EXTREMOPHILES AS SOURCES OF ...

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In: Handbook of Carbohydrate Polymers… Editor: Ryouichi Ito and Youta Matsuo, pp. 605-619

ISBN: 978-1-60876-367-2 © 2010 Nova Science Publishers, Inc.

Chapter 19

EXTREMOPHILES AS SOURCES OF EXOPOLYSACCHARIDES Hande Kazak 1, Ebru Toksoy Öner 1,* and Robert F. H. Dekker 2 1

Department of Bioengineering, Faculty of Engineering, Marmara University, Istanbul, TURKEY. 2 Biorefining Research Initiative, Lakehead University, Thunder bay, Ontario, CANADA, P7B 5E1.

ABSTRACT Extreme environments, generally characterized by atypical temperatures, pH, pressure, salinity, toxicity and radiation levels, are inhabited by various microorganisms specifically adapted to these particular conditions. These microorganisms, called extremophiles, are of significant biotechnological importance as their enzymes (extremozymes) and biopolymers possess unique properties that offer insights into their biology and evolution. The enthusiastic search for novel extremophiles has largely been stimulated by the uniqueness of their survival mechanisms. This uniqueness can be transformed into valuable applications ranging from wastewater treatment to the diagnosis of infectious and genetic diseases. One adaptation strategy of particular importance to extremophiles is the production of extracellular polymeric substances (EPSs) that envelop the cell as a barrier protecting them against environmental extremes such as desiccation, temperature, pressure, salinity, acidity, heavy metals, and radiation. Due to their many interesting physicochemical and rheological properties, these biopolymers possess novel functionality that is generally superior to petrochemicalderived polymers in aspects that embrace biodegradability, and environmental and human compatibility. Consequently, biopolymers of extremophiles are widely used in foods, cosmetics, pharmaceutical products, textiles, detergents, adhesives, oil-recovery from wells, brewing and waste treatment processes. In this chapter, we present a brief overview of life under extreme environmental conditions. This is followed by a discussion of extremophilic microorganisms and their adaptation mechanisms, and *

Corresponding author: Marmara University, Faculty of Engineering, Department of Bioengineering, Goztepe 34722 Istanbul, Turkey, E-mail: [email protected], Tel: +90 216 348 0292 ext. 726, Fax: +90 216 348 0293.

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Hande Kazak, Ebru Toksoy Öner and Robert F. H. Dekker specifically focuses on the production of EPSs and their ecological and physiological functions. The application areas of industrially important EPSs from various extremophilic producer strains are also mentioned.

INTRODUCTION The last 50 years have been marked by an astounding growth in the use of plastics and polymers to provide packaging for safer foods, lighter weight and longer lasting appliances, safer cars, and an increased general level of convenience in all manner of applications. These advantages have created a global industry providing 160 billion kilograms per year of materials valued at over $US 250 billion. Yet the vast majority of these materials are based upon the extraction and processing of fossil carbon, typically oil and gas, leading ultimately to increases in greenhouse gases in the atmosphere and the accumulation of persistent plastic materials in the environment. During recent years, a variety of biopolymers have become available for use in many applications that are not only compatible with human lifestyle but are also environmental friendly. In nature, biopolymers often play important roles in maintaining cell viability by conserving genetic information, by storing carbon-based macromolecules, by producing either energy or reducing power, and by defending an organism against attack from hazardous environmental factors [30]. At present, polymers derived from microbial origin represent only a small fraction of the current global polymer market, but the number and their applications are gradually increasing. Xanthan, dextran and pullulan are examples of microbial polysaccharides with a considerable market due to their exceptional properties. Biopolymers with properties superior to the commercial ones cannot preserve their functions under industrial process conditions like extremes of temperature, salinity or pH. Hence, most research is focused on the identification of EPS-producing extremophiles with the idea that as these microorganisms survive environmental extremes of desiccation, temperature, pressure, salinity, acidity, heavy metals, and radiation, it is expected that their biopolymers will also have some unique properties to adapt to such extreme conditions. In this chapter, after a brief description of life under extreme conditions and extremophilic microorganisms, the adaptation mechanisms of these microorganisms are mentioned with a special focus on the production of EPSs by extremophiles and their ecological and physiological functions followed by a discussion on the application areas of industrially important EPSs from various extremophilic producer strains.

EXTREMOPHILES Human life is sustained under moderate environments which are generally described by conditions with pH near neutral, temperatures between 4 and 40°C, pressure around 1 atm, water, nutrients and salts at adequate levels, and low hydrostatic pressure and ionizing radiations. Thus ecological systems such as hot springs, salt and soda lakes, deserts and ocean beds and deep thermal vents that are not compatible with the growth and survival of human beings are considered as being “extreme” [10, 63]. Extreme conditions can refer to physical

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extremes such as temperature, pressure or radiation, but also to geochemical extremes such as salinity, desiccation, oxygen tension and pH. A variety of microorganisms, known as extremophiles, not only tolerate such extreme conditions, but usually require such environmental extremes for their survival and growth. The term “extremophile” was first coined by MacElroy in 1974, and from the mid 1970’s onwards, a growing number of extreme environments, formerly inaccessible due to technical limitations, are now being explored, which has led to the isolation and identification of a wealth of new organisms [23, 54]. Extremophiles are classified and named according to the conditions under which they exist (Table 1). The discovery of extreme environments and studies of extremophiles not only offered important insights into biology and evolution but also provided clues to extraterrestrial life since these microorganisms are widely accepted to represent the earliest forms of life. Moreover, taxonomic studies on extremophilic microorganisms enabled Archaea to find its place as the third domain in the evolutionary tree of life [54, 61]. Higher organisms were believed to be unable to withstand extreme environments most probably because of their cellular complexity and compartmentalization. Therefore research on extremophiles has focused mainly on organisms belonging to the Archaea and Bacteria kingdoms. However, a significant number of eukaryotes, both unicellular and multicellular, have evolved to live and thrive in extreme environments (e.g. Alvinella pompejana, Tetrahymena thermophila and Dunaliella salina). Hence, extremophiles span all three domains of life: Archaea, Bacteria and Eukarya [61]. The upper temperature limit of bacteria and eukaryotic life is around 90 and 60 °C, respectively. Whereas all hyperthermophiles are members of the Archaea and Bacteria, while eukaryotes are common among the psychrophiles, acidophiles, alkalophiles, piezophiles, xerophiles and halophiles [61]. There are thermophiles among the phototrophic bacteria (cyanobacteria, purple and green bacteria), bacteria (Bacillus, Clostridium, Thiobacillus, Desulfotomaculum, Thermus, lactic acid bacteria, actinomycetes, spirochetes and numerous other genera), and the Archaea (Pyrococcus, Thermococcus, Thermoplasma, Sulfolobus and the methanogens) [1, 61, 37, 54]. Among halophilic microorganisms are a variety of heterotrophic and methanogenic archaea; photosynthetic, lithotrophic, and heterotrophic bacteria; and photosynthetic and heterotrophic eukaryotes. There are a variety of organisms that can tolerate extreme desiccation, including bacteria, yeast, fungi, plants, insects, tardigrades, mycophagous nematodes and the shrimp Artemia salina. Organisms that can withstand high levels of radiation are Deinococcus radiodurans, two Rubrobacter species, and the green algae Dunaliella bardawil [61, 37, 54]. As a result of adaptation to their environment, many extremophilic microorganisms have evolved unique properties of considerable biotechnological importance and, therefore, commercial significance. It is therefore widely accepted that extremophiles provide a valuable resource for exploitation in novel biotechnological processes [48]. The applications of extremophiles, their enzymes (extremozymes) and biopolymers are as varied as their environments. Oil degrading extremophiles are used as oil recovery surfactants, methanogens in methane production, psychrophiles in plant frost protection and sulfur oxidizing ones are used in bioleaching, coal and waste gas desulfurization. The marine microalgae Dunaliella salina in whole dried form is taken as a natural dietary supplement for carotenoids and daily nutrients. Whereas S-layer protein and lipids of hyperthermophiles are employed as molecular sieves, as liposomes in drug delivery and cosmetic packaging, bacteriorhodopsin of

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halophiles are utilized in bioelectronic optical switches and photocurrent generators. Additionally, biodegradable plastics from extremophilic microorganisms have found many applications in waste treatment and in the biomedical sector. Ectoine, an osmoprotectant accumulated by many halophilic bacteria, is used both as a stabilizer of enzyme preparations and as an additive to skin treatment cosmetics [11]. Industrially important extremozymes include DNA polymerases (such as Taq DNA polymerase) and alkaline phosphatases that are used in diagnostics procedures, while amylases, pullulanases, proteases and lipases are utilized in cheese manufacturing, dairy processing and detergents. Amylolytic enzymes are used in starch processing, proteases find applications in contact-lens cleaning solutions, in gelatin removal from X-ray films, and in de-hairing animal hides for leather production, and oxidases are used in the preparation of environmental biosensors [37, 16].

ADAPTATION TO THE EXTREME Extremophiles cope with environmental extremes in two general ways. Either the stress is excluded from their cytoplasm by the unusual properties of the cell membrane or all intracellular components have to be functional at the extreme conditions. For the first strategy, only the cytoplasmic membrane, periplasmic and excreted proteins of the whole cell require a special adaptation to the environmental stress. T able 1. C lassification of extr emophiles (r estr uctur ed fr om H or ikoshi and G r ant, 1998). Environmental Parameter Temperature

Radiation Pressure Gravity Vacuum Desiccation Salinity pH Oxygen tension

Osmotic pressure Chemicals

Type Hyperthermophile Thermophile Mesophile Psychrophile Radiophiles Barophile Piezophile Hypergravity Hypogravity Xerophiles Halophile Alkalophile Acidophile Anaerobe Microaerophile Aerobe Osmophile Metallophile Toxitolerant

Definition Growth >80 °C Growth 60–80°C 15–60 °C 1g 9 Low pH-loving Cannot tolerate O2 Tolerates O2 deprivation Requires O2 survive in high sugar environments Able to tolerate high concentrations of metal Tolerates toxic and xenobiotic chemicals like benzene

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Halophilic or halotolerant organisms can grow over a wide range of salt concentrations (2 to 5 M NaCl) by two strategies. One is based on the intracellular accumulation of salts at concentrations close to those in the outside medium, while the other is based upon exclusion of salts from the cytoplasm to a large extent and production or accumulation of organic solutes such as ectoine and glycine-betaine, to balance the osmotic pressure of the medium [46, 70]. Acidophiles and alkalophiles retain the intracellular pH at a value close to neutrality by the very low permeability of their membranes to protons. Whereas high levels of polyunsaturated fatty acids are found in the membranes of some psychrophiles, some UV radiation-resistant radiophiles either have UV-absorbing pigments (scytonemin) in the sheath that surrounds the cell, or accumulate UV-absorbing pigments (mycosporine-like amino acids) within their cytoplasm, to protect their DNA from radiation damage [13]. Hyperthermophiles that grow at temperatures above 100 °C are all Archaea since their lipids contain ether bonds that are far more stable than the ester bond-based bacterial and eukaryal lipids. Also the monolayer membrane in which the glycerol moieties at both sides of the membrane are bridged by covalent bonds confer Archaea with additional stability towards high temperatures [54]. Besides all the above mentioned adaptation strategies, microbially-produced extracellular polysaccharides are also known to be involved in protecting cells from numerous external stresses [72, 45]. Exopolysaccharide (EPS) is a term first used by Sutherland (1972) to describe high molecular weight, structurally diverse carbohydrate polymers produced by many marine bacteria [67]. Since then, EPS has also been used to indicate more broadly defined extracellular polymeric substances [45]. Different classes of EPS can be distinguished based upon the mechanism of biosynthesis and the precursors required. The first class comprises the extracellularly produced homo-polysaccharides like dextran, levan and mutan. The polymerization reaction in these cases proceeds via extracellular glycosyltransferases, which transfer a monosaccharide residue from a disaccharide to a growing polysaccharide chain. The other categories comprises homo- and hetero- polysaccharides with (ir)regular repeating units that are synthesized from intracellular sugar nucleotide precursors. Some of these sugar nucleotides serve as precursors for EPS biosynthesis. However, they are also involved in the biosynthesis of several cell wall components and can therefore be considered essential for growth [9]. EPSs can absorb water and form a highly hydrated matrix. The presence of such a gelled polysaccharide layer around the cell may affect the diffusion of compounds both into and out of the cell, and hence act as a layer of protection to cells against toxic compounds [8, 24], very high acidity [2], UV radiation [71] or digestion by other organisms [12]. The specific role of the EPS depends on the natural environment of the microorganism. Changes in pH and salinity have little effect on the viscosity and stability of the EPS layer. Besides their buffering capacity, EPSs may also reduce the rate of water loss during desiccation and assist with water uptake during rehydration. Polymers made up of fructose residues (fructans) are known to be involved in the protection of plants during drought, salt or cold stress by preventing membrane damage [57]. High concentrations of EPS with high polyhydroxyl content would decrease the freezing point of water in the low temperature, high salinity brine channels and hence act as a cryoprotectant. For example EPS from a fungal strain, Phoma herbarum, isolated from Antarctic soil provides a cryoprotective role in the harsh Antarctic environment where the availability of liquid water and temperatures are extremely low [65].

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Microbial EPSs serve countless ecological functions like stabilizing marine sediments and preventing erosion, improvement of water holding capacity of soil, detoxification of heavy metals and removal of solid matter from water reservoirs [7]. They form biofilms and aggregates facilitating intracellular interaction and biochemical exchange between organisms [47, 73]. EPSs also contribute to various physiological activities in human beings as antitumor, anti-viral, and anti-inflammatory agents, and can act as inducers for interferon, platelet aggregation inhibition, and colony stimulating factor synthesis [29].

EXTREMOPHILES AS EPS PRODUCERS Because of their many interesting physicochemical and rheological properties with novel functionality, microbial EPSs in general act as new biomaterials and find wide ranges of applications in many industrial sectors like textiles, detergents, adhesives, microbial enhanced oil recovery (MEOR), wastewater treatment, dredging, brewing, downstream processing, cosmetology, pharmacology, and food additives [29]. On the other hand, EPSs can act as an emulsifier and bioflocculant, and they have the function of sorption of inorganic ions which constitute metabolic elements for bacteria. The microorganisms used as industrial or technical producers of extracellular polysaccharides are chiefly the bacteria. Species of Xanthomonas, Leuconostoc, Pseudomonas, Alcaligenes which produce xanthan, dextran, gellan, curdlan are the best known and most industrially used. Actually, much attention is lately accorded to the EPSs produced by lactic acid bacteria which are already accepted as GRAS (Generally Recognised As Safe) and are most adequate for the food industry. Fungal polysaccharides are still somewhat limited, with pullulan from Aureobasidium pullulans and scleroglucan produced by Sclerotium glucanicum being the most known and already obtained at technical scales. While dextran (synthesized by certain lactic-acid bacteria such as Leuconostoc mesenteroides and 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 [68]. Xanthan gum (the EPS from Xanthomonas campestris pv. campestris bacterium) is already well established by modern biotechnology and has a sizable market due to its exceptional qualities as a rheology control agent in aqueous systems and as a stabilizer for emulsions and suspensions [60]. Another example is pullulan (a fungal EPS produced by Aureobasidium pullulans), which has been used extensively in the food industry and as a pharmaceutical bulking agent for more than 20 years. Only recently, there is an attempt to explore the potential uses of this non-toxic, immunoceutical, non-mutagenic and non-carcinogenic EPS for various applications such as targeted drug and gene delivery and surface modification [55]. Until now, very few biopolymers have been produced on a commercial basis due to two major facts. Firstly, their production costs are much higher when compared with chemically synthesized polymers that possess similar material characteristics. Consequently, much effort has been devoted to the development of processes for biopolymer production by optimizing the upstream to downstream engineering strategies including the metabolic and cellular engineering of host cells, efficient fermentation and recovery processes, and post-production modification of the biopolymers obtained [30]. The second fact is that from a variety of

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microbial polysaccharides characterized so far, only those with physicochemical properties similar to those of plant (cellulose, pectin and starch) and seaweed (alginate and carrageenan) are used in industry [62]. Although many researchers claim to have found polysaccharides ‘of superior viscosity to xanthan’, in reality few match the robustness of the Xanthomonas polysaccharide and few can maintain their physical properties in the presence of salts, at higher temperatures or extremes of pH [30]. Therefore, most research is focused on the identification of EPS producing extremophiles with the idea that as these microorganisms survive environmental extremes of desiccation, temperature, pressure, salinity, acidity, heavy metals, and radiation, it is to be expected that their biopolymers will also have some unique properties to adapt to such extreme conditions. Since 1991, EPS producing extremophilic microorganisms have been isolated from deepsea hydrothermal vents characterized by extreme pressure and temperature, high concentrations of H2S and heavy metals. These include some bacteria such as Alteromonas macleodii sub spec fijiensis, Vibrio diabolicus, Alteromonas infernos [53, 58], Thermotoga maritime [56] and the psychrophile Pseudoalteromonas sp. SM9913 [31]. EPS producing bacteria from marine Mediterranean shallow vents were also described [41, 64]. EPS produced by moderately halophilic bacteria of the genus Halomonas that include Halomonas maura [4], Halomonas ventosae and Halomonas anticariensis [39], Halomonas eurihalina F2-7 [38], Halomonas cerina [21], Halomonas sp. GT-83 strain [19], the haloalkalitolerant Halomonas alkaliantarctica sp. [50] and the halophilic levan-producer Halomonas sp. AAD6 [51] strains. The two haloalkalophilic microorganisms, Bacillus sp. I-450 [29] and Salinivibrio costicola subsp. alcaliphilus [59] were also reported as good EPS producers. Additional examples include two halophilic Cyanobacteria, namely Cyanothece sp. 113 [14] and Aphanocapsa halophytica [40] and also Halobacterium sp. SM5 [34]. High molecular weight glucan produced by the thermophile Geobacillus tepidamans V264 isolated from a Bulgarian hot spring has been attributed with promising biotechnological applications due to its unusual stability and good biological activity [25]. Other EPSs isolated from thermophilic bacteria include EPS with high immunomodulatory and antiviral activity isolated from Geobacillus thermodenitrificans B3-72 strain [3], EPSs from Bacillus thermoantarcticus isolated from Mount Melbourne, Antarctica [36], Anoxybacillus amylolyticus isolated from Mount Rittmann, Antarctica [49] and Streptococcus thermophilus strains [52]. EPS production is not that common among Archaea, however, the halophilic Haloferax and Haloarcula and thermophilic Thermococcus are examples of producer genera. Whereas Haloferax gibbonsii and Haloferax denitrificans are known to produce a neutral and an acidic EPS, respectively, sulfated EPSs have been isolated from Haloferax mediterranei and Haloarcula japonica [44]. EPS production by the hyperthermophilic archaeon Thermococcus litoralis has been reported by Rinker and Kelly (2000). Although less is known about eukoaryotic life in extreme environments in comparison to prokaryotic extremophiles, advances in genomics and in comprehensive, high-throughput metabolic profiling techniques have provided new insight into the metabolic adaptations of eukaryotes living under extreme conditions. Recently, isolation and chemical characterization of EPSs produced by the halophilic micro-algea Dunaliella salina under salt stress were reported and were found to make emulsions with stability comparable to bacterial EPSs [42]. Duan et al. (2008) proposed a pathway for pullulan biosynthesis by the halotolerant

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Aureobasidium pullulans Y68 yeast strain and also reported their latest studies on the improvement of EPS yield. Monitoring the diversity, colonization rates, and seasonal variations of eukaryotic organisms in Rio Tinto, an extremely acidic environment (pH ca. 2) in Spain, revealed that biofilm formation by the two acidophiles Dunaliella and Cyanidium provided them nutritional advantages and less severe surrounding conditions [2].

APPLICATION AREAS OF EPSS FROM EXTREMOPHILES Commercially, the most important microbial EPS is xanthan gum, a complex polymer that is very efficiently produced by the phytopathogen Xanthomonas campestris and has been used for decades in food and non-food applications. Acetan is structurally related to xanthan and is produced by Acetobacter xylinum, a strain that is used in the food industry for the production of a sweet confectionery (Nata) and vinegar. Sphingans, capsular polysaccharides secreted by Sphingomonas strains, including gellan, wellan, rhamsan, and sphingan S-88, have special rheological properties and can be used in foods as gelling agents, stabilizers or suspending agents. In the dairy industry, EPS-producing lactic acid bacteria (LAB), including the genera Streptococcus, Lactobacillus and Lactococcus, are used in situ to improve the texture of fermented dairy products such as yoghurt and cheese. Numerous studies have been conducted for developing and implementing innovative technology to clean up contamination with petroleum hydrocarbons [26]. Bioemulsifiers are able of emulsifying these pollutants much more effectively than do chemical surfactants. Halophilic EPS producers are considered as an interesting source for microbial enhanced oil recovery (MEOR) where polymers act as emulsifiers and mobility controllers. Active emulsification of petroleum has been noted for six strains, close to Halobacterium salinarium, Haloferax volcanii, and Halobacterium distributum [27]. Martínez-Checa et al. (2007) studied the characteristics of the bioemulsifier V2-7 synthesized by strain F2-7 of Halomonas eurihalina and they found that it has the ability of emulsifying a wide range of hydrocarbons i.e. n-tetradecane, n-hexadecane, n-octane, xylene mineral light and heavy oils, petrol and crude oil. Flocculating agents are generally categorized into three major groups, namely, inorganic flocculants such as aluminum sulfate and polyaluminum chloride (PAC); organic synthetic polymer flocculants such as polyacrylamide derivatives and polyethylene; and naturally occurring biopolymer flocculants such as chitosan and algin. Among these, organic synthetic polymers are the most widely used flocculants, since they are the most economical and highly effective. However, the use of these flocculants sometimes causes environmental and health problems, because they are not readily biodegradable and some of their degraded monomers, such as acrylamide, are neurotoxic and even potent human carcinogens. On the other hand, naturally occurring biopolymers (bioflocculants), which are produced by microorganisms during their growth, have special advantages such as safety, produce a strong effect, are biodegradable and harmlessness to humans and the environment, so they may potentially be applied in drinking and wastewater treatment, downstream processing, and fermentation processes [33, 18]. In recent years, many bioflocculant-producing microorganisms including bacteria, fungi and actinomyces have been reported to produce extracellular polymeric substances, such as

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polysaccharides, functional proteins and glycoprotein, which function as bioflocculant. Flocculants produced by a haloakalophilic Bacillus sp. I-471 [28], a psychrophilic bacterium Pseudoalteromonas sp. SM9913 [31] are polysaccharides. Moreover, Li et al. (2008) reported that EPS SM9913 showed better flocculation performance than an inorganic aluminum based flocculant at low temperature (5 – 15 °C) or in high-salinity (5 –100 %) water suggesting its probable use as an effective flocculant for wastewater treatment at low temperature and/or salinity. However, only few of these bioflocculants have been practically applied in industry because of their low flocculating capability and large dosage requirement. To utilize bioflocculants widely in industrial fields, it is desirable to find various microorganisms with high bioflocculant-producing ability and improve the flocculating efficiency of the bioflocculant [74]. EPSs also have the function of sorption of inorganic ions which constitute metabolic elements for bacteria. Their composition is complex but EPS are mainly composed of polysaccharide, protein, humic substances, uronic acids, nucleic acids and lipids, containing ionizable functional groups such as carboxyl, phosphoric, amine, and hydroxyl groups. These functional groups represent potential binding sites for the sequestration of metal ions. It is assumed that metal biosorption involves a physicochemical interaction between the metal and functional groups on the cell surface, based on physical adsorption, ion exchange, complexation and precipitation. Moreover, metal biosorption performance depends on external factors, such as pH, other ions in bulk solutions (which may be in competition), organic material in bulk solution and temperature. These properties are potentially of great importance in sewage treatment processes for the removal of toxic heavy metal pollutants [15, 32, 22, 69]. Though highly unusual in bacterial polysaccharides, EPSs of halophilic bacteria Halomonas ventosae and Halomonas anticariensis were found not only to have a high capacity for binding cations but also to incorporate considerable quantities of sulfates [39]. Removal of vanadate by biosorption with Halomonas sp. GT-83 [43] and considerably high heavy metal binding capacity of biofilms produced by eukaryotic extremophiles isolated from acidic environments in Rio Tinto (SW, Spain) were also reported [2]. The importance of various EPSs for pharmaceutical purposes has a long historical background and increased considerably during the last decades. Many interesting areas have opened in the past which include their role in drug delivery, in wound treatment, in cancer therapy, and the diagnosis, prevention, and treatment of bacterial and viral diseases. Some polysaccharides form integral components of vaccines, usually when coupled to a suitable protein. Thus, meningitis vaccines have been prepared in this way and multivalent polysaccharide vaccines have been formulated against Streptococcus pneumoniae and Klebsiella spp. However, these are expensive to prepare and only use very small amounts of material. Possibly of much greater significance is the role of certain microbial EPSs in tumour suppression and immunostimulation. A homopolymer named ‘scleroglucan’ or schizophyllan, from several fungal species, adopts a triple helix conformation that strongly influences biological activity and appears to be very effective against some types of cancers when applied in the ordered, triple helical form. These β-linked glucans are therefore the subject of much current study and have already been tested clinically in Japan, proving effective against certain types of tumors [20, 68]. Dextran, although no longer used as a food ingredient, is the base from which the ‘Sephadex’ range of biochemical adsorbents is prepared. Dextran solutions can also be used as a plasma substitute, being very poorly antigenic and having the correct physical properties

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[68]. Polysaccharides also contribute to various physiological activities in human beings as antitumor, antiviral, and anti-inflammatory agents, and can act as inducers for interferon, platelet aggregation inhibition, and colony stimulating factor synthesis [29]. Recently there is an attempt to explore pullulan for various biomedical applications including targeted drug and gene delivery and tissue engineering due to its non-toxic, nonimmunogenic, non-mutagenic and non-carcinogenic nature. The early observation on this exopolymer was made by Bauer in 1938 and this exopolysaccharide was named pullulan by Bender et al in 1959 [5, 6]. Pullulan and its derivatives can be used as a denture adhesive. It can also be used for pharmaceutical coatings, including sustained release formulations. Novel preparations such as tablets, pills, granules or the like, which contain pullulan in the sugar layer serve the purpose of preventing brownish color change of the composition. Oral care products have been commercialized based on pullulan films. The colorless, transparent and edible pullulan film has also attracted a great deal of interest for other uses such as a nonpolluting wrapping material [66, 55].

CONCLUSION It is widely accepted that extremophiles offer important insights into biology and evolution of many organisms, and they provide a valuable resource for exploitation in novel biotechnological processes. Despite the vast amount of extremophiles that are being isolated and identified so far, literature reports concerning their EPSs are still very limited. In recent years, significant progress has been made in discovering and developing novel and functional EPSs from extremophilic producer strains. These natural, non-toxic and biodegradable polymers show considerable diversity in their composition and structure. There is a huge gap between our knowledge of the relationship between the composition and structure of EPSs and the ability to predict their physical and health-promoting properties and thus their potential applications. Such knowledge is essential to increase the range of biopolymers with desirable functions and the present gap seems to be filled in the near future due to the current interest in glycobiology and the application of new analytical methods. Microbial EPS production and the structure, composition and viscosity of EPSs are greatly influenced by fermentation conditions, such as the type of strain, composition of the nutrient medium, pH, temperature, oxygen concentration and agitation. Hence, by manipulating the producer microorganism, feedstock and process conditions, fermentation allows a wide and reproducible range of different biomaterials with very good control over their characteristics. Currently, EPSs can be produced under controlled conditions to the specifications required at industrial levels. For example, xanthan, dextran and pullulan are well known industrial microbial polysaccharides with numerous applications and a considerable and sizable market. However, when compared with the synthetic polymers, natural origin polymers still represent only a small fraction of the current polymer market, mostly due to their costly production processes. Therefore, much effort has been devoted to the development of cost-effective and environmentally-friendly production processes such as investigating the potential use of cheaper fermentation substrates. Considering the wide spread use of these biopolymers in various industries as well as their potential applications in novel biotechnological processes, more studies need be

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conducted not only to minimize the production costs, but also to elucidate their production mechanisms, chemical structure and rheological properties.

ACKNOWLEDGEMENTS Hande Kazak gratefully acknowledges TUBITAK (Project 108M193) for a Fellowship.

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