Bioresource Technology 100 (2009) 3382–3386

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Bioresource Technology

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Isolation and characterization of extracellular polymeric substancesfrom micro-algae Dunaliella salina under salt stress

Avinash Mishra *, Bhavanath JhaDiscipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute (CSMCRI), Council of Scientific and Industrial Research (CSIR), G.B.Marg, Bhavnagar 364002, Gujarat, India

a r t i c l e i n f o

Article history:Received 14 November 2008Received in revised form 3 February 2009Accepted 3 February 2009Available online 9 March 2009

Keywords:DunaliellaEPSFT-IRPolysaccharideSalinity

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.02.006

* Corresponding author. Tel.: +91 278 2567760x64E-mail address: avinash@csmcri.org (A. Mishra).

a b s t r a c t

Extracellular polymeric substances (EPSs), produced by Dunaliella salina strain, increase concomitantlywith salt concentration and maximum (944 mg/l) were obtained at 5 M NaCl, whereas minimum(56 mg/l) at 0.5 M salinity. Emulsifying activity was measured in terms of strength to retain the emulsionand comparatively 85.76% retention was observed at 0.5 M salinity thereafter it intends to decline. TheFT-IR-spectra reveal characteristic functional groups NAH stretching, asymmetrical CAH stretchingvibration of aliphatic CH2-group, CAC stretching of aromatic, CAN stretch of aliphatic amine, NAH wagof primary amine and CAX stretch of alkyl-halides with a stretching of CAOAC, CAO corresponding tothe presence of carbohydrates. The FT-IR-spectra substantiated the presence of primary amine-group,aromatic-compound, halide-group, aliphatic alkyl-group and polysaccharides. Four monosaccharides(glucose, galactose, fructose and xylose) were also detected by HPLC analysis. Production of EPSs mayallow further exploration of D. salina as potential EPSs producer and make it as a promising candidatefor biotechnological and industrial exploitation.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The ability of cells to survive and flourish in saline environmentunder the influence of osmotic stress has received considerableattention and Dunaliella is recognized as the only eukaryotic andphotosynthetic organism, which grows in wide range of salt con-centration, ranging from 0.05 M to saturation (5.0 M) (Mishraet al., 2008). This biflagellate unicellular alga is responsible formost of the primary production in hyper-saline environment.Extracellular polymeric substances (EPSs) are renewable resourcerepresenting an important class of polymeric materials of biotech-nological importance with a wide variety of potentially usefulapplications (De Philippis et al., 2001; Kumar et al., 2007). Gener-ally, EPSs have been observed in bacteria (Freitas et al., 2009)and cyanobacteria (De Philippis et al., 2001; Parikh and Madam-war, 2006; Chi et al., 2007), however it is also reported in Crypto-phyta (Bermúdez et al., 2004), mushroom (Zou et al., 2006), yeast(Duan et al., 2008) and basidiomycete (Manzoni and Rollini,2001; Chi and Zhao, 2003). Despite EPSs of freshwater micro-algae,Scenedesmus acuminatus (Chlorophyceae), were explored for itsmetal complexing properties (Lombardi et al., 2005), however inDunaliella, there is no report of EPSs production and the effect ofsalinity. The polysaccharides of biological response modifiers can

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be isolated from bacteria, fungi, brown algae and photosyntheticplants (Leung et al., 2006).

Numerous bacterial polysaccharides are potentially available,known to be involved in pathogenesis, symbiosis, biofilm forma-tion, protection from phagocytic predation and stress resistance(Parikh and Madamwar, 2006). In recent years, interest in theexploitation of valuable EPSs has been increasing for various indus-trial applications and the attention towards polysaccharide pro-ducing bacteria and cyanobacteria has greatly increased. EPSs areregarded as abundant source of structurally diverse polysaccha-rides, some of which may possess unique properties for specialapplications. Antitumor, antiviral and immunostimulant activitiesof polysaccharides produced by marine Vibrio sp. and Pseudomonassp. have been reported (Okutani, 1984), however, heparin-like exo-polysaccharide exhibiting anticoagulant property has been isolatedfrom Alteromonas infernus (Colliec et al., 2001). Apart from bacte-rial EPSs applications, various other uses such as improvement ofwater holding capacity of soil, detoxification of heavy metals andradionuclides contaminated water and removal of solid matterfrom water reservoirs have been proposed for cyanobacterial EPSs(Bender and Phillips, 2004). The increased demand of natural poly-mers for various industrial applications in recent years has led tosway interest in EPSs production by new sources and marine mi-cro-algae, which is used as a source of products of high aggregatedvalue such as pigments, osmoprotectant, metabolites, fatty acidsand proteins, may also be exploited for the EPSs.

A. Mishra, B. Jha / Bioresource Technology 100 (2009) 3382–3386 3383

The present work involves isolation and characterization ofEPSs from the micro-algae Dunaliella salina, surviving in varyingsalt concentration and studies on the effect of salinity on EPSs pro-ductivity both qualitatively and quantitatively. Despite of b-caro-tene, glycerol and other metabolites, EPSs make Dunaliella morepromising candidate which will play an important role in its bio-technological and industrial application.

Fig. 1. Amount of crude EPSs extracted from the 20 days old Dunaliella culturegrown over a salinity gradient. The exopolymers were precipitated with equalvolume of cold methanol and dialysed against tap and distilled water.

2. Methods

2.1. Unialgal culture and extraction of EPSs

Axenic culture of D. salina, isolated from experimental salt farm(Mishra et al., 2008), was grown in 500 ml of De Walne’s culturemedia (Orset and Young, 1999) containing different salt concentra-tions; 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 M under controlled laboratoryconditions at 25 ± 2 �C under 12:12 h (light/dark cycle) with whitefluorescent lamp of 38 l photons m�2 s�1 light intensity. Twentydays grown Dunaliella culture was centrifuged at 15,000g for20 min at 4 �C to remove cells and other precipitates (debris).The supernatant was filtered (Whatmann, UK) twice and concen-trated to one forth volume on magnetic stirrer at 60 �C for10–12 h (Parikh and Madamwar, 2006). The exopolymer was pre-cipitated by gradually adding equal volume of cold methanol toconcentrated supernatant and kept at 4 �C for 12 h. After removalof residual methanol, the precipitate was washed with absoluteethanol and re-dissolved in Milli-Q water (Millipore, USA). Dis-solved EPSs were dialysed against tap water for 2 days, followedby distilled water for 1 day to remove ions and salt. Dialysed EPSswere frozen at �20 �C and lyophilized at �70 �C for 10–12 h(Parikh and Madamwar, 2006; Chi et al., 2007).

2.2. Emulsifying activity

Lyophilized EPSs (1 mg), dissolved in 0�5 ml deionized water,were heated at 100 �C for about 15–20 min and allowed to coolto room temperature (25 ± 2 �C). The volume was made up to2 ml using phosphate-buffered saline (PBS). The sample was vor-texed for 1 min after the addition of 1 ml hexadecane. The absor-bance at 540 nm was read immediately before and aftervortexing (A0). The fall in absorbance was recorded after incubationat room temperature for 30 min (At). A control was run simulta-neously with 2 ml PBS and 1 ml hexadecane. The emulsificationactivity was expressed as the percentage retention of emulsionduring incubation for time t: At/A0 � 100 (Bramhachari and Dubey,2006).

2.3. Fourier-transformed infrared spectroscopy

The major structural groups of purified EPSs were detectedusing Fourier-transformed infrared (FT-IR) spectroscopy. Pelletsfor infrared analysis were obtained by grinding a mixture of 2 mgEPSs with 200 mg dry KBr, followed by pressing the mixture intoa 16-mm-diameter mould. The FT-IR spectra were recorded inthe region of 4000–400 cm�1 on a Perkin–Elmer spectrum GX FT-IR system (Perkin–Elmer, USA).

2.4. HPLC analysis

Lyophilized EPSs were hydrolyzed with 2 M H2SO4 for 5–6 h at121 �C in sealed glass test tube. After complete hydrolysis, contentwas neutralized with BeCO3 and filtered (Whatmann, UK). Mono-saccharide composition of the hydrolysate was determined byHPLC (Waters Alliance, 2996-seperation module) using Supelcogel 610H column (30 cm � 7.8 mm) and RI (2414) detector with

flow rate 0.4 ml/min at temperature 30 �C and mobile phase,0.17% H3PO4 in water (Meisen et al., 2008). The relative proportionof the peak area was calculated to estimate the monomercomposition.

3. Results and discussion

3.1. Extraction of EPSs

Extracellular polymeric substances (EPSs) are biopolymers, gen-erally observed in bacteria (Freitas et al., 2009) and/or cyanobacte-ria (De Philippis et al., 2001; Parikh and Madamwar, 2006; Chiet al., 2007). Apart from these, exopolysaccharides have also beenreported from marine micro-alga Chroomonas sp.; Cryptophyta(Bermúdez et al., 2004), medicinal mushroom Phellinus linteus(Zou et al., 2006), yeast Aureobasidium pullulans (Duan et al.,2008) and basidiomycete Daedalea quercina (Manzoni and Rollini,2001) and Rhodotorula bacarum (Chi and Zhao, 2003). The nativeEPSs produced by D. salina, a micro-algae (Chlorophyceae), wereisolated at 20th day of inoculation and it were found increasingconcomitantly with salt concentration (Fig. 1). Maximum EPSs(944 mg/l) were obtained from Dunaliella culture grown in 5 M saltconcentration while minimum (56 mg/l) from 0.5 M salt contain-ing media. The yield of EPSs was far low in comparison to that pro-duced by the cyanobacteria Cyanothece sp. 113 which releases22.34 g/l of exopolysaccharide into the medium in 11 days and thiswas claimed highest exopolysaccharide yield by cyanobacteria re-ported so far (Chi et al., 2007). However, similar amount of EPSs(700, 685 and 870 mg/g) were isolated from cyanobacteria; Oscill-atoria, Nostoc and Cyanothece, respectively (Parikh and Madamwar,2006). The production of EPSs was investigated under differentgrowth conditions including salinity in algae and cyanobacteriaand it was observed that only Mg2+ shortage causes a significantenhancement of EPS production while salinity has no effect (DePhilippis et al., 1991). In contrast, the synthesis of carbohydrates(in EPS) was neither enhanced by increasing salinity (sea-water en-riched with NaCl in the range 0–2.0 M) nor by Mg2+, K+ or Ca2+ defi-ciencies in cyanobacteria Cyanothece sp. (De Philippis et al., 1993).However, in halotolerant bacterium R. acidophila (Sheng et al.,2006) and medicinal mushroom P. linteus (Zou et al., 2006), the to-tal EPS content increased with NaCl concentration, suggesting aprotective response by the bacterium (Sheng et al., 2006). Thismay be the reason why high level of NaCl enhances the flocculationin photosynthetic bacteria (Sheng et al., 2006).

In Dunaliella, EPSs production remain unchanged up to 1.0 MNaCl concentration and thereafter a significant increase was ob-

3384 A. Mishra, B. Jha / Bioresource Technology 100 (2009) 3382–3386

served. As far as previous literature is concerned, EPS productionand its variation with salt concentration has not been reportedyet in this micro-algae. D. salina is known as the only eukaryoticphotosynthetic organism, having tremendous ability to grow inwide range (0.5–5.5 M) of salt concentration (Mishra et al., 2008).The cell of Dunaliella is enclosed in a thin elastic plasma membranesurrounded by mucus ‘‘surface coat” (Ben-Amotz and Avron, 1990).Dunaliella has typical cellular organelles like other green alga butlacks rigid polysaccharide cell wall. The ability of Dunaliella cellto survive in wide range of salt concentrations may be attributedto the release of EPSs with salinity which may provide protection.The presence of salt creates an osmotic pressure in the broth whichprovokes the micro-organisms to protect themselves from it andthe production of exopolysaccharides is an additional mechanismto keep themselves safe from salinity (Abbasi and Amiri, 2008).Most of the functions ascribed to exopolysaccharides are of protec-tive nature and the presence of gel like polysaccharide layeraround the cell may have paramount effect and may reduce thepenetration of ions through the cell surface (Kumar et al., 2007).Previously, we have also observed total intra-cellular sugar, pro-line, glycine betaine and total protein increased concomitantlywith salt concentration and a significant increase was observedin high salt stress 5.5 M NaCl (Mishra et al., 2008).

3.2. Emulsifying stability

The emulsifying activity of Dunaliella exopolymer was deter-mined by its strength in retaining the emulsion. The emulsifyingactivity of EPSs decreased from 85.76% (approx.) retention to66.37% with salinity (Fig. 2). In contrast, EPSs of Enterobacter cloa-cae were steady in the presence of sodium chloride and there wereno significant differences in varying concentrations of sodiumchloride (Abbasi and Amiri, 2008). The emulsion of exopolymerfrom Dunaliella is comparatively stable compared to that of otherEPSs, produced by bacteria (Bramhachari and Dubey, 2006).

3.3. FT-IR spectroscopy of EPSs

Infrared spectroscopy reveals the fact that molecules possessspecific frequencies at which they can rotate or vibrate corre-sponding to discrete energy levels (vibrational modes). These res-onant frequencies are determined by the shape of the molecularpotential energy surface, the mass of the atoms and by the associ-ated vibronic coupling. Variation in stretching and bending modesof vibration with single functional group is usually coupled withthe vibration of adjacent group, as well as with the number of sub-

EPSs Isolated from the culture grown De Walne’ s media with different salt concentrations

Fig. 2. Emulsifying activity of crude EPSs extracted from the 20 days old Dunaliellaculture grown over a salinity gradient. Hexadecane, 1 ml, was added to EPSs (1 mg)diluted to 2 ml with PBS, vortexed for 1 min and the absorbance monitored at540 nm.

stitution(s) taking place on the molecule itself. This leads to theshifting or overlapping of the peaks of two or more functionalgroups in the same region of IR spectrum. The interpretation ofinfrared spectra involves the correlation of absorption bands inthe spectrum of an unknown compound with the known absorp-tion frequencies for different type of bonds.

The FT-IR spectrum of EPSs, obtained from Dunaliella culture,reveals characteristic functional groups (Fig. 3). The mediumstretch and medium bend of frequency range 3500–3300 cm�1

and 1650–1580 cm�1, respectively, is assigned to NAH stretching.Weak absorptions at 2915–2935 cm�1 are attributed to asymmet-rical CAH stretching vibration of aliphatic CH2-group. Mediumstretch observed in the range 1400–1500 cm�1 is assigned toCAC stretching of aromatic i.e. in ring, while CAN stretch of ali-phatic amine tumbled in the range of 1250–1020 cm�1. A stretch-ing of CAOAC, CAO at 1000–1200 cm�1 corresponds to thepresence of carbohydrates (Bremer and Geesey, 1991; Bramhachariand Dubey, 2006). Absorption peaks centered around 910–665 and690–515 cm�1 correspond to NAH wag of primary amine and CAXstretch of alkyl-halides, respectively. IR peak observed in the rangeof 2350–2360 cm�1 may be because of CO2 adsorption (Nabievet al., 1976) or asymmetric stretching of group AN@C@OA (Pandaand Sadafule, 1996). The FT-IR spectrum of EPSs confirms the pres-ence of primary amine-group, aromatic-compound, halide-group,aliphatic alkyl-group and polysaccharides (carbohydrates). Conse-quently, IR spectra may be attributed to the presence of Alkylamine and/or cyclic amine with polysaccharides in the EPSs. Com-parative IR spectrum of EPSs (Fig. 3), obtained from Dunaliella cul-ture grown in varying salt concentration, do not show relevantshifting of peaks and it may be depicted that salinity has no effecton the composition of EPSs of D. salina.

3.4. Monosaccharide composition of EPSs

The monosaccharides were analysed after acid hydrolysis (2 MH2SO4) for 5–6 h at 121 �C and subsequent HPLC. After completeacidic hydrolysis of obtained EPSs, only four constituent monosac-charides viz. galactose, glucose, xylose and fructose were detectedin different combinations and ratio by HPLC analysis. Monosaccha-rides detected in EPSs of Dunaliella can be categorized as aldohex-oses (glucose and galactose), ketohexose (fructose) and pentose(xylose) sugars. The presence of pentose sugar, which is usually ab-sent in polysaccharides of prokaryotic origin and is quite uniqueamong cyanobacteria (Parikh and Madamwar, 2006) is remarkable.Monosaccharide composition was found contrast with most of thecyanobacterial EPSs (6–12 monosaccharides; De Philippis et al.,2001; Rainer et al., 2007), however EPSs with the composition offour monosaccharides (ribose, xylose, glucose and mannose) werealso detected in cyanobacteria; Oscillatoria, Nostoc and Cyanothece(Parikh and Madamwar, 2006). In Dunaliella, all four monosaccha-rides were found in almost same frequency and maximum amountof monosaccharides (106–159 lg) was obtained from the culturecontaining 3 M salt concentration whereas maximum percentageof the monosaccharide (0.17–0.25%), with respect to total amountof EPSs, was detected in culture with 0.5 M salinity (Fig. 4). Fruc-tose amount was detected comparatively higher to galactose, xy-lose and glucose.

Monosaccharide’s ingredient of EPSs, isolated from Dunaliellaculture grown in media containing different salt concentration, in-creased with salinity and observed maximum at 3 M salt concen-tration thereafter declined to minimum at 5 M salinity (Fig. 4).However, total amount of crude EPSs was found increasing con-comitantly with salt concentration (Fig. 1). A sudden increase inmonosaccharide constituent was observed (Fig. 4) at 3 molar saltconcentration which later on intend to decrease. It may be explainedas Dunaliella is a halotolerant alga responding to high salinity by

2931 1M3422 2364

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1618 14121124

6242915

1618 14123437 2M

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4492928

1622 14113419 3M1622 1411

11362357628

1620 1412628

11363437 4M

235929152915

T

1616 1412 6275192 5192

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tanc

e

5M11353437

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5M

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29152915 635

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5192 5192 6353431 0.5M

2351 144816271118

0.5M

29152915

4000.0 3000 2000 1500 1000 400.02000 1500 1000 400.0

Wavenumbers (cm-1)

Fig. 3. Comparative FT-IR spectra of EPSs extracted from the 20 days old Dunaliella culture grown over a salinity gradient (0.5–5.0 M).

Fig. 4. Monosaccharide composition and percentage amount to total EPSs extracted from the 20 days old Dunaliella culture grown over a salinity gradient.

A. Mishra, B. Jha / Bioresource Technology 100 (2009) 3382–3386 3385

enhancing photosynthetic CO2 assimilation and, by diversion ofcarbon and energy resources. In addition to this, a significant in-crease in chlorophyll content at 3 M NaCl was also observed byus (Mishra et al., 2008), which is remarkable and supports compar-atively higher rate of photosynthesis. This may be the reason ofgetting higher amount of monosaccharides in EPSs, obtained fromthe Dunaliella culture grown at 3 M salt concentration.

4. Conclusion

Intraspecific physiological variability has been reported in D.salina, which may lead to erroneous assumptions related to its

industrial potential. EPSs of D. salina can play an important rolein its biotechnological and industrial application. In this study weisolated EPSs from D. salina grown under salt stress and it maybe the first report. The extracellular salt stress affects the releaseof EPSs concomitantly and significant advancement in the EPSsproduction was observed at higher salinity. The emulsion of iso-lated exopolymer is more stable compared to other EPSs, producedby bacteria, but its emulsifying activity slightly reduces with salin-ity. The FT-IR-spectra of the EPSs reveal the presence of primaryamine-group, aromatic-compound, halide-group, aliphatic alkyl-group and polysaccharides (carbohydrates), as a resultant presenceof alkyl amine and/or cyclic amine with polysaccharides in the

3386 A. Mishra, B. Jha / Bioresource Technology 100 (2009) 3382–3386

EPSs can be depicted. Four monosaccharides (galactose, glucose,xylose and fructose) were detected by HPLC analysis and an abruptincrease was determined at 3 M salinity. EPSs may allow furtherexploration of D. salina and make it as a promising candidate forthe commercial exploitation. However, further work is requiredto determine its (EPSs) biotechnological uses including environ-mental bioremediation, medicinal applications, gelling ability, bio-logical recovery probability, bioflocculants and source ofmonosaccharides.

Acknowledgements

Discipline of Analytical Science of the institute is duly acknowl-edged for sustaining central instrumentation facilities. The finan-cial support of CSIR, Govt. of India and Department of Scienceand Technology, Govt. of India (Under SERC Fast Track scheme videOrder No.: SR/FT/L-25/2005 dated January 02, 2006) for carryingout this project is thankfully acknowledged.

References

Abbasi, A., Amiri, S., 2008. Emulsifying behavior of an exopolysaccharide producedby Enterobacter cloacae. African J. Biotechnol. 7, 1574–1576.

Ben-Amotz, A., Avron, M., 1990. The biotechnology of cultivating the halotolerantalgae Dunaliella. Trends Biotechnol. 8, 121–125.

Bender, J., Phillips, P., 2004. Microbial mats for multiple applications in aquacultureand bioremediation. Biores. Technol. 94, 229–238.

Bermúdez, J., Rosales, N., Loreto, C., Briceño, B., Morales, E., 2004.Exopolysaccharide, pigment and protein production by the marine microalgaChroomonas sp. in semicontinuous cultures. World J. Microbiol. Biotechnol. 20,179–183.

Bramhachari, P.V., Dubey, S.K., 2006. Isolation and characterization ofexopolysaccharide produced by Vibrio harveyi strain VB23. Lett. Appl.Microbiol. 43, 571–577.

Bremer, P.J., Geesey, G.G., 1991. An evaluation of biofilms development utilizingnon-destructive attenuated total reflectance Fourier transform infraredspectroscopy. Biofouling 3, 89–100.

Chi, Z., Su, C.D., Lu, W.D., 2007. A new exopolysaccharide produced by marineCyanothece sp. 113. Biores. Technol. 98, 1329–1332.

Chi, Z., Zhao, S., 2003. Optimization of medium and cultivation conditions forpullulan production by a new pullulan-producing yeast. Enzyme Microb.Technol. 33, 206–211.

Colliec, J.S., Chevolot, L., Helley, D., Ratiskol, J., Bros, A., Sinquin, C., Roger, O., Fischer,A.M., 2001. Characterization, chemical modifications and in vitro anticoagulantproperties of an exopolysaccharide produced by Alteromonas infernos. Biochim.Biophys. Acta 1528, 141–151.

De Philippis, R., Margheri, M.C., Pelosi, E., Ventura, S., 1993. Exopolysaccharideproduction by a unicellular cyanobacterium isolated from a hypersaline habitat.J. Appl. Phycol. 5, 387–394.

De Philippis, R., Sili, C., Paperi, R., Vincenzini, M., 2001. Exopolysaccharide-producing cyanobacteria and their possible exploitation: a review. J. Appl.Phycol. 13, 293–299.

De Philippis, R., Sili, C., Tassinato, G., Vincenzini, M., Materassi, R., 1991. Effects ofgrowth conditions on exopolysaccharide production by Cyanospira capsulata.Biores. Technol. 38, 101–104.

Duan, X., Chi, Z., Wang, L., Wang, X., 2008. Influence of different sugars on pullulanproduction and activities of a-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis inAureobasidium pullulans Y68. Carbohydr. Polym. 73, 587–593.

Freitas, F., Alves, V.D., Pais, J., Costa, N., Oliveira, C., Mafra, L., Hilliou, L., Oliveira, R.,Reis, M.A., 2009. Characterization of an extracellular polysaccharide producedby a Pseudomonas strain grown on glycerol. Biores. Technol. 100, 859–865.

Kumar, A.S., Mody, K., Jha, B., 2007. Bacterial exopolysaccharides – a perception. J.Basic Microbiol. 47, 103–117.

Leung, M.Y.K., Liu, C., Koon, J.C.M., Fung, K.P., 2006. Polysaccharide biologicalresponse modifiers. Immunol. Lett. 105, 101–114.

Lombardi, A.T., Hidalgo, T.M.R., Vieira, A.A.H., 2005. Copper complexing propertiesof dissolved organic materials exuded by the freshwater microalgaeScenedesmus acuminatus (Chlorophyceae). Chemosphere 60, 453–459.

Manzoni, M., Rollini, M., 2001. Isolation and characterization of theexopolysaccharide produced by Daedalea quercina. Biotechnol. Lett. 23, 1491–1497.

Meisen, S., Wingender, J., Telgheder, U., 2008. Analysis of microbial extracellularpolysaccharides in biofilms by HPLC. Part I: development of the analyticalmethod using two complementary stationary phases. Anal. Bioanal. Chem. 391,993–1002.

Mishra, A., Mandoli, A., Jha, B., 2008. Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan. J. Ind.Microbiol. Biotechnol. 35, 1093–1101.

Nabiev, B.A., Lafer, L.I., Yakerson, V.I., Rubinshtein, A.M., 1976. IR spectra of catalystsand adsorbed molecules. Russ. Chem. Bull. 25, 1398–1402.

Okutani, K., 1984. Antitumor and immunostimulant activities of polysaccharidesproduced by a marine bacterium of the genus Vibrio. Bull. Jpn. Soc. Sci. Fish 50,1035–1037.

Orset, S., Young, A.J., 1999. Low-temperature-induced synthesis of a-carotene in themicroalga Dunaliella salina (Chlorophyta). J. Phycol. 35, 520–527.

Panda, S.P., Sadafule, D.S., 1996. FTIR spectral evaluation of polyurethane adhesivebonds in perspex canopies of aircraft. Defence Sci. J. 46, 171–174.

Parikh, A., Madamwar, D., 2006. Partial characterization of extracellularpolysaccharides from cyanobacteria. Biores. Technol. 97, 1822–1827.

Rainer, B.V., Venzke, K., Blaschek, W., 2007. Structural investigation of apolysaccharide released by the cyanobacterium Nostoc insulare. J. Appl.Phycol. 19, 255–262.

Sheng, G.P., Yu, H.Q., Yue, Z., 2006. Factors influencing the production ofextracellular polymeric substances by Rhodopseudomonas acidophila. Int.Biodeterior. Biodegrad. 58, 89–93.

Zou, X., Sun, M., Guo, X., 2006. Quantitative response of cell growth andpolysaccharide biosynthesis by the medicinal mushroom Phellinus linteus toNaCl in the medium. World J. Microbiol. Biotechnol. 22, 1129–1133.