Ability ol Aureobasidium pullulans to synthesize

Ability ol Aureobasidium pullulans to synthesize pullulan upon selected sources of carbon and nitrogen Thomas P. West and Beth Reed-Hamer Olson Biochemistry Laboratories, Department of Chemistry, South Dakota State University, Brookings, South Dakota 57007, U.S.A.

Abstract The effect of carbon and nitrogen sources upon the level of pullulan synthesized by Aureobasidium pullulans ATCC 42023 was examined. ln addition, cell dry weight and the final pH of the culture medium was investigated after fungal growth upon the selected carbon source or nitrogen sources. The carbon sources studied included sucrose, glucose,

fructose, maltose and corn syrup while the nitrogen sources studied were ammonium nitrate, ammonium sulphate, ammonium tartrate, sodium nitrate, asparagine and urea. After 5 days at 30oC, it was found that the level of pullulan found was maximal upon sucrose while the lowest level was noted on maltose. Cell dry weight was found to remain relatively constant independent of the carbon source used while the pH of the culture medium dropped significantly. lt was demonstrated that the amount of pullulan synthesized was not proportional to the concentration of carbon source present. Pullulan synthesis seemed to be inhibited when the carbon source concentration was increased in batch culture. With respect to the nitrogen sources screened, ammonium tartrate and asparagine stimulated maximal pullulan synthesis while urea allowed little pullulan

synthesis.

Introduction

\.

Pullulan is a complex polysaccharide synthesized by the polymorphic fungus Aureobasidium pullulans (Bernier, 1958; Bender et a1.,1959; Ueda et a|.r 1963). It is composed of primarily maltotriose units arranged in a fashion that provides it with unique physical properties (Bouveng et a|.r 1962; Sowa er a\.r 1963; Catley, 1970; Taguchi et al., 1973; Zajic and LeDuy, 1973, 1977; Catley et al., 1986). A number of carbon sources have been shown to initiate pullulan synthesis by the fungus. These carbon sources include sucrose, maltose, glucose, fructose, corn starch and corn meal hydrolysate (Catley, 1971a; Zaiic and LeDuy, 1973; Leal-Serrano et al., 1980; Imshenetskii et al., 1981, 1985; Aleksanyan and Markosyan, 1985; Leathers et al., 1988). When the sucrose concentration exceeds 5Vo (wlv), it has been noted that an inhibition of pullulan synthesis occurs (Shin ar al.,1987). The pH ofthe culture medium decreases following the addition of a carbon source (Ueda er aL.r 1963; Catley, l97la;Zajic and LeDuy, 1973; Leal-Serrano et al., 1980). Pullulan synthesis was found to begin once a pH of 5 for the medium was attained. This coincides with the fungal cells entering their late exponential phase of growth and is still ongoing even after the onset of their stationary phase of growth (Catley, l97la).It appears that as the fungal growth rate is diminished, pullulan synthesis by the cells increases (Catley, 1973). The presence ofoxygen is required for polysaccharide synthesis (Rho er al., 1988).

117 Microbios 67 117-124

1991

Published anO @ t ggt by The Faculty Press 88 Regent Street, Cambridge, Great Britain

The onset of pullulan biosynthesis is also related to nitrogen availability (Catley,

797tb, 1973). It appears that a depletion ofavailable nitrogen is necessary for pullulan synthesis to begin (Seviour and Kristiansen, 1983). Polysaccharide synthesis can be delayed by the addition ofexogenous nitrogen. It has been shown that this is related to inhibition of protein synthesis by the fungus (Bulmer et al., 1987). Few studies have screened various nitrogen sources as to their effect upon pullulan synthesis (Imshenetskii et al., 1981). Althoughl. pullulans ATCC 42023 was one ofthe first pullulan synthesizing strains isolated and characterized(Zajic,1967;Zaiic and LeDuy, 1973), sources ofcarbon and nitrogen do not appear to have been compared with respect to their effect upon pullulan elaboration by this strain. In this report, the effect ofselected carbon and nitrogen sources upon the level ofpullulan synthesis by ATCC 42023 are observed. This included measuring fungal cell dry weight as well as the final pH of the culture medium. In addition, the influence of carbon source concentration upon polysaccharide synthesis was investigated.

Materials and methods Strain and growth conditions

Aureobasidium pullulans ATCC 42023 was the fungal strain used in this study (Zaiic,1967). The minimal medium contained 0.5% K2HPO a,A.lVo NaCl, 0.02% MgSOo, 0.04Vo yeast extract and 0.0670 ammonium sulphate (Ueda et al.,1963). The pH of the culture medium was adjusted to 6.0 and autoclaved. The carbon

source concentration added to this medium after autoclaving varied from 2.5Vo-lOVo (w/v). I7hen ammonium sulphate was replaced by an alternative nitrogen source in the minimal medium, its final concentration remained 0.06Vo (w/v). Batch cultures (100 ml) were inoculated using overnight cultures (1 ml) containing the same medium. Each culture was shaken (200 rpm) for a period

of 5 days at 30oC.

Pullulan, dry weight and pH determinations Samples (5 ml) of each culture were removed and centrifuged at low speed. The

supernatant containing pullulan was retained fqr further analysis following centrifugation. The cell pellet present *m *r'rh.d with water and. again centrifuged. The pellet was resuspended in water and then filtered on Millipore 0.45 pm HVLP filters (47 mm diameter). These preweighed filters were dried to constant weight at 105oC and weighed again to determine the cell dry weight (Lacroix et a1.,1985). Pullulan was determined using alcohol precipitation. For each volume of pullulan, two volumes of 95Vo ethanol was added to precipitate the polysaccharide. The precipitated pullulan was collected on preweighed Millipore 0.45 pm HVLP filters (25 mm diameter). The filters were dried to constant weight at l05oc and then reweighed to ascertain the content of pullulan (op.

'l

18

cit.). Microbios

T. P. West and B. Reed-Hamer

Chemicals

The source of sucrose was Schwarz-Mann, Cambridge, Massachusetts, U.S.A. Corn syrup was obtained from ICN Biomedicals, Cleveland, Ohio, U.S.A. Glucose, maltose and fructose were products of the Sigma Chemical Company, St Louis, Missouri, U.S.A. Filters were purchased from the Millipore Corporation, Bedford, Massachusetts, U.S.A. Yeast extract was obtained from

Difco Laboratories, Detroit, Michigan, U.S.A. All other chemicals were of analytical grade.

Results The ability of Aureobasidium pullulans ATCC 42023 to utilize a number of carbon sources was initially investigated. This included determining the concentration of pullulan synthesized, the cell dry weight and the final pH of the culture medium after 5 days at 30'C. The carbon sources screened were sucrose, maltose, fructose, glucose and corn syrup where the initial four carbon sources have previously been shown to promote pullulan synthesis when included in the culture medium (Catley, l97la; Leal-Serrano et al., 1980; Imshenetskii et al., l98l; Aleksanyan and Markosyan, 1985). Of these carbon sources, sucrose has been reported to allow significant pullulan formation (Catley, l97la; Aleksanyan and

Markosyan, 1985).

In this study, it is clear that sucrose was superior to the other carbon sources tested with respect to its ability to stimulate pullulan formation (Table 1). Its cell dry weight was comparable to those observed for the other carbon sources investigated (Table l). Corn syrup was found to promote greater pullulan synthesis than the remaining carbon sources which were explored (Table 1). The final pH of the culture medium did not appear to differ significantly regardless of the carbon source present (Table I).

Table 1 Pullulan level, celldry weight and finalpH of culture medium after Aureobasidium pullulans growth on selected carbon sources (5 days at

300c) Carbon source

Pullulan (g/l)

Dry weight (g/l)

pH

2.39 2. s7 2.56 2.59 2.63

Sucrose

7.OO (0.05)

4.83 (0.06)

Corn syrup Fructose

3.55 (0.23) 2.43 (O.121

5.OO (O.28)

Glucose

2.O2 lO.16l 1.95 (0. 13)

Maltose

4.89 (0.13) 4.21 lO.15l 4.34 lO.O2l

(0.01) (0.01

)

(O)

(0.01) (0.01)

Each value represents the mean of three separate determinations (standard deviation). Each carbon source was present at a concentration of 2.5% (w/v) in the culture medium where 0.O6016 (w/v) ammonium sulphate served as the nitrogen source.

1

19

Pullulan synthesis on carbon and nitrogen sources

2 Determination of pullulan level, cell dry weight and final pH of culture medium alter Aureobasidium pullulans growth on selected concentrations of sucrose (5 days at 30oC)

Table

Concn (%)

Pullulan (g/l)

Dry weight (g/l)

pH

2.60 2.39 2.41 2.57

2.5

7.OO (O.05)

5.0 7.5

8.O5 (0.22) 1 1 .02 (0.14)

4.83 (0.06) 6.31 (0.14) 3.81 (0.31)

10.0

16.22 fi.151

1.50 (O.13)

(0.01) (O.01) (0.011

(0.01)

Each value represents the mean of three separate determinations (standard deviation). The source of nitrogen

in the medium was 0.O60/o (w/v) ammonium sulphate.

Considering that sucrose and corn syrup were the two most effective carbon in enhancing pullulan production by this strain, the effect of varying their final concentration in the culture medium was studied. As the sucrose concentration was elevated, it was found that the concentration of pullulan did increase (Table 2). It should be noted that the pullulan level did not increase proportionately. In general, cell dry weight was observed to decrease as the sucrose concentrations increased (Table 2). In much the same fashion, the pullulan levels observed after growth on corn syrup were not proportional to concentration (Table 3). Rather, the pullulan levels appeared to become static. Cell dry weights diminished slightly as the concentration of corn syrup was raised (Table 3). In comparison with the dry weights of sucrose-grown cells, cell dry weights after growth on corn syrup were generally higher. The pH of the culture medium after fungal growth on either sucrose (Table 2) or corn syrup (Table 3) did not sources

seem to be an indicator for predicting final pullulan levels.

Table

3

6on6p

(o/o)

Study of pullulan level, cell dry weight and final pH of culture medium after growth of fungus on various corn syrup concentrations (5 days at 3OoC)

2.5 5.0 7.5 10.0

Pullulan (g/l)

Dry weight (g/l)

pH

3.55 (O.23) 3.92 (0.13) 4.45 (0.40)

5.00 (0.28) 4.47 l0.19l 4.29 (O.08) 3.81 (O. 18)

2.57 2.54 2.54 2.71

s.32 (0.36I

(O.O1l (O.O1l

(0.01) lO.O1l

Each value represents the mean of three separate determinations (standard deviation). The culture medium contained 0.060/o (w/v) ammonium sulphate as the nitrogen source

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Table 4 Effect of nitrogen source upon pullulan level, cell dry weight and final pH of culture medium after Aureobasidium pullulans growth on 2.5o/o

sucrose as a carbon source (5 days at 30oC) Nitrogen source

Pullulan (g/l)

Dry weight (g/l)

pH

Ammonium tartrate

10.o5 (0.13)

4.73 lO.12l

4.1

9.57 (0.23) 7.00 (0.05) 6.70 (0.23) 4.57 l0.37l 3.43 (O.20)

4.74 (0.09) 4.83 (0.06) 6.88 (0.29)

3.86 2.60 4.21 3.20 3.1 9

Asparagine Ammonium sulphate Sodium nitrate

Ammonium nitrate Urea

4.s9 (0.03) 4.41 (0.30)

I

(0.03) (0.01) (O.01)

(0.06) (0.08) (0.07)

Each value represents the mean of three separate determinations (standard deviation). Each nitrogen source

was included at a concentration of O.06% (w/v) in the culture medium.

The influence of nitrogen source upon the level of pullulan produced by the

N

organism was also explored in relation to the carbon sources sucrose and corn syrup. It appeared that ammonium tartrate and asparagine were the best nitrogen sources for stimulating pullulan production on sucrose (Table 4) or corn syrup (Table 5) as a carbon source. Ammonium sulphate as a nitrogen source did not promote optimal pullulan synthesis by A. pullulazs when sucrose (Table 4) or corn syrup (Table 5) was supplied as a source of carbon. For either sucrose (Table 4) or corn syrup (Table 5), urea promoted the lowest pullulan concentration ofthe nitrogen sources screened. The type ofnitrogen source added to the culture medium did seem to affect the fungal cell weight. Sodium nitrate was found to increase the cell weight substantially when sucrose served as the carbon source

Table

5

lnfluence of nitrogen source upon pullulan level, cell dry weight

and final pH of culture medium alter Aureobasidium pullulans growth on 2.5o/o corn syrup as a carbon source (5 days at 3OoC)

Nitrogen source

Pullulan (g/l)

Dry weight (g/l)

pH

Ammonium tartrate Asparagine Ammonium nitrate Ammonium sulphate

5.63 5.08 4.27 3.55 2.22 0.85

3.82 5.25 4.94 5.00 4.30 6.61

5.54 3.96 3.99 2.57 4.50 3.81

Sodium nitrate Urea

(1

.25)

(0.1 5) @.461

(0.23)

(0.06l (O.74)

(0.1 1) (0. 10)

(O.63)

(0.28) (0.47], (0.19)

(0.O1)

(0.04) (0.50) (0.01) (0.15) (0.07)

Each value represents the mean of three separate determinations (standard deviation). Each nitrogen source

was added to the culture medium at a final concentration of O.06% (w/v).

121


(Table 4). '$fith respect to corn syrup, urea-grown cells exhibited the highest cell weight (Table 5). The final pH of each culture medium was found to fluctuate according to the nitrogen source utilized. If ammonium tartrate served as the nitrogen source, the final pH of the culture medium was generally less acidic than that observed for the other nitrogen sources tested when grown on sucrose

(Table 4) or corn syrup (Table

5).

Discussion After comparing the carbon sources examined in this study for their ability to stimulate fungal pullulan synthesis under the given culture conditions, it was concluded that fungal pullulan levels were maximal after growth on sucrose. This is in agreement with previous investigations where sucrose has been shown to allow pullulan to accumulate at high concentrations (Catley, l97la; Aleksanyan and Markosyan, 1985). Other than sucrose, corn syrup was shown to enhance pullulan synthesis more than the carbon source glucose, fructose or maltose. The pullulan level produced after A. pullulans growth on 2.5Vo corn syrup was 51% of the value obtained using 2.570 sucrose. After comparing fungal growth on the carbon sources, the cell dry weights did not vary significantly while the pH of each culture medium was found to have decreased from its initial pH. Similar results have been shown in other investigations (Catley, 197lb; LealSerrano et al.,1980; Aleksanyan and Markosyan, 1985). Although corn meal hydrolysate and corn starch have been reported to allow pullulan synthesis (Imshenetskii et aI.r 1985; Leathers et al.r l988), corn syrup has not been previously examined as a potential carbon source. The influence of carbon source concentration upon final pullulan levels has not been fully explored. In this work, increasing sucrose or corn syrup concentrations did not proportionately increase pullulan levels. Instead, pullulan elaboration seemed to diminish, particularly for corn syrup, as the carbon source concentration was elevated. Similarly, it has been found that pullulan synthesis by the fungus decreased ifthe concentration ofsucrose exceeded 590 (w/v) in the culture medium (Shin er al., 1987). However, fungal cell growth has been reported to be inhibited in medium containing 590 sucrose (Shin et a1.,1987).

)

In this investigation, fungal dry weight decreased above a concentration of 5Vo sucrose in the culture medium. The screening of potential nitrogen sources, in relation to pullulan formation, indicated that ammonium sulphate was not the optimal nitrogen source. Rather,

it was ammonium tartrate and asparagine which stimulated more pullulan formation than ammonium sulphate when sucrose or corn syrup served the carbon source. In addition if corn syrup was the carbon source, ammonium nitrate proved

to be a better nitrogen source than ammonium sulphate in promoting pullulan formation. IJrea was the least effective nitrogen source in initiating pullulan synthesis by the fungus. A previous study has indicated that if sucrose was added as a carbon source then sodium nitrate was more effective as a nitrogen source

122

Microbios


than urea or ammonium sulphate in promoting pullulan synthesis (Imshenetskii et al., 1981).In this investigation, sodium nitrate was a superior source of nitrogen to urea but was inferior to ammonium sulphate in aflecting pullulan synthesis. In conclusion, it was determined that both carbon source and nitrogen source

do influence the level of pullulan synthesized by Aureobasidium pullulans. Moreover, the amount of pullulan produced by the fungus in batch culture will not necessarily be proportional to the carbon source concentration. Acknowledgements This investigation is paper 2543, Journal Series, South Dakota Agricultural Experiment Station. We wish to thank the South Dakota CUC and the South Dakota

Agricultural Experiment Station for supporting this study.

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Y. N. and Byun S. M' 1987. Production of pullulan by fed-batch fermentation. Biotechnol. Letters 9 621 -4. SOWA W., Blackwood A. C. and Adams G. A. 1963. Neutral extracellular glucan of Pullularia pullulans (de Bary) Berkhout. Can. J. Chem. 41 2314-9. TAGUCHI R., Kikuchi Y., Sakano Y. and Kobayashi T. 1973. Structural uniformity of pullulan produced by several strains of Pullularia pullulans. Agric. Biol. Chem.37 1583-8. UEDA S., Fujita K., Komatsu K. and Nakashima Z. 1963. Polysaccharide produced by the genus Pullularia. l. Production of polysaccharide by growing cells. Appl. Microbiol. 11 211-5. ZAJIC J. E. 1967. Polysaccharide flocculating agent. U.S. Patent 3,32O,136. ZAJIC J. E. and LeDuy A. 1973. Flocculant and chemical properties of polysaccharide from Pullularia pullulans. Appl. Microbiol. 25 628-35. ZAJIC J. E. and LeDuy A. 1977. Pullulan. /n Encyclopedia of Polymer Science and Technology. Supplement 2, pp 643-52. John Wiley and Sons, lnc., New York. U.S.A. SHIN Y. C., Kim Y. H.. Lee H. S., Kim

/

Accepted 1O February 1991

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