Mycol. Res.

101 (6) : 650652 (1997)

Printed in Great Britain

650

The ability of the yeast form of Aureobasidium pullullans to elaborate exopolysaccharide in chemostat culture at various pH values

M. R E E S L E V*, T. S T R M, B. J E N S E N A N D J. O L S E N
Department of General Microbiology, University of Copenhagen, Slvgade 83 H DK-1307 Copenhagen, Denmark

The yeastmycelial dimorphic Aureobasidium pullulans var. pullulans was grown in chemostat culture under zinc-limitation at pH values in the range 27. Steady state was obtained and the growth yield with respect to zinc was independent of pH. The culture grew entirely as yeast, independently of the pH in the pH interval 37. The exopolysaccharide (EPS) production from the yeast cells was highest in the pH range 36 with a maximum at pH 4n0. Only little EPS was produced a pH 7n0 and no EPS was produced at pH 2n1. At pH 2n1 about 20 % of the total biomass was in the lamentous growth form and some chlamydospores were present. A change in the carbon source from glucose to sucrose resulted in a more than doubling of the steady-state EPS concentration from 3n1 to 6n9 g l". A simple mass balance on carbon was constructed which could account for more than 95 % of the carbon.

Aureobasidium pullulans is able to grow both as yeast and as mycelium and has a number of intermediary and resting stages like swollen cells, pseudomycelium and chlamydospores in its life cycle (Ramos & Garcia-Acha, 1975). It can elaborate the exopolysaccharide (EPS) pullulan. Pullulan synthesis is inducible and is believed to be dierently expressed by the two growth forms, the yeast cells (blastospores) being the main producers (Catley, 1980). pH has been shown to aect both the EPS production as well as the ratio between yeast and mycelium in batch (Seviour, Kristiansen & Harvey, 1984 ; Heald & Kristiansen, 1985) and in chemostat culture (McNeil, Kristiansen & Seviour, 1989). Thus, these results raise the question as to whether the variable EPS elaboration at dierent pH values is due to a direct eect of pH on the ability of the cells to elaborate EPS or to the eect of pH on the proportion of yeast and mycelium, or to a combination of both. In chemostat culture performed at pH 5n0 the proportion of the two growth forms could be completely reversed, being more than 90 % yeast in zinc-limited cultures and more than 90 % mycelial in cultures with a surplus of zinc (carbon-limited) (Reeslev, Jrgensen & Jrgensen, 1993). The aim of the present work was to investigate whether it would be possible to obtain a culture of pure yeast cells growing under steady-state conditions independent of pH, by keeping the chemostat culture zinclimited. This would allow evaluation of the eect of pH on the production of EPS by the yeast form.

MATERIALS AND METHODS Microbial strain and culture media Aureobasidium pullulans (deBary) G. Arnaud var. pullulans QM 3092 was used in all experiments. This strain was also used by Reeslev et al. (1991), where it regrettably was named ATCC 3092. Continuous culture was performed in a simple mineral medium containing (g l" distilled water) : glucose 15n0, (NH ) SO 3n6, KH PO 2n98, NaCl 1n5, MgSO ;7H O 0n2, %# % # % % # citric acid monohydrate 0n34 and the following components (mg l" distilled water) : FeCl 6H O 4n80, ZnCl 1n04, $ # # CaCl ;2H O 18n60, MnCl ;2H O 1n11, CuCl ;2H O 0n27, # # # # # # NaMoO ;2H O 0n22, CoCl ;6H O 0n28, H BO 0n40, Kl % # # # $ $ 0n06. Antifoaming agent Synperonic PE\L61 (J. Lorentzen A\S, / Kvistgard, Denmark) was added to a concentration of 0n07 g l". In experiments with dierent carbon sources, glucose, sucrose, fructose or a 1 : 1 mixture of glucose and fructose were used in concentrations as stated in Results and Discussion. The distilled water was prepared by distillation in an all-glass apparatus from water that had been demineralized by passage through an ion exchange column. The pH of the medium in the growth tank was adjusted to 2n5 using HCl, which prevented precipitation of e.g. Fe(OH) . Except for $ Synperonic, all chemicals used were of analytical grade. The carbon source was autoclaved separately and added to the sterile medium. The medium was sterilized by autoclaving at 121 mC.

* Corresponding author.

M. Reeslev and others

Biomass & exopolysaccharide (g D.W. 11 )

651
6 5 4 3 2 1 2 3 4 pH 5 6 7 6 5 4 3 2 1 Residual glucose (g 11 )

Cultural conditions The chemostat experiments employed a 2 l fermenter (LH Fermentation Ltd, U.K.) with two six-bladed impellers. The dilution rate was 0n08 h" ; temperature, 27m ; working volume was typically about 1600 ml ; rate of aeration, 1n6 l min" ; rate of agitation, 1200 rpm. At each steady state the pH was maintained constant by automatic titration with 2n0 aqueous NaOH. At least 10 mean residence times were allowed to pass after inoculation and at least six after changes of the external pH, before sampling was initiated. Samples were collected on three consecutive days after which the constancy of the data was evaluated. The performance of the experiments at dierent pH values was made in random order. Quantitative determination of biomass dry weight, morphology and EPS The dry weight (..) of EPS and total biomass were determined as described previously (Reeslev et al., 1991). No attempts were made to determine the pullulan content of the EPS. Yeast and mycelium were separated by gravity ltration through a nylon mesh of 41 m square porosity (Reeslev et al., 1991). From microscopical observations of the ltrate it was ensured that only yeast cells were present. Determinations of the dry weight of the yeast cells were made from measurements of the absorbance at 550 nm (Reeslev et al., 1991). The concentration of glucose in the culture supernatant was measured using the hexokinase\ glucose 6-phosphate dehydrogenase method (Gluco-quant Test-Combination, Boehringer Mannheim, Germany). RESULTS AND DISCUSSION Growth and carbon balance A. pullulans was grown at various pH values in chemostat culture in a dened medium ; steady state was obtained. The experiments were designed in order to obtain zinc-limited growth. Under the present experimental conditions this was obtained by using 0n85 ZnCl in the growth medium, as # control experiments with steady-state culture at pH 3n0, 5n0 and 7n0 had shown that an increase in ZnCl to 1n3 resulted # in an increase in the total biomass from about 5n0 to about 7n2 g .. l". The apparent discrepancy between the results from the present experiments and those of Reeslev, Jrgensen & Jrgensen (1993) in which a concentration of 0n80 ZnCl # resulted in primarily glucose-limited growth may be explained by dierences in the contribution of zinc from dierent levels of impurities from compounds of the medium. Thus, Failla & Weinberg (1977) found that the mineral salts contributed approximately 0n1 zinc to their medium while a contribution of 0n6 was determined by Lawford et al. (1980). Furthermore, the contribution of zinc to the medium from the corrosion of metal parts of the bioreactor may dier as dierent bioreactors were used. Due to the very high growth yield with respect to zinc even small changes in the level of zinc impurities will, when the culture is growing under zinclimitation, alter the steady-state biomass considerably. At constant pH in the interval from pH 2n1 to 7n0 the

Fig. 1. The inuence of pH on morphology and EPS production of A. pullulans grown in zinc-limited chemostat culture with a feed concentration of glucose of 15 g l". Steady-state levels of : total biomass (
), yeast biomass ( ), exopolysaccharide (>), and residual glucose ($). Each point represents the mean of three measurements performed on three subsequent days ; the bars give the highest and lowest values measured and when not shown were smaller than the dimensions of the symbols.

steady-state total biomass concentration was constant at about 5 g .. l" (Fig. 1), which shows that pH did not inuence the growth yield with respect to zinc. At pH 8 no steady state was obtained as the biomass continued to decrease and the fungus was ultimately washed out. At pH 2n1 some chlamydospores were present which gave the broth, which was otherwise white, a light greyish-green colour. No attempts were made to cultivate below pH 2n1. We have calculated the carbon balance by using the following equation : X Si l Ssj jP, Y where Si and Ss are the feed and the steady-state concentrations of glucose, X is the steady-state biomass, Y is the growth yield with respect to glucose i.e. g biomass .. (g glucose)" and P is the amount of glucose bound in EPS (equal to the dry weight of the EPS minus the water released from the polymerization of glucose). A value of Y of 0n49 g biomass .. (g glucose)" was used for the calculations. This value was determined for cultures growing under carbon-limitation where no EPS was produced (Reeslev et al., 1993). By using these assumptions 95n399n5 % of the carbon could be accounted for in the pH interval 36. The ATP consumption for EPS synthesis must therefore be quite low and\or EPS synthesis functions as overow metabolism, dissipating excess ATP under the particular growth conditions (zinc-limitation and surplus of glucose) as suggested by Linton (1991). Less of the carbon could be accounted for at pH 2n1 (94n3 %) and pH 7n0 (87n9 %). This could be due to an increased requirement of the cells for maintenance energy in order to maintain the pH homeostasis. Morphology and exopolysaccharide production The results presented in Fig. 1 clearly show that the growth form of A. pullulans in chemostat culture under zinc-limitation is as yeast since 91100 % of the total biomass .. was found

Elaboration of exopolysaccharide by A. pullulans

Table 1. The eect of the carbon source in the growth medium on the steady-state concentration of biomass and exopolysaccharide (EPS). Aureobasidium pullulans was grown in chemostat culture at pH 4n0 at a dilution rate of 0n08 h" Steady-state concentration of Feed concentration (g l") Glucose Glucose Sucrose Sucrose Fructosejglucose 15 20 15 20 10j10 Biomass EPS (g l") (g l") 4n8 5n1 3n7 3n9 4n1 3n1 3n3 6n9 9n1 8n4 Specic EPS production (g g" h") 0n05 0n05 0n15 0n19 0n16

652 15 g l" of glucose as the feed concentration in the medium, a two-fold increase in the steady-state concentration of EPS was obtained, whereas the steady-state concentration of biomass decreased by about 23 %. By increasing the feed concentration of sucrose to 20 g l" the steady-state concentration of EPS increased further by about 32 %, while no signicant increase in the concentration of biomass was found. A similar increase in the EPS production was not found when the feed concentration of glucose was increased from 15 to 20 g l". When a 1 : 1 mixture of glucose and fructose (total feed concentration of 20 g l") was used in the medium, the specic EPS production obtained was comparable to that found for sucrose, which was three to four fold higher than for glucose. The use of dierent carbon sources did not aect the morphology which remained as yeast form in all the steadystate cultures. The specic EPS production of 0n19 g EPS g" biomass .. h" (Table 1) is the highest one reported from chemostat culture with A. pullulans. McNeil et al. (1989), who used the same strain of A. pullulans and 20 g l" of sucrose, obtained a specic EPS production in chemostat culture of about 0n11 g EPS g" biomass .. h" at pH 4n5. This dierence in the specic EPS production might be explained by dierences in the yeast fraction of the biomass in the cultures which in the present study was about 100 % as compared to about 47 % in the study by McNeil et al. (1989). REFERENCES
Catley, B. J. (1980). The extracellular polysaccharide, pullulan, produced by Aureobasidium pullulans : a relationship between elaboration rate and morphology. Journal of General Microbiology 120, 265268. Failla, M. L. & Weinberg, E. D. (1977). Cyclic accumulation of zinc by Candida utilis during growth in batch culture. Journal of General Microbiology 99, 8597. Heald, B. & Kristiansen, B. (1985). Synthesis of polysaccharide by yeast-like forms of Aureobasidium pullulans. Biotechnology and Bioengineering 27, 15161519. Lawford, H. G., Pik, J. R., Lawford, G. R., Williams, T. & Kligerman, A. (1980). Physiology of Candida utilis in zinc-limited chemostat culture. Canadian Journal of Microbiology 26, 6470. Linton, J. D. (1991). Metabolite production and growth eciency. Antonie van Leeuwenhoek 60, 293312. McNeil, B., Kristiansen, B. & Seviour, R. J. (1989). Polysaccharide production and morphology of Aureobasidium pullulans in continuous culture. Biotechnology and Bioengineering 33, 12101212. Ramos, S. & Garcia-Acha, I. (1975). A vegetative cycle of Pullularia pullulans. Transactions of the British Mycological Society 64, 129135. Reeslev, M., Jrgensen, B. B. & Jrgensen, O. B. (1993). Inuence of Zn#+ on yeastmycelium dimorphism and exopolysaccharide production by the fungus Aureobasidium pullulans grown in a dened medium in continuous culture. Journal of General Microbiology 139, 30653070. Reeslev, M., Nielsen, J. C., Olsen, J., Jensen, B. & Jacobsen, T. (1991). Eect of pH and the initial concentration of yeast extract on regulation of dimorphism and exopolysaccharide formation of Aureobasidium pullulans in batch culture. Mycological Research 95, 220226. Seviour, R. J., Kristiansen, B. & Harvey, L. (1984). Morphology of Aureobasidium pullulans during polysaccharide elaboration. Transactions of the British Mycological Society 82, 350356.

in the yeast form, and that the growth as yeast was independent of the external pH in the interval pH 37. In several of the previous studies with batch culture (Seviour et al., 1984 ; Heald & Kristiansen, 1985) and with chemostat culture (McNeil, Kristiansen & Seviour, 1989) the mycelial form prevailed at low pH values (2n03n5) and the yeast form at higher pH values (6n06n5). In those studies a concentration of yeast extract of 0n4 g l" was used. Reeslev et al. (1991), however, showed that a mycelial culture could be obtained even at pH 6n5 when the concentration of yeast extract in the medium was increased from 0n4 to 4n0 g l". When the concentration of yeast extract was 4n0 g l" the duration of the exponential growth phase was extended and the mycelialyeast transition was delayed. Thus, it appears that the nutritional requirements of the mycelial growth forms vary at dierent pH values and we suggest that more Zn#+ is required in order to maintain mycelial growth at high pH values than at low pH values. This hypothesis would explain the variation in the yeastmycelial ratio with varying pH described in previous studies and would not contradict the results of the present study. The homogeneous yeast cultures that were obtained independent of the external pH in the present investigation make it possible to evaluate the direct eect of pH on the ability of the yeast cells to produce EPS. The maximum EPS production was obtained at pH 4n0, whereas no EPS was produced at pH 2n1 and only little at pH 7n0 (Fig. 1). At pH 4n0 the EPS production was almost twice that obtained at pH 3n0, which is not in accordance with the conclusion drawn from the study with batch cultures by Heald & Kristiansen (1985), who found that pullulan production by yeast cells is independent of pH in the interval from pH 3n0 to 6n3. A pH optimum for EPS production at pH 4n0 corresponds well with the results of McNeil, Kristiansen & Seviour (1989), who, using chemostat cultures, found a pH optimum at pH 4n5. The steady-state concentrations of biomass and EPS were related to the carbon source used for growth and production (Table 1). Thus, by using 15 g l" of sucrose instead of
(Accepted 17 October 1996)