Microbiol. Res. (2001) 156, 285–288 http://www.urbanfischer.de/journals/microbiolres
Short Communication Polysaccharide production by immobilized Aureobasidium pullulans cells in batch bioreactors Thomas P. West, Beth Strohfus Olson Biochemistry Laboratories, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA Accepted: April 16, 2001
Abstract Cells of the fungus Aureobasidium pullulans ATCC 201253 were entrapped within 4% agar cubes or 5% calcium alginate beads and were examined for their production of the polysaccharide pullulan in batch bioreactors. The batch bioreactors were utilized twice for 168 hours of polysaccharide production in medium containing corn syrup as a carbon source. The agar-entrapped cells produced nearly equivalent pullulan concentrations during both production cycles. The alginateentrapped cells produced higher polysaccharide levels during the second cycle compared to the levels observed during the initial cycle. The agar-entrapped cells elaborated a polysaccharide with a higher pullulan content than did the alginateentrapped cells during both production cycles. Key Words: Aureobasidium pullulans – pullulan – immobilization – bioreactor – polysaccharide
Introduction The polymorphic fungus Aureobasidium pullulans has been shown to elaborate an extracellular polysaccharide called pullulan (Bernier 1958 ; Ueda et al. 1963; Sowa et al. 1963 ; Zajic and LeDuy 1973). Cross-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 Corresponding author: T. P. West e-mail: [email protected]
pullulan (Yuen 1974), it is considered to be a commercially-emerging gum (Simon et al. 1995). The adsorption of A. pullulans cells to diatomaceous earth or sponge cubes and the use of these immobilized cells for pullulan production has been documented (Mulchandani et al. 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 al. 1994). The results indicated a pullulan yield of 29.7% by the entrapped cells (Lebrun et al. 1994). A prior investigation has shown that A. pullulans ATCC 201253 cells immobilized by entrapment in agar cubes or calcium alginate beads were capable of producing polysaccharide when shaken in batch cultures containing a corn syrup-containing medium (West and Strohfus 1998). In this study, batch polysaccharide production by A. pullulans ATCC 201253 cells immobilized in agar cubes or calcium alginate beads during two cycles of 168 h in an aerated column bioreactor was compared. It was of interest to learn whether entrapment of A. pullulans cells could be effective in the production of authentic pullulan for more than one cycle using an aerated column bioreactor because of the high molecular weight of this biopolymer.
Materials and methods Strain and culture media. In this study, Aureobasidium pullulans ATCC 201253 (strain RP-1) was the strain utilized (West and Reed-Hamer 1993). The fungus was Microbiol. Res. 156 (2001) 3
grown in batch cultures (50 ml) containing a phosphate-buffered minimal medium (pH 6.0) where corn syrup (2.5%, w/v) served as the carbon source (Ueda et al. 1963 ; West and Reed-Hamer 1994). Polysaccharide determinations. Polysaccharide levels were determined by removing a sample of culture medium (5 ml) and centrifuging it at 14,600 × 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 µm HVLP filter (25 mm). After drying to constant weight at 105 °C, each filter was reweighed to determine the pullulan concentration (West and ReedHamer 1993). Pullulan content of the polysaccharide produced by the entrapped cells was determined using pullulanase sensitivity. Ethanol-precipitated polysaccharide was resuspended into 0.05 M sodium acetate buffer (pH 5.0) at a concentration of 1 mg/ml. Pullulanase (EC 188.8.131.52) from Klebsiella pneumoniae was added at a final concentration of 0.22 U/ml to each polysaccharide sample (West and Reed-Hamer 1993). The samples were subjected to digestion 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). Column bioreactor production protocol. Cells of A. pullulans ATCC 201253 grown in the phosphatebuffered (pH 6.0) medium containing 2.5% corn syrup were collected and entrapped in agar cubes or calcium alginate beads as previously described (West and Strohfus 1998). The resultant 540 mm3 cubes (approximately 107 g wet weight) or 3 mm calcium alginate beads (approximately 207 g wet weight) were rinsed
and then resuspended in phosphate-buffered minimal medium (pH 6.0) containing 2.5% corn syrup prior to use. The cubes or beads were aseptically added to a sterile column (5 cm × 20 cm) that had a 19.6 cm2 cross-sectional area. The total volume of the column was 393 ml. The agar cube column (5 cm × 10.5 cm) or alginate bead column (5 cm × 12.5 cm) was aerated with filtered air at a rate of 500 ml/min provided at the base of the column. Each column was maintained at 25°C for a period of 168 h. Production of the polysaccharide from the corn syrup-containing medium by the immobilized cells in each column was measured daily using 3 separate determinations during each period of 168 h. After the initial production cycle, each column was drained of medium and washed twice with 0.85% NaCl (360 ml). Corn syrup-containg minimal medium was used to refill the column for the second production cycle. After each cycle, cell leakage from the supports was monitored by removing culture medium (5 ml) and centrifuging the sample at 14,600 × g for 30 min at 4°C. The leaked cells were washed, centrifuged and collected on preweighed Millipore 0.45 µm HVLP filters (47 mm). After drying to constant weight at 105°C, the filters were reweighed to measure the weights of the leaked cells. To determine the viable cell concentration entrapped in the cubes or beads, representative samples were crushed aseptically, suspended in 0.85% NaCl (2 ml) and vigorously mixed. The viable cell concentrations in the samples were determined after the suspensions were diluted and spread onto potato dextrose agar plates to derive colony-forming units. Dry weights were quantitated by collecting the crushed supports on preweighed Millipore 0.45 µm 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.
Table 1. Polysaccharide concentrations produced by immobilized ATCC 201253 cells in a batch bioreactor during 2 cycles of use for 168 h. Polysaccharide concentrations, determined by ethanol precipitation, are given as mg polysaccharide/ml where each value represents the mean of three separate trials ± SD. Hours
Cycle and polysaccharide concentration: Agar
0 24 48 72 96 120 144 168 286
0.00 ± 0.00 0.30 ± 0.09 1.95 ± 0.10 3.30 ± 0.05 3.52 ± 0.16 3.50 ± 0.05 3.70 ± 0.00 3.90 ± 0.05
0.00 ± 0.00 0.15 ± 0.13 1.10 ± 0.05 1.83 ± 0.10 2.92 ± 0.10 3.27 ± 0.12 3.42 ± 0.08 3.95 ± 0.05
0.00 ± 0.00 2.45 ± 0.25 3.17 ± 0.18 3.22 ± 0.28 3.12 ± 0.23 3.33 ± 0.15 3.32 ± 0.25 3.40 ± 0.23
0.00 ± 0.00 1.13 ± 0.15 3.67 ± 0.42 4.90 ± 0.09 6.02 ± 0.28 5.55 ± 0.17 5.93 ± 0.55 5.62 ± 0.77
Microbiol. Res. 156 (2001) 3
Results and discussion The degree of immobilization of A. pullulans ATCC 201253 cells in the two supports was investigated. It was determined at 0 h that 9.6 × 104 colony forming units/g dry weight of support were entrapped in the agar cubes while the degree of immobilization in the calcium alginate beads was calculated to be 5.2 × 105 colony forming units/g dry weight of support. The ability of the agar-entrapped or alginate-immobilized A. pullulans cells in the batch bioreactor to produce polysaccharide was examined during both cycles (Table 1). During the initial cycle of polysaccharide production by the agarentrapped cells, the highest polysaccharide level was detected after 168 h (Table 1). Polysaccharide production by the alginate-entrapped cells appeared to be maximal after 48 h of the initial cycle (Table 1). After the initial cycle, cell leakage from the agar-entrapped cells was determined to be 2.5 mg cells/g cubes while leakage from the calcium alginate-entrapped cells was observed to be 2.7 mg cells/g beads. Although the agar-entrapped cells produced a higher polysaccharide level after 168 h during the second cycle of production compared to the first cycle, pullulan production was generally lower during the second cycle then the initial cycle (Table 1). Relative to the initial cycle of production, the alginate-entrapped cells elaborated higher polysaccharide levels after 24 h of the second production cycle (Table 1). The polysaccharide concentration produced by the alginate-entrapped cells was maximal after 96 h of the second cycle (Table 1). During the initial cycle of pullulan production using the entrapped cells, the agar cubes produced a higher polysaccharide concentration than did the calcium alginate beads (Table 1). Polysaccharide levels elaborated by the alginate-entrapped cells were higher than the levels synthesized by the agar-entrapped cells during the second production cycle (Table 1). For the agar-immobilized cells, polysaccharide production was comparable during both cycles while polysaccharide production by the alginate-immobilized cells was higher during the second cycle than the first cycle (Table 1). Following the second production cycle, cell leakage of the agar-entrapped cells was found to be 7.8 mg cells/g cubes while the leakage from the alginate-entrapped cells was 9.3 mg cells/g beads. Thus, an increase in cell leakage from the bioreactors after the second cycle was noted but was still less than the cell leakage observed in shake flask cultures (West and Strohfus 1998). During the initial production cycle, the agar-entrapped cells were more effective in producing polysaccharide than the alginate-entrapped cells (Table 2). During the second production cycle, the agar-immobilized and agarose-entrapped cells were equally productive (Table 2). The pullulan content
Table 2. Productivity of entrapped ATCC 201253 cells and the pullulan content of the polysaccharide synthesized during each cycle. Productivity is stated as mg polysaccharide/ (g cells × h). The highest productivity observed for the agarimmobilized cells during cycle 1 or 2 was observed after 72 or 96 h, respectively. The highest productivity witnessed for the alginate-entrapped cells during cycle 1 or 2 was noted after 24 or 48 h, respectively. The pullulan content of the polysaccharide produced, which is given in %, was calculated by measuring its sensitivity to pullulanase digestion. Each value represents the mean of three experiments ± SD Support
1 2 1 2
0.67 ± 0.01 0.45 ± 0.02 0.57 ± 0.06 0.43 ± 0.05
51 ± 4 66 ± 4 26 ± 8 36 ± 9
of the polysaccharide elaborated by the agar-entrapped cells during either cycle was significantly higher than the polysaccharide elaborated by the alginateentrapped cells (Table 2). It has been suggested that calcium ions may stimulate the synthesis of an alternate polysaccharide by the alginate-immobilized cells of A. pullulans (Simon et al. 1993; West and Strohfus 1998). Little of the extracellular material precipitated by ethanol was shown to be protein but was likely another polysaccharide (such as beta-1,3glucan) or polyol (Simon et al. 1993; Leal-Serrano et al. 1980). When agar cubes containing immobilized A. pullulans ATCC 201253 were used for pullulan production in batch cultures containing 2.5% (w/v) corn syrup, the findings were different from what was observed using the batch bioreactor. The agar-immobilized cells utilized in shake flask cultures elaborated higher levels of polysaccharide during the first or second cycle of 168 h than did the agar-entrapped cells used in the batch bioreactor (West and Strohfus 1998). Polysaccharide production by the calcium alginate-immobilized cells was comparable during the initial cycle of 168 h for the immobilized cells used in batch cultures or in the batch bioreactor (West and Strofus 1998). In contrast, polysaccharide elaboration was found to be higher by the alginate-immobilized cells used in the batch bioreactor for a second cycle of 168 h compared to production by the alginate-immobilized cells in batch cultures (West and Strohfus 1998). As was observed in this study using the batch bioreactors, the agar-entrapped ATCC 201253 cells exhibited a higher productivity in batch cultures than did the alginate-entrapped cells (West and Strohfus 1998). Also, polysaccharide synthesized by the agarimmobilized A. pullulans cells had a higher pullulan content than did the polysaccharide elaborated by the Microbiol. Res. 156 (2001) 3
alginate-immobilized ATCC 201253 cells during either production cycle of 168 h in batch cultures or in batch bioreactors (West and Strohfus 1998). Relative to other immobilization supports, sponge-immobilized, 2.5% (w/v) corn syrup-grown A. pullulans cells produced comparable polysaccharide levels to those produced by the agar-entrapped cells during the two production cycles of 168 h (West and Strohfus 1996). The pullulan content of the polysaccharide produced by the spongeimmobilized cells in a corn syrup-containing medium was higher than the pullulan content of the polysaccharide elaborated by the agar- or alginate-entrapped cells (West and Strofus 1996). Polyurethane foam entrapment of 5% (w/v) sucrose-grown cells of A. pullulans strain 2552 has proved successful since they could be used for four cycles of polysaccharide production with little cell leakage (Mulchandani et al. 1989). The immobilization of 5% (w/v) glucose-grown A. pullulans CNCM 1726.88 cells on a 0.5% (w/v) agar layered membrane filter resulted in a low level of polysaccharide elaboration due to the fouling of the membrane filter by cells leaking from the support (Lebrun et al. 1994). Overall, entrapped A. pullulans cells can be used in batch bioreactors for semi-continuous pullulan production where cell leakage is less of a problem than noted in batch culture production. The feasibility of a largescale bioreactor process for pullulan production using entrapped fungal cells depends upon whether the pullulan content of the polysaccharide elaborated can be increased.
Acknowledgements Published as paper 3219, 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 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.
Microbiol. Res. 156 (2001) 3
References Bernier, B. (1958): The production of polysaccharides by fungi active in the decomposition of wood and forest litter. Can. J. Microbiol. 4, 195–204. Catley, B. J. (1970): Pullulan, a relationship between molecular weight and fine structure. FEBS Lett. 10, 190–193. Dygert, S., Li, L. H., Florida, D., Thoma, J. A. (1965): Determination of reducing sugar with improved precision. Anal. Biochem. 13, 367–374. Leal-Serrano, G., Ruperez, P., Leal, J. A. (1980): Acidic polysaccharide from Aureobasidium pullulans. Trans. Br. Mycol. Soc. 75, 57–62. Lebrun, L., Junter, G. A., Jouenne, T., Mignot, L. (1994): Exopolysaccharide production by free and immobilized microbial cultures. Enzyme Microbiol. Technol. 16, 1048–1054. Mulchandani, A., Luong, J. H. T., LeDuy, A. (1989) : Biosynthesis of pullulan using immobilized Aureobasidium pullulans. Biotechnol. Bioeng. 33, 306–312. Simon, L., Caye-Vaugien, C., Bouchonneau, M. (1993): Relation between pullulan production, morphological state and growth conditions in Aureobasidium pullulans: new observations. J. 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. J. Microbiol. 40, 35–45. Sowa, W., Blackwood, A. C., Adams, G. A. (1963): Neutral extracellular glucan of Pullularia pullulans (de Bary) Berkhout. Can. J. Chem. 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. Microbiol. 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 pullans. 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. West, T. P., Strohfus, B. (1998): Polysaccharide production by Aureobasidium pullulans cells immobilized by entrapment. Microbiol. Res. 153, 253–256. Yuen, S. (1974) : Pullulan and its applications. Process Biochem. 9, 7–9. Zajic, J. E., LeDuy, A. (1973): Flocculant and chemical properties of a polysaccharide from Pullularia pullulans. Appl. Microbiol. 25, 628–635.