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Production of kojic acid by membrane-surface liquid culture of Aspergillus oryzae NRRL484
JOURNALOF FERMENTATION AND
BIOENGINEERING
Vol. 80, No. 1, 41-45. 1995
Production of Kojic Acid by Membrane-Surface Liquid Culture of Aspergillus oryzae NRRL484 AKINORI
OGAWA,
YASUSHI
WAKISAKA, TAKAAKI TANAKA, KAZUHIRO NAKANISHI*
TAKAHARU
SAKIYAMA,
AND
Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700, Japan Received 2 February 1995/Accepted 17 April 1995 Membrane-surface liquid culture (MSLC) was applied to the production of kojic acid using Aspergillus oryzae NRRL484. The characteristics of kojic acid fermentation by MSLC were compared particularly with those by shaking culture. The maximum concentration of kojic acid produced and the production rate of kojic acid by the MSLC were usually higher than those by the shaking culture. In the shaking culture, the kojic acid production was the highest with 0.0%0.25% yeast extract and the maximum concentration was around 20 mg/ml. In the MSLC, the highest kojic acid concentration of about 30 mg/ml was obtained with 0.25-0.5 % yeast extract. By addition of powdered glucose at a final concentration of lo%, 2-3 times at appropriate intervals during the batch MSLC, the concentration of kojic acid increased to over 100 mg/ml and kojic acid crystals formed in the medium. Repeated fed-batch production of kojic acid by MSLC was quite successful. The concentration of kojic acid produced in each batch was maintained at 75 mg/ml or more with a yield of around 50% for 10 batches and 75 d when the medium contained 0.25% yeast extract. The kojic acid productivity by the repeated fed-batch MSLC was 14.2 g/Z/d, about 6 times higher than that by the shaking culture with the medium containing 0.25% yeast extract. After the 11th batch, the production rate decreased, probably due to an increased amount of cells formed on the membrane. [Key words: Aspergillus oryzae, kojic acid, membrane, mold, solid-state culture] the pH was adjusted to 6.0 with 1 N HCl. The medium composition was similar to that reported previously (3) except for the yeast extract concentration. The cultivation was carried out at 30°C. Usually, 5,000 spores were used for inoculation. In all the cultures, the pH of the medium was not controlled during the cultivation. In the Membrane-surface liquid culture (MSLC) batch MSLC, several cultures were carried out in plastic dishes (9 cm x 1.4 cm) under the same cultivation conditions (2). About 20ml of the liquid medium was poured into a bottom dish. On the surface of the liquid medium, an SE 20 membrane made of polysulfone with a nominal pore size of 0.2/1m (Fuji Photo Film Co., Tokyo) was supported by four pieces of rectangular polyurethane foam. The bottom dish was covered with a top dish. Twenty microliters of a suspension of spores was inoculated uniformly onto the membrane surface and incubated statically. The molds grew on the surface of the membrane. At appropriate times during cultivation, an aliquot of the cultures was withdrawn for analysis of kojic acid and glucose concentrations, dry cell weight and pH. In the fed-batch and repeated fed-batch MSLC, an apparatus made of polycarbonate was used, which was the same as that reported previously (1, 2). About 23 ml of the liquid medium was added into the cylindrical groove (inner diameter, 7 cm; depth, 6 mm), and the remaining 2ml was poured into a lOO-ml Erlenmeyer flask (2). At appropriate times during the fed-batch MSLC, the entire liquid medium was transferred from the cylindrical groove into the flask using a peristaltic pump (MP-3, Tokyorikakikai, Tokyo), and 2ml of the sample was withdrawn. The volume of medium withdrawn for analysis and that lost due to evaporation were replaced with the same volume of the medium without yeast extract and glucose and pure water, respectively. Then, some of
In a previous study (l), we developed a novel cultivation method for molds, named membrane-surface liquid culture (MSLC), in which molds are grown on a microporous membrane surface exposed to the air with the other side of the membrane in contact with liquid medium. We showed that Aspergillus oryzae IAM produces much more protease in MSLC than in shaking culture (1, 2), and that repeated-batch culture for production of protease is also possible (2). We suggested that the advantageous features of MSLC are due to the growth on the surface of a porous material and to an efficient oxygen uptake. In this study, we applied MSLC to the production of kojic acid using A. oryzae NRRL484 as a model of secondary metabolite fermentation. We compared the characteristics of kojic acid fermentation by MSLC with those by shaking culture. We also conducted repeated-batch MSLC to produce kojic acid, which is usually difficult by conventional liquid culture methods. MATERIALS
AND METHODS
Microorganisms and media A. oryzae NRRL484 was used to produce kojic acid. The vegetative cells were cultivated at 30°C for about 10d on a PDA slant medium adjusted to pH 6.0 (100 g squeezed potato, 10 g glucose and log agar dissolved in 500ml ion-exchanged water) to obtain spores. The spores thus obtained were suspended aseptically in 0.9% saline with vigorous shaking and filtered through a glass filter (G-3 type, Hario Glass Co. Ltd., Tokyo). The liquid medium for batch culture usually contained 1OOg glucose, 0.5-log yeast extract, 1 g K2HP04, 0.5 g MgS04.7Hz0, 0.5 g KC1 and 0.01 g FeSO,. 7H20 in 1 I of ion-exchanged water and *
Corresponding author.
41
42
OGAWA ET AL.
J. FERMENT.BIOENG.,
the medium (about 23 ml) was returned to the MSLC apparatus. When the glucose concentration in the medium was sufficiently low, the entire liquid medium was transferred into the flask as described above. Then, powdered glucose (2.5 g) which had been irradiated with a UV lamp to give a 10% final concentration, was aseptically dissolved in the liquid medium, and again some of the liquid medium (about 23 ml) was returned to the MSLC apparatus. These procedures were repeated 2 to 5 times. In the repeated fed-batch MSLC, the entire medium was exchanged with fresh medium after each batch culture. Shaking culture Sakaguchi flasks (100 ml) containing 20 ml of the medium inoculated with a suspension of spores were incubated at 30°C on a reciprocal shaker (120 strokes/min). At appropriate times during cultivation, an aliquot of the cultures was withdrawn and analyzed. In one experiment, 5 g powdered glucose was added into the medium at a final concentration of 10% after 7 d of cultivation. During long-term cultivation, some of the water in the medium evaporated and was replaced with the same volume of pure water before an aliquot of the culture was analyzed. The concentrations of glucose and kojic Analysis acid were measured by a glucose oxidase/peroxidase method using a Glucostat reagent kit (Toyobo Co., Osaka) and Bentley’s method (4), respectively. The growth of the microorganism was indicated by the dry weight of the mycelia. The cell weight was measured as follows (2). During the cultivation, an aliquot of the culture was withdrawn at appropriate cultivation times. In the MSLC, the cells were quite easily removed from the membrane surface with a spatula. The cells were weighed without additional washing. In the shaking culture, about 50ml of ion-exchanged water was added to remove the cells adhering to the wall of the flask and the suspension was filtered with suction onto a nylon bolting cloth (mesh size: 5 pm; Nytal HD5 p; Tanaka Sanjiro Co. Ltd., Fukuoka) and rinsing. The recovered cells were dried at 105°C to a constant weight. RESULTS AND DISCUSSION Effect of yeast extract concentration on kojic acid production Kojic acid production usually depends on the concentration of the nitrogen source (5). In this study, we used yeast extract as a nitrogen source, which was the most suitable for the kojic acid fermentation. In Table 1, the effect of yeast extract concentration on the
kojic acid production in the presence of 10% glucose is summarized. As the yeast extract concentration increased, the time required to reach the maximum concentration of kojic acid decreased in both the shaking culture and MSLC. In the shaking culture, the maximum concentration of kojic acid and particularly its yield with respect to the amount of glucose consumed increased with decreasing yeast extract concentration, and kojic acid production was the highest with 0.05-0.25% yeast extract. With 0.05% yeast extract, an appreciable amount of glucose remained at 12 d of cultivation; its residual concentration was about 63 mg/ml, and the kojic acid concentration in the medium continued to increase gradually up to around 20mg/ml. In this case, glucose tended not to be utilized for cell growth but predominantly for production of kojic acid, and thus the conversion yield was high. The dry cell weight was about 40 mg at 12 d of cultivation. These findings were similar in tendency to those reported by Kwak and Rhee (3); they cultivated the same strain of microorganism as used in this study using the same medium containing 0.05% yeast extract in a 5-f jar fermentor. On the other hand, with higher yeast extract concentrations, glucose tended not to be converted to kojic acid but to be utilized for cell growth. With 0.5% yeast extract, the residual glucose concentration was nearly zero at 8 d of cultivation and the dry cell weight was 2OOmg, which was much higher than that with 0.05% yeast extract. The maximum yield of kojic acid was only 13%. With 1% yeast extract, the amount of kojic acid produced was extremely low. In the MSLC, the maximum concentration of kojic acid produced with 0.05% yeast extract was similar to that in the shaking culture. However, with 0.25 or 0.5% yeast extract, it increased further to about 30mg/ml in contrast to the result obtained in the shaking culture. This value was around 50% higher compared to the highest amount obtained in the shaking culture with 0.05-0.25% yeast extract. The production rate was slightly higher with 0.5% yeast extract than that with 0.25% yeast extract. With 1% yeast extract, the kojic acid concentration greatly decreased, although it was much higher than that in the shaking culture. These different dependences of kojic acid production on the yeast extract concentration in the shaking culture and MSLC should be studied further. Courses of kojic acid fermentation by shaking culture and MSLC Figures la and lb show courses of kojic
TABLE 1. Effect of yeast extract concentration on kojic acid production by shaking culture and MSLC” Yeast extract concentration (%; w/v)
Maximum kojic acid
Shaking culture
0.05 0.10 0.25 0.5 1.0
Batch MSLC
0.05 0.10 0.25 0.5 1.0
Cultivation mode
Cultivation time (d)b
Yield’ (%)
19.0 20.5 20.6 12.0 0.5
12 12 9 8 6
51 42 30 13 0.7
18.4 24.0 29.0 28.3 5.0
13 11 10 8 4
46 36 41 36 6.4
concentration (mglml)
a The glucose concentration was 10% (w/v). b The time required to reach the maximum kojic acid concentration. c The yield of kojic acid with respect to the amount of glucose consumed.
VOL. 80, 1995
125r
PRODUCTION
. . . . v
25,
. . . I
30
Cultivation ,looo
,
25
10
0
0
Culkation
10 time (d)
43
. . . 11000
Cultivation time (d) 150 r
OF KOJIC ACID BY MSLC
15
FIG. 1. Changes of kojic acid concentration, dry cell weight, residual glucose concentration, and pH in shaking culture (a) and MSLC (b). Symbols: A, kojic acid concentration; 0, dry cell weight; 0, residual glucose concentration; A, pH of the medium. The concentrations of yeast extract and glucose in the medium were 0.5% and lo%, respectively, both for shaking culture and MSLC.
acid fermentation by shaking culture and MSLC, respectively with the medium containing 0.5% yeast extract and 10% glucose. In both cultivation methods, the pH of the solution was decreased to around 2.5 after 2-3 d of cultivation. In the shaking culture, the glucose concentration gradually decreased with concomitant increase in the kojic acid concentration. The kojic acid concentration reached about 12 mg/ml at 8 d of cultivation, and then gradually decreased. Such behavior of kojic acid fermentation is usually observed (5, 6). In the MSLC, the growth rate of mycelia and consumption rate of glucose were higher than those in the shaking culture, probably because of the high availability of oxygen. At 6 d of cultivation, the glucose supply was exhausted. The kojic acid concentration reached a maximum (about 28 mg/ ml) at 8 d of cultivation, and then rapidly decreased. At 13 d of cultivation, only a small amount of kojic acid was detected. The decrease in the kojic acid concentration after reaching a maximum was more pronounced in the MSLC than in the shaking culture (Fig. la), for an as yet unknown reason. Production of kojic acid by fed-batch MSLC In the batch MSLC, the concentration of kojic acid started to decrease when the glucose supply was exhausted as described previously. Thus, we added powdered glucose
time
(d)
Courses of kojic acid production in the fed-batch MSLC. The concentrations of yeast extract and glucose of the medium were 0.5% and lo%, respectively. During thecultivation, 2.5 g of powdered glucose was added at the times shown by arrows ( 1). Symbols: A, kojic acid concentration; 0, residual glucose concentration; A, pH of the medium. FIG.
2.
(2.5 g) to give a 10% (w/v) final concentration during the batch MSLC. The cultivation was started with the medium containing 0.5% yeast extract and 10% glucose as in the case of the batch MSLC. As shown in Fig. 2, the kojic acid concentration increased almost linearly up to about 80 mg/ml at 16 d of cultivation after 2 additions of glucose, and then the kojic acid started to precipitate as needle-like crystals after 3 additions of glucose. The final kojic acid concentration was over 100 mg/ml. This concentration exceeds the solubility limit of kojic acid (about 80mg/ml) because we measured the kojic acid concentration after dissolving the crystals. In the shaking culture using 0.5% yeast extract and 10% glucose, we added 2.5 g powdered glucose (10% final concentration) into the liquid medium after 7 and 18 d of cultivation. However, the kojic acid concentration was only 38 mg/ml after 22 d of cultivation (data not shown). When the yeast extract concentration in the medium was 0.25% in the MSLC, the kojic acid concentration reached lOOmg/ml at 20d of cultivation after 2 additions of glucose (data not shown), since less glucose was utilized for growth than in the case with 0.5% yeast extract. In the fed-batch shaking culture, the kojic acid concentration was about 55 mg/ml at 30 d of cultivation when powdered glucose was added once at 9 d of cultivation. Production of kojic acid by repeated fed-batch MSLC We conducted a repeated fed-batch MSLC using the exchange medium with or without yeast extract as a nitrogen source. During one batch culture, 2.5 g powdered glucose was added several times in the same manner as described previously. Figure 3 shows the result for the repeated-batch culture using a medium containing 0.25% yeast extract and 10% glucose as the exchange medium as well as the starting medium. The yeast extract concentration was set at 0.25% to decrease the cell growth rate. The powdered glucose was added usually twice during each batch culture. When kojic acid in the medium started to crystallize, the entire medium was exchanged with fresh medium, and cultivation was continued for lOOd, in which 12 batch MSLCs were carried out including the
44
J. FERMENT.BIOENG.,
OGAWA ET AL. TABLE 2.
Comparison of yield and productivity using various cultivation method@
Cultivation method Shaking culture Batch MSLC Fed-batch MSLC Repeated fed-batch MSLCb
Yield (%) 51 45 56 51
Productivity (n/l/d) 1.6 2.9 5.0 14.2
a The yeast extract concentration was 0.25% (w/v) except in the case of the shaking culture (0.05%; w/v). b The yield and productivity were calculated from the data of the 2nd to 10th batches. 5.0
X
2.5 a 0
20
40
Cultivation
60
80
time (d)
FIG. 3. Courses of kojic acid production in the repeated fedbatch MSLC using the exchange medium containing yeast extract. The cultivation was started with the medium containing 0.25% yeast extract and 10% glucose. During each batch culture, 2.5 g powdered glucose (final concentration of 10%) was added usually twice (1st to 9th batch) and 3 times (10th to 12th batch). At the times shown by arrows the entire medium was exchanged with the fresh medium containing 0.25% yeast extract and 10% glucose. Symbols: A, kojic acid concentration; A, pH of the medium.
first batch. The production rate of kojic acid was much higher in the second batch than the first batch since molds had grown over the entire membrane surface after the first batch. From the 2nd to 10th batches, the kojic acid production rate was almost constant. In every batch, the kojic acid concentration reached 75 mg/ml or more. From the 11th batch, the production rate decreased. One possible reason for this decrease is that the amount of mycelia formed on the membrane surface increased, which might inhibit oxygen transfer to the inside of the mycelial mat formed on the membrane. Thus, some mycelia that were exposed on the membrane
Cultivation
time
(d)
FIG. 4. Courses of kojic acid production in the repeated fedbatch MSLC using the exchange medium without yeast extract. The cultivation was started with the medium containing 0.5% yeast extract and 10% glucose. During each batch culture, 2.5 g powdered glucose (final concentration of 10%) was added 3 times (1st to 4th batches) and 4 times (5th batch). At the times shown by arrows the entire medium was exchanged with the fresh medium containing 10% glucose without yeast extract. Symbols: A, kojic acid concentration; A, pH of the medium.
surface were autolyzed. The dry cell weight was about 3,300 mg at 100 d of cultivation. Figure 4 shows results obtained using an exchange medium without yeast extract. In this case, the starting medium contained 0.5% yeast extract and 10% glucose. The powdered glucose was added usually 3 times. In the first 4 batches, the kojic acid concentration increased linearly up to around 80 mg/ml. From the 5th batch, the production rate decreased. This indicates that molds require a nitrogen source (yeast extract) to maintain enzymatic activity. Thus, the optimum yeast extract concentration for long-term repeated fed-batch culture exists. Bajpai et al. (6) showed that the mycelia of A. flaws that had grown in the medium were resuspended in a buffer containing only glucose produced almost the same amount of kojic acid as those cultivated in the growth medium. However, they did not try the reaction repeatedly. Kwak and Rhee attempted repeated-batch production of kojic acid with immobilized A. oryzae cells in a chemically defined buffer medium containing only glucose as carbon source; however, the production rate was greatly decreased (3). Comparison of productivities using various cultivation Table 2 summarizes the yield of kojic acid methods with respect to the amount of glucose consumed and overall kojic acid productivity for various cultivation methods. The productivity was defined as the amount of kojic acid produced in one liter of the medium per day. In the batch culture, the yield and overall productivity were calculated at the time at which the maximum amount of kojic acid was detected. In the shaking culture with the medium containing 0.05% yeast extract, the yield was around 50%. This value was similar to that obtained in the case of a jar fermentor using the same strain, as described previously (3). In the batch MSLC with 0.25% yeast extract, the yield was slightly lower than that in the shaking culture presumably because more glucose was utilized for the growth of the cells. The yields in the fed-batch MSLC and repeated fedbatch MSLC with 0.25% yeast extract were similar to or slightly higher than that in the shaking culture. The overall productivity in the repeated fed-batch MSLC for the 2nd to 10th batches was 14.2g/l/d, which was about 6 times higher than that for the shaking culture with 0.25% yeast extract (calculated to be 2.3 g/l/d from Table 1). This value was also several times higher than that in the batch MSLC or in the fed-batch MSLC (Table 2), since in the initial stage of the MSLC several days were necessary for mycelia to grow over the membrane surface. Kwak and Rhee immobilized A. oryzae NRRL484 by entrapping it in calcium alginate gel beads to produce
VOL. 80, 1995
kojic acid (3). The kojic acid concentration reached 83 mg/ml in the case of batch culture feeding of glucose. They also conducted repeated-batch culture. However, the kojic acid production rate decreased early, from the 3rd batch culture. The productivity for repeated-batch culture with the immobilized cells was only 3.7 g/I/d, which was nearly l/4 that obtained in the repeated fedbatch MSLC in this study (Table 2). Secondary metabolite fermentation using molds is usually conducted either by submerged liquid culture using liquid medium or by solid-state culture using water-insoluble solid substrates. However, in these cultivation methods, it is difficult to conduct repeated-batch culture. With the MSLC, repeated-batch production of kojic acid was fairly successful. Without any appreciable decrease in production rate 10 batch cultures could be conducted with the medium containing 0.25% yeast extract as a nitrogen source. Even using the medium without yeast extract several repeated-batch cultures were possible. Cells suspended in liquid medium usually suffer from irreversible loss in activity when incubated for a long time. In kojic acid production, repeated-batch culture with suspended cells was difficult in the shaking culture (data not shown). We showed in a previous study that repeated-batch production of neutral protease is possible using MSLC (2). Thus, we speculate that molds are more stable against autolysis when cultivated by MSLC. Molds are typical aerobes and thus require oxygen, favor atmospheres under somewhat low water activity conditions (less than 0.99) for production of specific substances (7), and grow on solid surfaces in the natural environment. Probably, such properties of molds help explain the high productivity observed in MSLC, in which they are grown on a membrane surface, obtaining oxygen directly from the atmosphere and at the same time obtaining nutrients from liquid medium through membrane pores. In this study, we showed that kojic acid could be
PRODUCTION
OF KOJIC ACID BY MSLC
45
produced efficiently from A. oryzae NRRL484 by MSLC following the production of neutral protease from A. oryzue IAM in previous studies (1, 2). We intend to apply MSLC to other fermentation systems using various molds and possibly other microorganisms. Furthermore, we are developing methods to apply MSLC to large-scale production as discussed in a previous report (2). ACKNOWLEDGMENTS We thank the Natl. Center for Agricultural Util. Research (University St. Peoria, IL, U.S.A.) for providing the strain of A. oryzue NRRL484. REFERENCES 1. Yasuhara, A., Ogawa, A., Tanaka, T., Sakiyama, T., and Nakanishi, K.: Production of neutral protease from Aspergillus oryzae by a novel cultivation method on a microporous membrane. Biotechnol. Techniq., 8, 249-254 (1994). 2. Ogawa, A., Yasuhara, A., Tanaka, T., Sakiyama, T., and Nakaaishi, K.: Production of neutral protease by membranesurface liquid culture of Aspergihs oryzae IAM2704. J. Ferment. Bioeng., 80, 35-40 (1995). 3. Kwak, M. Y. and Rhee, S.: Cultivation characteristics of immobilized Aspergiks oryzae for kojic acid production. Biotechnol. Bioeng., 39, 903-906 (1992). 4. Bentley, R.: Preparation and analysis of kojic acid, p. 238-241. In Colowick, S. P. and Kaplan, N. 0. (ed.), Methods in enzymology, vol. 3. Academic Press, New York (1957). 5. Kitada, M., Ueyama, H., Suzuki, E., and Fukimbara, T.: Studies on kojic acid fermentation. I. Cultural conditions in submerged culture. J. Ferment. Technol., 45, 1101-1107 (1967). 6. Bajpai, P., Agrawala, P. K., and Vishwanthan, L.: Production of kojic acid by resuspended mycelia of Aspergihs pavus. Can. J. Microbial., 28, 1340-1346 (1982). 7. Johns, M. R.: Production of secondary metabolites, p. 341352. In Doelle, H. W., Mitchell, D.A., and Rolz, C. E. (ed.), Solid substrate cultivation. Elsevier Applied Science, London and New York (1992).
BIOENGINEERING
Vol. 80, No. 1, 41-45. 1995
Production of Kojic Acid by Membrane-Surface Liquid Culture of Aspergillus oryzae NRRL484 AKINORI
OGAWA,
YASUSHI
WAKISAKA, TAKAAKI TANAKA, KAZUHIRO NAKANISHI*
TAKAHARU
SAKIYAMA,
AND
Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700, Japan Received 2 February 1995/Accepted 17 April 1995 Membrane-surface liquid culture (MSLC) was applied to the production of kojic acid using Aspergillus oryzae NRRL484. The characteristics of kojic acid fermentation by MSLC were compared particularly with those by shaking culture. The maximum concentration of kojic acid produced and the production rate of kojic acid by the MSLC were usually higher than those by the shaking culture. In the shaking culture, the kojic acid production was the highest with 0.0%0.25% yeast extract and the maximum concentration was around 20 mg/ml. In the MSLC, the highest kojic acid concentration of about 30 mg/ml was obtained with 0.25-0.5 % yeast extract. By addition of powdered glucose at a final concentration of lo%, 2-3 times at appropriate intervals during the batch MSLC, the concentration of kojic acid increased to over 100 mg/ml and kojic acid crystals formed in the medium. Repeated fed-batch production of kojic acid by MSLC was quite successful. The concentration of kojic acid produced in each batch was maintained at 75 mg/ml or more with a yield of around 50% for 10 batches and 75 d when the medium contained 0.25% yeast extract. The kojic acid productivity by the repeated fed-batch MSLC was 14.2 g/Z/d, about 6 times higher than that by the shaking culture with the medium containing 0.25% yeast extract. After the 11th batch, the production rate decreased, probably due to an increased amount of cells formed on the membrane. [Key words: Aspergillus oryzae, kojic acid, membrane, mold, solid-state culture] the pH was adjusted to 6.0 with 1 N HCl. The medium composition was similar to that reported previously (3) except for the yeast extract concentration. The cultivation was carried out at 30°C. Usually, 5,000 spores were used for inoculation. In all the cultures, the pH of the medium was not controlled during the cultivation. In the Membrane-surface liquid culture (MSLC) batch MSLC, several cultures were carried out in plastic dishes (9 cm x 1.4 cm) under the same cultivation conditions (2). About 20ml of the liquid medium was poured into a bottom dish. On the surface of the liquid medium, an SE 20 membrane made of polysulfone with a nominal pore size of 0.2/1m (Fuji Photo Film Co., Tokyo) was supported by four pieces of rectangular polyurethane foam. The bottom dish was covered with a top dish. Twenty microliters of a suspension of spores was inoculated uniformly onto the membrane surface and incubated statically. The molds grew on the surface of the membrane. At appropriate times during cultivation, an aliquot of the cultures was withdrawn for analysis of kojic acid and glucose concentrations, dry cell weight and pH. In the fed-batch and repeated fed-batch MSLC, an apparatus made of polycarbonate was used, which was the same as that reported previously (1, 2). About 23 ml of the liquid medium was added into the cylindrical groove (inner diameter, 7 cm; depth, 6 mm), and the remaining 2ml was poured into a lOO-ml Erlenmeyer flask (2). At appropriate times during the fed-batch MSLC, the entire liquid medium was transferred from the cylindrical groove into the flask using a peristaltic pump (MP-3, Tokyorikakikai, Tokyo), and 2ml of the sample was withdrawn. The volume of medium withdrawn for analysis and that lost due to evaporation were replaced with the same volume of the medium without yeast extract and glucose and pure water, respectively. Then, some of
In a previous study (l), we developed a novel cultivation method for molds, named membrane-surface liquid culture (MSLC), in which molds are grown on a microporous membrane surface exposed to the air with the other side of the membrane in contact with liquid medium. We showed that Aspergillus oryzae IAM produces much more protease in MSLC than in shaking culture (1, 2), and that repeated-batch culture for production of protease is also possible (2). We suggested that the advantageous features of MSLC are due to the growth on the surface of a porous material and to an efficient oxygen uptake. In this study, we applied MSLC to the production of kojic acid using A. oryzae NRRL484 as a model of secondary metabolite fermentation. We compared the characteristics of kojic acid fermentation by MSLC with those by shaking culture. We also conducted repeated-batch MSLC to produce kojic acid, which is usually difficult by conventional liquid culture methods. MATERIALS
AND METHODS
Microorganisms and media A. oryzae NRRL484 was used to produce kojic acid. The vegetative cells were cultivated at 30°C for about 10d on a PDA slant medium adjusted to pH 6.0 (100 g squeezed potato, 10 g glucose and log agar dissolved in 500ml ion-exchanged water) to obtain spores. The spores thus obtained were suspended aseptically in 0.9% saline with vigorous shaking and filtered through a glass filter (G-3 type, Hario Glass Co. Ltd., Tokyo). The liquid medium for batch culture usually contained 1OOg glucose, 0.5-log yeast extract, 1 g K2HP04, 0.5 g MgS04.7Hz0, 0.5 g KC1 and 0.01 g FeSO,. 7H20 in 1 I of ion-exchanged water and *
Corresponding author.
41
42
OGAWA ET AL.
J. FERMENT.BIOENG.,
the medium (about 23 ml) was returned to the MSLC apparatus. When the glucose concentration in the medium was sufficiently low, the entire liquid medium was transferred into the flask as described above. Then, powdered glucose (2.5 g) which had been irradiated with a UV lamp to give a 10% final concentration, was aseptically dissolved in the liquid medium, and again some of the liquid medium (about 23 ml) was returned to the MSLC apparatus. These procedures were repeated 2 to 5 times. In the repeated fed-batch MSLC, the entire medium was exchanged with fresh medium after each batch culture. Shaking culture Sakaguchi flasks (100 ml) containing 20 ml of the medium inoculated with a suspension of spores were incubated at 30°C on a reciprocal shaker (120 strokes/min). At appropriate times during cultivation, an aliquot of the cultures was withdrawn and analyzed. In one experiment, 5 g powdered glucose was added into the medium at a final concentration of 10% after 7 d of cultivation. During long-term cultivation, some of the water in the medium evaporated and was replaced with the same volume of pure water before an aliquot of the culture was analyzed. The concentrations of glucose and kojic Analysis acid were measured by a glucose oxidase/peroxidase method using a Glucostat reagent kit (Toyobo Co., Osaka) and Bentley’s method (4), respectively. The growth of the microorganism was indicated by the dry weight of the mycelia. The cell weight was measured as follows (2). During the cultivation, an aliquot of the culture was withdrawn at appropriate cultivation times. In the MSLC, the cells were quite easily removed from the membrane surface with a spatula. The cells were weighed without additional washing. In the shaking culture, about 50ml of ion-exchanged water was added to remove the cells adhering to the wall of the flask and the suspension was filtered with suction onto a nylon bolting cloth (mesh size: 5 pm; Nytal HD5 p; Tanaka Sanjiro Co. Ltd., Fukuoka) and rinsing. The recovered cells were dried at 105°C to a constant weight. RESULTS AND DISCUSSION Effect of yeast extract concentration on kojic acid production Kojic acid production usually depends on the concentration of the nitrogen source (5). In this study, we used yeast extract as a nitrogen source, which was the most suitable for the kojic acid fermentation. In Table 1, the effect of yeast extract concentration on the
kojic acid production in the presence of 10% glucose is summarized. As the yeast extract concentration increased, the time required to reach the maximum concentration of kojic acid decreased in both the shaking culture and MSLC. In the shaking culture, the maximum concentration of kojic acid and particularly its yield with respect to the amount of glucose consumed increased with decreasing yeast extract concentration, and kojic acid production was the highest with 0.05-0.25% yeast extract. With 0.05% yeast extract, an appreciable amount of glucose remained at 12 d of cultivation; its residual concentration was about 63 mg/ml, and the kojic acid concentration in the medium continued to increase gradually up to around 20mg/ml. In this case, glucose tended not to be utilized for cell growth but predominantly for production of kojic acid, and thus the conversion yield was high. The dry cell weight was about 40 mg at 12 d of cultivation. These findings were similar in tendency to those reported by Kwak and Rhee (3); they cultivated the same strain of microorganism as used in this study using the same medium containing 0.05% yeast extract in a 5-f jar fermentor. On the other hand, with higher yeast extract concentrations, glucose tended not to be converted to kojic acid but to be utilized for cell growth. With 0.5% yeast extract, the residual glucose concentration was nearly zero at 8 d of cultivation and the dry cell weight was 2OOmg, which was much higher than that with 0.05% yeast extract. The maximum yield of kojic acid was only 13%. With 1% yeast extract, the amount of kojic acid produced was extremely low. In the MSLC, the maximum concentration of kojic acid produced with 0.05% yeast extract was similar to that in the shaking culture. However, with 0.25 or 0.5% yeast extract, it increased further to about 30mg/ml in contrast to the result obtained in the shaking culture. This value was around 50% higher compared to the highest amount obtained in the shaking culture with 0.05-0.25% yeast extract. The production rate was slightly higher with 0.5% yeast extract than that with 0.25% yeast extract. With 1% yeast extract, the kojic acid concentration greatly decreased, although it was much higher than that in the shaking culture. These different dependences of kojic acid production on the yeast extract concentration in the shaking culture and MSLC should be studied further. Courses of kojic acid fermentation by shaking culture and MSLC Figures la and lb show courses of kojic
TABLE 1. Effect of yeast extract concentration on kojic acid production by shaking culture and MSLC” Yeast extract concentration (%; w/v)
Maximum kojic acid
Shaking culture
0.05 0.10 0.25 0.5 1.0
Batch MSLC
0.05 0.10 0.25 0.5 1.0
Cultivation mode
Cultivation time (d)b
Yield’ (%)
19.0 20.5 20.6 12.0 0.5
12 12 9 8 6
51 42 30 13 0.7
18.4 24.0 29.0 28.3 5.0
13 11 10 8 4
46 36 41 36 6.4
concentration (mglml)
a The glucose concentration was 10% (w/v). b The time required to reach the maximum kojic acid concentration. c The yield of kojic acid with respect to the amount of glucose consumed.
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125r
PRODUCTION
. . . . v
25,
. . . I
30
Cultivation ,looo
,
25
10
0
0
Culkation
10 time (d)
43
. . . 11000
Cultivation time (d) 150 r
OF KOJIC ACID BY MSLC
15
FIG. 1. Changes of kojic acid concentration, dry cell weight, residual glucose concentration, and pH in shaking culture (a) and MSLC (b). Symbols: A, kojic acid concentration; 0, dry cell weight; 0, residual glucose concentration; A, pH of the medium. The concentrations of yeast extract and glucose in the medium were 0.5% and lo%, respectively, both for shaking culture and MSLC.
acid fermentation by shaking culture and MSLC, respectively with the medium containing 0.5% yeast extract and 10% glucose. In both cultivation methods, the pH of the solution was decreased to around 2.5 after 2-3 d of cultivation. In the shaking culture, the glucose concentration gradually decreased with concomitant increase in the kojic acid concentration. The kojic acid concentration reached about 12 mg/ml at 8 d of cultivation, and then gradually decreased. Such behavior of kojic acid fermentation is usually observed (5, 6). In the MSLC, the growth rate of mycelia and consumption rate of glucose were higher than those in the shaking culture, probably because of the high availability of oxygen. At 6 d of cultivation, the glucose supply was exhausted. The kojic acid concentration reached a maximum (about 28 mg/ ml) at 8 d of cultivation, and then rapidly decreased. At 13 d of cultivation, only a small amount of kojic acid was detected. The decrease in the kojic acid concentration after reaching a maximum was more pronounced in the MSLC than in the shaking culture (Fig. la), for an as yet unknown reason. Production of kojic acid by fed-batch MSLC In the batch MSLC, the concentration of kojic acid started to decrease when the glucose supply was exhausted as described previously. Thus, we added powdered glucose
time
(d)
Courses of kojic acid production in the fed-batch MSLC. The concentrations of yeast extract and glucose of the medium were 0.5% and lo%, respectively. During thecultivation, 2.5 g of powdered glucose was added at the times shown by arrows ( 1). Symbols: A, kojic acid concentration; 0, residual glucose concentration; A, pH of the medium. FIG.
2.
(2.5 g) to give a 10% (w/v) final concentration during the batch MSLC. The cultivation was started with the medium containing 0.5% yeast extract and 10% glucose as in the case of the batch MSLC. As shown in Fig. 2, the kojic acid concentration increased almost linearly up to about 80 mg/ml at 16 d of cultivation after 2 additions of glucose, and then the kojic acid started to precipitate as needle-like crystals after 3 additions of glucose. The final kojic acid concentration was over 100 mg/ml. This concentration exceeds the solubility limit of kojic acid (about 80mg/ml) because we measured the kojic acid concentration after dissolving the crystals. In the shaking culture using 0.5% yeast extract and 10% glucose, we added 2.5 g powdered glucose (10% final concentration) into the liquid medium after 7 and 18 d of cultivation. However, the kojic acid concentration was only 38 mg/ml after 22 d of cultivation (data not shown). When the yeast extract concentration in the medium was 0.25% in the MSLC, the kojic acid concentration reached lOOmg/ml at 20d of cultivation after 2 additions of glucose (data not shown), since less glucose was utilized for growth than in the case with 0.5% yeast extract. In the fed-batch shaking culture, the kojic acid concentration was about 55 mg/ml at 30 d of cultivation when powdered glucose was added once at 9 d of cultivation. Production of kojic acid by repeated fed-batch MSLC We conducted a repeated fed-batch MSLC using the exchange medium with or without yeast extract as a nitrogen source. During one batch culture, 2.5 g powdered glucose was added several times in the same manner as described previously. Figure 3 shows the result for the repeated-batch culture using a medium containing 0.25% yeast extract and 10% glucose as the exchange medium as well as the starting medium. The yeast extract concentration was set at 0.25% to decrease the cell growth rate. The powdered glucose was added usually twice during each batch culture. When kojic acid in the medium started to crystallize, the entire medium was exchanged with fresh medium, and cultivation was continued for lOOd, in which 12 batch MSLCs were carried out including the
44
J. FERMENT.BIOENG.,
OGAWA ET AL. TABLE 2.
Comparison of yield and productivity using various cultivation method@
Cultivation method Shaking culture Batch MSLC Fed-batch MSLC Repeated fed-batch MSLCb
Yield (%) 51 45 56 51
Productivity (n/l/d) 1.6 2.9 5.0 14.2
a The yeast extract concentration was 0.25% (w/v) except in the case of the shaking culture (0.05%; w/v). b The yield and productivity were calculated from the data of the 2nd to 10th batches. 5.0
X
2.5 a 0
20
40
Cultivation
60
80
time (d)
FIG. 3. Courses of kojic acid production in the repeated fedbatch MSLC using the exchange medium containing yeast extract. The cultivation was started with the medium containing 0.25% yeast extract and 10% glucose. During each batch culture, 2.5 g powdered glucose (final concentration of 10%) was added usually twice (1st to 9th batch) and 3 times (10th to 12th batch). At the times shown by arrows the entire medium was exchanged with the fresh medium containing 0.25% yeast extract and 10% glucose. Symbols: A, kojic acid concentration; A, pH of the medium.
first batch. The production rate of kojic acid was much higher in the second batch than the first batch since molds had grown over the entire membrane surface after the first batch. From the 2nd to 10th batches, the kojic acid production rate was almost constant. In every batch, the kojic acid concentration reached 75 mg/ml or more. From the 11th batch, the production rate decreased. One possible reason for this decrease is that the amount of mycelia formed on the membrane surface increased, which might inhibit oxygen transfer to the inside of the mycelial mat formed on the membrane. Thus, some mycelia that were exposed on the membrane
Cultivation
time
(d)
FIG. 4. Courses of kojic acid production in the repeated fedbatch MSLC using the exchange medium without yeast extract. The cultivation was started with the medium containing 0.5% yeast extract and 10% glucose. During each batch culture, 2.5 g powdered glucose (final concentration of 10%) was added 3 times (1st to 4th batches) and 4 times (5th batch). At the times shown by arrows the entire medium was exchanged with the fresh medium containing 10% glucose without yeast extract. Symbols: A, kojic acid concentration; A, pH of the medium.
surface were autolyzed. The dry cell weight was about 3,300 mg at 100 d of cultivation. Figure 4 shows results obtained using an exchange medium without yeast extract. In this case, the starting medium contained 0.5% yeast extract and 10% glucose. The powdered glucose was added usually 3 times. In the first 4 batches, the kojic acid concentration increased linearly up to around 80 mg/ml. From the 5th batch, the production rate decreased. This indicates that molds require a nitrogen source (yeast extract) to maintain enzymatic activity. Thus, the optimum yeast extract concentration for long-term repeated fed-batch culture exists. Bajpai et al. (6) showed that the mycelia of A. flaws that had grown in the medium were resuspended in a buffer containing only glucose produced almost the same amount of kojic acid as those cultivated in the growth medium. However, they did not try the reaction repeatedly. Kwak and Rhee attempted repeated-batch production of kojic acid with immobilized A. oryzae cells in a chemically defined buffer medium containing only glucose as carbon source; however, the production rate was greatly decreased (3). Comparison of productivities using various cultivation Table 2 summarizes the yield of kojic acid methods with respect to the amount of glucose consumed and overall kojic acid productivity for various cultivation methods. The productivity was defined as the amount of kojic acid produced in one liter of the medium per day. In the batch culture, the yield and overall productivity were calculated at the time at which the maximum amount of kojic acid was detected. In the shaking culture with the medium containing 0.05% yeast extract, the yield was around 50%. This value was similar to that obtained in the case of a jar fermentor using the same strain, as described previously (3). In the batch MSLC with 0.25% yeast extract, the yield was slightly lower than that in the shaking culture presumably because more glucose was utilized for the growth of the cells. The yields in the fed-batch MSLC and repeated fedbatch MSLC with 0.25% yeast extract were similar to or slightly higher than that in the shaking culture. The overall productivity in the repeated fed-batch MSLC for the 2nd to 10th batches was 14.2g/l/d, which was about 6 times higher than that for the shaking culture with 0.25% yeast extract (calculated to be 2.3 g/l/d from Table 1). This value was also several times higher than that in the batch MSLC or in the fed-batch MSLC (Table 2), since in the initial stage of the MSLC several days were necessary for mycelia to grow over the membrane surface. Kwak and Rhee immobilized A. oryzae NRRL484 by entrapping it in calcium alginate gel beads to produce
VOL. 80, 1995
kojic acid (3). The kojic acid concentration reached 83 mg/ml in the case of batch culture feeding of glucose. They also conducted repeated-batch culture. However, the kojic acid production rate decreased early, from the 3rd batch culture. The productivity for repeated-batch culture with the immobilized cells was only 3.7 g/I/d, which was nearly l/4 that obtained in the repeated fedbatch MSLC in this study (Table 2). Secondary metabolite fermentation using molds is usually conducted either by submerged liquid culture using liquid medium or by solid-state culture using water-insoluble solid substrates. However, in these cultivation methods, it is difficult to conduct repeated-batch culture. With the MSLC, repeated-batch production of kojic acid was fairly successful. Without any appreciable decrease in production rate 10 batch cultures could be conducted with the medium containing 0.25% yeast extract as a nitrogen source. Even using the medium without yeast extract several repeated-batch cultures were possible. Cells suspended in liquid medium usually suffer from irreversible loss in activity when incubated for a long time. In kojic acid production, repeated-batch culture with suspended cells was difficult in the shaking culture (data not shown). We showed in a previous study that repeated-batch production of neutral protease is possible using MSLC (2). Thus, we speculate that molds are more stable against autolysis when cultivated by MSLC. Molds are typical aerobes and thus require oxygen, favor atmospheres under somewhat low water activity conditions (less than 0.99) for production of specific substances (7), and grow on solid surfaces in the natural environment. Probably, such properties of molds help explain the high productivity observed in MSLC, in which they are grown on a membrane surface, obtaining oxygen directly from the atmosphere and at the same time obtaining nutrients from liquid medium through membrane pores. In this study, we showed that kojic acid could be
PRODUCTION
OF KOJIC ACID BY MSLC
45
produced efficiently from A. oryzae NRRL484 by MSLC following the production of neutral protease from A. oryzue IAM in previous studies (1, 2). We intend to apply MSLC to other fermentation systems using various molds and possibly other microorganisms. Furthermore, we are developing methods to apply MSLC to large-scale production as discussed in a previous report (2). ACKNOWLEDGMENTS We thank the Natl. Center for Agricultural Util. Research (University St. Peoria, IL, U.S.A.) for providing the strain of A. oryzue NRRL484. REFERENCES 1. Yasuhara, A., Ogawa, A., Tanaka, T., Sakiyama, T., and Nakanishi, K.: Production of neutral protease from Aspergillus oryzae by a novel cultivation method on a microporous membrane. Biotechnol. Techniq., 8, 249-254 (1994). 2. Ogawa, A., Yasuhara, A., Tanaka, T., Sakiyama, T., and Nakaaishi, K.: Production of neutral protease by membranesurface liquid culture of Aspergihs oryzae IAM2704. J. Ferment. Bioeng., 80, 35-40 (1995). 3. Kwak, M. Y. and Rhee, S.: Cultivation characteristics of immobilized Aspergiks oryzae for kojic acid production. Biotechnol. Bioeng., 39, 903-906 (1992). 4. Bentley, R.: Preparation and analysis of kojic acid, p. 238-241. In Colowick, S. P. and Kaplan, N. 0. (ed.), Methods in enzymology, vol. 3. Academic Press, New York (1957). 5. Kitada, M., Ueyama, H., Suzuki, E., and Fukimbara, T.: Studies on kojic acid fermentation. I. Cultural conditions in submerged culture. J. Ferment. Technol., 45, 1101-1107 (1967). 6. Bajpai, P., Agrawala, P. K., and Vishwanthan, L.: Production of kojic acid by resuspended mycelia of Aspergihs pavus. Can. J. Microbial., 28, 1340-1346 (1982). 7. Johns, M. R.: Production of secondary metabolites, p. 341352. In Doelle, H. W., Mitchell, D.A., and Rolz, C. E. (ed.), Solid substrate cultivation. Elsevier Applied Science, London and New York (1992).