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Environmental Technology
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Exopolysaccharides from extremophiles: from fundamentals to biotechnology
Barbara Nicolausa; Margarita Kambourovab; Ebru Toksoy Onerc a Istituto di Chimica Biomolecolare (ICB), CNR via Campi Flegrei 34,80078, Pozzuoli (Na), Italy b Department of Extremophilic Bacteria, Institute of Microbiology, BAS, Sofia, Bulgaria c Department of Bioengineering, Marmara University, Istanbul, Turkey Online publication date: 16 June 2010
To cite this Article Nicolaus, Barbara , Kambourova, Margarita and Oner, Ebru Toksoy(2010) 'Exopolysaccharides from
extremophiles: from fundamentals to biotechnology', Environmental Technology, 31: 10, 1145 — 1158 To link to this Article: DOI: 10.1080/09593330903552094 URL: http://dx.doi.org/10.1080/09593330903552094
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Environmental Technology Vol. 31, No. 10, September 2010, 1145–1158
Exopolysaccharides from extremophiles: from fundamentals to biotechnology Barbara Nicolausa*, Margarita Kambourovab and Ebru Toksoy Onerc a Istituto di Chimica Biomolecolare (ICB), CNR via Campi Flegrei 34,80078, Pozzuoli (Na), Italy; bDepartment of Extremophilic Bacteria, Institute of Microbiology, BAS, Sofia, Bulgaria; cDepartment of Bioengineering, Marmara University, Istanbul, Turkey
(Received 30 October 2009; Accepted 11 December 2009 )
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10.1080/09593330903552094
Exopolysaccharides (EPSs) make up a substantial component of the extracellular polymers surrounding most microbial cells in extreme environments like Antarctic ecosystems, saline lakes, geothermal springs or deep sea hydrothermal vents. The extremophiles have developed various adaptations, enabling them to compensate for the deleterious effects of extreme conditions, e.g. high temperatures, salt, low pH or temperature, high radiation. Among these adaptation strategies, EPS biosynthesis is one of the most common protective mechanisms. The unusual metabolic pathways revealed in some extremophiles raised interest in extremophilic microorganisms as potential producers of EPSs with novel and unusual characteristics and functional activities under extreme conditions. Even though the accumulated knowledge on the structural and rheological properties of EPSs from extremophiles is still very limited, it reveals a variety in properties, which may not be found in more traditional polymers. Both extremophilic microorganisms and their EPSs suggest several biotechnological advantages, like short fermentation processes for thermophiles and easily formed and stable emulsions of EPSs from psychrophiles. Unlike mesophilic producers of EPSs, many of them being pathogenic, extremophilic microorganisms provide non-pathogenic products, appropriate for applications in the food, pharmaceutical and cosmetics industries as emulsifiers, stabilizers, gel agents, coagulants, thickeners and suspending agents. The commercial value of EPSs synthesized by microorganisms from extreme habitats has been established recently. Keywords: exopolysaccharide; sugar composition; extremophile; exopolysaccharide production; ecology
General features Exopolysaccharides are high-molecular-weight polymers that are composed of sugar residues and are secreted by microorganisms into the surrounding environment. Exopolysaccharide is a term first used by Sutherland [1] to describe high-molecular-weight carbohydrate polymers produced by marine bacteria. Microorganisms synthesize a wide spectrum of multifunctional polysaccharides including intracellular polysaccharides, structural polysaccharides and extracellular polysaccharides or exopolysaccharides (EPS). Exopolysaccharides can be found as in capsular material or as dispersed slime in the surrounding environment with no obvious association to any one particular cell [2]. They generally constitute of monosaccharides and some non-carbohydrate substituents (such as acetate, pyruvate, succinate and phosphate). Owing to the wide diversity in composition, EPSs have found multifarious applications in various food and pharmaceutical industries. Many microbial EPSs provide properties that are almost identical to the gums currently in use. With innovative approaches, efforts are underway *Corresponding author. Email:
[email protected] ISSN 0959-3330 print/ISSN 1479-487X online © 2010 Taylor & Francis DOI: 10.1080/09593330903552094 http://www.informaworld.com
to supersede the traditionally used plant and algal gums by their microbial counterparts. Moreover, considerable progress has been made in discovering and developing new microbial EPSs that possess novel industrial significance [3].
Microbial exopolysaccharides structure Exopolysaccharides synthesized by microbial cells vary greatly in their composition and hence in their chemical and physical properties. Some are neutral macromolecules, but the majority are polyanionic due to the presence of either uronic acids (D-glucuronic acid being the commonest, although D-galacturonic and D-mannuronic acids are also found) or ketal-linked pyruvate. Inorganic residues, such as phosphate or, rarely, sulphate, may also confer polyanionic status [4]. A few EPSs may even be polycationic, as exemplified by the adhesive polymer obtained from Staphylococcus epidermidis strains associated with biofilms [5]. The composition and structure of the polysaccharides determines their primary conformation. Furthermore, the
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ordered secondary configuration frequently takes the form of aggregated helices. In some of these polymers, the backbone, composed of sequences of 1,4-ß- or 1,3ß linkages, may confer considerable rigidity, as is seen in the cellulosic backbone of xanthan from Xanthomonas campestris. Other linkages in polysaccharides may yield more flexible structures such as the α1,2 or α1,6 linkages found in many dextrans. The transition in solution from random coil to ordered helical aggregates is often greatly influenced by the presence or absence of acyl substituents such as O-acetyl or O-succinyl esters or pyruvate ketals [4]. Polysaccharides are essentially very long, thin molecular chains with molecular masses of 0.5–2.0 × 106 Da, but they can associate in a number of different ways. In several preparations, they have been visualized as fine strands attached to the bacterial cell surface and forming a complex network surrounding the cell. In most natural and experimental environments, at lower temperatures and in the presence of salts, EPSs are found in ordered configurations. Whereas electrostatic and hydrogen bonds are the dominant forces involved, ionic interactions may also be involved, but are more subtle. Chain–chain complex formation in which one macromolecule ‘fits’ into the other may result in the formation of flocs, strong or weak gels, or networks with poor solubility in aqueous solvents [6].
Structural studies Studies on the EPS structure are crucial not only to understand their physico-chemical and biological properties, but also for the exploitation of EPS-producing microorganisms in industrial or medical applications. Several chemical and physical techniques are used to determine the primary structure of EPSs. Chemical degradation and derivatization combined with chromatographic methods, often coupled to mass spectrometry (MS), are used to determine the sugar composition, together with the absolute configuration, their positions of substitution and the substituent composition. The main steps involved in the recovery, purification and structural characterization of microbial EPSs are shown in Figure 1. Nuclear magnetic resonance (NMR), in particular two-dimensional 1H and 13 C NMR, is the most powerful technique to obtain information about the nature and configuration of sugar residues, their interconnectivity, and the nature and location of substituents, which in turn is used to ultimately determine the sequence of the repeating unit. Sometimes, chemical or enzymatic fractionation of the polysaccharide is required to produce smaller fragments that are more easily analysed by NMR or MS [7–9]. Figure 1.
Flow chart of the main steps in the structural elucidation of microbial EPSs.
Though structural studies on EPSs from mesophiles have a long history, reports on the elucidation of the chemical structure of EPSs produced by extremophiles started to appear only recently with the first one being the high-molecular-weight sulphated EPS from the halophilic archaeon Haloferax mediterranei [7]. The structure of the repeating unit of this polymer was determined by a combination of glycose, methylation, and sulphate analysis, periodate oxidation, and 1D and 2D NMR spectroscopic analysis of the native and periodate-oxidized/ reduced polysaccharides. The location of the sulphate group was established from the 1H and 13C NMR data. In a recent study on the chemical characterization of the EPSs produced by a novel halophilic Halomonas sp. isolate, sugar analysis by HPAE–PAD (high performance anion exchange chromatography with pulsed amperometric detection), methylation studies and NMR analysis indicated that the repeating unit of this polysaccharide was composed of -(2,6)-D-fructofuranosyl residues. The presence of fructose was clearly shown by GC–MS analysis. After a mild acid hydrolysis, the EPS was reduced and acetylated. The reduction of the prochiral keto group led to the formation of the diastereoisomers mannitol and glucitol, which, after acetylation, were revealed in the GC–MS chromatogram. The linkage positions of the monosaccharides were determined by methylation analysis. The chromatogram showed the presence of 1,3,4,6-tetra-O-methyl(2,5-diO-acetyl)-mannitol and -glucitol, 1,3,4-tri-Omethyl(2,5,6-tri-O-acetyl)-mannitol and -glucitol and 3,4-di-O-methyl(1,2,5,6-tetra-O-acetyl)-mannitol and glucitol, which corresponded to t-Fruf, 6-Fruf and 1,6Fruf, with mole percentages of partially methylated alditol acetates as 1%, 10% and 1%, respectively. The reduction at C-2 of fructose was performed by using NaBD4. This allowed 6-Fruf and 1-Fruf to be differentiated. The 13C NMR spectrum of polysaccharide showed the presence of six well-resolved peaks. The carbon chemical shifts were attributed to β-configuration fructofuranose units, by comparison with the carbon chemical shifts of the standard methyl glycoside. The presence of a downfield shifted signal at 65.9 ppm confirmed a β-(2-6) backbone structure (levan-type) for the EPS [10]. Similar techniques were also applied for the structural characterization of β-glucan type EPSs secreted by the ascomyceteous fungus Botryosphaeria rhodina (botryosphaerans) grown on different carbon sources, and, with the results of this study, the rational control of the degree and frequency of side branching of EPSs by modifying the composition of the nutrient medium was established [11]. In another study, the specificity of enzymatic hydrolysis in revealing the fine chemical structures of EPSs in the characterization of new heteropolysaccharides (HePS) produced by Lactobacillus sp. was reported, where enzymatic
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Figure 1.
Flow chart of the main steps in the structural elucidation of microbial EPSs.
hydrolysis of the HePS with pullulanase and alphaamylase indicated the presence of 1,6-α and traces of 1,4-α linkages, respectively [12]. In addition to these classical chemical procedures, new and powerful tools
such as Raman Microspectroscopy and Atomic Force Microscopy (AFM) techniques have been applied to investigate the cultivation-time dependence of bacterial cellular surface biopolymers at the single cell level [13].
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Mechanism of biosynthesis and its regulation Different classes of EPSs can be distinguished based on the mechanism of biosynthesis and the precursors required. In the first class, comprising the extracellularly produced homopolysaccharides like dextran, levan and mutan, the polymerization reaction proceeds via extracellular glycosyltransferases, which transfer a monosaccharide from a disaccharide to a growing polysaccharide chain. The other categories comprise homo- and heteropolysaccharides with repeating units that are synthesized from intracellular sugar nucleotide precursors. The repeating unit is first assembled by the sequential transfer of sugar residues on to a lipophilic carrier by specific glycosyltransferases. Subsequently, the completed repeating unit is exported and polymerized. The length of the polymers produced is controlled by a complex polymerization mechanism. Modifications include such reactions as acetylation or pyruvylation, and the addition of phosphate or sulphate substituents. On the other hand, the structure of the repeating units of bacterial heteropolysaccharides is determined by the action of glycosyltransferase enzymes. Hence, mapping the number and type of the glycosyltransferase genes has been useful in predicting the structure of the repeating unit. In lactic acid bacteria, a wide variety of polysaccharide structures is synthesized most probably as a result of gene transfer and recombination events [14–16]. Investigations on the diversity in enzymatic capability of lactic acid bacteria may thus facilitate the design of novel polysaccharides for food and pharmaceutical applications. Numerous regulatory circuits require reversible phosphorylation events. Also, recently, kinase and phosphatase activities have been shown to modulate polysaccharide production at the polymerization level. [16]. In general, the Wzy-dependent pathway is widely used, where the genes that are necessary for high-level polymerization and surface assembly encode for an outer-membrane protein (Wza), an acid phosphatase (Wzb) and an inner-membrane tyrosine autokinase (Wzc). In fact, these genes are highly conserved within many eps and cps operons, suggesting a common biosynthetic mechanism [17]. Physiological studies have revealed the effect of such factors as temperature, pH, oxygen tension and carbon/nitrogen ratio on the availability of the sugar nucleotide precursors and thus on EPS production. However, the regulation of EPS biosynthesis is still very poorly understood. More knowledge on the genetics and biochemistry of EPS biosynthesis is necessary to be able to successfully engineer polysaccharide properties by modifying composition and chain length.
Functional properties and biological activity In nature, bacterial EPSs fulfil a variety of diverse functions including cell protection, adhesion of bacteria to solid surfaces, and participating in cell-to-cell interactions. Exopolysaccharides also contribute to the mouthfeel, texture and taste perception of fermented dairy products. Capsular polysaccharides can protect pathogenic bacteria and contribute to their pathogenicity. Attachment of nitrogen-fixing bacteria to plant roots and soil particles, which is important for colonization of the rhizosphere and roots and for infection of the plant, can be mediated by polysaccharides. Extracellular polysaccharides synthesized by extremophilic microorganisms play an important role in microbial adaptation, creating an environment that allows cell adhesion, retention of water and concentration of nutrients [18]. Mediating cell adhesion, they form a matrix of cells, commonly referred to as a biofilm. The mechanical stability of biofilms is determined by EPSs [19]. Polymers keep the biofilm bacteria together and protect them against severe environmental conditions. Hence, the formation of biofilms is an essential step in the survival of bacterial populations [20]. Basically, extremophilic biofilm formation appears to proceed in much the same way as for mesophiles, and involves the initial attachment of cells to a solid support by EPS assistance followed by EPS production and biofilm development [21]. The EPS matrix also binds and accumulates biodegradable compounds and cations from the bulk water phase, which is a particularly important mechanism in oligotrophic environments such as many of the extreme environments. Moreover, it was also reported that, when there is a lack of nutrients in the environment, the production of EPS is increased [22]. As centres of high bacterial activity, cell aggregates are believed to have a major role in the downward transport of carbon [23]. Sulphates and uronic acids contained in many of the EPSs are ionizable and interact with cations such as metals. Qin et al. [24] showed that EPS was able to bind a range of metal cations, such as Fe2+, Zn2+ and Co2+, indicating that it might function in concentrating helpful metal ions around the cell. Sand and Gehrke [25] found that there was a correlation between the presence of Fe3+ ions within the EPS layer and the extent of metabolism in acidophilic bacteria. The polysaccharides can also enhance the immobilization of heavy metals such as Pb2+ and Cu2+ and may have significant ecological implications. The important role of EPSs in the removal of heavy metals from the environment is due to their involvement in flocculation and their ability to bind metal ions from solutions. The formation of intricate EPS-heavy metal complexes via coordinating bonds between the OH
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and COOH radicals of the EPS and metal cations could allow these bacteria to survive in their native habitat [26]. Extreme thermophilic Sulfolobus, Thermococcus, and Thermotoga species produce EPSs that can act indirectly as extracellular storage polymers in their natural extreme environments where no other organic source has been identified [27,28]. In addition, EPSs also protect microbial cells against environmental stress and dehydration such that EPS production was registered as a survival cryoprotectant strategy [29]. Biofilms also defend the cells against cytotoxic compounds. Anti-cytotoxic activity against avarol was proved for the polymer from a halophilic bacterium Halomonas sp. [10] and a thermophilic bacterium Geobacillus tepidamans [30]. A commonly accepted hypothesis for the thermophilic origin of life considers thermophiles as direct heirs of ancient ancestors. Horizontal gene transfer may be facilitated as a result of the close contact of aggregated cells and DNA accumulation in the EPS matrix, which has already been demonstrated in pure culture biofilms [31,32]. That way, by assisting in gene exchange, EPSs seem to contribute also to the development of evolutionary diversity. EPS-producing extremophiles Extracellular polysaccharide producers are found in different classes of two domains, Bacteria and Archaea,
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inhabiting a variety of extreme environments. Considerably less is known about the eukaryotic life in extreme environments and their metabolic adaptation mechanisms. Therefore, only prokaryotic and archaeal EPS producers will be covered in this review. Prokaryotic producers of EPS EPS from thermophiles Thermophilic (‘heat loving’) microorganisms can be found in every phylum of bacteria and archaea so far described. Among the various thermophilic ecosystems that could provide EPS producers, marine hot springs, both deep and shallow, and terrestrial hot springs have served as sources for isolation of microbial producers of EPS. The thermophilic bacilli Bacillus thermoantarcticus, Geobacillus thermodenitrificans and B. licheniformis were isolated from hot marine shallow vents as producers of large amounts of EPS [33,34]. Figure 2 shows the scanning electron microscopy image of Bacillus thermoantarcticus producing an EPS slime [33]. Microbial communities associated with deep-sea hydrothermal vents were found to produce EPSs [35– 38]. These ecosystems are characterized by extremely high pressure and temperature and high levels of toxic elements such as sulphur and heavy metals, and the EPSs could serve as enhancers for bacterial survival. The extremely thermophilic fermentative anaerobe Thermotoga maritima [39] and cocultures of T. maritima and the Figure 2.
Scanning electron microscopy of Bacillus thermoantarcticus , producing an EPS slime, grown on glucose medium, in a fermenter at 65 °C. The sample morphology was observed using a SEM Philips XL20 series microscope.
Figure 2. Scanning electron microscopy of Bacillus thermoantarcticus, producing an EPS slime, grown on glucose medium, in a fermenter at 65 °C. The sample morphology was observed using a SEM Philips XL20 series microscope.
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H2-consuming methanogen Methanococcus jannaschii [40] were found to develop significant biofilms. The EPS, synthesized by G. tepidamans, isolated from a terrestrial hot spring expressed unusual high thermostability: it started to decompose at about 280 °C [30]. EPS from psychrophiles Cold environments – fresh and marine waters, polar and high alpine soils, waters and glaciers – dominate the biosphere. Most deep sea environments are influenced by high pressure, low temperature and low nutrient concentrations [24]. As a rule, isolates from deep ocean environments are also barophiles. Studies of bacteria growing in cold aquatic systems, such as marine sediments, aggregates and detrital particles, show that nearly all the cells are surrounded by EPSs [18]. Many studies have focused on extreme marine environments as novel sources of EPS, and their research revealed a number of different species of marine bacteria producing EPS. Exopolymer production was reported for Pseudoalteromonas sp. of the Gamma Proteobacteria [24,41] and Olleya marilimosa from the family Flavobacteriaceae [42]. In marine environments, bacterial EPSs provide protection and ecosystem stability. The enhanced production of a high-molecular-weight polyanionic EPS at suboptimal incubation temperatures lends support to theories that EPS may serve as a cryoprotectant both for organisms and their enzymes. The cryoprotectant role of EPS was established in the psychrophilic Gamma Proteobacterium Colwellia psychrerythraea grown at increased pressure or temperatures from −8 to −14 °C [29]. The EPS from the psychrotolerant bacterium Pseudoalteromonas could enhance the stability of the cold-adapted protease secreted by the same strain through preventing its autolysis, avoiding enzyme diffusion, and helping the strain in enriching the proteinaceous particles and trace metals in the deep-sea environment [24]. EPS from halophilic bacteria Although the oceans are the largest saline body of water, hypersaline environments are generally defined as those containing salt concentrations in excess of seawater. Many hypersaline bodies are derived from the evaporation of seawater with salt concentration of about 3–3.5 mol L−1 NaCl. Many halophilic microorganisms possess an EPS capsule around the cell, which helps in protecting membrane integrity. Various Gamma-Proteobacteria were established as being EPS producers. The commonest halophilic producers belong to the genus Halomonas, most importantly H. maura [43], H. eurihalina [44], H. ventosae, H. anticariensis [45], and Halomonas sp. [10].
Expolysaccharides synthesized by Halomonas strains had an unusually high sulphate content and a significant amount of uronic acid determining their good gellifying properties [44]. Other good halophilic EPS producers belonging to the Gamma-Proteobacteria were the genera Idiomarina [46] and Alteromonas, namely Alteromonas hispanica [47]. Also the Alpha-Proteobacteria Salipiger mucosus and Palleronia marisminoris [48] were reported as EPS producers. It has been known for a long time that Cyanobacteria, the most widely distributed photosynthetic prokaryotes in nature, produce large amounts of EPSs. Moreover, EPSs from Cyanospira capsulate and Aphanothece halophytica show rheological properties similar to those of xanthan [49,50]. EPS from acidophilic bacteria The primary areas with a pH lower than 3.0 are those where relatively large amounts of sulphur or pyrite are exposed to oxygen. These areas are quite toxic due to high concentrations of heavy metal sulphides, but they also are rich in valuable or precious metals. Oxidizing processes increased 106 times through the activity of acidophiles – active participants in bioleaching processes [51,52]. Microbial EPS production is observed during bioleaching [53–55]. The microbial consortia involved in bioleaching are composed mainly of acidophilic, autotrophic iron- and/or sulphur-oxidizing bacteria. Acidophilic bacteria such as Acidithiobacillus ferrooxidans, A. thiooxidans and Leptospirillum ferrooxidans [57] and sulphobacilli [58] are major agents in the bioleaching processes. Sand and Gehrke [25] demonstrated that EPSs play a role in the attachment of bacteria to the sulphide surface and in the concentration of iron. Michel et al. [56] noted a relationship between EPS presence and bioleaching efficiency in continuously stirred bioreactors although there was not a direct relationship between the amount of EPSs that could be measured and bioleaching activity. This underlines the fact that the bioleaching role played by EPSs is not just dedicated to bacterial attachment to pyrite: it is possible that part of the EPS might play a protective role against the stress conditions that characterize bioleaching media (low pH, presence of metals, stirring conditions, complex medium, etc.). EPS from alkaliphilic bacteria An increase in pH in alkaline environments is due to microbial ammonification and sulphate reduction and by water derived from leached silicate minerals. Soda lakes represent the most alkaline naturally occurring environments on earth, with pH values generally greater than
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10. Exopolysaccharide from an alkaliphilic Bacillus was found to have a quite unusual structure containing D-galactopyranuronic acid, diacetamido-trideoxy-Dglucopyranose, acetamido-deoxy D-mannopyranuronic acid and one uncommon unit of D-galactopyranuronic acid with the carboxyl group amide-linked to glycine [59]. Many haloalkaliphilic species of the genus Halomonas produced different EPSs [60]. Archaeal producers of EPS Exopolysaccharides were identified in different groups of Archaea, predominantly in halophiles and thermophiles. Massive amounts of EPSs are excreted by members of the halophilic genera Haloferax, Haloarcula, Halococcus, Natronococcus and Halobacterium [7–9,61]. Various thermoacidophilic archaea, including members of the genera Thermococcus and Sulfolobus, were observed to accumulate storage polysaccharides, such as glycogen and mannan, or sulphated heteropolysaccharide [62,27]. Archaeoglobus fulgidus and Thermococcus litoralis accumulated significant amounts of EPSs as biofilms [62,63]. Production of EPS from extremophiles The enthusiastic search for novel extremophiles has been largely stimulated by the industry’s interest in the fact that the survival mechanisms of these microorganisms could be transformed into valuable applications ranging from wastewater treatment to the diagnosis of infectious and genetic diseases. One such survival mechanism is the production of EPSs, and these biopolymers with diverse functionalities have found a wide range of applications in many industrial sectors. However, no matter how high the market potential of new EPS, it cannot find its place on the polymer market unless it can be produced economically. Hence, to achieve high production yields as well as to compete with synthetic petrochemical products in performance and cost, the design of an optimal cost-effective production process is a prerequisite. Fermentation is an extremely versatile process technology for producing value-added products such as microbial biopolymers, and optimization of important fermentation parameters is considered to be very important in process development because of their high impact upon the bioprocess viability and economics. Particularly, microbial polysaccharide production is greatly influenced by fermentation conditions. The structure, composition and viscosity of EPSs depend on several factors, such as the composition of the culture medium, carbon and nitrogen sources and precursor molecules, mineral salts, trace elements, type of strain, and fermentation conditions such as pH, temperature,
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oxygen concentration and agitation. Hence, by manipulating the producer microorganism, feedstock and process conditions, fermentation allows production of a wide and reproducible range of different biomaterials with very good control over their characteristics [64]. Microorganisms used as industrial or technical producers of extracellular polysaccharides are mainly pathogenic bacteria. Species of Xanthomonas, Leuconostoc, Pseudomonas and Alcaligenes which produce xanthan, dextran, gellan and curdlan, respectively, are the most well known and most industrially used. Actually, a great deal of interest has been accorded to the EPSs produced by lactic acid bacteria (LAB), which are already accepted as GRAS (Generally Recognised As Safe) and the most suitable for the food industry. They are widely used in the dairy industry since in situ production of their EPSs improves the texture of fermented dairy products and also confers health benefits as a result of their immunostimulatory, antitumoral or cholesterol-lowering activity [65]. While dextran (synthesized by certain LAB such as Leuconostoc mesenteroides and the mesophilic dental pathogen Streptococcus mutans) was the first microbial polysaccharide to be commercialized and to receive approval for food use, several such polymers now have a variety of commercial uses [66]. Xanthan gum (the EPS from the plant pathogen Xanthomonas campestris pv. campestris bacterium) is already well established in modern biotechnology and has a sizable market because of its exceptional qualities as a rheology control agent in aqueous systems and as a stabilizer for emulsions and suspensions [67]. Gellan, produced by the non-pathogenic bacterium Pseudomonas elodea, and curdlan, produced by the alkaline tolerant mesophilic pathogen Alcaligenes faecalis, are xanthan-like commercial polysaccharides that recently entered the market. Gellan is gaining increasing attention because of its novel property of forming thermo-reversible gels, and it has great commercial potential in the food and pharmaceutical industries and, predominantly, in environmental bioremediation [68]. On the other hand, aqueous suspensions of curdlan can be thermally induced to produce high-set gels, which will not return to the liquid state upon reheating. This unique feature, as well as its immunostimulatory effect, attracted the attention of the food industry, and after 1989 curdlan entered the food industry as a formulation aid, processing aid, stabilizer, and thickener or texture modifier [69]. Because of the pathogenicity of the commercial EPS-producing strains, in recent years significant progress has been made in discovering and developing novel and functional EPSs from extremophilic producer strains. For example, when the EPS production abilities of LAB strains were compared, thermophilic strains
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(Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Streptococcus macedonicus and Streptococcus thermophilus) were found to produce more EPS than the mesophilic strains (Enterococcus faecalis, Enterococcus faecium, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus) [70]. However, natural production levels of these EPSs are about a thousand times less than those of other food-grade polysaccharides such as xanthan and curdlan, and improving their production levels, therefore, is a constant challenge [71]. On the other hand, low EPS production yields reported for the thermophilic strain Geobacillus tepidamans V264 were compensated by the unusually short production periods, which in turn significantly increased the commercial potential of this fast EPS-producing strain [30]. Besides thermophiles, halophiles are also known to have the ability to produce EPSs with novel physical and chemical characteristics. Among these, mauran, the EPS produced by Halomonas maura, is notable for its high viscosifying capacity, similar to that of xanthan, and for the pseudoplastic and thixotropic behaviour of its solutions. In addition, the stability of its functional properties under a wide range of pH, saline and freezing–thawing conditions makes this polymer a good candidate for use in foodstuffs, pharmaceutical products and other fields of biotechnology [43]. Also recently, large quantities of levan production by a Halomonas sp. AAD6 strain was reported with yields exceeding earlier reports even in minimal growth media [10]. Currently, despite the vast number and biodiversity of the extremophilic producers of EPS, these natural, non-toxic and biodegradable polymers represent only a small fraction of the current polymer market not only because of their high production costs but also because of their poor physico-chemical properties when compared with those of industrial EPSs from plant (guar gum, gum Arabic, cellulose, pectin and starch) and seaweed (alginate and carrageenan) origin [72,73]. Thus, to overcome these limitations, development of processes for EPS production, by applying various upstream and downstream engineering strategies that include metabolic and cellular engineering of host cells, efficient fermentation and recovery processes, and postproduction modification of the biopolymers obtained have been active areas of research. Fermentations for EPS production are batch, fedbatch or continuous processes depending on the microbial system used. Production may or may not be associated with growth, and nutrient availability is an important factor in EPS synthesis for which a significant amount of carbon and electrons are channelled by the metabolism. In batch cultures, polysaccharide synthesis takes place when the medium is depleted with one or more nutrients, and it is often maximal in
media with a high carbon/nitrogen ratio [6]. However, generalizations regarding the fermentation conditions should be avoided because of the diversity of the nutritional and environmental requirements of the EPSproducing strains. For some EPS-producing bacteria, such as Xanthomonas, Pseudomonas and Rhizobium meliloti, nitrogen-limiting conditions resulted in increased EPS production. On the other hand, Gorret et al. [74] demonstrated that addition of yeast extract to the medium improved both growth and EPS production by Propionibacterium acidi-propoinici. The type of carbon source influences not only the yield but also the size of the EPS produced [75], as reported for high molecular weight alginate production on fructose and glucose [76] and also for EPSs of varying size produced by Lactobacillus delbruckii. On the other hand, sugar composition of the EPSs produced by L. delbruckii and Streptococcus thermophilus was not affected by the carbon source used [77,78]. Whereas non-growth-associated EPS production has been reported for two L. delbrueckii subsp. bulgaricus strains and for Alteromonas macleodii subsp. fijiensis, that of S. thermophilus 1275 was shown to be growth associated [79]. The relationship of the EPS production rate to the substrate consumption rate is also subject to significant controversy. Fermentation period also affects the molecular mass of the EPSs, as reported for L. acidophilus [80]. In aerobic microorganisms, molecular oxygen plays a key role in their life cycle by regulating pathways of primary or secondary metabolism and by enabling the oxidative reactions for nutrient utilization and energy generation. Since the microbial polysaccharide production process is aerobic, the supply of the liquid media with oxygen during the fermentation is of great importance. As a consequence of better availability of oxygen and nutrients, enhanced EPS production at high agitation and aeration rates was reported for the marine bacterium Hahella chejuensis [81]. During the course of fermentation, considerable changes in the rheological properties occur because of EPS production resulting in a highly viscous and nonNewtonian broth, which in turn may not only cause serious problems of mixing, heat transfer and oxygen supply but also give rise to instabilities in the quality of the end product [75]. Whereas bacterial biomass has a negligible role in broth rheology, fungal biomass contributes significantly to it. Technical difficulties originating from highly viscous broth are common in commercial xanthan and pullulan production processes. On the other hand, these difficulties are mitigated by the use of thermophilic producer strains where the viscosity of the culture medium is considerably lower at the high fermentation temperatures [30].
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Temperature and pH are important parameters affecting both culture growth and EPS synthesis. In most cases, optimum values of these parameters for maximum EPS yield differ from those for optimum growth. Therefore, developed fermentation strategies usually start with a growth phase followed by a production phase where the conditions are switched to promote EPS production [75]. In the case of xanthan production, Moraine and Rogovin [82] observed that culture pH influenced polysaccharide production. The EPS production by P. acidipropionici [74] was reported to be possible only between pH 5.3 and 6.5 and increased at lower temperatures, whereas Garcia-Garibay and Marshall [14] reported an increase in production of EPS with increase in temperature in the case of L. delbrueckii subsp. bulgaricus. The fermentation medium can represent almost 30% of the cost for a microbial fermentation. Complex media commonly employed for growth and production are not economically attractive because of their high amount of expensive nutrients such as yeast extract, peptone and salts. Hence to achieve high production yields as well as to compete with synthetic petrochemical products in performance and cost, it is a prerequisite to design an optimal cost-effective production medium. The greatest expense in producing biopolymers has traditionally been the substrate which is used as the fermentation feedstock. Until the late 1990s, researchers working on the development of microbial systems to produce biopolymers generally focused on using a single type of microorganism, with a minimum component synthetic feedstock to achieve defined culture conditions. However, to maximize the cost-effectiveness of the process, recent work shifted to using multi-component feedstock systems, and the synthetic media were replaced by cheaper alternatives such as cheese whey and molasses [64]. Stredansky and Conti [83] reported the use of spent malt grains, apple pomace, grape pomace and citrus peels for xanthan production by solid state fermentation, and their yields with apple pomace were comparable to those obtained from conventional submerged cultivation. Use of olive mill wastewater, which is the liquid waste by-product of the olive oil extraction process, in xanthan production [84] and an EPS production by Paenibacillus jamilae [85] have been investigated. Molasses, owing to its high sucrose and other nutrient contents, low cost and ready availability and ease of storage, is also considered to be a promising fermentation substrate for EPS production. Molasses was successfully used for fermentative production of commercial polysaccharides such as curdlan [53], xanthan [86], dextran [87], scleroglucan [88] and gellan [89]. Another potential area of economic benefit is new
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uses of milk sugar, which presents a challenge to dairy research and the industry. Indeed, the market for lactose in the pharmaceutical industry is over-saturated, and all the routes for chemically modifying this sugar in products such as lactulose, lactitol and detergents involve only small markets. The dairy industry worldwide is investigating markets for by-products containing a high lactose content, such as whey [90]. Cheese whey could be a promising fermentation substrate for EPS production by L. delbrueckii subsp. bulgaricus [91] and Streptococcus thermophilus [92]. Research and development on EPSs over the last few decades was dedicated towards understanding the basic science underlying the biosynthesis of biopolymers and important precursors which can be subjected to polymerization reactions. Insight into these molecular processes enabled the metabolic engineering of microorganisms, which in turn may lead to the production of tailor-made biopolymers as well as to the development of optimized microbial production strains, also called cell factories, for industrial biotechnology. Important applications of EPSs from extremophiles A huge variety of biopolymers, such as polysaccharides, polyesters and polyamides, are produced by microorganisms. These products range from viscous solutions to plastics. Genetic manipulation of microorganisms has permitted the biotechnological production of biopolymers with tailored material properties suitable for high-value medical applications such as tissue engineering and drug delivery. Industrial microbiology can be used for the biosynthesis of xanthan, alginate, cellulose, cyanophycin, poly(gamma-glutamic acid), levan, hyaluronic acid, organic acids, oligosaccharides, polysaccharides, and polyhydroxyalkanoates, and some of these are already produced on an industrial scale with annually increasing consumption in the food industry, medicine, agriculture and wastewater treatment. An example of industrial use of EPSs is the application of dextran in the bakery industry [3,16,87,93]. The producers of microbial EPSs with industrial value are mainly mesophilic microorganisms. Information concerning polysaccharides produced by extremophilic prokaryotes is scarce, and exploitation of their unusual properties is a question for future industrial processes. It is now widely accepted that extremophilic microorganisms will provide a valuable resource not only for exploitation in novel biotechnological processes but also as models for investigating how biomolecules are stabilized when subjected to extreme conditions. Microbial isolates from extreme environments, as compared with anywhere else in the biosphere, offer a great diversity in the chemical and physical properties of their EPSs [94]. Table 1 lists the chemical compositions
Haloferax mediterranei
Sulfolobus solfataricus
Thermococcus litoralis
Archaea Haloarcula strain T5 Haloferax gibbonsii
EPS-MT4
EPS T5
Mauran
EPS EPS EPS
Halomonas anticariensis strain FP36 Halomonas ventosae strain Al16 Halomonas alkaliantarctica
Halomonas maura Halomonas eurihalina
Levan
EPS 4001
EPS 1 EPS 2 EPS 3
EPS 1
EPS
Halophiles Halomonas sp. AAD6
Geobacillus sp. strain 4001 Psychrophiles Pseudoalteromonas CAM025 and CAM036 Hahella chejuensis gen. nov., sp. nov.
Geobacillus sp.
Geobacillus tepidamans V264 Thermotoga maritima Bacillus thermoantarticus
Name of EPS
Man is the only monosaccharidic constituent Glc/Man/Glucosamine/Gal (1:0.8:0.1:0.05) Man/Glc/Gal/Amino sugars/Uronic acids, Man as major component
Gluc. Ac./Man/Gal (1:0.6:0.3) Man/Glc/Gal/Rha (0.6:0.3:1:0.3)
Fru is the main monosaccharidic residue Glc/Man/Gal.Ac. Glc/Man/Gal Glc/Fru/Glucosamine/Galactosamine (1:0.7:0.3:0.2) Man/Gal/Glc (1:0.6:0.2) High sulphate content and significant amounts of uronic acid
Neutral sugars and uronic acids with sulphates Gal/Glc/Xyl/Rib
Man/Glc/Gal (0.5:1:0.3) Man/Glc/Gal (1:0.3:trace) Gal/Man/Glucosamine/Arab (1:0.8:0.4:0.2) Man/Glc/Gal/Mannosamine
Glc/Gal/Fuc/Fru (1:0.07:0.04:0.02) Glc/Rib/Man (1:0.05:0.02)
Chemical composition of EPS
EPSs produced by thermophiles, psychrophiles, halophiles and archaea.
Thermophiles Bacillus licheniformis
Producers
Table 1.
A β-D-Glc-(1,6)-α-D-Man--(1,4)-β-D-Glc repeating unit with acetyles and sulphates on both Glc residue
A pentasaccharide repeating unit A heptasaccharide repeating unit containing two branches
Unknown
Unknown Unknown A hexasaccharide repeating unit
[7,61]
[27]
[62]
[9] [8]
[43] [44]
[45] [45] [96]
[10]
[81]
Unknown A β-(2,6)-D-fructofuranosyl repeating unit
[93]
[34]
[26]
[30] [39] [33]
[95]
References
Unknown
A mannan with a complex primary structure
A pentasaccharide repeating unit
A repeating unit constituted by four different α-D-Mans and three different β-D-Glcs
A tetrasaccharide repeating unit and a mannopyranosidic configuration A galacto-glucan with α-glicosidic linkage
Characteristics of chemical structure
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1154 B. Nicolaus et al.
Environmental Technology
and bibliographic reference for EPSs produced by thermophiles, psychrophiles, halophiles and archaea. Several EPSs produced by microbes from these extreme environments show biotechnological promise. By examining the chemical characteristics of these carbohydrate polymers, it is possible to begin to understand the ecological role of these natural products as well as to gain insight into their commercial potential [93,95,96]. In fact their specific rheological properties either in the presence or absence of monovalent and divalent ions, biological activities, metal binding capabilities and novel chemical compositions mean that these EPSs are expected to find many applications in the near future [94].
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Acknowledgements This study was partly funded by Bilateral Projects: BAN Academy-CNR and TUBITAK-CNR. The authors thank Dr. GianLuca Anzelmo for his helpful assistance.
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