World Journal of Microbiology & Biotechnology 20: 435439, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

435

Production and characterization of an exopolysaccharide by yeast

K. Pavlova1,*, L. Koleva2, M. Kratchanova3 and I. Panchev2 1 Laboratory of Applied Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria 2 University of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria 3 Institute 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 salmonicolor AL1 strain was selected. It was studied for exopolysaccharide biosynthesis on dierent carbon and nitrogen sources. The investigations showed that sucrose and ammonium sulphate were suitable culture medium components for polymer biosynthesis. Exopolysaccharide formation by the yeast strain was accompanied by a decrease in the culture medium pH value from the initial pH 5.3 to pH 1.72.0. During the biosynthetic process, the dynamic viscosity of the culture broth increased to the maximum value of 15.37 mPa s and the polysaccharide yield reached 5.63 g/l on a culture medium containing 5.00% sucrose and 0.25% ammonium sulphate at a temperature of 22 C for 120 h. The crude polysaccharide obtained from Sp. salmonicolor AL1 featured high purity (90.16% of carbon content) and consisted of glucose (54.1%), mannose (42.6%) and fucose (3.3%). Pure mannan containing 98.6% of mannose was isolated from it. Introduction Microorganisms of dierent taxonomic groups possess the ability to synthesize exopolysaccharides with interesting physicochemical properties and functional characteristics. Some microbial polysaccharides are characterised by high viscosity and good gelating properties; they have a synergistic eect when interacting with other polysaccharides and combine with dierent salts within a wide pH and temperature range. The dierences in the structure and properties of individual polysaccharides used as food additives account for the diverse functions they perform as thickeners, emulsiers, leavenings, and frothers. Commercially used biopolymers are bacterial and fungal products such as xanthan, dextran and scleroglucan (Shimada et al. 1977; Margaritis & Pace 1985; Paul et al. 1986; Crescenzi 1995; Sutherland 1998). Yeasts belonging to the dierent Cryptococcus, Hansenula, Rhodotorula, Lipomyces, Bullera, Aureobasidium, and Sporobolomyces genera can synthesize exopolysaccharides. The polymer types reported for yeast producers 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 yeastproduced exopolysaccharides are more easily separated from 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 composition and the growth conditions. Studies on the eect of growth-limiting substrates on exopolysaccharide synthesis clearly demonstrate that the growth medium composition can dramatically aect the specic rate of polymer synthesis (Heald & Kristiansen 1985; Lacroix et al. 1995). The amount of carbon substrate converted to polymer by the microbial cells depends on the growth medium composition. Generally, media containing a high carbon-to-limiting-nutrient ratio are favoured for polysaccharide production (Sutherland 1998). The carbon and nitrogen sources used do not aect the qualitative monosaccharide composition of the polysaccharide, although the introduction of ammonium salts into the medium can cause a change in the monosaccharide ratio in the side chains of the polymer (Vitovskaya et al. 1989). The growth medium composition can also indirectly aect the polymer yield, for example, by governing the pH change which can occur during fermentation without pH control (Heald & Kristiansen

436 1985; Lacroix et al. 1995). In our previous papers, a Rh. acheniorum MC strain was selected as a suitable mannan exopolysaccharide producer, an optimal culture medium was established and their rheology in a mixture with xanthan was investigated (Grigorova et al. 1999; Pavlova & Grigorova 1999). The object of this paper was to study the exopolysaccharide-producing capacity of psychrophilic yeast, to select an active producer, to investigate the patterns in the biosynthetic process, the dynamic viscosity and its relationship to cell physiology and polymer composition.

K. Pavlova et al. The exopolysaccharides in the culture supernatants were precipitated with 2 vol. of 96% ethanol at 4 C for 18 24 h. The resultant supernatant was discarded and the pellet was washed twice with ethanol, dried and weighed. Analytical methods The dry weight of the yeast biomass was determined by heating at 105 C until constant weight. The residual sugar concentration was measured by the 3,5 dinitrosalicylic acid method (Chaplin & Kennedy 1986). The total carbohydrate amount in the crude exopolysaccharides was determined using the phenolsulphuric acid method (Dubois et al. 1956). The protein amount in the solution of non-hydrolyzed polysaccharides was determined according to the Lowry method. The ash content was estimated after calcination for 2 h and glowing the polymer at 550 C for 3 h. The dynamic viscosity of the cell-free culture broth was measured using a Rheotest-2 Hoppler type viscometer at a temperature of 25 C, K 0.00463212, P 10 g/cm2. The data were statistically analysed using Sigma 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-2 rheoviscometer, Germany, using measuring cylinder N of the device N (R/r) 1.02. The numerical values of the velocity gradient Dr and the modulus of shearing were processed using the Oswald de Waele equation (Holdsworth 1993) on a REO computer program written in FORTRAN 77. The carbohydrate composition was determined after hydrolysis of the crude and pure exopolysaccharides with M H2SO4 at 105 C for 8 h, neutralization with barium hydroxide followed by centrifugation and elimination of the Ba ions using a Wofatit KPS cation exchanger. The monosaccharide content was determined with HPLC on Waters with an R 401 dierential refractometer on a carbohydrate column. The mobile phase was acetonitrile:water (87:13), with ow rate of 0.6 ml/min and ambient temperature.

Materials and methods Microorganisms The Sporobolomyces salmonicolor AL1 strain was chosen by multistage selection out of 38 yeast strains isolated from soil lichen, moss and soil taken from the region of the Bulgarian base on Livingston Island, Antarctica. The selection was made according to the ability of the strains to synthesize polysaccharides on synthetic media with glucose as a carbon source. The exopolysaccharideproducing strain was identied using the yeast classication criteria proposed by Kurtzman & Fell (1998) and was registered in the National Bank for Industrial Microorganisms and Cell Culture, Bulgaria. The culture was maintained on a malt slant agar at 4 C. Media and growth conditions The basal medium for the testing of carbon sources contained (w/v): 0.20% (NH4)2SO4, 0.1% KH2PO4, 0.05% MgSO47H2O, 0.01% NaCl, 0.01% CaCl22H2O and 0.1% yeast extract. Glucose, sucrose and fructose were tested as carbon sources and were added to the basal medium in dierent concentrations. The basal medium for the testing of nitrogen sources contained (w/v): 4.0% sucrose, 0.1% KH2PO4, 0.01% NaCl, 0.05% MgSO47H2O, 0.01% CaCl22H2O, 0.1% yeast extract and dierent nitrogen sources. (NH4)2SO4, NH4Cl and (NH4)NO3 were tested as nitrogen sources and were added to the basal medium in dierent concentrations. The initial pH was adjusted to pH 5.3 and the media were sterilized at 112 C for 30 min. The inoculum from Sp. salmonicolor AL1 was obtained on a rotary shaker (220/min) in 500 ml Erlenmeyer asks containing 50 ml of Sabouraud medium (Merck,Germany) at 22 C for 48 h. The fermentation media were inoculated with 1.0% w/v of inoculum. The cultivation was carried out in 500 ml Erlenmeyer asks containing 50 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 for 30 min to separate the yeast cells from the supernatant.

Results and discussion Out of the 38 Antarctic yeast strains investigated for exopolysaccharide biosynthesis, eight were selected. They were representatives of the Sporobolomyces and Cryptococcus genera reported for polysaccharide producers by other authors as well (Adami & Cavazzoni 1990; Elinov et al. 1992). The yeast was cultivated on a basal medium with sucrose, glucose and fructose as carbon sources in 4.0% concentration and the results showed that the strains synthesized dierent amounts of polysaccharides extracellularly (Table 1). For the cultures investigated, sucrose proved to be the most suitable carbon source for polysaccharide biosynthesis,

Exopolysaccharide by yeast
Table 1. Biosynthesis of polysaccharides on media with dierent sugars. Strains Sucrose 4% Polysacch. (g/l) Sporobolomyces salmonicolor AL1 Sporobolomyces salmonicolor AL36 Cryptococcus albidus 16-1 Cryptococcus laurentii 16-2 Cryptococcus allbidus AL3 Cryptococcus sp. AL14 Cryptococcus sp. Al18 Cryptococcus sp. AL27 5.18 5.03 4.58 4.06 4.10 3.90 3.92 3.98 Final pH 1.83 2.02 2.20 2.01 1.98 1.72 2.05 2.18 Glucose 4% Polysacch. (g/l) 3.12 3.76 1.26 2.34 2.68 3.40 1.20 1.82 Final pH 1.94 2.11 2.22 2.16 2.27 1.68 2.10 2.24 Fructose 4% Polysacch. (g/l) 4.80 4.68 2.72 2.52 2.72 3.46 2.28 3.57

437

Final pH 1.88 2.02 2.13 2.04 2.32 1.65 2.08 2.18

and the highest yields were obtained by the Sp. salmonicolor AL1 strain: 5.18 g/l, and the Sp. salmonicolor AL36 strain: 5.03 g/l. Both strains also synthesized considerable amounts of polysaccharide on a fructosecontaining medium, i.e. Sp. salmonicolor AL1: 4.80 g/l, Sp. salmonicolor AL36: 4.68 g/l, whereas on a glucosecontaining medium the yields were 3.12 g/l and 3.76 g/l respectively. The strains of the Cryptococcus genus synthesized lower amounts of biopolymer on a sucrose-containing medium. The pH values at the end of the process were similar for all cultures studied (pH 1.682.27). The Sp. salmonicolor AL1 strain, being the best exopolysaccharide producer, was studied dynamically on a medium containing 3.0, 4.0 and 5.0% of sucrose for exopolysaccharide biosynthesis, biomass accumulation, carbon source assimilation and pH change during the strain development process (Figure 1AC). The polymer biosynthesis started at the twenty-fourth hour corresponding to the end of the logarithmic phase or the beginning of the stationary phase of the producer development. The maximum exopolysaccharide formation was observed at the hundred-and-twentieth hour, then the polymer content decreased slowly, except for the medium containing 3.0% of sucrose. In the course of the polysaccharide biosynthesis by Sp. salmonicolor AL1, sucrose assimilation was observed. The most complete 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 carbon source was left unassimilated at the end of the fermentation 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 exopolysaccharides by yeast is the signicant pH change, which proves to be a regulating factor in their biosynthesis (Heald & Kristiansen 1985; Adami & Cavazzoni 1990; Elinov et al. 1992). In the course of its metabolism, the Sp. salmonicolor AL1 strain studied changed the culture medium pH from the initial pH 5.3 to pH 1.72.0 after 24 h, and these pH values were preserved until fermentation was complete. Thus the polysaccharide biosynthesis took place at low pH values. The eect of several nitrogen sources on exopolysaccharide production by Sp. salmonicolor AL1 was exam-

5 Exopolysaccharide (g/L) 4 3 pH 2 1 0

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 0 24 48 72 96 120 144 168 Time (h)

6 5 Biomass dw (g/L) 4 3 2 1

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Residual sugar (%)

(A)

6 Exopolysaccharide (g/L) 5 4 pH 3 2 1 0

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

6 5 4 3 2 1 0 Biomass dw (g/L)

5 Residual sugar (%) Residual sugar (%) 4 3 2 1 0

(B)
0 24 48 72 96 120 144 168 192 Time (h)

6 Exopolysaccharide (g/L) 5 4 pH 3 2 1 0

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

7 6 Biomass dw (g/L) 5 4 3 2 1

6 5 4 3 2 1 0

(C)
0 24 48 72 Time (h)

0 96 120 144 168 192

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.

ined (Table 2). The yeast strain was incubated at 22 C for 168 h in the media mentioned previously. The comparison of the inuence of dierent ammonium salts on the yeast growth and metabolic production showed that NH played a role in nitrogen metabolism 4

438
Table 2. Eect of several nitrogen sources on polysaccharide production. Nitrogen source (NH4)2SO4 Concentration (%) 0.100 0.150 0.200 0.250 0.300 0.100 0.150 0.200 0.250 0.300 0.100 0.150 0.200 0.250 0.300 Final pH 2.20 2.08 2.00 1.98 1.88 2.07 1.94 1.88 1.74 1.60 2.00 1.98 1.88 1.83 1.77 Biomass (g/l)a 6.53 5.87 5.60 5.33 4.50 6.42 6.12 6.04 5.80 5.00 6.24 6.03 5.82 5.43 5.07 0.12 0.13 0.10 0.11 0.13 0.20 0.16 0.14 0.15 0.16 0.13 0.15 0.14 0.14 0.13

K. Pavlova et al.

Polysaccharides (g/l)a 4.80 5.03 5.32 5.63 5.40 1.62 2.42 2.88 3.08 3.12 2.30 3.01 4.23 4.50 4.45 0.09 0.08 0.10 0.11 0.09 0.08 0.09 0.10 0.11 0.11 0.08 0.08 0.10 0.12 0.13

NH4Cl

NH4NO3

Values represent two determinations per analysis.

as the form in which nitrogen is incorporated into organic cell components (biomass). As seen from the polysaccharide yields, a low concentration of ammonium salts ensured the highest biomass production but exopolysaccharide extraction was impossible. The growth with NH was accompanied by a rapid decrease 4 in pH values when NH components were utilized. The 4 highest exopolysaccharide yield and viscosity of the culture broth were obtained with a 0.25% concentration of ammonium salts but no correspondence with the biomass yield was established. The dynamic viscosity values of the cell-free culture broth during the exopolysaccharide synthesis by Sp. salmonicolor AL1 on media containing dierent sucrose percentages and 0.25% of (NH4)2SO4 are shown in Figure 2. The culture broth had low viscosity when the strain was cultivated on a 3.0% sucrose-containing medium. Dynamic viscosity appeared to be associated with cell physiology and exopolysaccharide production. Carbon sources in low concentrations were not suitable

18 16 Dynamic viscosity (MPa.s) 14 12 10 8 6 4 2 0 0 24 48 72 96 120 Time (h) 144 168 192 5% 4% 3%

Figure 2. Dynamic viscosity of the cell-free culture broth during polysaccharide synthesis on dierent percentages of sucrose.

for exopolysaccharide synthesis by Sp. salmonicolor AL1, because the carbon was almost all used for biomass production. The cell-free culture broth exhibited high viscosity values during exopolysaccharide synthesis in the 4.0 and 5.0% sucrose containing media. The viscosity reached a maximum of 14.948 and 15.367 mPa s after 120 h of yeast incubation on these substrates and coincided with the highest polysaccharide and biomass synthesis. The dynamic viscosity of the culture broth containing the exopolysaccharide synthesized by the Sp. salmonicolor AL1 psychrophilic strain was 1.5 times as high as the viscosity obtained by the mesophilic Rh. acheniorum MC (Grigorova et al. 1999), the fermentation of both strains having occurred under identical circumstances regarding carbon and nitrogen sources and at dierent temperatures of 22 and 26 C depending on the taxonomic identication of the strains. The investigation of the rheological properties of the exopolysaccharide synthesized is related to its potential application as a thickener or gelatinizer in the food industry, therefore its ow behaviour kinetics was established. The Power Law Model for a non-Newtonian ow is known as the Oswald de Waele equation s K Dn r where K is the consistency coecient (factor) Pa Sn, Dr is the shear rate per second, s is the shear stress Pa, n is the ow behaviour index. The value of n 0.481 shows that the water solution of the polysaccharide is characterized by a decrease in the apparent viscosity with the increase in Dr. ga KDn1 , ga 4.01D0:4811 r r 4.01D0:519 . The resultant ow behaviour index value r characterizes the yeast product as a polysaccharide with pseudoplastic behaviour and provides the reason for its being studied as a thickener (Figure 3). The chemical composition of the crude and pure exopolysaccharide is shown in Table 3. As seen from the data, the crude polysaccharide is of exceptionally high purity: 90.16%, and contains mostly glucose and man-

Exopolysaccharide by yeast
160 140 120 100 (Pa) 80 60 40 20 0 0 200 400 600 Dr (s-1) 800
K = 4.01 1.0 Pa.S n = 0.481 0.0028 R = 0.992
n

439 References
Adami, A. & Cavazzoni, V. 1990 Exopolysaccharides produced by some yeast strains. Annali di Microbiologia ed Enzimologia 40, 245 253. Chaplin, M.F. & Kennedy, J.F. 1986 Carbohydrate Analysis: A Practical Approach UK: IRL Press. ISBN 0-94794644-6. Chiura, H., Iizuka, M. & Yamamoto, T. 1982a A glucomannan as an extracellular product of Candida utilis, I. Production and characterization of a glucomannan. Agricultural and Biological Chemistry 46, 17231733. Chiura, H., Iizuka, M. & Yamamoto, T. 1982b A glucomannan as an extracellular product of Candida utilis II. Structure of a glucomannan: characterization of olygosaccharides obtained by partial hydrolysis. Agricultural and Biological Chemistry 46, 17331742. Crescenzi, V. 1995 Microbial polysaccharides of applied interest: ongoing research activities in Europe. Biotechnology Progress 11, 251259. Dubois, M., Gilles, K., Hamilton, Y., Robers, P. & Smith, F. 1956 Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350356. Elinov, N.P., Ananyeva, E.P. & Vitovskaya, G.A. 1992 Features of the biosynthesis and characteristics of exoglycans in yeasts of the genus Sporobolomyces Microbiology (Engl. transt. of Mikrobiologiya) 60, 466470. Grigorova, D., Pavlova, K. & Panchev, I. 1999 Preparation and preliminary characterization of exopolysaccharides by yeast Rhodotorula acheniorum MC. Applied Biochemistry and Biotechnology 81, 181191. Heald, P.J. & Kristiansen, B. 1985 Synthesis of polysaccharide by yeast-like forms of Aureobasidium pullulans. Biotechnology and Bioengineering 27, 15161519. Holdsworth, S.D. 1993 Rheological models used for the prediction of the ow properties of food products. Transactions of the Institute of Chemical Engineers part C, 71, 139179. Kurtzman, C.P. & Fell, J.W. 1998 The Yeast: A Taxonomic Study, 4th edn. Amsterdam (Netherlands): Elsevier Scientic Publisher. ISBN 0-444-81312-8. Lacroix, C., LeDuy, A., Noel, G. & Choplin, L. 1995 Eect of pH on the batch fermentation of pullulan from sucrose medium. Biotechnology and Bioengineering 27, 202207. Margaritis, A. & Pace, G.W. 1985 Microbial polysaccharides. Comprehensive Biotechnology, vol. 3, pp. 10051041. Oxford, UK: Pergamon Press. Paul, F., Morin, P. & Monsan, P. 1986 Microbial polysaccharides with actual potential industrial applications. Biotechnology Advances 4, 45259. Pavlova, K. & Grigorova, D. 1999 Production and properties of exopolysaccharides by Rhodotorula acheniorum MC. Food Research International 32, 473477. Peterson, G.R., Nelson, G.A., Cathey, C.A. & Fuller, G.G. 1989 Rheologically interesting polysaccharides from yeasts. Applied Biochemistry and Biotechnology 20/21, 845867. Peterson, G.R., Schubert, W.W., Richards, G.F. & Nelson, G.A. 1990 Yeasts producing exopolysaccharides with drag-reducing activity. Enzyme Microbiology and Technology 12, 255259. Shimada, A., Nanata, H. & Nakamura, I. 1977 Acidic exopolysaccharide produced by Enterobacter sp. Journal of Fermentation and Bioengineering 84, 113118. Sutherland, I.W. 1998 Novel and established applications of microbial polysaccharides. Trends in Biotechnology 16, 4145. Vitovskaya, G.A., Samarkina, G.M., Ananyeva, E.P. & Sinitskaya, A. 1989 Synthesis of extracellular heteroglucans by yeasts of the genus Cryptococcus Microbiology (Engl. transt. of Mikrobilogiya) 58, 185190. Vorotynskaya, S.L., Vitovskaya, G.A. & Ananyeva, E.P. 1992 Studies on the properties of polysaccharides produced by the yeasts Cryptococcus luteolus (Saito) Skinner. Mikologiya i Fitopatologiya 26, 367371.

1000

1200

1400

Figure 3. Determining the ow behaviour constants in the Oswald de Waele equation s KDn . r

Table 3. Chemical composition of the exopolysaccharides synthesized by Sp. salmonicolor AL1. Polysaccharide Carbohydrate (%) Protein (%) Ash (%) Monosaccharides (% of carbohydrate content) Glucose Mannose Fucose Crude 90.2 1.9 5.3 0.25 4.5 3.0 Puried 98.9 0.2 0.8 0.1 0.3 0.1

54.1 42.6 3.3

1.4 98.6

Values represent two determinations per analysis.

nose. Proteins and minerals are present in minimum quantities of 5.30 and 4.54% respectively. The quantitative monosaccharide composition comprises 54.1% of glucose, 42.6% of mannose and 3.3% of fucose. Mannan containing 98.6% of mannose was isolated through precipitation using a Fehling reagent. Our results coincide with the ones obtained for purifying a polysaccharide synthesized by Sp. salmonicolor BKMY-679 using a Fehling reagent (Elinov et al. 1992). In conclusion, under the cultivation conditions established, with suitable carbon and nitrogen sources and low culture broth pH during fermentation, the Sp. salmonicolor AL1 strain synthesized a heteropolysaccharide. The rheological properties of the polysaccharide (culture broth dynamic viscosity and ow behaviour index) suggest that it may have applicability to the food industry.

Acknowledgements This research was nanced by the National Science Foundation of Bulgaria.