- Email: [email protected]
Production of Monascus pigments by a solid-liquid state culture method
JOURNAL OF FERMENTATION AND BIOENCINEERING Vol. 79, No. 5, 516-518. 1995
Production YUAN-KUN
of Monascus Pigments by a Solid-Liquid Culture Method
LEE,‘* DUAN-CHENG
CHEN,’ SOMCHAI CHAUVATCHARIN,2 TOSHIOMI YOSHIDA2
State
TATSUJI
SEKI,*
AND
Department of Microbiology, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511’ and International Center of Cooperative Research in Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan* Received
2 November
1994/Accepted
16 February
1995
In a simple batch culture of Monuscus growing on 50 g/Z tapioca starch as the carbon substrate, the biomass dry weight reached a maximum value of 8 g/Z. The red and yellow polyketide pigments continued to be produced after cell growth had leveled off at 40 h, and reached the concentrations of 31 and 26.5 optical density (OD) units, respectively. When a 200 g/l starch medium was continuously fed into a A4onuscus culture after being grown in batch mode for 60 h, the biomass dry weight reached a concentration of 16 g/l at 140 h. At this point in time, the red and yellow pigment concentrations were 70 and 60 OD units, respectively. A two-state (solid-liquid) batch culture system was constructed using a solid state of 400 g/Z gelatinized starch cake and a liquid state which contained all the culture substrates except starch, in which the mycelia were cultured. The starch block was digested gradually through the amylolytic activity of enzymes produced by the MO~USCUS culture. The growth rate of the culture was almost linear up to 170 h, and a cell concentration of 37.5 g/Z and pigment concentrations of about 145 OD units for both the red and yellow pigments were achieved. [Key words:
Monascus, pigments,
culture
method]
The red polyketide pigments of the fungus Monascus are widely used in Oriental countries as natural food colorants. The annual consumption of Monascus pigments in Japan alone amounted to 600 tons and was valued at 1,440 million yen according to a survey done in 1992 (1). For the production of Monascus pigments, the fungus has been traditionally cultured on steamed rice using solid-state fermentation (2). Lately, Monascus has been successfully cultured in submerged culture systems (3-6) and most of the industrial production today is achieved using submerged systems. Tapioca starch, on the other hand, might be an ideal alternative substrate for Monascus fermentation, for it is (i) affordable, (ii) colorless and (iii) readily digested and utilized by the fungus. However, there are a few technical difficulties in the cultivation of Monascus in submerged culture using starch: (i) The high viscosity of gelatinized crude starch prevents practical use of a high initial starch concentration in the culture medium, resulting in relatively low final cell and pigment concentrations. Moreover, the low mass transfer rate in a viscous culture does not permit sufficient supply of oxygen to maintain high Monascus pigment productivity (7). (ii) For the same reason, the starch concentration in medium fed to a fed-batch culture cannot be very high, and resulting in relatively low final cell and pigment concentrations. Some of the technical difficulties caused by the rheological properties of crude starch could be overcome by pre-digesting the substrate using starch hydrolytic enzymes prior to fermentation. However, this entails additional manipulation, resulting in increased production cost and possibility of microbial contamination. It is common knowledge that a gelatinized starch cake is not easily dissolved in water. Thus the addition of one starch block to a culture system will not greatly influence * Corresponding author.
the rheological property of the liquid phase, and the hydrolysis of the starch by enzymes produced by the Monascus culture would result in a continuous supply of glucose as carbon source for growth and pigment production. The Monascus sp. B683 used in this study was a high pigment producer isolated in Singapore from angkak (red rice), and produced loose colonies on Sabouraud dextrose agar at 30°C. The basal culture medium contained the following (g/l): tapioca starch, 50; monosodium glutamate, 20; MgS04.7Hz0, 15; KH2P04, 10; 3N-morpholino-propanesulfonic acid buffer, 74; pH 6.5. The Monascus was cultured in a 3-1 stirred tank fermentor (either Eyela model MBF or M-100, Tokyo Rikakikai Co. Ltd., Tokyo) maintained at 30°C and stirred at 350 rpm. Lower agitation rates resulted in low DO concentration whereas higher agitation rates caused cell damage and lower growth rates (data not shown). The buffer system maintained the pH of the cultures between 6.5 and 5.5. The DO concentration of the cultures was measured using a galvanic electrode and maintained between 5 and 6 ppm by manually adjusting the air supply rate. Dry cell weight was measured after drying (SO’C) a known sample volume of the mycelia collected by centrifugation, in centrifuge tubes. The concentrations of extracellular red and yellow pigments were estimated from the relative absorbance of filtrates at 500 and 400nm, respectively, with a l-cm light path. To estimate the concentrations of intracellular pigments, lOm1 of the culture was centrifuged and the collected mycelia were washed with distilled water. The washed mycelia were then extracted with 50 ml of pure ethanol for 2 h with continuous agitation. The extraction procedure was repeated as necessary. The concentrations of red and yellow pigments were again estimated from the absorbance of extracts at 500 and 400 nm, respectively, taking the dilution factor into con-
VOL. 79, 1995
NOTES
517
30 ‘3 -_ 2 9
8% 8
*O
z 2
B’ 0
I
15
40
8
20
gg
2
8O 4
0
10
40
120
00
Time
0
0
(h)
FIG. 1. Growth kinetics of Monascus in simple batch culture. Symbols: 0, biomass dry weight; n , OD SOOnm; 0, OD 400nm; 0, residual glucose. sideration. The two values (extracellular and intracellular) were added to give the total pigment concentration. The ratio of the extracellular to intracellular pigments measured in this study was always about 1 : 5. The residual glucose was determined using a YSI Biochemistry Analyzer model 2700 Select (Yellow Springs Instrument Co., Ohio, USA). The quantity of residual gelatinized crude starch in the culture vessel was estimated from the graduation on the vessel wall. The amount of soluble starch in the culture supernatant was determined by the polarization method (8). In the present study using tapioca starch as the carbon substrate, the highest culture growth rate and pigment yield of Monascus in a simple batch culture were observed in medium containing 5Og/f of the starch. At higher starch concentrations, the productivities of cell mass and pigments were severely decreased, possibly due to shear stress created by the high viscosity of the highstarch medium. It was also difficult to maintain a high DO concentration in the high-starch culture. Oxygen molecules are essential for the biosynthesis of polyketides (9). Low oxygen partial pressure was found to inhibit Monascus pigment production in both solid-state (10) and submerged fermentations (7, 11). As shown in Fig. 1, as a consequence of low initial starch concentration (50 g/l) in the culture medium, the final cell and pigment concentrations achievable in a simple batch culture were 8 g/l and 30 OD units, respectively, and were too low for a practical industrial production process. The residual glucose concentration in the culture was high during the first 10 h of incubation, indicating rapid hydrolysis of starch, and then declined very rapidly due to its uptake by the microorganism. No residual glucose was detected after 80 h of incubation. A fed-batch culture system with continous feeding of starch would overcome the problem of low cell and product concentrations. However, a starch gel prepared using more than 200 g/Z of starch granules is almost completely solid. Even at 2OOg/l starch concentration, the semisolid gel could only be fed into the culture at a low speed and with difficulty using a peristaltic pump. In this study, the Monascus culture was first cultured in a batch mode, and the fed-batch mode began after 60 h of incu-
0
40
0
80
Time
120
(h)
FIG. 2. Growth kinetics of Monascus in fed-batch culture. Feed medium containing 2OOg/l tapioca starch was fed from the time indicated by the arrow. Symbols: 0, biomass dry weight; H , OD 500 nm; q , OD 400 nm; 0, residual glucose.
bation as shown in Fig. 2. The feed medium contained 2OOg/l tapioca starch in concentrated basal medium. The biomass dry weight continued to increase to a concentration of 16 g/l at 140 h. At 140 h, the culture volume had increased from 1 1 to 1.28 1 due to the addition of starch to the medium. The properties and concentration of the feed stock limit the flexibility of the culture system. The pigment concentrations reached 70 and 60 OD units at 140 h for the red and yellow pigments, respectively. The residual glucose concentration was negligible during the fed-batch period, suggesting that the uptake of glucose was faster than the release of glucose through the hydrolysis of the starch being fed. The two-state culture system proposed here takes advantage of the fact that gelatinized crude starch does not dissolve readily in water even with agitation. In this culture system, the culture vessel was autoclaved with 1 1 of basal medium containing 400g of tapioca starch in place. After cooling to the cultivation temperature of 3O”C, another liter of medium, which contained all the nutrient components of the basal medium except starch and was autoclaved separately, was fed into the vessel. Before inoculation of the culture, the medium was agitated continuously for 4 h and the concentration of soluble starch measured in the medium was only 11 g/l. The gelatinized starch block remained stuck to the bottom of the culture vessel and did not mix with the liquid phase throughout the course of the study. The growth kinetics of the Monascus culture are shown in Figs. 3A and 3B. The Monascus biomass increased throughout the course of the cultivation in a rather linear fashion. The nearlinear growth kinetics of the Monascus culture suggests that growth rate of the culture was limited by the of supply of substrate available to the culture, possibly the glucose produced by the hydrolysis of the starch. Indeed, the estimated volume of the solid starch in the culture system appeared to decrease almost linearly after 50 h of incubation and the residual soluble starch and glucose concentrations in the culture medium remained low after 50 h. Thus, in theory the productivity of the Monascus culture could be improved by using a high
518
J. FERMENT. BIOENG.,
LEE ET AL.
30
120 ‘3
z 9 j g20 z
8o
2
0
g 10
40
starch block and were able to achieve a cell concentration of 37 g/l. In practice, it is possible to use a much higher concentration of starch block, thus achieving a much higher product concentration if the oxygen demand of the culture is met. The highest red and yellow pigment concentrations measured in the two-state culture system were 147 and 140 OD units, respectively.
0
This research collaboration was made possible through a Japan Society for Promotion of Science (JSPS) Programme between the Osaka University and the National University of Singapore. REFERENCES
0
40
80
120
Time
(h)
Time
(h)
160
0
B
Growth kinetics of Monuscus in two-state batch (A) Accumulation of biomass dry weight (0), red ( n , OD and yellow ( q , OD 400 nm) pigments, and (B) changes in the volume of solid starch block (A), the residual soluble starch glucose (0 ) concentrations. FIG.
3.
culture. 500 nm) residual (A) and
amylase producing strain of Monascus, or a system which provides a larger solid starch-liquid interface area such as by coating the inner surface of the fermentation vessel with gelatinized starch or cutting the starch block into cubes. In this study, we started the culture with a 40% (w/v)
1. Marketing research for food colors. Monthly J. Food Chem., 11, 48-61 (1992). (in Japanese) 2. Steinkraus, K. (ed.): Handbook of indigenous fermented foods. Marcel Dekker, New York (1978). 3. Lin, C.: Isolation and culture conditions of Monascus sp. for the production of pigment in a submerged culture. J. Ferment. Technol., 51, 407-414 (1973). 4. Yoshimura, M., Yamanaka, S., Mitsugi, K., and Hirose, Y.: Production of Monascus pigment in a submerged culture. Agric. Biol. Chem., 39, 1789-1795 (1975). of extracellular pigment 5. Lin, C. F. and Iizuka, H.: Production by a mutant of Monascus kaoling nov. Appl. Environ. Microbial., 43, 671-676 (1982). of Monus6. Lin, T. F. and Demain, A. L. C.: Effect of nutrition cus sp. on formation of red pigments. Appl. Microbial. Biotechnol., 36, 70-75 (1991). I. Lee, Y. K., Lim, B. L., Ng, A. L., and Chen, D. C.: Production of polyketide pigments by submerged culture of Monascm: effect of substrate limitations. Asia Pacific J. Mol. Biol. Biotechnol., 2, 21-26 (1994). 8. A.O.A.C.: Polarization method, 15th ed., p. 783. Helrich, K. (ed.), Official Methods of Analysis by Association of Official Analytical Chemists. A.O.A.C., Arlington (1990). metabolites, p. 136-139. Academic 9. Turner, W. B.: Fungal Press, London (1971). 10. Han, 0. and Mudgett, R.E.: Effect of oxygen and carbon dioxide partial pressures on Monascus growth and pigment production in solid-state fermentations. Biotechnol. Prog., 8, 5010 (1992). 11 Lee, Y. K., Ng, A. L., and Lim, B. L.: The kinetics of growth and pigment production of Monascus cultures, p. 334-338. In Matangkasombut, P. and Oshima, Y. (ed.), Microbial utilization of renewable resources, vol. 8. International Centre of Cooperative Research in Biotechnology, Osaka (1992).
Production YUAN-KUN
of Monascus Pigments by a Solid-Liquid Culture Method
LEE,‘* DUAN-CHENG
CHEN,’ SOMCHAI CHAUVATCHARIN,2 TOSHIOMI YOSHIDA2
State
TATSUJI
SEKI,*
AND
Department of Microbiology, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511’ and International Center of Cooperative Research in Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan* Received
2 November
1994/Accepted
16 February
1995
In a simple batch culture of Monuscus growing on 50 g/Z tapioca starch as the carbon substrate, the biomass dry weight reached a maximum value of 8 g/Z. The red and yellow polyketide pigments continued to be produced after cell growth had leveled off at 40 h, and reached the concentrations of 31 and 26.5 optical density (OD) units, respectively. When a 200 g/l starch medium was continuously fed into a A4onuscus culture after being grown in batch mode for 60 h, the biomass dry weight reached a concentration of 16 g/l at 140 h. At this point in time, the red and yellow pigment concentrations were 70 and 60 OD units, respectively. A two-state (solid-liquid) batch culture system was constructed using a solid state of 400 g/Z gelatinized starch cake and a liquid state which contained all the culture substrates except starch, in which the mycelia were cultured. The starch block was digested gradually through the amylolytic activity of enzymes produced by the MO~USCUS culture. The growth rate of the culture was almost linear up to 170 h, and a cell concentration of 37.5 g/Z and pigment concentrations of about 145 OD units for both the red and yellow pigments were achieved. [Key words:
Monascus, pigments,
culture
method]
The red polyketide pigments of the fungus Monascus are widely used in Oriental countries as natural food colorants. The annual consumption of Monascus pigments in Japan alone amounted to 600 tons and was valued at 1,440 million yen according to a survey done in 1992 (1). For the production of Monascus pigments, the fungus has been traditionally cultured on steamed rice using solid-state fermentation (2). Lately, Monascus has been successfully cultured in submerged culture systems (3-6) and most of the industrial production today is achieved using submerged systems. Tapioca starch, on the other hand, might be an ideal alternative substrate for Monascus fermentation, for it is (i) affordable, (ii) colorless and (iii) readily digested and utilized by the fungus. However, there are a few technical difficulties in the cultivation of Monascus in submerged culture using starch: (i) The high viscosity of gelatinized crude starch prevents practical use of a high initial starch concentration in the culture medium, resulting in relatively low final cell and pigment concentrations. Moreover, the low mass transfer rate in a viscous culture does not permit sufficient supply of oxygen to maintain high Monascus pigment productivity (7). (ii) For the same reason, the starch concentration in medium fed to a fed-batch culture cannot be very high, and resulting in relatively low final cell and pigment concentrations. Some of the technical difficulties caused by the rheological properties of crude starch could be overcome by pre-digesting the substrate using starch hydrolytic enzymes prior to fermentation. However, this entails additional manipulation, resulting in increased production cost and possibility of microbial contamination. It is common knowledge that a gelatinized starch cake is not easily dissolved in water. Thus the addition of one starch block to a culture system will not greatly influence * Corresponding author.
the rheological property of the liquid phase, and the hydrolysis of the starch by enzymes produced by the Monascus culture would result in a continuous supply of glucose as carbon source for growth and pigment production. The Monascus sp. B683 used in this study was a high pigment producer isolated in Singapore from angkak (red rice), and produced loose colonies on Sabouraud dextrose agar at 30°C. The basal culture medium contained the following (g/l): tapioca starch, 50; monosodium glutamate, 20; MgS04.7Hz0, 15; KH2P04, 10; 3N-morpholino-propanesulfonic acid buffer, 74; pH 6.5. The Monascus was cultured in a 3-1 stirred tank fermentor (either Eyela model MBF or M-100, Tokyo Rikakikai Co. Ltd., Tokyo) maintained at 30°C and stirred at 350 rpm. Lower agitation rates resulted in low DO concentration whereas higher agitation rates caused cell damage and lower growth rates (data not shown). The buffer system maintained the pH of the cultures between 6.5 and 5.5. The DO concentration of the cultures was measured using a galvanic electrode and maintained between 5 and 6 ppm by manually adjusting the air supply rate. Dry cell weight was measured after drying (SO’C) a known sample volume of the mycelia collected by centrifugation, in centrifuge tubes. The concentrations of extracellular red and yellow pigments were estimated from the relative absorbance of filtrates at 500 and 400nm, respectively, with a l-cm light path. To estimate the concentrations of intracellular pigments, lOm1 of the culture was centrifuged and the collected mycelia were washed with distilled water. The washed mycelia were then extracted with 50 ml of pure ethanol for 2 h with continuous agitation. The extraction procedure was repeated as necessary. The concentrations of red and yellow pigments were again estimated from the absorbance of extracts at 500 and 400 nm, respectively, taking the dilution factor into con-
VOL. 79, 1995
NOTES
517
30 ‘3 -_ 2 9
8% 8
*O
z 2
B’ 0
I
15
40
8
20
gg
2
8O 4
0
10
40
120
00
Time
0
0
(h)
FIG. 1. Growth kinetics of Monascus in simple batch culture. Symbols: 0, biomass dry weight; n , OD SOOnm; 0, OD 400nm; 0, residual glucose. sideration. The two values (extracellular and intracellular) were added to give the total pigment concentration. The ratio of the extracellular to intracellular pigments measured in this study was always about 1 : 5. The residual glucose was determined using a YSI Biochemistry Analyzer model 2700 Select (Yellow Springs Instrument Co., Ohio, USA). The quantity of residual gelatinized crude starch in the culture vessel was estimated from the graduation on the vessel wall. The amount of soluble starch in the culture supernatant was determined by the polarization method (8). In the present study using tapioca starch as the carbon substrate, the highest culture growth rate and pigment yield of Monascus in a simple batch culture were observed in medium containing 5Og/f of the starch. At higher starch concentrations, the productivities of cell mass and pigments were severely decreased, possibly due to shear stress created by the high viscosity of the highstarch medium. It was also difficult to maintain a high DO concentration in the high-starch culture. Oxygen molecules are essential for the biosynthesis of polyketides (9). Low oxygen partial pressure was found to inhibit Monascus pigment production in both solid-state (10) and submerged fermentations (7, 11). As shown in Fig. 1, as a consequence of low initial starch concentration (50 g/l) in the culture medium, the final cell and pigment concentrations achievable in a simple batch culture were 8 g/l and 30 OD units, respectively, and were too low for a practical industrial production process. The residual glucose concentration in the culture was high during the first 10 h of incubation, indicating rapid hydrolysis of starch, and then declined very rapidly due to its uptake by the microorganism. No residual glucose was detected after 80 h of incubation. A fed-batch culture system with continous feeding of starch would overcome the problem of low cell and product concentrations. However, a starch gel prepared using more than 200 g/Z of starch granules is almost completely solid. Even at 2OOg/l starch concentration, the semisolid gel could only be fed into the culture at a low speed and with difficulty using a peristaltic pump. In this study, the Monascus culture was first cultured in a batch mode, and the fed-batch mode began after 60 h of incu-
0
40
0
80
Time
120
(h)
FIG. 2. Growth kinetics of Monascus in fed-batch culture. Feed medium containing 2OOg/l tapioca starch was fed from the time indicated by the arrow. Symbols: 0, biomass dry weight; H , OD 500 nm; q , OD 400 nm; 0, residual glucose.
bation as shown in Fig. 2. The feed medium contained 2OOg/l tapioca starch in concentrated basal medium. The biomass dry weight continued to increase to a concentration of 16 g/l at 140 h. At 140 h, the culture volume had increased from 1 1 to 1.28 1 due to the addition of starch to the medium. The properties and concentration of the feed stock limit the flexibility of the culture system. The pigment concentrations reached 70 and 60 OD units at 140 h for the red and yellow pigments, respectively. The residual glucose concentration was negligible during the fed-batch period, suggesting that the uptake of glucose was faster than the release of glucose through the hydrolysis of the starch being fed. The two-state culture system proposed here takes advantage of the fact that gelatinized crude starch does not dissolve readily in water even with agitation. In this culture system, the culture vessel was autoclaved with 1 1 of basal medium containing 400g of tapioca starch in place. After cooling to the cultivation temperature of 3O”C, another liter of medium, which contained all the nutrient components of the basal medium except starch and was autoclaved separately, was fed into the vessel. Before inoculation of the culture, the medium was agitated continuously for 4 h and the concentration of soluble starch measured in the medium was only 11 g/l. The gelatinized starch block remained stuck to the bottom of the culture vessel and did not mix with the liquid phase throughout the course of the study. The growth kinetics of the Monascus culture are shown in Figs. 3A and 3B. The Monascus biomass increased throughout the course of the cultivation in a rather linear fashion. The nearlinear growth kinetics of the Monascus culture suggests that growth rate of the culture was limited by the of supply of substrate available to the culture, possibly the glucose produced by the hydrolysis of the starch. Indeed, the estimated volume of the solid starch in the culture system appeared to decrease almost linearly after 50 h of incubation and the residual soluble starch and glucose concentrations in the culture medium remained low after 50 h. Thus, in theory the productivity of the Monascus culture could be improved by using a high
518
J. FERMENT. BIOENG.,
LEE ET AL.
30
120 ‘3
z 9 j g20 z
8o
2
0
g 10
40
starch block and were able to achieve a cell concentration of 37 g/l. In practice, it is possible to use a much higher concentration of starch block, thus achieving a much higher product concentration if the oxygen demand of the culture is met. The highest red and yellow pigment concentrations measured in the two-state culture system were 147 and 140 OD units, respectively.
0
This research collaboration was made possible through a Japan Society for Promotion of Science (JSPS) Programme between the Osaka University and the National University of Singapore. REFERENCES
0
40
80
120
Time
(h)
Time
(h)
160
0
B
Growth kinetics of Monuscus in two-state batch (A) Accumulation of biomass dry weight (0), red ( n , OD and yellow ( q , OD 400 nm) pigments, and (B) changes in the volume of solid starch block (A), the residual soluble starch glucose (0 ) concentrations. FIG.
3.
culture. 500 nm) residual (A) and
amylase producing strain of Monascus, or a system which provides a larger solid starch-liquid interface area such as by coating the inner surface of the fermentation vessel with gelatinized starch or cutting the starch block into cubes. In this study, we started the culture with a 40% (w/v)
1. Marketing research for food colors. Monthly J. Food Chem., 11, 48-61 (1992). (in Japanese) 2. Steinkraus, K. (ed.): Handbook of indigenous fermented foods. Marcel Dekker, New York (1978). 3. Lin, C.: Isolation and culture conditions of Monascus sp. for the production of pigment in a submerged culture. J. Ferment. Technol., 51, 407-414 (1973). 4. Yoshimura, M., Yamanaka, S., Mitsugi, K., and Hirose, Y.: Production of Monascus pigment in a submerged culture. Agric. Biol. Chem., 39, 1789-1795 (1975). of extracellular pigment 5. Lin, C. F. and Iizuka, H.: Production by a mutant of Monascus kaoling nov. Appl. Environ. Microbial., 43, 671-676 (1982). of Monus6. Lin, T. F. and Demain, A. L. C.: Effect of nutrition cus sp. on formation of red pigments. Appl. Microbial. Biotechnol., 36, 70-75 (1991). I. Lee, Y. K., Lim, B. L., Ng, A. L., and Chen, D. C.: Production of polyketide pigments by submerged culture of Monascm: effect of substrate limitations. Asia Pacific J. Mol. Biol. Biotechnol., 2, 21-26 (1994). 8. A.O.A.C.: Polarization method, 15th ed., p. 783. Helrich, K. (ed.), Official Methods of Analysis by Association of Official Analytical Chemists. A.O.A.C., Arlington (1990). metabolites, p. 136-139. Academic 9. Turner, W. B.: Fungal Press, London (1971). 10. Han, 0. and Mudgett, R.E.: Effect of oxygen and carbon dioxide partial pressures on Monascus growth and pigment production in solid-state fermentations. Biotechnol. Prog., 8, 5010 (1992). 11 Lee, Y. K., Ng, A. L., and Lim, B. L.: The kinetics of growth and pigment production of Monascus cultures, p. 334-338. In Matangkasombut, P. and Oshima, Y. (ed.), Microbial utilization of renewable resources, vol. 8. International Centre of Cooperative Research in Biotechnology, Osaka (1992).