Production and characterization of an exopolysaccharide by yeast

K. Pavlova1,*, L. Koleva2, M. Kratchanova3 and I. Panchev21Laboratory of Applied Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, 26 Maritza Blvd.,4002 Plovdiv, Bulgaria2University of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria3Institute of Organic Chemistry with Phytochemistry Centre, Bulgarian Academy of Sciences, 95 V. Aprilov Blvd.,P.O. Box 27, 4002 Plovdiv, Bulgaria*Author for correspondence: Tel.: +359-32-603-831, Fax: +359-32-440-102/2-700-109,E-mail: lbpmbas@plov.omega.bg

Received 24 September 2003; accepted 6 December 2003

Keywords: Antarctic yeasts, biosynthesis, exopolysaccharides, mannan, Sporobolomyces salmonicolor AL1

Summary

Antarctic yeast strains were investigated for exopolysaccharide biosynthesis and the Sporobolomyces salmonicolorAL1 strain was selected. It was studied for exopolysaccharide biosynthesis on different carbon and nitrogen sources.The investigations showed that sucrose and ammonium sulphate were suitable culture medium components forpolymer biosynthesis. Exopolysaccharide formation by the yeast strain was accompanied by a decrease in theculture medium pH value from the initial pH 5.3 to pH 1.7–2.0. During the biosynthetic process, the dynamicviscosity of the culture broth increased to the maximum value of 15.37 mPa s and the polysaccharide yield reached5.63 g/l on a culture medium containing 5.00% sucrose and 0.25% ammonium sulphate at a temperature of 22 �Cfor 120 h. The crude polysaccharide obtained from Sp. salmonicolor AL1 featured high purity (90.16% of carboncontent) and consisted of glucose (54.1%), mannose (42.6%) and fucose (3.3%). Pure mannan containing 98.6% ofmannose was isolated from it.

Introduction

Microorganisms of different taxonomic groups possessthe ability to synthesize exopolysaccharides with inter-esting physicochemical properties and functional char-acteristics. Some microbial polysaccharides arecharacterised by high viscosity and good gelatingproperties; they have a synergistic effect when interact-ing with other polysaccharides and combine withdifferent salts within a wide pH and temperature range.The differences in the structure and properties ofindividual polysaccharides used as food additivesaccount for the diverse functions they perform asthickeners, emulsifiers, leavenings, and frothers.Commercially used biopolymers are bacterial and

fungal products such as xanthan, dextran and sclero-glucan (Shimada et al. 1977; Margaritis & Pace 1985;Paul et al. 1986; Crescenzi 1995; Sutherland 1998).Yeasts belonging to the different Cryptococcus, Hansen-ula, Rhodotorula, Lipomyces, Bullera, Aureobasidium,and Sporobolomyces genera can synthesize exopoly-saccharides. The polymer types reported for yeastproducers include mannans, glucans, glucomannans,galactomannans and phosphomannans (Chiura et al.1982a, b; Peterson et al. 1989; Vitovskaya et al. 1989;Adami & Cavazzoni 1990; Peterson et al. 1990; Elinov

et al. 1992; Vorotynskaya et al. 1992). The yeast-produced exopolysaccharides are more easily separatedfrom the culture broth than those produced by bacteria,therefore they are attractive for large-scale production(Peterson et al. 1989).The structure and physical properties of microbial

polysaccharides depend on the culture medium compo-sition and the growth conditions. Studies on the effect ofgrowth-limiting substrates on exopolysaccharide syn-thesis clearly demonstrate that the growth mediumcomposition can dramatically affect the specific rate ofpolymer synthesis (Heald & Kristiansen 1985; Lacroixet al. 1995). The amount of carbon substrate convertedto polymer by the microbial cells depends on the growthmedium composition. Generally, media containinga high carbon-to-limiting-nutrient ratio are favouredfor polysaccharide production (Sutherland 1998). Thecarbon and nitrogen sources used do not affect thequalitative monosaccharide composition of the polysac-charide, although the introduction of ammonium saltsinto the medium can cause a change in the monosac-charide ratio in the side chains of the polymer (Vitovs-kaya et al. 1989). The growth medium composition canalso indirectly affect the polymer yield, for example, bygoverning the pH change which can occur duringfermentation without pH control (Heald & Kristiansen

World Journal of Microbiology & Biotechnology 20: 435–439, 2004. 435� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

1985; Lacroix et al. 1995). In our previous papers, aRh. acheniorum MC strain was selected as a suitablemannan exopolysaccharide producer, an optimal culturemedium was established and their rheology in a mixturewith xanthan was investigated (Grigorova et al. 1999;Pavlova & Grigorova 1999).The object of this paper was to study the exopolysac-

charide-producing capacity of psychrophilic yeast, toselect an active producer, to investigate the patterns inthe biosynthetic process, the dynamic viscosity and itsrelationship to cell physiology and polymer composition.

Materials and methods

Microorganisms

The Sporobolomyces salmonicolor AL1 strain was chosenby multistage selection out of 38 yeast strains isolatedfrom soil lichen, moss and soil taken from the region ofthe Bulgarian base on Livingston Island, Antarctica.The selection was made according to the ability of thestrains to synthesize polysaccharides on synthetic mediawith glucose as a carbon source. The exopolysaccharide-producing strain was identified using the yeast classifi-cation criteria proposed by Kurtzman & Fell (1998) andwas registered in the National Bank for IndustrialMicroorganisms and Cell Culture, Bulgaria. The culturewas maintained on a malt slant agar at 4 �C.

Media and growth conditions

The basal medium for the testing of carbon sourcescontained (w/v): 0.20% (NH4)2SO4, 0.1% KH2PO4,0.05% MgSO4Æ7H2O, 0.01% NaCl, 0.01% CaCl2Æ2H2Oand 0.1% yeast extract. Glucose, sucrose and fructosewere tested as carbon sources and were added to thebasal medium in different concentrations.The basal medium for the testing of nitrogen sources

contained (w/v): 4.0% sucrose, 0.1% KH2PO4, 0.01%NaCl, 0.05% MgSO4Æ7H2O, 0.01% CaCl2Æ2H2O, 0.1%yeast extract and different nitrogen sources. (NH4)2SO4,NH4Cl and (NH4)NO3 were tested as nitrogen sourcesand were added to the basal medium in differentconcentrations. The initial pH was adjusted to pH 5.3and the media were sterilized at 112 �C for 30 min.The inoculum from Sp. salmonicolorAL1 was obtained

on a rotary shaker (220/min) in 500 ml Erlenmeyer flaskscontaining 50 ml of Sabouraud medium (Merck,Ger-many) at 22 �C for 48 h. The fermentation media wereinoculated with 1.0% w/v of inoculum. The cultivationwas carried out in 500 ml Erlenmeyer flasks containing50 ml of the tested medium on a rotary shaker (220/min)at 22 �C for 168 h.

Isolation of crude exopolysaccharides

Whole cell cultures were centrifuged at 6000 · g for30 min to separate the yeast cells from the supernatant.

The exopolysaccharides in the culture supernatants wereprecipitated with 2 vol. of 96% ethanol at 4 �C for 18–24 h. The resultant supernatant was discarded and thepellet was washed twice with ethanol, dried andweighed.

Analytical methods

The dry weight of the yeast biomass was determined byheating at 105 �C until constant weight. The residualsugar concentration was measured by the 3,5 dinitro-salicylic acid method (Chaplin & Kennedy 1986). Thetotal carbohydrate amount in the crude exopolysaccha-rides was determined using the phenol–sulphuric acidmethod (Dubois et al. 1956). The protein amount in thesolution of non-hydrolyzed polysaccharides was deter-mined according to the Lowry method. The ash contentwas estimated after calcination for 2 h and glowing thepolymer at 550 �C for 3 h.The dynamic viscosity of the cell-free culture broth

was measured using a Rheotest-2 Hoppler type visco-meter at a temperature of 25 �C, K ¼ 0.00463212,P ¼ 10 g/cm2. The data were statistically analysed usingSigma Plot (Version 100) and the standard deviations(SDs) were determined.The dynamic viscosity of the aqueous polysaccharide

solution was measured by means of a Rheotest-2rheoviscometer, Germany, using measuring cylinder Nof the device N (R/r) ¼ 1.02. The numerical values ofthe velocity gradient Dr and the modulus of shearingwere processed using the Oswald de Waele equation(Holdsworth 1993) on a REO computer programwritten in FORTRAN 77.The carbohydrate composition was determined after

hydrolysis of the crude and pure exopolysaccharideswith M H2SO4 at 105 �C for 8 h, neutralization withbarium hydroxide followed by centrifugation and elim-ination of the Ba ions using a Wofatit KPS cationexchanger. The monosaccharide content was determinedwith HPLC on Waters with an R 401 differential ref-ractometer on a carbohydrate column. The mobilephase was acetonitrile:water (87:13), with flow rate of0.6 ml/min and ambient temperature.

Results and discussion

Out of the 38 Antarctic yeast strains investigated forexopolysaccharide biosynthesis, eight were selected.They were representatives of the Sporobolomyces andCryptococcus genera reported for polysaccharide pro-ducers by other authors as well (Adami & Cavazzoni1990; Elinov et al. 1992). The yeast was cultivated on abasal medium with sucrose, glucose and fructose ascarbon sources in 4.0% concentration and the resultsshowed that the strains synthesized different amountsof polysaccharides extracellularly (Table 1). For thecultures investigated, sucrose proved to be the mostsuitable carbon source for polysaccharide biosynthesis,

436 K. Pavlova et al.

and the highest yields were obtained by the Sp. salmo-nicolor AL1 strain: 5.18 g/l, and the Sp. salmonicolorAL36 strain: 5.03 g/l. Both strains also synthesizedconsiderable amounts of polysaccharide on a fructose-containing medium, i.e. Sp. salmonicolor AL1: 4.80 g/l,Sp. salmonicolor AL36: 4.68 g/l, whereas on a glucose-containing medium the yields were 3.12 g/l and 3.76 g/lrespectively. The strains of the Cryptococcus genussynthesized lower amounts of biopolymer on a su-crose-containing medium. The pH values at the end ofthe process were similar for all cultures studied (pH1.68–2.27).The Sp. salmonicolor AL1 strain, being the best

exopolysaccharide producer, was studied dynamicallyon a medium containing 3.0, 4.0 and 5.0% of sucrose forexopolysaccharide biosynthesis, biomass accumulation,carbon source assimilation and pH change during thestrain development process (Figure 1A–C). The polymerbiosynthesis started at the twenty-fourth hour corre-sponding to the end of the logarithmic phase or thebeginning of the stationary phase of the producerdevelopment. The maximum exopolysaccharide forma-tion was observed at the hundred-and-twentieth hour,then the polymer content decreased slowly, except forthe medium containing 3.0% of sucrose. In the courseof the polysaccharide biosynthesis by Sp. salmonicolorAL1, sucrose assimilation was observed. The mostcomplete assimilation occurred after 72 h on a 3.0%sucrose-containing medium, after 168 h on a 4.0%sucrose-containing medium, whereas 1.0% of the car-bon source was left unassimilated at the end of thefermentation on a 5.0% sucrose-containing medium. Sp.salmonicolor AL1 was most productive on a 5.0%sucrose-containing medium: 5.53 g of polysaccharide/l.A typical feature of the formation of exopolysaccha-

rides by yeast is the significant pH change, which provesto be a regulating factor in their biosynthesis (Heald &Kristiansen 1985; Adami & Cavazzoni 1990; Elinovet al. 1992). In the course of its metabolism, the Sp.salmonicolor AL1 strain studied changed the culturemedium pH from the initial pH 5.3 to pH 1.7–2.0 after24 h, and these pH values were preserved until fermen-tation was complete. Thus the polysaccharide biosyn-thesis took place at low pH values.The effect of several nitrogen sources on exopolysac-

charide production by Sp. salmonicolor AL1 was exam-

ined (Table 2). The yeast strain was incubated at 22 �Cfor 168 h in the media mentioned previously. Thecomparison of the influence of different ammoniumsalts on the yeast growth and metabolic productionshowed that NHþ

4 played a role in nitrogen metabolism

Table 1. Biosynthesis of polysaccharides on media with different sugars.

Strains Sucrose 4% Glucose 4% Fructose 4%

Polysacch. (g/l) Final pH Polysacch. (g/l) Final pH Polysacch. (g/l) Final pH

Sporobolomyces salmonicolor AL1 5.18 1.83 3.12 1.94 4.80 1.88

Sporobolomyces salmonicolor AL36 5.03 2.02 3.76 2.11 4.68 2.02

Cryptococcus albidus 16-1 4.58 2.20 1.26 2.22 2.72 2.13

Cryptococcus laurentii 16-2 4.06 2.01 2.34 2.16 2.52 2.04

Cryptococcus allbidus AL3 4.10 1.98 2.68 2.27 2.72 2.32

Cryptococcus sp. AL14 3.90 1.72 3.40 1.68 3.46 1.65

Cryptococcus sp. Al18 3.92 2.05 1.20 2.10 2.28 2.08

Cryptococcus sp. AL27 3.98 2.18 1.82 2.24 3.57 2.18

Time (h)

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Figure 1. Time course of polysaccharide synthesis, pH, cell growth

and sucrose consumption. (A) the cultural medium with 3% sucrose,

(B) the cultural medium with 4% sucrose, (C) the cultural medium

with 5% sucrose. (-m-) exopolysaccharide; (-,-) pH; (-s-) biomass;

(-d-) residual sugar.

Exopolysaccharide by yeast 437

as the form in which nitrogen is incorporated intoorganic cell components (biomass). As seen from thepolysaccharide yields, a low concentration of ammo-nium salts ensured the highest biomass production butexopolysaccharide extraction was impossible. Thegrowth with NHþ

4 was accompanied by a rapid decreasein pH values when NHþ

4 components were utilized. Thehighest exopolysaccharide yield and viscosity of theculture broth were obtained with a 0.25% concentrationof ammonium salts but no correspondence with thebiomass yield was established.The dynamic viscosity values of the cell-free culture

broth during the exopolysaccharide synthesis by Sp. sal-monicolor AL1 on media containing different sucrosepercentages and 0.25% of (NH4)2SO4 are shown inFigure 2. The culture broth had low viscosity whenthe strain was cultivated on a 3.0% sucrose-containingmedium. Dynamic viscosity appeared to be associatedwith cell physiology and exopolysaccharide production.Carbon sources in low concentrations were not suitable

for exopolysaccharide synthesis by Sp. salmonicolorAL1, because the carbon was almost all used forbiomass production. The cell-free culture broth exhi-bited high viscosity values during exopolysaccha-ride synthesis in the 4.0 and 5.0% sucrose containingmedia. The viscosity reached a maximum of 14.948 and15.367 mPa s after 120 h of yeast incubation on thesesubstrates and coincided with the highest polysaccharideand biomass synthesis.The dynamic viscosity of the culture broth containing

the exopolysaccharide synthesized by the Sp. salmoni-color AL1 psychrophilic strain was 1.5 times as high asthe viscosity obtained by the mesophilic Rh. acheniorumMC (Grigorova et al. 1999), the fermentation of bothstrains having occurred under identical circumstancesregarding carbon and nitrogen sources and at differenttemperatures of 22 and 26 �C depending on the taxo-nomic identification of the strains.The investigation of the rheological properties of the

exopolysaccharide synthesized is related to its potentialapplication as a thickener or gelatinizer in the foodindustry, therefore its flow behaviour kinetics wasestablished.The Power Law Model for a non-Newtonian flow is

known as the Oswald de Waele equation s ¼ K ÆDnr

where K is the consistency coefficient (factor) Pa ÆSn, Dr

is the shear rate per second, s is the shear stress Pa, n isthe flow behaviour index. The value of n ¼ 0.481 showsthat the water solution of the polysaccharide is charac-terized by a decrease in the apparent viscosity withthe increase in Dr. ga ¼ KÆDn�1

r , ga ¼ 4.01ÆD0:481�1r ¼

4.01ÆD�0:519r . The resultant flow behaviour index value

characterizes the yeast product as a polysaccharide withpseudoplastic behaviour and provides the reason for itsbeing studied as a thickener (Figure 3).The chemical composition of the crude and pure

exopolysaccharide is shown in Table 3. As seen from thedata, the crude polysaccharide is of exceptionally highpurity: 90.16%, and contains mostly glucose and man-

Table 2. Effect of several nitrogen sources on polysaccharide production.

Nitrogen source Concentration (%) Final pH Biomass (g/l)a Polysaccharides (g/l)a

(NH4)2SO4 0.100 2.20 6.53 ± 0.12 4.80 ± 0.09

0.150 2.08 5.87 ± 0.13 5.03 ± 0.08

0.200 2.00 5.60 ± 0.10 5.32 ± 0.10

0.250 1.98 5.33 ± 0.11 5.63 ± 0.11

0.300 1.88 4.50 ± 0.13 5.40 ± 0.09

NH4Cl 0.100 2.07 6.42 ± 0.20 1.62 ± 0.08

0.150 1.94 6.12 ± 0.16 2.42 ± 0.09

0.200 1.88 6.04 ± 0.14 2.88 ± 0.10

0.250 1.74 5.80 ± 0.15 3.08 ± 0.11

0.300 1.60 5.00 ± 0.16 3.12 ± 0.11

NH4NO3 0.100 2.00 6.24 ± 0.13 2.30 ± 0.08

0.150 1.98 6.03 ± 0.15 3.01 ± 0.08

0.200 1.88 5.82 ± 0.14 4.23 ± 0.10

0.250 1.83 5.43 ± 0.14 4.50 ± 0.12

0.300 1.77 5.07 ± 0.13 4.45 ± 0.13

a Values represent two determinations per analysis.

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Figure 2. Dynamic viscosity of the cell-free culture broth during

polysaccharide synthesis on different percentages of sucrose.

438 K. Pavlova et al.

nose. Proteins and minerals are present in minimumquantities of 5.30 and 4.54% respectively. The quanti-tative monosaccharide composition comprises 54.1% ofglucose, 42.6% of mannose and 3.3% of fucose.Mannan containing 98.6% of mannose was isolated

through precipitation using a Fehling reagent. Ourresults coincide with the ones obtained for purifying apolysaccharide synthesized by Sp. salmonicolor BKM-Y-679 using a Fehling reagent (Elinov et al. 1992).In conclusion, under the cultivation conditions estab-

lished, with suitable carbon and nitrogen sources andlow culture broth pH during fermentation, the Sp.salmonicolor AL1 strain synthesized a heteropolysaccha-ride. The rheological properties of the polysaccharide(culture broth dynamic viscosity and flow behaviourindex) suggest that it may have applicability to the foodindustry.

Acknowledgements

This research was financed by the National ScienceFoundation of Bulgaria.

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Table 3. Chemical composition of the exopolysaccharides synthesized

by Sp. salmonicolor AL1.

Polysaccharide Crude Purified

Carbohydrate (%) 90.2 ± 1.9 98.9 ± 0.2

Protein (%) 5.3 ± 0.25 0.8 ± 0.1

Ash (%) 4.5 ± 3.0 0.3 ± 0.1

Monosaccharides

(% of carbohydrate content)

Glucose 54.1 1.4

Mannose 42.6 98.6

Fucose 3.3 –

Values represent two determinations per analysis.

Dr (s-1)

0 200 400 600 800 1000 1200 1400

0

20

40

60

80

100

120

140

160

K = 4.01 ± 1.0 Pa.Sn

n = 0.481 ± 0.0028

R = 0.992

τ (P

a)

Figure 3. Determining the flow behaviour constants in the Oswald de

Waele equation s ¼ KÆDnr .

Exopolysaccharide by yeast 439