Polysaccharide production by Aureobasidium pullulans cells immobilized by entrapment

Polysaccharide production by Aureobasidium pullulans cells immobilized by entrapment

Microbiol. Res. (1998) 153,253-256 © Gustav Fischer Verlag Short communication Polysaccharide production by Aureobasidium pullulans cells immobiliz...

626KB Sizes 0 Downloads 14 Views

Microbiol. Res. (1998) 153,253-256


Gustav Fischer Verlag

Short communication Polysaccharide production by Aureobasidium pullulans cells immobilized by entrapment Thomas P. West, Beth Strohfus I

Olson Biochemistry Laboratories, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA, Telephone: (605)688-5469, FAX: (605) 688 -6295

Accepted: May 21, 1998

Abstract Cells of the fungus Aureoba sidium pullulan s ATCC 201253 were entrapped within 4% agar cube s or 5% calcium alginate beads and were examined for their production of the polysaccharide pullulan. The immobil ized fungal cells were utilized twice in batch culture s for 7 days of polysaccharide production in medium containing com syrup as a carbon source . The entrapped cells produced higher pullulan levels during the second cycle when compared to the first cycle polysaccharide levels. The agar-entrapped cells exhib ited a higher level of productivity and pullulan content than did the alginate-entrapped cells during both production cycle s.

Key words: Aureobasidium pullulans - pullulan - immobi lization - alginate - agar

The adsorption of A. pul/ulans cells to diatomaceous earth or sponge cubes and the use of these immobilized cells for pullulan production has been documented (Mulchandani et at. 1989 ; West and Strohfus 1996). Another study has examined the use of A. pullulans cells entrapped in a composite agar layer-membrane matrix for polysaccharide production (Lebrun et at. 1994). The results indicated relati vely little polysaccharide synthe sis by the entrapped cells. The objective of this study was to immobilize A. pullulans ATCC 201253 cells by entrapment in agar cubes or calcium alginate beads and to compare their levels of polysaccharide production in batch cultures for two cycles with each cycle consisting of a period of 7 d.


Materials and methods

The polymorphic fungus Aureobasidium pul/ulans has been shown to elaborate an extracellular polysaccharide called pullulan (Bernier 1958 ; Veda et at. 1963; Sowa et at. 1963; Zajic and LeDu y 1973). Cro ss-linked maltotriose units comprise the majority of its polysaccharide structure although a small proportion of maltotetraose units have been detected (Catley 1970; Zajic and LeDuy 1973). Due to the number of possible industrial applications that have been developed for pullulan (Yuen 1974), it is considered to be a commercially-emerging gum (Simon et at. 1995).

Strain and culture media. In this study, Aureobasidium pul/ulans ATCC 201253 (strain RP-l ) was the strain utilized (West and Reed-Hamer 1993). The fungu s was grown in batch culture s (50 ml) containing a phosphatebuffered minimal medium (pH 6.0) where corn syrup (2.5%, w/v) served as the carbon source (Veda et al. 1963 ; West and Reed-Hamer 1994).

Corresponding author: T. P. West

Polysaccharide determinations. Pullulan levels were determined by removing a sample of culture medium (5 ml) and centrifuging it at 14,600 x g for 30 min at 4 °C. Following centrifugation, pullulan was precipitated from the supernatant using ethanol and the precipitate was collected on a preweighed Millipore 0.45 urn HVLP filter (25 mm). After drying to con stant weight at 105°C, Microbiol. Res. 153 (1998) 3


each filter was reweighed to determine the pullulan concentration (West and Reed-Hamer 1993). Pullulan content of the polysaccharide produced by the entrapped cells was determined using pullulana se sensitivity. Ethanol-precipitated polysaccharide was resuspended into 0.05 M sodium acetate buffer (pH 5.0) at a concentration of 1 mg/ml. Pullulan ase (EC 1)from Klebsiella pn eumoniae was added at a final concentration of 0.22 Vlml to each polysaccharide sample (West and Reed-H amer 1993). The samples were allowed to digest at 25° C for 21 h. As a control, authentic pullulan was also digested. Pullulan content was derived from the glucose reducing equivalents determined using a previously described reducing sugar assay (Dygert et al. 1965). Immobilization procedure. To entrap the fungal cells in agar cubes (Matsunaga et al. 1985), about 105 cells of ATCC 20 1253 were used to inoculate corn syrup-containing minimal medium (50 mI) batch cultures that were aerated by shaking at 200 rpm for 48 h at 30 °C. The cells were collected by centrifugation at 7,700 x g for 20 min at 4 °C, washed and suspended in 0.85% NaCl (5 mI). The resuspended cells from each culture (0.74 g) were added to an autoclaved solution of agar (cooled to 40 °C) to give a final concentration of 4% (w/v) . The 4% agar-cell suspension was added in 1 ml volume increments to a sterile plastic rack serving as a mold to allow cube formation. After cooling at 4 °C, the 540 rnm'' cubes (approximately 14 g wet weight) were aseptically added to sterile Erlenmeyer flasks. To entrap the fungal cells in calcium alginate beads, approximately 105 cells of ATCC 201253 were utilized to inoculate com syrupcontain ing medium (50 ml) in batch cultures. These cultures were grown for 48 h at 30° C by shaking at 200 rpm. The cells were collected by centrifugation at 7,700 x g for 20 min at 4 °C and washed once with 0.85% NaC!. The cells (0.77 g) were added to an autoclaved solution of 5% (w/v) sodium alginate (cooled to 40 °C). The mix was placed in a 20 ml syringe and the drops were collected in a sterile 50 ml solution of 0.3 M CaCl 2 in a sterile Erlenmeyer flask (Dainty et al. 1986). The resultant beads (3 mm diameter) were kept at 4 °C for 1 h. The CaC!2 solution was drained and the beads (about 20 g wet weight) were rinsed with 50 ml of phosphatebuffered minimal medium (pH 6.0). The cubes or beads were placed in 250 ml Erlenm eyer flasks to which 50 ml of minimal medium (pH 6.0) containing 2.5% corn syrup had been added. For a period of 7 d at 30 °C, the cubes or beads in each flask were shaken at 125 rpm to provide aeration. Production of the polysaccharide was determined daily. After this initial cycle, the cubes or beads were rinsed with 0.85% NaCI and again suspended in com syrup-containing minimal medium for another 7 d of pullulan producti on at 30° C. After each 7 d 254

Microbial. Res. 153 (1998) 3

cycle, cell leakage from the supports was determined by removing culture medium (5 ml) and centrifuging the sample at 14,600 x g for 30 min at 4 °C. The leaked cells were washed, centrifu ged and collected on preweighed Millipore 0.45 urn HVLP filters (47 mm). After drying to constant weight at 105°C, the filters were reweighed to determine the weights of the leaked cells. To determine the viable cell concentration entrapped in the cubes or beads, representati ve samples were crushed aseptically, suspended in 0.85% NaCl (2 ml) and vigorously mixed. The viable cell concentrations in the samples were determined after appropriate dilution s of the suspensions on potato dextrose agar plates by determining colony-forming units. Dry weights were quantitated by collecting the crushed supports on preweighed Millipore 0.45/lm HVLP filters (47 mm). The filters were dried to constant weight at 105°C and reweighed to calculate the dry weight levels of the supports.

Results and discussion Initially, the degree of immobili zation of A. pullulans ATCC 20 1253 cells in the agar cubes was investigated. It was determined that 1.15 x 105 colony formin g units/g dry weight of support (SO =0.05 X 105) were entrapped in the agar cubes while the degree of immob ilization in the calcium alginate bead s was calculated to be 2.11 x 107 colony forming units/g dry weight of support (SO = 0.38 X 107) . Pullul an production by the agar-entrapped or alginate-immobil ized A. pullulans cells in batch culture s was investigated during both cycles (Table 1). During the initial cycle, it was observed that pullulan production by the agar-entrapped cells was essentially complete by day 6 with the highest polysaccharide levels being detected on days 6 and 7 (Table 1). The alginate-entrapped cells produced the Table 1. Effect of recycling entrapped ATCC 201253 cells upon polysaccharide production. Polysaccharide levels are expressed as mg/ml where each value represents the mean of three separate determinations ± SD. Cycle and polysaccharide level : Agar Day

a 1 2 3 4 5 6 7

Alginate 2

0.0 ± 0.0 1.8 ± 0.8 2.6 ± 0.2 2.8 ± 0.1 3.2 ± 0.1 4.0 ± 0.1 4.4 ± 0.1 4.4 ± 0.1

0.0 ± 0.0 1.4 ± 0.5 3.3 ± 0.6 5.4 ± 0.8 6.4 ± 0.4 6.7 ± 0.7 6.8 ± 0.5 6.8 ± 0.6

2 0.0 ± 0.0 2.3 ± 0.4 3.1 ± 0.4 3.4 ± 0.5 3.7 ± 0.4 3.9 ± 0.4 3.8 ± 0.4 4.3 ± 0.7

O.O±0.0 2.3 ±O.l 3.2 ± 0.2 3.7 ± 0.1 3.8 ± 0.0 4.2 ± 0.3 4.2 ± 0.2 4.8 ± 0.3

entrapment of the fungal cells in 5% calcium alginate stimulated the production of an alternate polysaccharide by A. pullulans (Simon et al. 1993). Sponge-immobilized, 2.5% (w/v) com syrup-grown cells of A. pullulans produced slightly lower polysaccharide levels than did the agar- or alginate-entrapped cells during the two 7 d production cycles (West and Strohfus 1996). Similar to what was observed for the entrapped fungal cells used in this study, polysaccharide elaboration by the sponge-immobilized cells was found to be higher during the second production cycle than during the first cycle (West and Strohfus 1996). The pullulan content of the polysaccharide produced by the sponge-immobilized cells was slightly higher than the pullulan content of the polysaccharide elaborated by the agar-entrapped cells (West and Strohfus 1996). Sucrose (5%, w/v)-grown cells of A. pullulans strain 2552 immobilized on diatomaceous earth produced pull ulan but exhibited a high degree of cell leakage due to shear (Mulchandani et al. 1989). Polyurethane foam entrapment of strain 2552 cells proved successful in that the immobilized cells could be used for four cycles of polysaccharide production with much less cell leakage from the foam (Mulchandani et al. 1989). Another study examined the immobilization of 5% (w/v) glucose-grown A. pullulans CNCM 1726.88 cells on a 0.5% (w/v) agar layered membrane filter (Lebrun et al. 1994). This composite support produced only 3.5 mg pullulan/ml and it was suggested that oxygen limitation may have been a major factor in the low level of polysaccharide elaboration (Lebrun et al. 1994). Cell leakage was stilI observed using the agar-layered membrane filter and fouling of the membrane was noted to occur (Lebrun et al. 1994). Relative to the findings of the prior investigations, polysaccharide production by the immobilized 2.5% com syrup-grown fungal cells in a 2.5% com syrup-containing medium would seem to be more effective than pullulan elaboration by the immobilized 5% sucrose- or glucose-grown fungal cells in the 5% sucrose- or glucose-containing medium. In conclusion, the use of entrapped A. pullulans cells for the semi-continuous production of pullulan appeared Table 2. Polysaccharide production of entrapped ATCC feasible although cell leakage from each support is stilI 201253 cells relative to support weight and pullulan content a concern. The development of such a process might induring each cycle of production. Productivity is expressed as volve agar-entrapped fungal cells since they maintained mg polysaccharide/g support. The pullulan content of the a high level of polysaccharide production and pullulan polysaccharide produced, which is givenin %, wasdetermined content during both cycles of use. by measuring its sensitivity to pullulanase digestion. Each value represents the mean of three experiments ± SD.

highest pullulan level on day 7 of the first cycle although polysaccharide synthesis appeared to have slowed by day 5 (Table 1). After the initial cycle, cell leakage from the agar-entrapped cells was determined to be 0.02 g cells/g cubes while leakage from the calcium alginateentrapped cells was observed to be 0.01 g cells/g beads. During the second cycle, polysaccharide elaboration by the agar-entrapped cells appeared to be greater than the initial cycle of production from the second day onward (Table 1). Polysaccharide production by the agar-entrapped cells during the second cycle was essentially complete by day 4 (Table 1). Relative to the initial cycle of utilization, the alginate-entrapped cells used for a second cycle of production exhibited higher polysaccharide levels by day 2 (Table 1). Polysaccharide elaboration by the alginate-entrapped cells appeared to be maximal on day 7 of the second cycle (Table I). During the initial cycle of pullulan production using the entrapped cells, a slightly higher concentration of polysaccharide was produced by the agar cubes than the calcium alginate beads (Table 1). This was unexpected since the cubes had a lower concentration of viable cells immobilized. Also, the level of polysaccharide produced by the agar-entrapped cells was higher than the alginate-entrapped cells during the second cycle (Table 1). For both types of immobilized cells, polysaccharide production was much higher during the second cycle than the initial cycle (Table 1). Following the second production cycle, cell leakage of the agar-entrapped cells was found to be 0.01 g cells/g cubes while the leakage from the alginateentrapped cells was also 0.01 g cells/g beads. During both production cycles, the agar-entrapped cells were more effective in producing polysaccharide per gram of support (wet weight) than were the alginate-entrapped cells (Table 2). This is despite the difference in the levels of viable cells entrapped in each support. In addition, the pullulan content of the polysaccharide elaborated by the agar-entrapped cells during either cycle was substantially higher than the polysaccharide synthesized by the alginate-entrapped cells (Table 2). It may be that the




Pullulan content



I 2 1 2

15.9 ± 0.4 24.7 ± 2.0 10.9 ± 1.8 12.3 ± 0.7

69 ± 8 63 ± 7 26±4 39 ± 5

Published as paper 2946, Journal Series, South Dakota AES. This work was supported by grants from the South Dakota Corn Utilization Council, the South Dakota Agricultural Experiment Station and U.S. Department of Agriculture Grant


Microbial. Res. 153 (1998) 3


No. 94-37501-0884. This paper reports results of research only and the mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture or South Dakota AES over others of a similar nature not mentioned.

References Bernier, B. (1958): The production of polysaccharides by fungi active in the decomposition of wood and forest litter. Can. 1. Microbiol. 4,195-204. Catley, B. J. (1970): Pullulan, a relationship between molecular weight and fine structure. FEBS Lett. 10, 190-193. Dainty, A. L., Goulding, K. H., Robinson, P. K., Simpkins, I., Trevan, M. D. (1986): Stability of alginate-immobilized algal cells. Biotechnol. Bioeng. 28, 210-216. Dygert, S., Li, L. H., Florida, D., Thoma, 1. A. (1965): Determination of reducing sugar with improved precision. Anal. Biochem. 13,367-374. Lebrun, L., Junter, G.-A., Jouenne, T., Mignot, L. (1994): Exopolysaccharide production by free and immobilized microbial cultures. Enzyme Microbiol. Technol. 16,1048-1054. Matsunaga, T., Matsunaga, N., Nishimura, S. (1985): Regeneration of NAD(P)H by immobilized whole cells of Clostridium butyricum under hydrogen high pressure. Biotechnol. Bioeng. 27,1277-1281. Mulchandani, A., Luong, 1. H. T., LeDuy, A. (1989): Biosynthesis of pull ulan using immobilized Aureobasidium pullulans. Biotechnol. Bioeng. 33, 306-312.


Microbiol. Res. 153 (1998) 3

Simon, L., Caye- Vaugien, C, Bouchonneau, M. (1993): Relation between pullulan production, morphological state and growth conditions in Aureobasidium pullulans: new observations. 1. Gen. Microbiol. 139,979-985. Simon, L., Bouchet, B., Caye-Vaugien, C, Gallant, D. J. (1995): Pullulan elaboration and differentiation of the resting forms in Aureobasidium pullulans. Can. 1. MicrobioI. 40, 35-45. Sowa, Blackwood, A. C, Adams, G. A. (1963): Neutral extracellular glucan of Pullularia pullulans (de Bary) Berkhout. Can. 1. Chern. 41, 2314-2319. Ueda, S., Fujita, K., Komatsu, K., Nakashima, Z. (1963): Polysaccharide produced by the genus Pullularia. I. Production of polysaccharide by growing cells. Appl. MicrobioI. 11, 211-215. West, T. P., Reed-Hamer, B. (1993): Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans. FEMS Microbiol. Lett. 113,345-349. West, T. P., Reed-Hamer, B. (1994): Elevated polysaccharide production by mutants of the fungus Aureobasidium pullulans. FEMS Microbiol. Lett. 124, 167-171. West, T. P., Strohfus, B. R.-H. (1996): Polysaccharide production by sponge-immobilized cells of the fungus Aureobasidium pullulans. Lett. Appl. Microbiol. 22, 162-164. Yuen, S. (1974): Pullulan and its applications. Process Biochern. 9,7 -9. Zajic, 1. E., LeDuy, A. (1973): Flocculant and chemical properties of a polysaccharide from Pullularia pullulans. Appl. Microbiol. 25,628-635.