Pigment Production from Immobilized Monascus sp. Utilizing ...

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Dec 5, 1983 - berlite XAD-7 resin was obtained from Rohm and Haas,. Philadelphia, Pa. Microorganism and culture medium. Monascus sp. NRRL. 1993 was ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1984, p. 1323-1326

Vol. 47, No. 6

0099-2240/84/061323-04$02.00/0

Pigment Production from Immobilized Monascus Polymeric Resin Adsorption

sp.

Utilizing

PATRICK J. EVANSt AND HENRY Y. WANG* Department of Chemical Engineering, The University of Michigan, Ann Arbor, Michigan 48109 Received

5

December 1983/Accepted 19 March 1984

Pigment production by the fungus Monascus sp. was studied to determine why Monascus sp. provides pigment in solid culture than in submerged culture. Adding a sterilized nonionic polymeric adsorbent resin directly to the growing submerged culture did not enhance the pigment production, thus indicating that pigment extraction is probably not a factor. Monascus cells immobilized in hydrogel were studied and exhibited decreased pigment production as a result of immobilization. This result is thought to be due to diffusional resistance of the pigment through the hydrogel beads. Addition of the adsorbent resin to the immobilized Monascus culture increased both the maximum pigment yield and the production rate above those of the free-cell fermentations. The provision of a support for the mycelium may explain enhanced pigment production by the solid-state culture. These results indicate that product diffusion from immobilized cell systems can be the limiting factor and that in situ extraction is one possible way to circumvent this problem. more

Of the 29 food colorants currently approved for use in the United States, 16 are chemically synthesized, and 13 arise from natural sources (4). Most precursors of the chemically based colorants are generally derived from petrochemicals, a fact which evokes considerable consumer concern over the safety of long-term ingestion of these substances. It is likely that increasingly stringent restriction in the near future will eliminate some of the currently approved synthetic colorants. Consequently, there is a need to find suitable alternative sources of food color. One alternative method of food colorant production is microbial fermentation. The fungus Monascus sp. has been utilized in the Orient for making red rice wine, red Shao-Hsing wine, red soybean cheese, and Ang-Khak (red chinese rice) (2, 5). Monascus sp. is traditionally cultivated by inoculating large quantities of moistened rice on flat trays. This method of solid-state culture is quite inefficient and space intensive. A more efficient approach is to utilize submerged fermentation. Unfortunately, Lin has demonstrated that pigment production in submerged culture is only 1/10 of that in solid-state culture (5). Mutants obtained from Monascus sp. F-2 produced an optical density at 500 nm (OD500) of 92.33 U in submerged culture, which is still less than in solid-state culture (6).. The pigment is actually a mnixture of red, yellow, and purple pigmented polyketides. They are produced via a pathway very similar to fatty acid biosynthesis (8) and are insoluble in acid solution (3). Since fungal fermentations are typically acidic, the pigment is expected to be retained inside the cell in the submerged fermentation (8). To mimic the solid-state fermentation, we initiated experiments to study free and immobilized Monascus cultures with in situ pigment extraction utilizing a nonionic polymeric adsorbent resin. MATERIALS AND METHODS Medium. Sodium alginate, type IV, was obtained from Sigma Chemical Co., St. Louis, Mo. Cerelose was obtained

from Corn Products, Englewood Cliffs, N.J. Neopeptone was obtained from Difco Laboratories, Detroit, Mich. Amberlite XAD-7 resin was obtained from Rohm and Haas, Philadelphia, Pa. Microorganism and culture medium. Monascus sp. NRRL 1993 was obtained from the Northern Regional Research Laboratory, Peoria, Ill. The stock medium consisted of 2% Cerelose and 1% neopeptone by weight. Fermentation medium consisted of 5% Cerelose and 1% neopeptone by weight. Both media were initially adjusted to pH 6.0 before sterilization. Cultivation conditions. The stock suspension culture of Monascus sp. was maintained by weekly transfers into 200 ml of the fresh stock medium. These were incubated at 28°C in 1-liter Erlenmeyer flasks on rotary shakers (300 rpm). The free-cell fermentation experiments were carried out in 500-ml Erlenmeyer flasks, each containing 100 ml of the fermentation medium. Each was inoculated with 1 ml of 48-h stock culture and was incubated at 28°C on shakers. Aeration was provided by operating the shakers at high speed (300 rpm). The immobilized-cell fermentations were carried out in a manner identical to the free-cell fermentations, except for the method of inoculation. Under aseptic conditions, 1% (wt/ wt) alginic acid was inoculated with 5% (vol/vol) 48-h stock culture. Portions of the gel solution (20 ml) were pumped into stirred 80-ml volumes of 4% (wt/wt) CaCl2 solution in water by using a peristaltic pump (Harvard Apparatus, Millis, Mass.). After 1 h of gelation time, the calcium chloride solution was decanted, and the beads were transferred by aseptic technique into the shake flasks containing fermentation medium. The average bead diameter was approximately 3 to 4 mm. The shake flasks were harvested daily for the time studies, and the experiments were terminated upon glucose depletion to prevent utilization of the pigments as an energy source (8). The resin was soaked in methanol overnight and rinsed with water, and after excess water was removed, was fractionated into predetermined wet weights, sterilized at 121°C for 20 min, and transferred into the cultures at 48 h. Assay methods. The pigment was extracted from the entire free-cell fermentation broth with methanol. For the immobi-

* Corresponding author. t Present address: Department of Chemical and Biochemical Engineering, Busch Campus, Rutgers University, New Brunswick, NJ 08903.

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APPL. ENVIRON. MICROBIOL.

EVANS AND WANG

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FIG. 1. Comparison of yellow (OD420) and red (OD500) absorbances of pigments produced in Monascus sp. culture with no resin and with 6 g of XAD-7 resin per 100 ml.

lized-cell experiments, the alginate gel was first dissolved in a solution of 4% (wt/wt) Na2HPO4 solution and then was extracted with methanol. The suspended cells were removed from the broth pigment samples by centrifugation. All of the

extractions with methanol, including those with the resin, were considered complete when the extracts became relatively clear. The samples were diluted to 50% (vol/vol) methanol, and absorbance was measured at 500 nm with an UV/VIS spectrophotometer (model DMS 90; Varian Associates, Palo Alto, Calif.). The total pigment production was based on the broth plus cell-gel bead volume; broth pigment concentrations were based on the broth voltime alone. The addition of the resin did not change relative concentrations of the produced pigments. The ratio of the red to yellow absorbances (500 and 420 nm, respectively) was 1.27 for pigment samples taken from the cultures both with and without resin (Fig. 1). The free-cell fermentations were centrifuged for 20 min at 2,700 rpm, and the packed cell volumes were measured. The volumes were correlated to the dry cell weight as a function of time so that the total dry cell weight could be recorded for these experiments. The ratio of the dry cell weight to the packed cell volume was found to increase linearly with time. The total dry cell weights of the immobilized-cell fermentations were measured after dissolving one bead in 4% (wt/wt) Na2HPO4 solution, washing the mycelium with distilled water, and drying. These weights were then multiplied by the appropriate number of beads. Glucose was measured with the Somogyi method (7). pH was measured with a Corning pH meter.

FIG. 2. Pigment production profile for free Monascus sp. culture.

RESULTS AND DISCUSSION Initial experiments were performed to compare the experimental data with the literature data for Monascus fermentation. The strain used for this study (NRRL 1993) produced a greater pigment concentration at a greater rate than strain F2 did (Table 1). However, the mutant of strain F-2 had pigment production capabilities superior to those of strain NRRL 1993 in terms of concentration and rate. The submerged cultivation of Monascus sp. NRRL 1993 was inferior to the solid-state culture in pigment production. The pigment production profile for the Monascus sp. NRRL 1993 fermentation is shown in Fig. 2. Two important observations can readily be made from these data. First, since the broth pigment concentration was much lower than

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TABLE 1. Comparison of data from free-cell fermentation and literature data Maximum total total Strain

Reference

pigment concn

(OD500) F-2 Mutant of F-2 NRRL 1993 Anka (solid-

state)

5 6 This paper 5

~Mean rate

production

(OD,,(x)

U/day)

24.0

6.0

92.0 62.0 240.0

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TIME (HRS)

FIG. 3. Pigment production profile for free Monascus sp. culture with 6 g of XAD-7 resin per 100 ml.

PIGMENT PRODUCTION FROM MONASCUS SP.

VOL. 47, 1984

the total pigment concentration, most of the pigment was accumulated within the mycelium. The distribution of a substance between the mycelium and the medium has been shown to be related to its solubility (8). Thus, intracellular localization of the pigment is most likely due to its poor solubility in the acidic fermentation broth (3). This fact poses the problem of extracting the pigment from the mycelium in a commercial process. It is desirable to reduce the number of required separation steps and thus reduce the processing cost and time. Second, after approximately 100 h, the pigment yield and production rate started to decrease. If the yield and rate decreases are due to product inhibition or repression, continuous extraction of the pigment should eliminate these problems. The extractive effect of the moistened rice in the solidstate culture can be mimicked by adding an adsorbent resin to the submerged culture. XAD-7 polymeric nonionic resin was found to be the most effective adsorbent for the pigments. Based on the loading characteristics of the resin, 6 g of resin per 100 ml of medium was used. The resin, however, was not added to the cultures until the growth phase was nearly complete at 48 h. Figure 3 shows the results from the above experiment. The most obvious and significant result was that the pigment was now predominantly extracellular, as shown by the small difference between the resin-plus-broth pigment concentration and the total concentration. This result is further illustrated in Fig. 4. The addition of adsorbent resin drastically redistributed the pigment toward the extracellular environment. Furthermore, the pigment was primarily adsorbed onto the resin and was not in the broth, as illustrated by the negligible pigment concentration in the broth. Apart from this redistribution, the data for the experiments with and without resin were reasonably similar. This similarity is further shown in Table 2; the maximum pigment yield and production rate for both cases were not significantly different. Since extraction of the pigment did not enhance the pigment production, it is not probable that the mechanism by which solid-state fermentation enhances pigment production is through pigment extraction. Rather, immobilization of the mycelium per se may be the mechanism. To test this hypothesis, whole cell immobilization was considered. In addition to providing a support for the mycelium, whole cell immobilization has numerous other advantages such as

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TABLE 2. Pigment yield and rate of production Maximum pigment

Expt

yield (based onU/g glucose)

(OD5!0

per ml)

Maximum pigment production rate U/h)

(OD5to

Free cells With resin Without resin

1.7 1.8

0.50 0.61

Immobilized cells With resin Without resin

3.0 0.19

0.78 0.05

prevention of lysis owing to shear, greater cellular density, greater applicability to continuous culture, and ease of cellresin separation. On the other hand, substrate and product mass transfer limitations need to be recognized as possible shortcomings of cell immobilization (1). The studies of immobilized Monascus sp. were done under conditions identical to those of the free-cell fermentation, except that the inoculum was immobilized in calcium alginate gel. The volume of the gel was 10% of the broth volume. Although the biomass production was not inhibited, the pigment production was (Fig. 5). Furthermore, the maximum pigment yield and production rate of the immobilized Monascus sp. were drastically reduced (Table 2). Possible explanations include oxygen or nutrient mass transfer limitations and product inhibition or repression arising from product mass transfer limitations. To ascertain specifically which of the above conditions were limiting, XAD-7 resin was added to the immobilizedcell fermentation in a manner identical to that for the freecell fermentation. The final pigment concentration, maximum yield, and maximum production rate were equal to or somewhat greater than those of the free-cell fermentations (Fig. 6 and Table 2). These results suggest that without resin addition, pigment mass transfer limitations caused product inhibition or repression and that this was the primary cause of the limited pigment productior. Addition of the resin enhanced removal of the pigment but would not be expected to influence the nutrient or oxygen transport. Mass transfer limitations are plausible because the pig¶ent is relatively insoluble in acid solution, which also explains the predominantly intracellular localization of the pigment. 0

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FIG. 5. Pigment production profile for immobilized Monascus sp. culture.

APPL. ENVIRON. MICROBIOL.

EVANS AND WANG

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FIG. 6. Pigment production profile for immobilized Monascus sp. culture with 6 g of XAD-7 resin per 100 ml.

Thus, addition of resin to the immobilized-cell system drastically increased the rate of pigment diffusion through the gel and allowed this system to behave much like the freecell system. The addition of the resin did not appear to alter the gel beads physically. The mycelium grows predominantly at the outer radius of the beads owing to the nutrient transport limitation. Since the gel immobilization shields the mycelium from the resin, physical contact between the resin and the mycelium is unlikely to be the mechanism for the increased pigment mass transfer. Furthermore, the ability of the resin to extract the pigment from the immobilized mycelium suggests that direct contact between the mycelium and resin is not necessary for the extraction. The improvement of the maximum pigment yield and production rate of the immobilized cells over those of the free-cell fermentation provides credible evidence that enhanced pigment production by solid-state culture is due to provision of a support for the mycelium. However, a final pigment concentration of 56 U (OD500) is nowhere near the 240 U for the solid-state culture (Table 1). Continuous processing may be appropriate for increased pigment production. Since product inhibition or repression affects only the immobilized-cell system, increasing amounts of the resin in the fermentation broth should increase the pigment production. This was found to be true (Fig. 7). Thus, for the immobilized-cell system, continuous processing may be appropriate because it would allow the broth pigment concentration to be maintained near zero, for example, in a dual resin column extraction scheme. With this system, one column could be extracting the fermentation broth while the other eluted the pigment. In summary, the red pigment production by Monascus sp. NRRL 1993 was found to be similar to that reported previously for the free-cell fermentation. Adsorbent resin was added to the fermentation medium to mimic the extractive effect of the moistened rice in the solid-state culture. No

increase in the pigment yield or production rate was observed. On the other hand, a drastic redistribution of the pigment toward the extracellular environment was observed. Next, the mycelium was immobilized in calcium alginate gel to mimic the action of the moistened rice as a physical support. The pigment production was drastically reduced, probably owing to limited pigment mass transfer and subsequent product inhibition or repression. Addition of the adsorbent resin to the immobilized-cell system circumvented the mass transfer limitation and resulted in a maximum pigment yield and production rate superior to those of the free-cell cultures. The possibility of increased pigment yield and production rate is good with a continuous immobilized Monascus sp.-adsorbent resin system. Studies with this type of operation are presently under way. ACKNOWLEDGMENTS We thank Frank Robinson and Jim Pochodylo for their technical assistance, Greg Payne and David Hettwer for their editorial assistance, and Cynthia Miller for typing the manuscript.

LITERATURE CITED 1. Abbott, B. J. 1977. Immobilized cells. Annu. Rep. Ferment. Process 1:205-233. 2. Gray, W. 1970. The use of fungi as food and in food processing, p. 56. CRC Press, Inc., Cleveland. 3. Haws, E. J., J. S. E. Holker, A. Kelly, A. D. G. Powell, and A. Robertson. 1959. The chemistry of fungi. XXXVII. The structure of rubropunctation. J. Chem. Soc. 1959:3598. 4. Institute of Food Technologists. 1980. Food colorants. Food Technol. 5:77. 5. Lin, C. 1973. Isolation and cultural conditions of Monascus spp. for the production of pigment in a submerged culture. J. Ferment. Technol. 51:407-414. 6. Lin, C., and S. J. Suen. 1973. Isolation of hyperpigment-productive mutants of Monascus spp. F-2. J. Ferment. Technol. 51:757759. 7. Somogyi, M. 1945. A new reagent for the determination of sugars. J. Biol. Chem. 155:61-68. 8. Turner, W. B. 1971. Fungal metabolites, p. 14-80. Academic Press, Inc. (London), Ltd., London.