic polysaccharides (Table 1). Among these, the only complete structure so far reported for the EPS produced by a polychaete annelid isolate is that from Vibrio diabolicus. It consists of a linear tetrasaccharide repeating unit with a structure containing glucuronic acid and rich in amino sugars (Table 1) (Romalde et al., 1999). This molecule resembles glycosaminoglycans and it has been tested as enhancers of bone healing. The tests revealed that the polymer was able to act as a filler of bone defects in rat calvaria, with new, well-structured woven bone. They have oriented collagen fibres, with osteoblast cells covering the bone surfaces (Zanchetta et al., 2003). In addition, the polymer did not show any inflammatory activity, thus encouraging its application in the healing process. Vibrio alginolyticus is a marine biofouling bacterium, able to produce large quantities of EPS (Muralidharan and Jayachandran, 2003). After purification, the polysaccharide was tested for viscosity measurements. It was revealed to be a pseudoplastic performance, i.e., the viscosity of the solution decreases concomitantly with the shear rate. Authors also reported for the polymer a good stability to pH range between 5 and 10, and the ability to regain its pseudoplastic nature on cooling from 90°C ((Muralidharan and Jayachandran, 2003). The glycosyl composition for the purified polymer indicated only neutral and amino sugars (Table 1). Instead, the EPS isolated from the strain CNCM I-4994 of V. alginolyticus was composed of galacturonic and N-acetyl glucosamine monosaccharides (Table 1) (Drouillard et al., 2015). This polymer is produced on an industrial scale as an ingredient for cosmetic applications for its mattifying and anti-inflammatory properties (Drouillard et al., 2015). The most interesting feature of the repeating unit is the amino acidic decoration, which has also been reported for both C. psychrerythraea 34H CPS and EPS (Carillo et al., 2015; Casillo et al., 2017). This is not surprising, because Vibrio and Colwellia are phylogenetically close to each other. Indeed, also the glycolipid portion of the LPS from C. psychrerythraea 34H (Casillo et al., 2017) has been found to be similar to that of Vibrio fisheri (Phillips et al., 2011). Vibrio harveji strain VB23 (Bramhachari et al., 2006) and Vibrio furnissii strain VB0S3 (Bramhachari et al., 2007) have both been isolated from the Mandovi and Zuari estuarine in Goa, on the west coast of India. They are both able to produce an acidic exopolysaccharide with emulsifying properties. Unfortunately, only neutral sugar composition has been reported for both polymers, thus hampering any comparison with other Vibrio EPSs. An anti-biofilm activity has been reported for Vibrio sp. QY101 EPS (Jiang et al., 2011). This marine bacterium, isolated from a decaying thallus of Laminaria, was initially supposed to inhibit the growth of Pseudomonas for the alginate-lyase activity of the culture supernatant. However, after elimination of the protein content, the anti-biofilm activity was lost. Instead, ion-exchange and gel filtration chromatographies afforded a pure polysaccharide able to inhibit cell aggregates of Pseudomonas aeruginosa and Staphylococcus aureus. The polysaccharide showed to contain more than 40% of uronic acids and about 10% of GlcNAc, revealing strong similarities with the EPS structures of alginolyticus and diabolicus species.
Other EPS-Producing Bacteria
Cobetia marina (DSMZ 4741), a slightly halophilic Gram-negative bacterium isolated from littoral seawater in USA (Cobet et al., 1971), is often found associated with microalgae. The exopolysaccharide, named L6, was produced at 20°C, pH 7.6 in a fermenter. Cobetia EPS, with a molecular weight of 2.7 x 105 Da, showed a disaccharide repeating unit containing ribose and a 7,8-pyruvilated Kdo (Table 1). The presence of Kdo, typically a component of the LPS molecule, has been reported for K-antigen polysaccharide from Escherichia coli. The EPS from Cobetia marina represented the first example of EPS with this type of structure. The unusual substitution of the Kdo could represent a possible way for the bacterium to escape the viral Kdo-hydrolases, which can be considered a resistance mechanism (Lelchat et al., 2015).
Members of the genus Flavobacterium are widely distributed in nature, occurring mostly in aquatic environments, from freshwater to seawater (Bernardet et al., 2006). Flavobacterium uliginosus MP-55 was isolated from the homogenates of sea weed collected in Japan. The marine bacterium grown in a medium supplemented with marine water produces a water-soluble polysaccharide. The polymer named marinactan contained glucose, mannose and fucose (Table 1), with a molecular weight higher than 1x106 Da. Biological assays showed that marinactan was an antitumoral molecule against sarcoma 180, in both solid and ascites forms (Umezawa et al., 1983).
Hahella chejuensis strain 96 CJ10356, a Gram-negative halophilic bacterium, was isolated from marine sediments from Cheju Island (Republic of Korea) (Lee et al., 2001). The monosaccharide analysis of EPS-R revealed a heteropolysaccharide consisting of glucose and galactose as main components, with xylose and ribose in minor amounts (Table 1). The molecular weight of EPS-R was approximately 2.2x103 KDa. The exopolysaccharide showed a good emulsifier activity (Yim et al., 2004).
Polaribacter sp. SM1127, a member of the genus Polaribacter, was isolated from the brown alga Laminaria (Dong et al., 2012). Bacteria of this genus have been isolated from different marine environments, mainly from Arctic and Antarctic regions. Polaribacter sp. SM1127 produces an exopolysaccharide containing Rha, Fuc, Man and Glc, together with a higher amount of GlcA and GlcN. The polysaccharide, with a molecular weight of 220 KDa, showed a good viscosity and antioxidant activity, thus representing a great potential in cosmetics as anti-ageing product. Furthermore, it showed moisture-adsorbing and retention ability superior to that of ialuronic acid and glycerine, both currently used in the cosmetic field. This enhanced activity could be related to the high percentage of GlcA, Fuc and GlcN residues (Table 1). In addition, the cryoprotectant role of EPS was tested on dermal fibroblast at 4°C. SM1127-EPS, displaying protection to fibroblast cells, could be used as component of cream to protect human skin from cold injury (Sun et al., 2015).
The bacterium Salipiger mucosus A3T, belonging to the alfaproteobacterium class, was isolated from a solar saltern on the Spanish Mediterranean seaboard (Martínez-Cánovas et al., 2004). The production of the EPS from the halophilic S. mucosus was observed with all carbon sources tested and the best yield was obtained at a sea-salt concentration of 2.5% (w/v). The cells, stained with ruthenium red and observed to TEM, revealed a different morphology over the time: at beginning, the polysaccharide appeared strictly associated to the cells, while from the third day of incubation, it was released in the surrounding medium as slime. The purified EPS, showed a molecular weight of 250 KDa and resulted to be composed by Glc, Man, Gal and the uncommon Fuc (Table 1). In addition, the presence of sulphate and phosphate groups make the polysaccharide interesting, since this EPS with high charge density have a great potential for removing toxic metals from environment. S. mucosus EPS showed interesting emulsifying activity with several hydrocarbons, compared with other molecules already used as surfactants. Furthermore, it showed a higher emulsifying activity than xanthan with crude oil (Llamas et al., 2010).
At first, bacteria belonging to the genus Zooglea were considered members of the Pseudomonadaceae family but later they were differentiated for the production of a gelatinous matrix. Indeed, the name Zooglea is derived from Greek and means animal glue, referring to the sticky feature of the zoogloeal matrix (Dugan et al., 1992). Bacterium Zooglea sp. KCCM 100376, isolated from the surface layers of the seaweed Undaria sp., was reported to produce two exopolysaccharides: a water-soluble polysaccharide (WSP), recovered in supernatant after centrifugation of growth broth and a cell-bound polysaccharide (CBP) obtained from the precipitate (Kwon et al., 1994).
BIOTECHNOLOGICAL APPLICATIONS OF MARINE EPS
Bacterial EPS have great biological activity and assume jobs in the control of cell division and separation, immune regulation, and additionally have antitumor, antioxidant, and antiviral activities (Zhang et al., 2003; Selim et al., 2018a, b). Umezawa et al. (1983) screened EPS of marine microorganisms for their anticancer activity against sarcoma- 180 strong tumor in mice. Lactic acid bacteria (LAB) for example, Lactobacillus, are imperative microorganisms in healthy human microbiotic environment (Macfarlane and Cummings, 2002). LAB are useful bacteria, which have been related with a few probiotic impacts in both people and animals (Fuller 1989; Yang et al., 2001; Iwai et al., 2004). Various reports have shown that both LAB and milk exert apply anticancer impacts (Biffi et al., 1997; Lee et al., 2004). Information from epidemiological and exploratory examinations have likewise shown that the ingestion of certain LAB strains, or of milk exert, may reduce the danger of particular sort of malignant growths and repress the development of tumors (Kato et al., 1994). Be that as it may, the exact component by which LAB applies anticancer impacts stays obscure. Enterobacter cloacae Z0206, a bacterial strain, can create a lot of EPSs. It has been accounted for that glycoproteins from E. cloacae demonstrated antitumor consequences for mice with S180 tumors (real), and F3, one of the glycoprotein parts, could particularly restrain QGY7703 (liver malignant growth), A549 (glandular cancer of the lungs), Kato III (gastric carcinoma), and Sw1116 (intestinal cancer) cell strains (Zhang et al., 2002). Ruiz-Ruiz et al., (2011) demonstrated that the novel halophilic bacterium Halomonas stenophila strain B100 emits EPSs with high sulfate substance. This hetero-EPS when over sulfated applied anticancer activity on T cell lines getting from acute lymphoblastic leukemia (ALL). Just tumor cells were vulnerable to apoptosis actuated by the sulfated EPS (B100S), while essential immune system cells were safe. In addition, naturally detached essential cells from the blood of patients with ALL were additionally susceptible to B100S-induced apoptosis. EPS are presently drawing in more prominent consideration inside the field of medicine. This is on the grounds that the EPS have various impacts that incorporate the capacity to animate T-cell formation and potentiate the induction of various sorts of antitumor effector cells, for example, cytotoxic T-cells, NK cells and macrophages (Wasser and Weis, 1999). Numerous immune-stimulating PS likewise have antitumor properties (Borchers et al., 1999; Ikewa, 2001). PS with antitumor activity have different chemical compositions, configurations, and also in their physical properties. Antitumor activity is shown by a wide scope of glycans reaching out from homo-polymers to very unpredictable heteropolymers (Ooi and Liu, 1999). The difference of EPS activity can be related with dissolvability in water, size of the particles, branching rate, and form.
Various sulfated EPS from marine bacteria, cyanobacteria, and green algae were depicted appearing inhibitory impacts against a few human and animal viruses (Luescher-Mattli, 2003; Damonte et al., 2004; Arad et al., 2006). Sulfated PS of synthetic origin has antiviral activity (Witvrouw and De Clercq, 1997). A portion of these macromolecules are now experiencing clinical assessment (Kleymann, 2005; McReynolds and Garvey-Hague, 2007). They have a promising point of view to be developed into a novel kind of antiviral medications. The antiviral activities are depending on the degree of sulfation, furthermore molecular weight (Hemmingson et al., 2006). Sulfated PS may hinder the connection of viruses with target particles on the cell surface (Ponce et al., 2003; Pilar et al., 2005). The viral connection peptides are very saved areas inside rather than factor platforms of viral surface glycoproteins. These peptides are just inadequately subject to adjustments by the common antigenic drift of viruses. Moreover, they are not expected to visit locales of medication-incited resistance mutation. Sulfated PS that is coordinated toward these objective peptides are in this manner favored possibility for antiviral medication improvement. Also, sulfated polymers appeared in vitro antiviral action against mutants resistant to nucleoside analogs (Adhikaria et al., 2006; Mandal et al., 2007).
Nguyen et al. (2008) built up a bacterially produced EPS (cellulose), containing nisin so as to control Listeria monocytogenes in foodstuff. Bacterial EPS was created by Gluconacetobacter xylinus K3. Nisin (2500 IU/mL) was joined into the polymer matrix. EPS decreased L. monocytogenes populaces on foodstuff of around 2 log CFU/g following 14 days of storage. Orsod et al. (2012) isolated two EPS from marine bacteria and screened their activities against Lysinibacillus and Paenibacillus sp. which represent Gram-positive bacteria and (Pseudomonas sp., Escherichia coli) as Gram-negative bacteria. The produced EPSs showed antimicrobial activities against all the tested organisms. Some studies exhibited the adequacy of lentinan administrated intraperitoneally before infection of Mycobacterium tuberculosis. The outcomes proposed that lentinan could assemble, have protection potential, and diminish Mycobacterium disease (Markova et al., 2003). Another exploration exhibited that lentinan prompted abnormal state of alveolar macrophage enactment showed through improved bactericidal impact against M. tuberculosis, which corresponded with the induction of responsive nitrogen intermediates, expanded movement of lysosomal enzymes (acid phosphates), and with powerful phagolysosomal combination pursued by decimation of Mycobacterium (Markova et al., 2005; Shanmugam et al., 2008; He et al., 2010).
Some carbohydrates have anticoagulant impacts by repressing thrombin or by initiating against thrombin III or by expanding the coagulating time. Additionally, these molecules can likewise have an antithrombotic action by blocking thrombin movement, interceded through the heparin cofactor II (Li et al., 2012; De Jesus Raposo et al., 2015). Yet, different researchers prove that they likewise interfere in the prothrombin (PT) pathway, and hence, are not ready to influence the outward coagulation pathway (Nishino et al., 1989; Silva et al., 2010; Wijesekara et al., 2011; Cao et al., 2019). Besides, a critical job of the substance in sulfate has been assigned in the anticoagulant activities, as the nearness of sulfate and its circulation design assume a vital job in the procedures of coagulation.
Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation (rheumatoid arthritis) or due to aging (arthrosis) are thus irreversible and may lead to the loss of the articular function. In order to repair cartilaginous defects, several surgical techniques have been developed mainly based on subchondral bone marrow, implantation of healthy cartilage or chondrogenic tissue. However, multiple disadvantages arising from these techniques led to development of a new therapeutic strategy, namely tissue engineering. Indeed, the association of cells (chondrocytes or mesenchymal stem cells, MSC) together with signaling molecules (growth factors) into biocompatible hydrogel matrix could lead to regeneration of the functional tissue. However, in order to guide the MSC differentiation into chondrocytes, the supply of growth factors during the differentiation phase is required. In this context, the encapsulation of growth factors within micro-matrices allowing both their protection against degradation conditions and their release leading to the enhancement of their bioactivity and bioavailability becomes an interesting approach. The study conducted by Zykwinska et al., 2018 was developing a growth factor delivery system based on a marine exopolysaccharide (EPS) displaying glycosaminoglycan (GAG)-mimetic properties that could further be used for stimulation of MSC chondrogenic differentiation in vitro and in vivo. To preserve its bioactivity, the growth factor was gently encapsulated inside two gelled polysaccharide-based matrices elaborated at micro-scale level using a capillary microfluidic method. The release of the growth factor from both systems was followed under in vitro conditions and the bioactivity of the released protein was assessed. It appears from this study that the micro-matrices based on a marine EPS may become promising candidates as growth factor delivery systems.
Treatment of Psoriasis
An exopolysaccharides/calcipotriol (EPS/CPT) emulsion was prepared using bacterial EPS as emulsifier, sunflower oil as an oil phase and CPT as the loaded drug, and the effect of this emulsion on psoriasis vulgaris treatment was evaluated by Song and al., 2019. An EPS composed of mannose (70.56%) and glucose (29.44%) was obtained from the marine mangrove bacteria Bacillus amyloliquefaciens ZWJ strain (Zhu et al., 2018). The EPS has significant emulsifying activity at the concentration of 1.5%. The prepared EPS/CPT emulsion has small and stable particle size, with a drug content of 0.00492%, and good spreading properties. The in vitro drug release results revealed that the emulsion showed a certain sustained release effect. In vitro and in vivo animal experiments show that the EPS/CPT emulsion can effectively treat psoriasis vulgaris by increasing the accumulation of CPT in psoriatic skin lesions and reducing the levels of inflammatory cells and inflammatory factors (TNF and IL6). Additionally, it has a certain effect on reducing the side effects associated with CPT. This study lays a foundation for the research of EPS in the topical application of medical materials and treatment of psoriasis.
Others biotechnological applications
V.ibrio diabolicus, a marine bacterium has been reported for the production of “Hyalurift” polysaccharides having properties that are similar to hyaluronic acid and known for its restoration of bone integrity (Nwodo et al., 2012; Onesti et al., 2013). Romano et al., (2007) and de Morais et al., (2010) have suggested the use of microbial polysaccharides for bone integrity. An acidic EPS released by Alteromonas sp. strain 1545 has interesting rheological properties and may be used as a thickening agent (Talmont et al., 1991); EPS secreted by A. madeodii sub sp. fijiensisbiovar has found application in cosmetics (patent number 94907582-4). The EPS secreted by Hahella chejuensis gen. nov., sp. nov., has emulsifying properties (Lee et al., 2001); polymer produced by Cyanothece sp. ATCC 51142 has the capability of gel formation and use in food industries (Shah et al., 2000). Kumar et al., (2007) isolated Planococcus maitriensis, which produced an EPS having biosurfactant properties. Microbial EPS are extensively used for Microbial Enhanced Oil Recovery (MEOR) and transport of polyaromatic and aliphatic hydrocarbons. The marine Pseudomonas sp. strain S9 was found to produce EPS in nutrient availability as well as in nutrient starvation conditions (Wrangstadh et al., 1990). An EPS capable of binding heavy metals was produced by the Alteromonas strain 1644 isolated from Alvinellidae collected from the East Pacific Rise (Bozzi et al., 1996). The Pseudoalteromonas strain SM9913 was isolated from deep-sea sediments in the Gulf of the Yellow Sea (China). EPS of this strain showed flocculating and biosorptive capacity (Qin et al., 2007; Li et al., 2008). Muralidharan and Jayachandran, 2003) described the physicochemical properties of bioadhesives produced by marine biofouling bacterium, Vibrio alginolyticus. The EPS of Artic marine bacterium Polaribacter sp. SM1127 showed antioxidant activity, moisture-retention ability and protective property on human dermal fibroblasts (HDFs) at low temperature. EPS has also promoted the skin wound healing and prevented the frostbite injury in Rat Skin (Sun et al., 2020).
HEAVY METAL REMEDIATION USING MARINE BACTERIA AND THEIR EPS
Biosorption is one of the mechanisms through which organisms remove or accumulate heavy metals. It is a rapid and passive process of metal uptake for which the cells need not be in a live state. Biosorption is a physicochemical process, which includes various mechanisms such as adsorption, absorption, intracellular or extracellular accumulation, redox reaction, ion exchange, surface complexation and precipitation (Gadd, 2010). Agricultural waste such as rice straw, wheat straw, soya bean straw, coconut husks, waste tea, waste coffee powders, dried plant leaves, wool, cork biomass, and cottonseed hulls are used for metal removal. Sewage, sludge and microbial cells such as bacteria, fungi and algae have been also used for their metal- binding capacity under various conditions (Abbas et al. 2014). Microbial EPS have the ability to bind with anion and cations, resulting in a candidate of choice for the bioremediation process (Saikia et al., 2013). In some remediation processes EPS modified by chemical processes such as acetylation, methylation, phosphorylation, and sulfonylation are used (Desbrieres et al., 2018). Acetylation of EPS decides the selectivity of metal-binding (Sutherland, 1983). The metal binding property of the EPS plays a significant role for metal remediation from the wastewater (Choi and Yun 2006).The reports of Gupta and Diwan (2017) demonstrated almost 85–95% of zinc, copper and chromium removal using consortium developed from activated sludge. They also reported that many Gram-negative bacterial consortia could remove 75–78% of zinc, lead, chromium, nickel, copper, cadmium, and cobalt within two hours. Immobilized EPS of Chryseomonas and Paenibacillus polymyxa showed the removal of cadmium, cobalt, copper, and lead (Ozdemir et al., 2005; Acosta et al., 2005). Dead cell-bound EPS of Bacillus cereus, Bacillus pumilus, Pentoea agglomerans showed 85.5–89% of chromium removal (Sultan et al., 2012). EPS of Acidithiobacillus ferrooxidans helps the organisms to bind with the mineral and thus extract metals from the sulphide ores (Yu et al., 2011). Salehizadeh and Shojaosadati (2003) reported the biosorption of copper (74.9%), lead (98.3%) and zinc (61.8%) by the EPS of Bacillus firmus. The EPS produced by Azotobacter chroococcum XU1 showed the sorption of lead (40.48%) and mercury (47.87%) (Rasulov et al., 2013). The EPS of Ensifer meliloti, showed 89, 85 and 66% of lead, nickel and zinc ion reduction respectively (Lakzian et al., 2008). Various marine bacteria are also reported for their metal removal ability. The specific structure and high uronic acid content impart an enhanced anionic property to marine bacterial EPS which may be responsible for metal removal. EPS of Marinobacter sp. showed sorption of metals like lead and copper (Bhaskar and Bhosle 2006). EPS from marine Enterobacter cloacae demonstrated the sorption of cadmium (65%), copper (20%) and hexavalent chromium (75%) (Iyer et al., 2004, 2005). Halomonas sp. associated with marine micro-alga was also reported to chelate metals such as calcium, aluminium, iron, and magnesium (Gutierrez et al., 2012). The EPS secreted by the Pseudoalteromonas sp. SM9913 showed the adsorption of Fe2+ (85.00 %), Zn2+ (58.15 %), Cu2+ (52.77 %), Co2+ (48.88 %), Mg2+ (30.69 %), Mn2+ (25.67 %) and Cr6+ (5.15 %) (Qin et al. 2007).
The marine biopolymers contribute only a small portion to the current polymer market. Mainly the high production costs of the EPS affect the profit margin at market level. The high production costs are mainly due to the use of expensive and specific nutrients in the preparation of fermentation media; this generally contributes about 30% of the cost for the fermentation process. To make the processes cost effective, cheaper alternative substrates such as cane molasses, sugarcane bagasse, corn steep liquor, fruit peels, potato peels etc. should be used for the large scale production. Some biopolymers like xanthan, curdlan, dextran, gellan have been produced by solid state fermentation using raw substrates like spent malt grains, vegetable and fruit wastes, citrus peels, olive mill waste water etc. But it requires lots of efforts to scale-up the process from lab level to industrial level for the production of a commercial product using cheaper or solid substrates (Poli et al., 2011; Casillo et al., 2018).
Although marine microbial EPS have been studied in recent times for their various industrial applications, there have been only a few reports highlighting their production and recovery. More detailed research in this field is needed to understand the properties of EPS in depth. To achieve the higher EPS yields, the marine bacterial strains can be improved using genetic engineering (use of mutagenic strains, gene manipulations) and also EPS having specific properties and structures can be produced using the same.
The present methods that are used for structural determination of EPS are labor-intensive and tedious. So suitable modifications can be incorporated in the existing protocols or novel strategies can be developed to make the process simpler. Moreover, the EPS extraction methods can be suitably modified in a cost-effective manner which would significantly lower down the overall cost of the downstream processes.
The review intends to provide information on Exopolysaccharide (EPS) producing marine bacteria, their unique properties, purification methods and applications in various fields. This also provided an insight of novel marine biopolymers of applied interest. There are several methods for recovery, extraction and purification of EPS, but they need to be considered critically depending upon the source of production and biochemical nature of the EPS. All the purification and recovery methods are having one or the other limitation and no universal extraction method is available due to wide variety of EPS specially from marine bacteria. Thus for the potential biotechnological and industrial applications of these polymers, further developments in the methods used for their recovery and purification are needed. Marine bacterial EPS can be a good source for metal remediation, MEOR as well as in the field of medicine thus can play role in maintaining environmental sustainability.
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