- Email: [email protected]
Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704
JOURNALOF FERMENTATION AND BIOENGINEEREW Vol. 80, No. 1, 35-40. 1995
Production
Liquid Culture
of Neutral Protease by Membrane-Surface of Aspergillus oryzae IAM
AKINORI OGAWA,
AKINORI YASUHARA, TAKAAK I TANAKA, AND KAZUHIRO NAKANISHI*
TAKAHARU
SAKIYAMA,
Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700, Japan Received 2 February 199UAccepted 24 April 1995
A membrane-surface liquid culture (MSLC), in which microorganisms were grown on a microporous membrane surface exposed to the air with the other side of the membrane in contact with liquid medium, was applied to the production of neutral protease from Aspergihs oryzue IAM2704. The amount of protease produced by batch MSLC was much higher than that produced by shaking culture, liquid-surface culture, or agar plate culture. In MSLC the amount of protease produced per milligram of dry cells as well as the amount of protease produced were dependent particularly on the glucose concentration. By decreasing the glucose concentration from 1.5% to 0.2%, the amount of protease produced and its specific amount were increased by 60% and around 7 times, respectively. Using a medium containing 0.2% glucose and 0.4% casein, long-term repeatedbatch production of the enzyme was conducted by exchanging the medium every 12 to 48 h with a fresh one. Protesse production continued in more tban 15 successive batches, although the amount of enzyme produced in each batch gradually decreased. When the medium was exchanged every 24 h, the degree of the decrease was the smallest, and the cumulative amount of enzyme produced was more than 20 and 400 times those obtained in the batch MSLC and shaking culture, respectively. [Key words: membrane,
Aspergillus oryzae, protease, mold, solid-state culture] was more than 10 ture. In this study, zae IAM2704. We concentrations of production of the term repeated-batch
Molds are one of the most useful microorganisms in many fields of biotechnology; they are used not only for production of traditional fermentative foods such as soy products and tempe but also for the production of enzymes, antibiotics, beverages, and volatile compounds. Physiological properties of the molds are as follows (1). (i) They grow well under lower moisture conditions than those required for optimal bacterial growth (2, 3). (ii) They require an aerobic environment for the production of specific products such as enzymes (2). (iii) They grow well on the surfaces of substances found in the natural environment. In practice, molds are cultivated by submerged liquid culture or by solid-state culture using water-insoluble solid substrates. Taking into consideration the physiological properties of the molds mentioned above, solid-state culture seems to be more suitable than liquid culture for production of specific fermentative substances as well as for growth. Many reports of mold production of useful substances in much larger amounts by solid-state culture than by shaking cultures have been published (2-5). However, the solid-state culture has several disadvantages (I), such as mass transfer limitation inside the solid substrate and difficulty of control of substrate concentration in as well as pH of the medium during cultivation. Furthermore, the medium is limited to solid substrates which are easily available. Thus, optimization of the medium composition is particularly difficult. To overcome these disadvantages of the solid-state culture, we developed a novel cultivation method, named membrane-surface liquid culture (MSLC) (6). In MSLC, 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 also showed that production of neutral protease from Aspergillus oryzae IAM by batch MSLC
times higher than that by shaking culwe cultivated the same strain, A. oryexamined the effects of changes in the carbon and nitrogen sources on the neutral protease and conducted longMSLC by exchanging the medium.
MATERIALS
AND METHODS
A. oryzae IAM Microorganisms and media was used to produce neutral protease. The vegetative cells were cultivated for about 10 d on a modified Czapek-Dox slant medium, using 1.5% glucose in place of sucrose, adjusted to pH 6.0 at 30°C 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). Usually, 2000 spores were used for inoculation. The medium for cultivation contained 1 to log casein (Hammarsten, E. Merck, Darmstadt, Germany), 0.5 to 15 g glucose, 1 g K2HP04, 0.5 g MgS04.7Hz0, 0.5 g NaCl, and 0.01 g FeS04. 7Hz0 in 1 I of ion-exchanged water and the pH was adjusted to 7. Casein induces the production of proteases. Figure 1 Membrane-surface liquid culture (MSLC) shows a schematic diagram of the cultivation system for the MSLC. The cultivation apparatus for the MSLC was similar to that used previously (6). The apparatus was made of polycarbonate and composed of a lower plate having a cylindrical groove (diameter, 7 cm; depth, 6 mm) and upper plate with a circular opening (diameter, 7 cm). About 23 ml of the liquid medium was added into the cylindrical groove of the lower plate. A microporous membrane made of polysulfone having a nominal pore size of 0.2 pm (SE20, Fuji Photo Film Co., Tokyo) was set in contact with the surface of the liquid medium through an O-ring. An Erlenmeyer flask
* Corresponding author. 35
36
J. FERMENT. BIOENG.,
OGAWA ET AL. 1 .
Air
, Molds
I
Sampling
/-
c
l-l-\
\
FIG. 1. Schematic diagram of the cultivation system for MSLC. 1, Plastic dish (cover dish); 2, upper plate; 3, O-ring; 4, microporous membrane; 5, stainless-steel pipe; 6, lower plate; 7, peristaltic pump; 8, Erlenmeyer flask (medium container). Molds are grown on the microporous membrane surface exposed to the air. At the same time they are in contact with a liquid medium via the membrane pores. Enzyme produced diffuses back into the medium.
was used as a medium container when the medium was exchanged. The entire cultivation system shown in Fig. 1 was placed in an aseptic box equipped with a UV lamp and an aseptic filter (MAC-lOFR, Air Tech Japan, Ltd., Saitama). The aseptic box was placed in a thermostatcontrolled room at 30°C. In the batch MSLC, several cultures were carried out under the same conditions to investigate the courses of cultivation. In this case, we mainly used plastic dishes (9 cm x 1.4 cm) for convenience in place of the cultivation apparatus shown in Fig. 1. About 20 ml of the liquid medium was poured into the bottom dish. An SE20 membrane was in contact with the surface of the medium in the bottom dish and supported by four pieces of rectangular polyurethane foam. Twenty microliters of a suspension of spores was inoculated uniformly onto the membrane surface, covered with the top dish and incubated statically. We confirmed that cultivation characteristics such as enzyme production and mycelial growth were similar to those obtained using the MSLC apparatus shown in Fig. 1. At appropriate times during cultivation, an aliquot of the cultures was withdrawn for analysis. Repeated-batch MSLC was carried out in the apparatus shown in Fig. 1. The cultivation was started in the same manner as that for the batch MSLC. After 4d of cultivation, molds grew over the entire membrane surface. Then, the cultivation was allowed to continue in the medium exchange mode, in which the liquid medium was exchanged every 12 to 48 h. The entire liquid medium was transferred from the cylindrical groove into the Erlenmeyer flask (medium container) with the aid of a peristaltic pump (Tokyorikakikai MP-3, Tokyo), and the same amount of fresh medium was supplied into the groove. Shaking culture Sakaguchi flasks (100 ml) contain-
ing 20 ml of the medium were inoculated by aseptically transferring a suspension of spores (20 ~1) and incubated at 30°C on a reciprocal shaker (140 strokes/min). At appropriate times during cultivation, an aliquot of the cultures was withdrawn for analysis. Liquid-surface culture A suspension of spores (20 ~1) was inoculated onto the surface of 20 ml of the liquid medium in plastic dishes (9 cm x 1.4 cm) and incubated statically at 30°C. At appropriate times during cultivation, an aliquot of the cultures was withdrawn. Agar plate culture A suspension of spores (20 ~1) was inoculated onto the surface of the agar plate medium (20 ml) in plastic dishes (9 cm x 1.4 cm) and incubated statically at 30°C. At appropriate intervals, an aliquot of the cultures was withdrawn. Twenty small cylindrical agar pieces were cut out from different places of the agar plate with a glass tube (inner diameter, 2mm), crushed and then immersed in 400 ml of 10 mM potassium phosphate buffer, pH 7.0 at 4°C for 12 h to extract the enzyme (6). Analysis The concentration of glucose was measured by a glucose oxidase/peroxidase method using a Glucostat reagent kit (Toyobo Co., Osaka). Neutral protease activity was measured by a modification of the method of Yano et al. (7). One unit of enzyme activity was defined as the amount of enzyme which catalyzes the solubilization of 1 /*g protein at pH 7.0 and 30°C in one minute measured by the Lowry method with bovine serum albumin as the standard. The dry cell weight was measured as follows. During the course of cultivation, an aliquot of the culture was withdrawn at appropriate cultivation times. In the MSLC and liquid-surface culture, the cells formed on the membrane surface and liquid surface could be removed quite easily with a spatula. The cells from the MSLC were directly subjected to measurement of the dry weight. The cells from the liquid surface culture were filtered with suction on a nylon bolting cloth (mesh size: 5 pm; Nytal HD5 p, Tanaka Sanjiro Co. Ltd., Fukuoka) and the dry weight was measured. In the shaking culture, about 50ml of ion-exchanged water was added into the shaking flask to remove the cells adhering to its wall and the suspension was filtered with suction through the nylon bolting cloth with rinsing with the water. In the agar plate culture, the entire agar plate was suspended in 1OOml of hot ion-exchanged water and heated in a microwave oven to dissolve the agar. The suspension was filtered through the nylon bolting cloth with rinsing with hot water. The recovered cells were dried at 105°C to a constant weight. EXPERIMENTAL
RESULTS
Batch MSLC In the previous study (6), we showed that the amount of neutral protease produced by MSLC was more than 10 times that produced by the shaking culture using 1.5% glucose and 0.4% casein. We also showed that the increase in the amount of the enzyme produced cannot be explained by the greater number of mycelia in the former case than in the latter case. In this study, we examined the effects of changes in the concentrations of glucose and casein on the amount of protease produced. We varied the glucose concentration in the range of 0.05-1.5% in the presence of 0.4% casein and the casein concentration in the range of 0.1-l% in the
PRODUCTION
VOL. 80, 1995 TABLE 1.
Concentration @ucose cm; w/v) 0.05 0.2 0.5 0.5 0.5 1.0 1.5
of
Concentration casein (%; w/v)
of
0.4 0.4 0.1 0.4 1.0 0.4 0.4
Amount of protease produced (units) 900 1550 1350 1500 1250 1200 1100
51500-
%I,
200
‘4
B
ij
g1000-
% B i a
c'
f
566 -
b)
3
5'
a The cultivation was carried out for 4 d.
presence of 0.5% glucose. After cultivation for 4 d in the medium with various concentrations of glucose and casein, we compared the amounts of protease produced. As shown in Table 1, the amount of protease produced depended on the concentrations of both glucose and casein. However, the dependence of the protease production on the casein concentration was weaker than that on the glucose concentration. In the presence of 0.5% glucose, the amount of protease produced was the highest with the casein concentration of 0.4%. In the presence of 0.4% casein, the amount of protease produced decreased with increasing glucose concentration above 0.2%, probably because glucose was utilized predominantly for growth. With 0.2 or 0.5% glucose in the presence of 0.4% casein, the amount of protease produced was the highest. With 0.05% glucose in the presence of 0.4% casein, it was the lowest presumably because of the small amount of mold which formed. Figure 2 shows the course of cultivation by MSLC in terms of the amount of protease produced and dry cell weight, using the medium containing 0.2% glucose and 0.4% casein. The pattern of the cultivation behavior was similar to that using 1.5% glucose and 0.4% casein (6). The maximum amount of protease (about 1750U) was observed at 5-6 d of cultivation. This value was more than 20 times that obtained by shaking culture using the same medium (about 70 U). By decreasing the glucose concentration from 1.5% to 0.2% in the presence of 0.4% casein, the maximum amount of protease produced by the MSLC was increased by around 60%; it was about 1100 U with 1.5% glucose (6). On the other
100
2.0
26lMlp
Effects of changes in concentrations of glucose and casein in the medium on protease productiona
37
OF NEUTRAL PROTEASE BY MSLC
o-
10 .$? p a
-,l.O ‘i; 5 s 3 0.5 I g s 0.0
;
6
2
4 Cultivation
6
6
10
12’
time (d)
FIG. 2. Courses of cultivation of A. oryzae IAM by MSLC. Symbols: A, dry cell weight; 0, concentration of glucose; 0, amount of protease.
hand, in shaking culture the amount of protease produced with 0.2% glucose was half that produced with 1.5% glucose in the presence of 0.4% casein. Figure 3 shows comparisons of the amount of protease produced, dry cell weight and specific amount of protease (amount of protease per mg dry cells) obtained by shaking culture, liquid-surface culture, agar plate culture and MSLC at the cultivation time at which the maximum amount of protease was observed, using medium containing 0.2% or 1.5% glucose in the presence of 0.4% casein. The cultivation time required to reach the maximum amount of protease produced was 4 to 5 d in each culture. Upon lowering of the glucose concentration from 1.5% to 0.2%, the dry cell weight decreased in all the cultures, the maximum amount of protease produced slightly decreased in the shaking culture and agar plate culture, and it increased in the liquid-surface culture and in the MSLC. Thus, in the MSLC and liquid-surface culture, the specific amount of protease produced increased to a great extent upon lowering of the glucose concentration. In particular, in the MSLC the extent of the increase was pronounced; the specific amount of protease produced increased by around 7
3 5o(c)
3 2 .s t
100
= 2 $
0
FIG. 3. Effect of glucose concentration on the dry cell weight (a), amount of protease produced (b), and specific amount of protease produced (c), by shaking culture (l), liquid-surface culture (2), agar plate culture (3), and MSLC (4). All the experimental data were obtained at the cultivation time at which the maximum amount of protease was observed during the batch culture, using 1.5% (left bar) or 0.2% (right bar) glucose. ma, 1.5% glucose; m, 0.2% glucose.
38
OGAWA ET AL.
J.
,
6ctoo-
.
.
.
.
0.6
0.8
1.0
FERMENT.BIOENG.,
(4 6060-
4000-
fi 2600-
0-o 0.0
0.2
0.4
Concentration
of casein
0-o 0.0
1.0
0.5
Concentration
(%)
1.5
of glucose
(%)
FIG. 4. Effects of changes in the casein and glucose concentrations in the medium on the dry cell weight and cumulative amount of protease produced. Symbols: A, dry cell weight; 0, cumulative amount of protease produced. Batch culture was repeated 4 times with a total cultivation time of 10d. After 4d of cultivation from the start of the 1st batch culture, the entire membrane surface was covered with mycelia. Then, the medium was exchanged every 2 d for a total of 3 times. (a) shows the effect of changes in the casein concentration in the presence of 0.5% glucose and (b) the effect of changes in glucose concentration in the presence of 0.4% casein.
times. In the MSLC, glucose might be readily consumed and utilized for growth at a high glucose concentration region probably because a large amount of oxygen is available under these conditions. On the other hand, at a lower glucose concentration, the microorganism might obtain energy by catabolyzing casein and accordingly produce a larger amount of the enzyme. Probably, in the mold grown in the MSLC, a switching mechanism to obtain energy from either glucose or casein catabolism is more developed than that in the mold grown in the shaking culture, although details of this possible mechanism are not known. One possible reason for the increase in the specific amount of protease produced in the liquid-surface culture is that the cell mass decreased and as a result the behavior of mycelia on the liquid surface became similar to that in the MSLC. Repeated-batch MSLC Repeated-batch production of protease was conducted by MSLC. To optimize the concentrations of glucose and casein in the medium for long-term repeated-batch culture, we performed 4 successive batch MSLCs with a total cultivation period of 10 d, using medium containing various concentrations of glucose and casein. After 4d of cultivation, the entire membrane surface was covered with mycelia. Then, the medium was exchanged every 2 d for a total of 3 times. We determined the optimal medium composition in terms of the cumulative amount of protease obtained in 4 successive batch cultures. Figure 4a shows the effect of changes in the casein concentration on the cumulative amount of protease produced and dry cell weight in the presence of 0.5% glucose. The dry cell weight increased with increasing casein concentration. However, the cumulative amount of protease produced was the highest at the casein concentration of 0.4%. Figure 4b shows the effect of changes in the glucose concentration in the presence of 0.4% casein. The dry cell weight increased with increasing glucose concentration and the cumulative amount of protease produced was the highest at the glucose concentrations of less than 0.2%. Based on these findings, long-term repeated-batch culture was conducted using medium containing 0.4% casein and 0.2% glucose.
Figure 5 shows results obtained with a medium exchange period of 48 h. Glucose was consumed almost completely in each batch culture. Although the amount of protease produced was high and nearly constant in the early stage of cultivation, it gradually decreased with increasing cultivation time. At 30 d of cultivation, the amount of protease produced in one batch was about l/S that of the initial value. However, this value was still more than 5 times higher than that obtained in the shaking culture. Figure 6 shows the results obtained with the medium exchange period of 24 h. The pattern of protease production was similar to that for the medium exchange period of 48 h (Fig. 5). The amount of protease produced in each batch was also similar in spite of halving the medium exchange period. This indicates that 24 h is sufficient for production of protease during the repeated-batch culture due to increased mycelial growth. The cumulative amount of protease increased to around 3O,OOOU, about 2 times that obtained with the medium I 2
20000 t
I 9
-I 2000
R
1 XNJ
0
10 Cultivation
0
20 time
30 (d)
FIG. 5. Courses of long-term repeated-batch culture by MSLC. The medium containing 0.2% glucose and 0.4% casein was exchanged every 48 h after 4 d of cultivation (1st batch). Symbols: 0, concentration of glucose; 0, amount of protease produced in each batch; 0 , cumulative amount of protease.
VOL. 80, 1995
PRODUCTION
Cultivation
time (d)
FIG. 6. Courses of long-term repeated-batch culture by MSLC. The medium containing 0.2% glucose and 0.4% casein was exchanged every 24 h after 4 d of cultivation (1st batch). Symbols: 0, concentration of glucose; 0, amount of protease produced in each batch; o , cumulative amount of protease.
exchange period of 48 h (Fig. 5). This amount was about 20 times that obtained in the batch MSLC and over 400 times that obtained in the shaking culture using the same medium. Figure 7 shows results obtained using a medium exchange period of 12 h. The amount of protease produced in each batch decreased more rapidly compared to that produced using a medium exchange period of 24 or 48 h. The reason for this rapid decrease might be as follows. Since the cultivation time was short, and the molds were grown in medium with a relatively high glucose concentration, energy obtained from glucose catabolism was likely to be utilized for growth as seen in Fig. 4. We increased the medium exchange period from 12 to 24 h at 14 d of cultivation. The amount of protease produced in each batch greatly increased, although it again started to decrease after 5 d of cultivation (Fig. 7). This supports the view that the molds were not damaged particularly in this culture system. In the case of the medium exchange period of 12 h, we examined the effect of stirring of the medium on the production of the enzyme, since the enzyme is transported into liquid medium by diffusion. However, no positive effects were observed, probably because the secretion rate of the enzyme was low and thus the overall production rate of the enzyme was not limited by diffusion. The specific amounts of protease obtained over the entire cultivation period were 27 and 43 U/mg (dry cell weights were 590mg and 679mg, respectively) for the medium exchange periods of 48 and 24 h, respectively. The specific amount of protease obtained in the case of the medium exchange period of 24 h was similar to that obtained in the batch MSLC (42.5 U/mg as shown in Fig. 3). We carried out a repeated-batch culture for 23 d using medium containing 0.1% glucose and 0.4% casein in order to suppress the growth further with the medium exchange period of 24 h. The amount of protease produced in each batch was slightly lower than that produced using the exchange medium containing 0.2% glucose and 0.4% casein (data not shown). The protease production was constant for about 2 weeks (10 batches) and then gradually decreased. The cumulative amount of enzyme produced (about 22000U) was lower than that produced using the medium containing 0.2% glucose and 0.4%
OF NEUTRAL PROTEASE BY MSLC
Cultivation
39
time (d)
FIG. 7. Courses of long-term repeated-batch culture by MSLC. The medium containing 0.2% glucose and 0.4% casein was exchanged every 12 h after 4 d of cultivation (1st batch). From 14 d of cultivation, the medium exchange period was extended to 24 h (shown by arrow [ 1 I). Symbols: 0, concentration of glucose; 0, amount of protease produced in each batch; q , cumulative amount of protease.
casein, although the specific amount of produced increased to 56U/mg with a dry cell of 395 mg. Thus, an appropriate growth rate of seems to be necessary more or less to increase mulative amount of protease produced in the long-term repeated-batch culture.
protease amount the cells the cucase of
DISCUSSION In this study, we showed that the molds produced much more protease in MSLC than in shaking culture, liquid-surface culture and agar plate culture, and that the molds were quite stable so as to allow a repeated-batch culture exchanging medium. In the case of shaking culture, it was difficult to conduct repeated-batch culture, since the mold formed a pellet, most of the inside of which was autolyzed after one batch cultivation. The molds seemed to be much more resistant to autolysis when cultivated by MSLC. The dependence of the enzyme production on the medium composition was also quite different between MSLC and shaking culture. Glucose as a carbon source was more easily utilized for growth in MSLC than shaking culture when its concentration was sufficiently high. Thus, glucose concentration should be appropriately low for efficient production of the enzyme particularly in MSLC. On the other hand, at a sufficiently low glucose concentration, the molds tended to utilize energy obtained from casein catabolism and to produce a large amount of the enzyme. In MSLC, molds grow on a membrane surface exposed to the air, which is similar to their growth conditions in the natural environment. Several studies in which molds produced specific substances at a higher yield in solid-state culture than in shaking or submerged liquid culture have been reported (2-5). Since the mechanisms behind the increased productivity in the solid-state culture and MSLC are probably similar, MSLC might be utilized as a model to examine the physiological properties of molds in the solid-state culture. It would also be interesting to clarify the differences in physiological properties of the molds cultivated by shaking culture and by MSLC in terms of differences in morphology, respiration ability, cellular
40
J. FERMENT.BIOENO.,
OGAWA ET AL.
mechanism (8) and others. MSLC is similar to the solid-state culture from the standpoint of growth on the surface of a solid material (a porous membrane). On the other hand, it can be regarded as a kind of liquid culture with respect to utilization of liquid medium. Thus, MSLC possesses advantages of both the solid-state and liquid cultures. Several approaches in which molds are grown on porous materials such as asbestos (9), sponge (lo), and urethane foam carrier (11) were reported. These methods are similar to MSLC in terms of cultivation on the surface of a porous material in contact with a liquid medium and exposed to the air. However, in these methods mycelia penetrate to the inside of the porous materials and also grow there. Kobayashi et al. cultivated A. oryzae B-3 on a urethane foam carrier to produce glucoamylase and recovered the enzyme by pressing the carrier (11). They repeated these procedures 13 times for 8 d; however, the cumulative amount of enzyme obtained increased by only about 3 times compared to the amount obtained in the batch culture, since only 13% of the medium was exchanged after each batch. MSLC is considered to be more advantageous than these methods, taking into consideration the ease of product recovery and of performing repeated-batch or continuous culture. The control of pH of and substrate concentration in the medium during the cultivation is also easy. Repeated-batch MSLC can be carried out efficiently without the need for any additional operations such as pressing. Separation of molds from the culture is quite easy. The liquid medium is clear when a membrane with an appropriate pore size is used. We carried out MSLC using membranes with different pore sizes. With the nominal pore size of 0.2 pm which was mainly used in this study, no mycelia were observed in the medium as described above. However, with a nominal pore size of 0.45 pm (SE45, Fuji Photo Film Co.), mycelia were observed in the liquid medium after 8 d of cultivation. With a pore size of 3 pm (SE300, Fuji Photo Film Co.), mycelia penetrated through the membrane pores from the initial stage of growth and grew all over the other side of the membrane. Since molds grow on the surface of the porous membrane, the composition of the membrane might also affect the growth behavior, which should be further investigated. MSLC can be utilized not only for the production of useful substances but also for a screening system of molds since production of metabolites can be enhanced and since the medium composition and pH can be varied in a manner similar to that in the case of the shaking culture. MSLC
might be used as a model of the solid-state culture to investigate the physiological properties of the molds as mentioned previously. To apply MSLC to industrial production, we must solve various problems related to scale-up of the apparatus, including types of membrane, kinds of modules, and methods for inoculation. ACKNOWLEDGMENT We thank Kikkoman Corp. (Noda, Japan) for providing strain of A. oryzae and manufacturing the MSLC apparatus.
the
REFERENCES 1
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Mitchell, D. A. and Lonsane, B. K.: Definition, characteristics and potential, p. 1-16. In Doelle, H. W., Mitchell, D. A., and Rolz, C. E. (ed.), Solid substrate cultivation. Elsevier Applied Science, London and New York (1992). Bajracharya, II. and Mudgett, R. E.: Effects of controlled gas environments in solid substrate fermentation of rice. Biotechnol. Bioeng., 22, 2219-2235 (1980). 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). &do, S., Ishikawa, T., Sato, K., and Oba, T.: Comparison of acid-stable a-amylase production by Aspergillus kawachii in solid-state and submerged cultures. J. Ferment. Bioeng., 77, 483489 (1994). Grajeck, W.: Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid-state fermentation. Appl. Microbial. Biotechnol., 26, 126-129 (1987). Yasuhara, A., Ogawa, A., Tanaka, T., Sakiyama, T., and Nakanishi, K.: Production of neutral protease from Aspergilhs oryzae by a novel cultivation method on a microporous membrane. Biotechnol. Techniq., 8, 249-254 (1994). Yano, T., Ashida, S., Tachiki, T., Kumagai, H., and Tochikura, T.: Development of a soft gel cultivation method. Agric. Biol. Chem., 55, 379-385 (1991). Pebexdy, J.F.: Protein secretion in filamentous fungi-trying to understand a highly productive black box. TIBTECH, 12, 50-57 (1994). Sakaguchi, K., Okazaki, H., and Takeuchi, M.: A note on the comparison of koji and submerged culture. Nippon Nougeikagaku Kaishi, 29, 349-353 (1955). (in Japanese) Fujishima, T., Uchida, K., and Yoshino, H.: Enzyme production by molds in sponge culture. J. Ferment. Technol., 50, 724-730 (1972). Kobayashi, T., Ozawa, S., Sato, K., Nagamune, T., and Endo, I.: Production of glucoamylase by a solid-state fermentation using urethane foam carrier as a semi-solid medium. Kagaku Kogaku Ronbunshu, 17,491-496 (1991). (in Japanese)
Production
Liquid Culture
of Neutral Protease by Membrane-Surface of Aspergillus oryzae IAM
AKINORI OGAWA,
AKINORI YASUHARA, TAKAAK I TANAKA, AND KAZUHIRO NAKANISHI*
TAKAHARU
SAKIYAMA,
Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700, Japan Received 2 February 199UAccepted 24 April 1995
A membrane-surface liquid culture (MSLC), in which microorganisms were grown on a microporous membrane surface exposed to the air with the other side of the membrane in contact with liquid medium, was applied to the production of neutral protease from Aspergihs oryzue IAM2704. The amount of protease produced by batch MSLC was much higher than that produced by shaking culture, liquid-surface culture, or agar plate culture. In MSLC the amount of protease produced per milligram of dry cells as well as the amount of protease produced were dependent particularly on the glucose concentration. By decreasing the glucose concentration from 1.5% to 0.2%, the amount of protease produced and its specific amount were increased by 60% and around 7 times, respectively. Using a medium containing 0.2% glucose and 0.4% casein, long-term repeatedbatch production of the enzyme was conducted by exchanging the medium every 12 to 48 h with a fresh one. Protesse production continued in more tban 15 successive batches, although the amount of enzyme produced in each batch gradually decreased. When the medium was exchanged every 24 h, the degree of the decrease was the smallest, and the cumulative amount of enzyme produced was more than 20 and 400 times those obtained in the batch MSLC and shaking culture, respectively. [Key words: membrane,
Aspergillus oryzae, protease, mold, solid-state culture] was more than 10 ture. In this study, zae IAM2704. We concentrations of production of the term repeated-batch
Molds are one of the most useful microorganisms in many fields of biotechnology; they are used not only for production of traditional fermentative foods such as soy products and tempe but also for the production of enzymes, antibiotics, beverages, and volatile compounds. Physiological properties of the molds are as follows (1). (i) They grow well under lower moisture conditions than those required for optimal bacterial growth (2, 3). (ii) They require an aerobic environment for the production of specific products such as enzymes (2). (iii) They grow well on the surfaces of substances found in the natural environment. In practice, molds are cultivated by submerged liquid culture or by solid-state culture using water-insoluble solid substrates. Taking into consideration the physiological properties of the molds mentioned above, solid-state culture seems to be more suitable than liquid culture for production of specific fermentative substances as well as for growth. Many reports of mold production of useful substances in much larger amounts by solid-state culture than by shaking cultures have been published (2-5). However, the solid-state culture has several disadvantages (I), such as mass transfer limitation inside the solid substrate and difficulty of control of substrate concentration in as well as pH of the medium during cultivation. Furthermore, the medium is limited to solid substrates which are easily available. Thus, optimization of the medium composition is particularly difficult. To overcome these disadvantages of the solid-state culture, we developed a novel cultivation method, named membrane-surface liquid culture (MSLC) (6). In MSLC, 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 also showed that production of neutral protease from Aspergillus oryzae IAM by batch MSLC
times higher than that by shaking culwe cultivated the same strain, A. oryexamined the effects of changes in the carbon and nitrogen sources on the neutral protease and conducted longMSLC by exchanging the medium.
MATERIALS
AND METHODS
A. oryzae IAM Microorganisms and media was used to produce neutral protease. The vegetative cells were cultivated for about 10 d on a modified Czapek-Dox slant medium, using 1.5% glucose in place of sucrose, adjusted to pH 6.0 at 30°C 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). Usually, 2000 spores were used for inoculation. The medium for cultivation contained 1 to log casein (Hammarsten, E. Merck, Darmstadt, Germany), 0.5 to 15 g glucose, 1 g K2HP04, 0.5 g MgS04.7Hz0, 0.5 g NaCl, and 0.01 g FeS04. 7Hz0 in 1 I of ion-exchanged water and the pH was adjusted to 7. Casein induces the production of proteases. Figure 1 Membrane-surface liquid culture (MSLC) shows a schematic diagram of the cultivation system for the MSLC. The cultivation apparatus for the MSLC was similar to that used previously (6). The apparatus was made of polycarbonate and composed of a lower plate having a cylindrical groove (diameter, 7 cm; depth, 6 mm) and upper plate with a circular opening (diameter, 7 cm). About 23 ml of the liquid medium was added into the cylindrical groove of the lower plate. A microporous membrane made of polysulfone having a nominal pore size of 0.2 pm (SE20, Fuji Photo Film Co., Tokyo) was set in contact with the surface of the liquid medium through an O-ring. An Erlenmeyer flask
* Corresponding author. 35
36
J. FERMENT. BIOENG.,
OGAWA ET AL. 1 .
Air
, Molds
I
Sampling
/-
c
l-l-\
\
FIG. 1. Schematic diagram of the cultivation system for MSLC. 1, Plastic dish (cover dish); 2, upper plate; 3, O-ring; 4, microporous membrane; 5, stainless-steel pipe; 6, lower plate; 7, peristaltic pump; 8, Erlenmeyer flask (medium container). Molds are grown on the microporous membrane surface exposed to the air. At the same time they are in contact with a liquid medium via the membrane pores. Enzyme produced diffuses back into the medium.
was used as a medium container when the medium was exchanged. The entire cultivation system shown in Fig. 1 was placed in an aseptic box equipped with a UV lamp and an aseptic filter (MAC-lOFR, Air Tech Japan, Ltd., Saitama). The aseptic box was placed in a thermostatcontrolled room at 30°C. In the batch MSLC, several cultures were carried out under the same conditions to investigate the courses of cultivation. In this case, we mainly used plastic dishes (9 cm x 1.4 cm) for convenience in place of the cultivation apparatus shown in Fig. 1. About 20 ml of the liquid medium was poured into the bottom dish. An SE20 membrane was in contact with the surface of the medium in the bottom dish and supported by four pieces of rectangular polyurethane foam. Twenty microliters of a suspension of spores was inoculated uniformly onto the membrane surface, covered with the top dish and incubated statically. We confirmed that cultivation characteristics such as enzyme production and mycelial growth were similar to those obtained using the MSLC apparatus shown in Fig. 1. At appropriate times during cultivation, an aliquot of the cultures was withdrawn for analysis. Repeated-batch MSLC was carried out in the apparatus shown in Fig. 1. The cultivation was started in the same manner as that for the batch MSLC. After 4d of cultivation, molds grew over the entire membrane surface. Then, the cultivation was allowed to continue in the medium exchange mode, in which the liquid medium was exchanged every 12 to 48 h. The entire liquid medium was transferred from the cylindrical groove into the Erlenmeyer flask (medium container) with the aid of a peristaltic pump (Tokyorikakikai MP-3, Tokyo), and the same amount of fresh medium was supplied into the groove. Shaking culture Sakaguchi flasks (100 ml) contain-
ing 20 ml of the medium were inoculated by aseptically transferring a suspension of spores (20 ~1) and incubated at 30°C on a reciprocal shaker (140 strokes/min). At appropriate times during cultivation, an aliquot of the cultures was withdrawn for analysis. Liquid-surface culture A suspension of spores (20 ~1) was inoculated onto the surface of 20 ml of the liquid medium in plastic dishes (9 cm x 1.4 cm) and incubated statically at 30°C. At appropriate times during cultivation, an aliquot of the cultures was withdrawn. Agar plate culture A suspension of spores (20 ~1) was inoculated onto the surface of the agar plate medium (20 ml) in plastic dishes (9 cm x 1.4 cm) and incubated statically at 30°C. At appropriate intervals, an aliquot of the cultures was withdrawn. Twenty small cylindrical agar pieces were cut out from different places of the agar plate with a glass tube (inner diameter, 2mm), crushed and then immersed in 400 ml of 10 mM potassium phosphate buffer, pH 7.0 at 4°C for 12 h to extract the enzyme (6). Analysis The concentration of glucose was measured by a glucose oxidase/peroxidase method using a Glucostat reagent kit (Toyobo Co., Osaka). Neutral protease activity was measured by a modification of the method of Yano et al. (7). One unit of enzyme activity was defined as the amount of enzyme which catalyzes the solubilization of 1 /*g protein at pH 7.0 and 30°C in one minute measured by the Lowry method with bovine serum albumin as the standard. The dry cell weight was measured as follows. During the course of cultivation, an aliquot of the culture was withdrawn at appropriate cultivation times. In the MSLC and liquid-surface culture, the cells formed on the membrane surface and liquid surface could be removed quite easily with a spatula. The cells from the MSLC were directly subjected to measurement of the dry weight. The cells from the liquid surface culture were filtered with suction on a nylon bolting cloth (mesh size: 5 pm; Nytal HD5 p, Tanaka Sanjiro Co. Ltd., Fukuoka) and the dry weight was measured. In the shaking culture, about 50ml of ion-exchanged water was added into the shaking flask to remove the cells adhering to its wall and the suspension was filtered with suction through the nylon bolting cloth with rinsing with the water. In the agar plate culture, the entire agar plate was suspended in 1OOml of hot ion-exchanged water and heated in a microwave oven to dissolve the agar. The suspension was filtered through the nylon bolting cloth with rinsing with hot water. The recovered cells were dried at 105°C to a constant weight. EXPERIMENTAL
RESULTS
Batch MSLC In the previous study (6), we showed that the amount of neutral protease produced by MSLC was more than 10 times that produced by the shaking culture using 1.5% glucose and 0.4% casein. We also showed that the increase in the amount of the enzyme produced cannot be explained by the greater number of mycelia in the former case than in the latter case. In this study, we examined the effects of changes in the concentrations of glucose and casein on the amount of protease produced. We varied the glucose concentration in the range of 0.05-1.5% in the presence of 0.4% casein and the casein concentration in the range of 0.1-l% in the
PRODUCTION
VOL. 80, 1995 TABLE 1.
Concentration @ucose cm; w/v) 0.05 0.2 0.5 0.5 0.5 1.0 1.5
of
Concentration casein (%; w/v)
of
0.4 0.4 0.1 0.4 1.0 0.4 0.4
Amount of protease produced (units) 900 1550 1350 1500 1250 1200 1100
51500-
%I,
200
‘4
B
ij
g1000-
% B i a
c'
f
566 -
b)
3
5'
a The cultivation was carried out for 4 d.
presence of 0.5% glucose. After cultivation for 4 d in the medium with various concentrations of glucose and casein, we compared the amounts of protease produced. As shown in Table 1, the amount of protease produced depended on the concentrations of both glucose and casein. However, the dependence of the protease production on the casein concentration was weaker than that on the glucose concentration. In the presence of 0.5% glucose, the amount of protease produced was the highest with the casein concentration of 0.4%. In the presence of 0.4% casein, the amount of protease produced decreased with increasing glucose concentration above 0.2%, probably because glucose was utilized predominantly for growth. With 0.2 or 0.5% glucose in the presence of 0.4% casein, the amount of protease produced was the highest. With 0.05% glucose in the presence of 0.4% casein, it was the lowest presumably because of the small amount of mold which formed. Figure 2 shows the course of cultivation by MSLC in terms of the amount of protease produced and dry cell weight, using the medium containing 0.2% glucose and 0.4% casein. The pattern of the cultivation behavior was similar to that using 1.5% glucose and 0.4% casein (6). The maximum amount of protease (about 1750U) was observed at 5-6 d of cultivation. This value was more than 20 times that obtained by shaking culture using the same medium (about 70 U). By decreasing the glucose concentration from 1.5% to 0.2% in the presence of 0.4% casein, the maximum amount of protease produced by the MSLC was increased by around 60%; it was about 1100 U with 1.5% glucose (6). On the other
100
2.0
26lMlp
Effects of changes in concentrations of glucose and casein in the medium on protease productiona
37
OF NEUTRAL PROTEASE BY MSLC
o-
10 .$? p a
-,l.O ‘i; 5 s 3 0.5 I g s 0.0
;
6
2
4 Cultivation
6
6
10
12’
time (d)
FIG. 2. Courses of cultivation of A. oryzae IAM by MSLC. Symbols: A, dry cell weight; 0, concentration of glucose; 0, amount of protease.
hand, in shaking culture the amount of protease produced with 0.2% glucose was half that produced with 1.5% glucose in the presence of 0.4% casein. Figure 3 shows comparisons of the amount of protease produced, dry cell weight and specific amount of protease (amount of protease per mg dry cells) obtained by shaking culture, liquid-surface culture, agar plate culture and MSLC at the cultivation time at which the maximum amount of protease was observed, using medium containing 0.2% or 1.5% glucose in the presence of 0.4% casein. The cultivation time required to reach the maximum amount of protease produced was 4 to 5 d in each culture. Upon lowering of the glucose concentration from 1.5% to 0.2%, the dry cell weight decreased in all the cultures, the maximum amount of protease produced slightly decreased in the shaking culture and agar plate culture, and it increased in the liquid-surface culture and in the MSLC. Thus, in the MSLC and liquid-surface culture, the specific amount of protease produced increased to a great extent upon lowering of the glucose concentration. In particular, in the MSLC the extent of the increase was pronounced; the specific amount of protease produced increased by around 7
3 5o(c)
3 2 .s t
100
= 2 $
0
FIG. 3. Effect of glucose concentration on the dry cell weight (a), amount of protease produced (b), and specific amount of protease produced (c), by shaking culture (l), liquid-surface culture (2), agar plate culture (3), and MSLC (4). All the experimental data were obtained at the cultivation time at which the maximum amount of protease was observed during the batch culture, using 1.5% (left bar) or 0.2% (right bar) glucose. ma, 1.5% glucose; m, 0.2% glucose.
38
OGAWA ET AL.
J.
,
6ctoo-
.
.
.
.
0.6
0.8
1.0
FERMENT.BIOENG.,
(4 6060-
4000-
fi 2600-
0-o 0.0
0.2
0.4
Concentration
of casein
0-o 0.0
1.0
0.5
Concentration
(%)
1.5
of glucose
(%)
FIG. 4. Effects of changes in the casein and glucose concentrations in the medium on the dry cell weight and cumulative amount of protease produced. Symbols: A, dry cell weight; 0, cumulative amount of protease produced. Batch culture was repeated 4 times with a total cultivation time of 10d. After 4d of cultivation from the start of the 1st batch culture, the entire membrane surface was covered with mycelia. Then, the medium was exchanged every 2 d for a total of 3 times. (a) shows the effect of changes in the casein concentration in the presence of 0.5% glucose and (b) the effect of changes in glucose concentration in the presence of 0.4% casein.
times. In the MSLC, glucose might be readily consumed and utilized for growth at a high glucose concentration region probably because a large amount of oxygen is available under these conditions. On the other hand, at a lower glucose concentration, the microorganism might obtain energy by catabolyzing casein and accordingly produce a larger amount of the enzyme. Probably, in the mold grown in the MSLC, a switching mechanism to obtain energy from either glucose or casein catabolism is more developed than that in the mold grown in the shaking culture, although details of this possible mechanism are not known. One possible reason for the increase in the specific amount of protease produced in the liquid-surface culture is that the cell mass decreased and as a result the behavior of mycelia on the liquid surface became similar to that in the MSLC. Repeated-batch MSLC Repeated-batch production of protease was conducted by MSLC. To optimize the concentrations of glucose and casein in the medium for long-term repeated-batch culture, we performed 4 successive batch MSLCs with a total cultivation period of 10 d, using medium containing various concentrations of glucose and casein. After 4d of cultivation, the entire membrane surface was covered with mycelia. Then, the medium was exchanged every 2 d for a total of 3 times. We determined the optimal medium composition in terms of the cumulative amount of protease obtained in 4 successive batch cultures. Figure 4a shows the effect of changes in the casein concentration on the cumulative amount of protease produced and dry cell weight in the presence of 0.5% glucose. The dry cell weight increased with increasing casein concentration. However, the cumulative amount of protease produced was the highest at the casein concentration of 0.4%. Figure 4b shows the effect of changes in the glucose concentration in the presence of 0.4% casein. The dry cell weight increased with increasing glucose concentration and the cumulative amount of protease produced was the highest at the glucose concentrations of less than 0.2%. Based on these findings, long-term repeated-batch culture was conducted using medium containing 0.4% casein and 0.2% glucose.
Figure 5 shows results obtained with a medium exchange period of 48 h. Glucose was consumed almost completely in each batch culture. Although the amount of protease produced was high and nearly constant in the early stage of cultivation, it gradually decreased with increasing cultivation time. At 30 d of cultivation, the amount of protease produced in one batch was about l/S that of the initial value. However, this value was still more than 5 times higher than that obtained in the shaking culture. Figure 6 shows the results obtained with the medium exchange period of 24 h. The pattern of protease production was similar to that for the medium exchange period of 48 h (Fig. 5). The amount of protease produced in each batch was also similar in spite of halving the medium exchange period. This indicates that 24 h is sufficient for production of protease during the repeated-batch culture due to increased mycelial growth. The cumulative amount of protease increased to around 3O,OOOU, about 2 times that obtained with the medium I 2
20000 t
I 9
-I 2000
R
1 XNJ
0
10 Cultivation
0
20 time
30 (d)
FIG. 5. Courses of long-term repeated-batch culture by MSLC. The medium containing 0.2% glucose and 0.4% casein was exchanged every 48 h after 4 d of cultivation (1st batch). Symbols: 0, concentration of glucose; 0, amount of protease produced in each batch; 0 , cumulative amount of protease.
VOL. 80, 1995
PRODUCTION
Cultivation
time (d)
FIG. 6. Courses of long-term repeated-batch culture by MSLC. The medium containing 0.2% glucose and 0.4% casein was exchanged every 24 h after 4 d of cultivation (1st batch). Symbols: 0, concentration of glucose; 0, amount of protease produced in each batch; o , cumulative amount of protease.
exchange period of 48 h (Fig. 5). This amount was about 20 times that obtained in the batch MSLC and over 400 times that obtained in the shaking culture using the same medium. Figure 7 shows results obtained using a medium exchange period of 12 h. The amount of protease produced in each batch decreased more rapidly compared to that produced using a medium exchange period of 24 or 48 h. The reason for this rapid decrease might be as follows. Since the cultivation time was short, and the molds were grown in medium with a relatively high glucose concentration, energy obtained from glucose catabolism was likely to be utilized for growth as seen in Fig. 4. We increased the medium exchange period from 12 to 24 h at 14 d of cultivation. The amount of protease produced in each batch greatly increased, although it again started to decrease after 5 d of cultivation (Fig. 7). This supports the view that the molds were not damaged particularly in this culture system. In the case of the medium exchange period of 12 h, we examined the effect of stirring of the medium on the production of the enzyme, since the enzyme is transported into liquid medium by diffusion. However, no positive effects were observed, probably because the secretion rate of the enzyme was low and thus the overall production rate of the enzyme was not limited by diffusion. The specific amounts of protease obtained over the entire cultivation period were 27 and 43 U/mg (dry cell weights were 590mg and 679mg, respectively) for the medium exchange periods of 48 and 24 h, respectively. The specific amount of protease obtained in the case of the medium exchange period of 24 h was similar to that obtained in the batch MSLC (42.5 U/mg as shown in Fig. 3). We carried out a repeated-batch culture for 23 d using medium containing 0.1% glucose and 0.4% casein in order to suppress the growth further with the medium exchange period of 24 h. The amount of protease produced in each batch was slightly lower than that produced using the exchange medium containing 0.2% glucose and 0.4% casein (data not shown). The protease production was constant for about 2 weeks (10 batches) and then gradually decreased. The cumulative amount of enzyme produced (about 22000U) was lower than that produced using the medium containing 0.2% glucose and 0.4%
OF NEUTRAL PROTEASE BY MSLC
Cultivation
39
time (d)
FIG. 7. Courses of long-term repeated-batch culture by MSLC. The medium containing 0.2% glucose and 0.4% casein was exchanged every 12 h after 4 d of cultivation (1st batch). From 14 d of cultivation, the medium exchange period was extended to 24 h (shown by arrow [ 1 I). Symbols: 0, concentration of glucose; 0, amount of protease produced in each batch; q , cumulative amount of protease.
casein, although the specific amount of produced increased to 56U/mg with a dry cell of 395 mg. Thus, an appropriate growth rate of seems to be necessary more or less to increase mulative amount of protease produced in the long-term repeated-batch culture.
protease amount the cells the cucase of
DISCUSSION In this study, we showed that the molds produced much more protease in MSLC than in shaking culture, liquid-surface culture and agar plate culture, and that the molds were quite stable so as to allow a repeated-batch culture exchanging medium. In the case of shaking culture, it was difficult to conduct repeated-batch culture, since the mold formed a pellet, most of the inside of which was autolyzed after one batch cultivation. The molds seemed to be much more resistant to autolysis when cultivated by MSLC. The dependence of the enzyme production on the medium composition was also quite different between MSLC and shaking culture. Glucose as a carbon source was more easily utilized for growth in MSLC than shaking culture when its concentration was sufficiently high. Thus, glucose concentration should be appropriately low for efficient production of the enzyme particularly in MSLC. On the other hand, at a sufficiently low glucose concentration, the molds tended to utilize energy obtained from casein catabolism and to produce a large amount of the enzyme. In MSLC, molds grow on a membrane surface exposed to the air, which is similar to their growth conditions in the natural environment. Several studies in which molds produced specific substances at a higher yield in solid-state culture than in shaking or submerged liquid culture have been reported (2-5). Since the mechanisms behind the increased productivity in the solid-state culture and MSLC are probably similar, MSLC might be utilized as a model to examine the physiological properties of molds in the solid-state culture. It would also be interesting to clarify the differences in physiological properties of the molds cultivated by shaking culture and by MSLC in terms of differences in morphology, respiration ability, cellular
40
J. FERMENT.BIOENO.,
OGAWA ET AL.
mechanism (8) and others. MSLC is similar to the solid-state culture from the standpoint of growth on the surface of a solid material (a porous membrane). On the other hand, it can be regarded as a kind of liquid culture with respect to utilization of liquid medium. Thus, MSLC possesses advantages of both the solid-state and liquid cultures. Several approaches in which molds are grown on porous materials such as asbestos (9), sponge (lo), and urethane foam carrier (11) were reported. These methods are similar to MSLC in terms of cultivation on the surface of a porous material in contact with a liquid medium and exposed to the air. However, in these methods mycelia penetrate to the inside of the porous materials and also grow there. Kobayashi et al. cultivated A. oryzae B-3 on a urethane foam carrier to produce glucoamylase and recovered the enzyme by pressing the carrier (11). They repeated these procedures 13 times for 8 d; however, the cumulative amount of enzyme obtained increased by only about 3 times compared to the amount obtained in the batch culture, since only 13% of the medium was exchanged after each batch. MSLC is considered to be more advantageous than these methods, taking into consideration the ease of product recovery and of performing repeated-batch or continuous culture. The control of pH of and substrate concentration in the medium during the cultivation is also easy. Repeated-batch MSLC can be carried out efficiently without the need for any additional operations such as pressing. Separation of molds from the culture is quite easy. The liquid medium is clear when a membrane with an appropriate pore size is used. We carried out MSLC using membranes with different pore sizes. With the nominal pore size of 0.2 pm which was mainly used in this study, no mycelia were observed in the medium as described above. However, with a nominal pore size of 0.45 pm (SE45, Fuji Photo Film Co.), mycelia were observed in the liquid medium after 8 d of cultivation. With a pore size of 3 pm (SE300, Fuji Photo Film Co.), mycelia penetrated through the membrane pores from the initial stage of growth and grew all over the other side of the membrane. Since molds grow on the surface of the porous membrane, the composition of the membrane might also affect the growth behavior, which should be further investigated. MSLC can be utilized not only for the production of useful substances but also for a screening system of molds since production of metabolites can be enhanced and since the medium composition and pH can be varied in a manner similar to that in the case of the shaking culture. MSLC
might be used as a model of the solid-state culture to investigate the physiological properties of the molds as mentioned previously. To apply MSLC to industrial production, we must solve various problems related to scale-up of the apparatus, including types of membrane, kinds of modules, and methods for inoculation. ACKNOWLEDGMENT We thank Kikkoman Corp. (Noda, Japan) for providing strain of A. oryzae and manufacturing the MSLC apparatus.
the
REFERENCES 1
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Mitchell, D. A. and Lonsane, B. K.: Definition, characteristics and potential, p. 1-16. In Doelle, H. W., Mitchell, D. A., and Rolz, C. E. (ed.), Solid substrate cultivation. Elsevier Applied Science, London and New York (1992). Bajracharya, II. and Mudgett, R. E.: Effects of controlled gas environments in solid substrate fermentation of rice. Biotechnol. Bioeng., 22, 2219-2235 (1980). 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). &do, S., Ishikawa, T., Sato, K., and Oba, T.: Comparison of acid-stable a-amylase production by Aspergillus kawachii in solid-state and submerged cultures. J. Ferment. Bioeng., 77, 483489 (1994). Grajeck, W.: Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid-state fermentation. Appl. Microbial. Biotechnol., 26, 126-129 (1987). Yasuhara, A., Ogawa, A., Tanaka, T., Sakiyama, T., and Nakanishi, K.: Production of neutral protease from Aspergilhs oryzae by a novel cultivation method on a microporous membrane. Biotechnol. Techniq., 8, 249-254 (1994). Yano, T., Ashida, S., Tachiki, T., Kumagai, H., and Tochikura, T.: Development of a soft gel cultivation method. Agric. Biol. Chem., 55, 379-385 (1991). Pebexdy, J.F.: Protein secretion in filamentous fungi-trying to understand a highly productive black box. TIBTECH, 12, 50-57 (1994). Sakaguchi, K., Okazaki, H., and Takeuchi, M.: A note on the comparison of koji and submerged culture. Nippon Nougeikagaku Kaishi, 29, 349-353 (1955). (in Japanese) Fujishima, T., Uchida, K., and Yoshino, H.: Enzyme production by molds in sponge culture. J. Ferment. Technol., 50, 724-730 (1972). Kobayashi, T., Ozawa, S., Sato, K., Nagamune, T., and Endo, I.: Production of glucoamylase by a solid-state fermentation using urethane foam carrier as a semi-solid medium. Kagaku Kogaku Ronbunshu, 17,491-496 (1991). (in Japanese)