Bioprocess Engineering 17 (1997) 7579 Springer-Verlag 1997

A new process for red pigment production by submerged culture of Monascus purpureus
M. Hamdi, P.J. Blanc, M.O. Loret, G. Goma

75 Abstract Formation of red pigment by Monascus purpureus via diauxic growth on glucose and ethanol in submerged culture was optimized based on inoculum preparation and culture medium. A vegetative inoculum was prepared from spores grown on ethanol. The optimized culture medium was low in phosphates, and had an initial pH of 5.5. The characteristics of Monascus purpureus grown on glucose and on ethanol were compared: the specic consumption rate of glucose (qG) was higher than the specic consumption rate of ethanol (qE), whereas the specic growth rate was greatest with ethanol. The specic production rate of red pigment (pOD) and pigment yield (YOD/s) with glucose was twice that with ethanol. A novel fermentation process was developed with M. purpureus initially grown with controlled ethanol formation, and consumption of the latter during pigment formation.
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cultures have been studied extensively in the past 10 years [1, 2, 3, 4]. Yields in submerged culture are affected by medium composition, pH and agitation [2, 3]. A chemically-dened medium supporting rapid and intensive red pigment production, using glutamate as nitrogen source was developed, and shifted the site of pigment from being predominantly cell-bound to being mainly extra cellular [4]. We focus on red pigment production by Monascus purpureus (this work) and M. ruber [5] grown in a submerged culture in both dened and complex media.

2 Material and methods 2.1 Microorganism and inoculum preparation Monascus purpureus CBS 10907 was maintained on potato dextrose agar (PDA) (Difco, Detroit, MI.). Spores were prepared by growth on PDA in an Erlenmeyer ask for 6 days at 30 C. Spores were washed from the agar with sterile water and glass beads. Cultures were inoculated with a spore suspension (0.5 ml) in 250-ml Erlenmeyer asks (40/250 ml) of optimized medium (see below, Table 1) and incubated on a rotary shaker (150 rpm) at 30 C. 2.2 Growth conditions Batch cultures were performed in 2 l fermentors (S.G.I., Toulouse, France) containing 1.5 l optimized medium (Table 1) at 30 C. The agitation system was a Rushton-like turbine. The dissolved oxygen concentration was kept constant or modied by changing the impeller speed or by aeration ow rate which was measured with a gas mass ow meter. The DO concentration was maintained between 60 and 80% of saturation level. The pH was not maintained constant.

DO qG qE pE pOD Yx/s YOD/s

(%) Dissolved oxygen (g/g.h) Specic consumption of glucose (g/g.h) Specic consumption of ethanol (h1) Maximum specic growth rate (g/g.h) Specic production rate of ethanol (ODU/g.h) Maximum specic production rate of red pigment (g/g) Yield of biomass on substrate (ODU/g) Yield of red pigment on ethanol

1 Introduction Study of the natural red food colorants has intensied and the objective being to replace certain synthetic ones. Monascus sp. red pigment has been recently utilized in the orient for making red rice wine, red soybean cheese, and red chinese rice [1]. The production of Monascus pigment as food coloring agent by both submerged and agar surface
Received: 23 September 1996

2.3 Analytical methods Biomass concentration was determined by ltering the P.J. Blanc, M.O. Loret, G. Goma Departement de Genie Biochimique et Alimentaire. mycelia through a glass ber membrane Whatman GF/C, UA CNRS N 544. INSA, Complexe Scientique de Rangueil. washed with water, dried under low pressure at 60 C for 31077 Toulouse Cedex, France 24 h, and then weighed. The water soluble red pigment were estimated by measuring the optical density at 480 nm M. Hamdi using a Kontron Uvikon spectrophotometer. Ecole Superieure des Industries Alimentaires, Ethanol concentration was determined using a Intersmat 58 Avenue Alain Savary. 1003 Tunis, Tunisia IGC 120 DFL gas chromatograph equipped with a ame Correspondence to: P.J. Blanc ionization detector: column (2 m 3.2 mm) packed with a

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Table 1. Media used for inoculum preparation and red pigment formation
Nutriments or salts Glucose (g/l) Ethanol (g/l) Monosodium glutamate (g/l) Potassium dihydrogenophosphate (g/l) Dipotassium hydrogenophosphate (g/l) Potassium chlorure (g/l) Magnesium sulfate (g/l) Iron sulfate (mg/l) Zinc sulfate (mg/l) Manganese sulfate (mg/l) pH Inoculum Inoculum preparation 25 4 2.5 2.5 0.5 1 10 10 3 5 Spores (105/ml) Red pigment formation 2050 213* 12.4 0.125 0.125 10 10 3 5.5 Free mycelium 2%(V/V)

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*Successive addition during red pigment formation: See Figs. 3 and 5

Poropack QS 80100 mesh, with nitrogen as the carrier gas. Injector, detector, and column temperatures were 260, 260, and 195 C respectively. Quantitative data were obtained by peak integration with an Intersmat ICR 1B after calibration with propanol (10 g/l) internal standard. Glucose analyses of broth supernatants (centrifuged and diluted) were by using a glucose analyzer (YSI Model 27, SGI, France). Dissolved oxygen (DO) concentrations were measured with an Ingold amperometric probe. Citric, succinic, acetic and malic acids were analysed by HPLC: Waters liquid chromatograph equipped with an ION organic acids column and a refractometer 410. Elution was carried out with an aqueous solution of H2SO4 10 mM at a ow rate of 0.4 ml/min.

3 Results and discussion 3.1 Optimization of media for inoculum preparation and red pigment formation Inocula can be crucial in fungus culture. Preliminary runs based on the factorial design experiments showed that the medium of Lin and Demain [4] is good for inoculum preparation when the monosodium glutamate (MSG) was reduced to 4 g/l, and source carbohydrates was changed to ethanol, and the initial pH changed from 5.5 into 5. The ethanol (23%) yields better vegetative growth than glucose. At higher concentration of ethanol, lesser biomass was produced with formation of larger pellets (Fig. 1). Pellets may originate from spores coalescing, freshly germinating spores aggregating, or by mycelial entanglement. Optimum concentration of spores to inoculate the ethanol medium is about 105 spores/ml whereas with glucose it is higher 107 spores/ml. The ethanol is more suitable source carbohydrate for inoculum preparation than glucose, because it gives twice mycelium concentration and it requires lesser spores for inoculation. The optimum initial pH of 5 favors spore germination, yields a free mycelium, higher biomass but lesser red pigment production (Fig. 1). Indeed, Lin [1] showed that maximum cell yield was obtained at pH 5, while the maximum pigment yield was at 6. At pH values above 5.5, cell walls of most microorganisms are negatively charged, tending to cause separation of the cells by electrostatic repulsion [6]. Under optimal conditions for inoculum preparation M. purpureus gave max-

Fig. 1. Inuence of initial concentration of ethanol and initial pH on the biomass ( ) and red pigment production ( ) by Monascus purpureus growth on ethanol

imum biomass in 4 days, a maximum specic growth rate of around 0.083 h1, with biomass yield of 8.9 g/l. The pH of the medium increased slowly from 5 up to 5.32 and red pigment was not greater than 0.7 ODU/ml. Optimization of red pigment production in Erlenmeyer asks using a vegetative inoculum showed 2% v/v as the optimal inoculum (Table 1). Relatively low amounts of potassium dihydrogenophosphate (50 mg/l) and dipotassium hydrogenophosphate (50 mg/l) were sufcient for high red pigment production. An initial pH of 5.5 favored mycelial growth, and as it rose it resulted in improved red pigment formation. Indeed, in submerged culture, pigments normally remain in the mycelium due to low solu-

M. Hamdi et al.: Production of MonascusI red pigment

bility in usually acidic medium [7]. Yet, Mak et al. [8] noted that high pH might facilitate the pigment removal from mycelium and was associated with the chemical conversion of orange to red. This optimized medium for red pigment formation was used in further experiments. In preliminary studies several carbon sources were compared with regard to red pigment production (data unreported). Glucose and ethanol appeared useful substrates. When the carbon source was a mixture of glucose and ethanol (Fig. 2), growth was in two stages diauxie. Glucose gave increase in biomass and ethanol, while ethanol resulted in growth of biomass plus pigment formation (Fig. 2).

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3.2 Red pigment production in fermentor Both substrates glucose and ethanol were studied for red pigment production in fermentor. Fermentors inoculated with a vegetative mycelial inoculum reduced the lag phase, and gave a higher specic growth rate than inocula of a spore suspension or vegetative pellets (data unreported). Culture parameters obtained with glucose are illustrated in Figs. 3 and 4. The biomass concentration increased in two stages corresponding to glucose consumption and ethanol formation for 75 h, and then ethanol consumption and red pigment formation. The specic consumption rate of glucose (qG) increased continuously from 0.1 to 0.3 g/ g.h. The specic production rate of ethanol from glucose (pE) ranged from 0.05 to 0.08 g/g.h, and could be increased by decreasing the DO concentration [9]. The specic growth rate on glucose decreases from 0.06 to 0.02 h1, whereas on the accumulated ethanol the mean specic growth rate was around 0.025 h1. After 80 h, the biomass decreases probably because of pigment removal from mycelium. The red pigment formation is initiated especially when glucose was completely exhausted and corresponding to the beginning of ethanol consumption

Fig. 3. Time courses of glucose ( ), ethanol ( ), biomass (), red pigment ( ) and pH () during Monascus purpureus growth on glucose in fermentor

Fig. 2. Time courses of glucose ( ), ethanol ( ), biomass (), and red pigment ( ), during Monascus purpureus growth on mixed ethanol and glucose in ask cultures

Fig. 4. Time courses of (), qG ( ), pE (), qE ( ) and pOD ( ) during Monascus purpureus growth on glucose in fermentor

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(Fig. 3). The specic consumption rate of ethanol (qE) was 0.050.08 g/g.h. The red pigment formation started after 50 h and reached 8.5 ODU/ml at the end of fermentation. The specic production rate of red pigment (pOD) continuously increased and reached 0.1 ODU/g.h. Growth of M. purpureus on glucose, showed that red pigment formation was not coupled to the glucose consumption but to ethanol consumption. The pH increases continuously during red pigment formation and reached 6.6 at the end of culture. Red water-soluble Monascus pigment is unstable at acidic pH [10]. With ethanol as sole carbon source, ethanol consumption, biomass and red pigment formation progressed simultaneously (Figs. 5 and 6). The specic consumption rates of ethanol (qE) were 0.2 g/g.h for the rst 50 h but only 0.1 g/g.h during red pigment production. The specic growth rate on ethanol ranged from 0.01 to 0.06 h1 during 50 h, whereas the mean specic growth rate during red pigment formation averaged 0.015 h1. The biomass decrease was lesser than with glucose and started after 100 h of culture. The red pigment formation began after 50 h and reached 7.8 ODU/ml at the end of fermentation. The Fig. 6. Time courses of (), qE ( ) and pOD ( ) during Monascus specic production rate of red pigment (pOD) continuously purpureus growth on ethanol in fermentor increased and reached 0.05 ODU/g.h. In contrast to the fermentations with glucose, the pH increased but gave a decrease after each addition of ethanol. Higher citric, malic, succinic and acetic acids concentrations were produced with ethanol fermentation than with glucose fermentation (Fig. 7). The growth of M. purpureus on glucose and on ethanol are summarized in Table 2. Glucose is more suitable for red pigment production than ethanol, but ethanol can be

Fig. 7. Time courses of citric acid ( ), malic acid ( ), succinic acid ( ) and acetic acid ( ) during Monascus purpureus growth on glucose (above) and on ethanol (below) in fermentor

Fig. 5. Time courses of ethanol ( ), biomass (), red pigment ( ) and pH () during Monascus purpureus growth on ethanol in fermentor

supplemented after exhaustion of ethanol initially synthetized from glucose. The specic consumption rate of glucose (qG) was higher than the specic consumption rate of ethanol (qE) during the growth step. The biomass from glucose was more favorable to red pigment formation than that obtained from ethanol. In both fermentations, the pigments were formed from the ethanol. The specic production rate of red pigment (pOD) and the yields (YOD/E) obtained with fermentation of glucose was twice of

M. Hamdi et al.: Production of MonascusI red pigment

Table 2. Growth characteristics of M. purpureus on glucose and on ethanol

Growth characteristics Specic consumption of glucose qG (g/g.h) Specic consumption of ethanol qE (g/g.h) Maximum specic growth rate (h1) Specic production rate of ethanol pE (g/g.h) Maximum specic production rate of pigments pOD (ODU/g.h) Yield of biomass on substrate Yx/s (g/g) Yield of red pigment on ethanol YOD/s (ODU/g) Glucose 0.10.3 0.050.08 0.04 0.05 -0.08 0.1 0.15* 0.81.1 Ethanol 0.10.2 0.06 0.05 0.3 0.50.7

*depending of the partial oxygen pressure during fermentation

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that obtained with ethanol fermentation, though the spe- References cic consumption of ethanol was higher with fermentation 1. Lin, C.F. 1973.: Isolation and cultural conditions of Monascus sp. for the production of pigment in submerged culture. J. Ferment. using ethanol as carbohydrate source (Table 2) probably Technol., 51: 407414 because the higher organic acids accumulated with ethanol 2. Carels, M.; Shepherd, D. 1979: The effect of changes in pH on (Fig. 7). The modication in the metabolites formation phosphates and potassium uptake by Monascus rubiginosus ATCC seems to be caused by the morphological type of myce16367 in submerged shaken culture. Can. J. Microbiol. 25: 1484 lium. In fact, the fungal hyphae extended and ramied to 1488 give smooth pellets with glucose while it gave lamentous 3. Broder, C.U.; Shepherd, D. 1980: Pigment production by Monascus purpureus with regard to quality and quantity. J. Food Science, suspension with ethanol. 45: 567569 A new process is under design based on glucose 4. Lin, T.F.; Demain, A.L. 1991: Effect of nutrition of Monascus sp. on (carbohydrates) as the initial carbon source, and subred pigment. sequent controlled ethanol addition for production of red 5. formation ofSanterre, A.L.;Appl. Microbiol. Biotechnol., 36: 7075 Fabre, C.E.; Loret, M.O.; Baberian, R.; Pareilleux, pigment. A.; Goma G.; Blanc, P.J. 1993.: Production and food applications of
the red pigment of Monascus ruber. J. Food Science, 58: 10991110 6. Braun, S.; Vecht-Lifshitz, S.E. 1991: Mycelial morphology and metabolite production. Tibtech. 9: 6368 7. Johns, M.; Stuart, D.M. 1991: Production of pigment by Monascus purporeus solid culture. J. Industrial Microbiol. 2328 8. Mak, N.K.; Fong, W.F.; Wong-Leung, Y.L. 1990: Improved fermentative production of Monascus pigment in roller bottle culture. Enzyme Microbiol Technol. 12: 965968 9. Hamdi, M.; Blanc, P.J.; Goma, G.: Effect of aeration conditions on the production of red pigments by Monascus purpureus growth on prickly pear juice. Process Biochem 31 (6): 543547 10. Wong, H.; Koehler, P.E. 1983: Production of red water-soluble Monascus pigment. J. Food Science, 48: 12001203