Growth and Pigmentation of Epicoccum nigrum

Transcription

1 APPLIED MICROBIOLOGY, June 1969, p Vol. 17, No. 6 Copyright 1969 American Society for Microbiology Printed in U.S.A. Growth and Pigmentation of Epicoccum nigrum in Submerged Culture L. TUTTOBELLO, F. H. FOPPEN, AND A. CARILLI Laboratori di Chimica Biologica, Istituto Superiore di Sanitai, Rome, Italy Received for publication 10 December 1968 Growth and pigmentation of Epicoccum nigrum was studied in submerged culture in various media. Red pigmentation of the mycelium and of fermentation broth was obtained only in a medium containing glucose and yeast autolysate. This pigmentation occurred at the time of maximal production of carotenoids. Observations on the submerged culture of this organism in flasks and in fermentors are described, and the details of a standardized procedure for the production of carotenoid pigments are given. The fungus Epicoccum nigrum Link has been MgSO4 7H0, 0.01 g of FeSO4-7H0, 40.0 g of studied by several investigators for its taxonomy glucose, 1.0 g of KHPO4, and 40.0 g of cornsteep in and pigmentation. Approximately 70 species of 1,000 ml of distilled water (ph 5.5). Medium 3 consisted of: 5.0 g of peptone, 5.0 g of KHPO4,.5 g of the genus Epicoccum were believed to exist until In that year, Schol-Schwarz (11) demonstrated that these species could be unified into ph 4.5. Medium 4 (modified Detmer) consisted of: MgSO4-7H0, and 0.0 g of sucrose in 1,000 ml of distilled water, adjusted with a-phosphoric acid to the single species E. nigrum, since the development g of Ca(NO3), g of MgSO4-7H0, and structure of the spores were similar in every g of KCI, g of KHPO4, 1 drop of FeCl3 strain. (1% in water), and 0.0 g of glucose in 1,000 ml of The production of pigment by Epicoccum distilled water (ph 6.0). Medium 5 was modified has also been extensively studied. Naumann (9) Czapek-Dox medium (6). Medium 6 was Rhaulinextracted an unidentified red pigment from the Thom medium (). Medium 7 consisted of 00 g of mycelium of a surface culture. A red pigment beans (boiled for 30 min) and 15.0 g of glucose in 1,000 ml of distilled water (ph 6.0). Medium 8 was extract from a surface culture was obtained by glucose-nutrient broth. Medium 9 consisted of 300 g Moreau and Moreau (8) and by Schol-Schwarz of potatoes (boiled for 30 min) and 30.0 g of glucose (11), but their physicochemical observations were in 1,000 ml of distilled water (ph 6.0). Medium 10 different from those reported by Naumann. was medium T, as described by Brian et al. (3). Bamford, Norris, and Ward (1) extracted a yellow Medium 11 consisted of 5.0 g of bakers' yeast autolysate and 10.0 g of glucose in 1,000 ml of distilled pigment from the submerged culture fluid of E. nigrum which was identified as flavipin (3,4,5- water (ph ). trihydroxy-6-methylphthalaldehyde) and had antifungal activity. Recently, the red pigments pronoid pigments, and quantitative determinations of the Extraction of the lipids, separation of the caroteduced by E. nigrum were reexamined and found separated carotenoids and ergosterol and of the dry mycelium weight have been described (7). to consist of four carotenoids (7). The glucose and sucrose contents of the fermentation broth were determined by the method of So- In the present paper, the growth and pigmentation in submerged culture of this fungus on a mogyi (1). laboratory and pilot-plant scale are described. Submerged cultures were grown in Erlenmeyer flasks under constant illumination of 100 to 110 lux MATERIALS AND METHODS at 4 C on a rotary shaker at 0 rev/min (10). Three 90-liter stainless-steel fermentors were Epicoccwn nigrum Link, strain 5-I-3, was received employed for submerged culture in pilot-plant scale; from E. Kuster, Laboratory for Industrial Microbiology, University of Dublin, Ireland. system with an air sparger were used (4, 5). Table 1 both the vortex system of aeration and the baffled Media. Medium 1 consisted of: 3.0 g of NaNOa, gives the mechanical characteristics of the fermentors 1.0 g of KHPO4, 0.5 g of MgS04.7H0, 0.5 g of and operating conditions. KCl, 0.01 g of FeSO4O7H0, 30.0 g of glucose, and 1.0 g of yeast autolysate in 1,000 ml of distilled water RESULTS AND DISCUSSION (ph 6.0). Medium (modified Czapek-Dox) consisted of: 3.3 g of NaNO3, 0.5 g of KC1, 0.5 g of "red pigmentation" are used throughout this Surface culture. The terms "red pigment" and 847 Downloaded from on July 7, 018 by guest

2 848 TUTTOBELLO, FOPPEN, AND CARILLI APPL. MICROBIOL. TABLE 1. Mechanical characteristics of the fermentors and operating conditions Fermentor Operating conditions 1 3 Liquid volume (liters) Liquid depth (height in mm) Fermentor diameter (diameter in mm) Height/diameter ratio Agitation system... Baffled Vortex Vortex Flat, eight-bladed turbine-type impeller No Diameter (mm) Blade (mm)... 3 X 0 3 X 0 3 X 0 Rev/min Oxygen diffusion rate (ml of 0 per 100 ml of liquid per hr) Baffles (0.1 mm diameter) Overpressure (atm) Air flow... By the sparger In the fermentor In the fermentor head head Temperature (C) study to indicate the entire pigment content of the organism. It was separated into orange-red, fat-soluble carotenoids and a wine-red, watersoluble pigment which was partially characterized as a protein with a molecular weight of 4,00 i 00 (A. Liuzzi, personal communication). Conditions for the surface culture of E. nigrum were described by Schol-Schwarz (11). This fungus grows irregularly when transferred from slants, but subsequent transfers result in a rapid and abundant mycelial development. A cottonlike, whitish mycelial layer is formed at first and becomes reddish to bright red after 3 days at 5 C, when the pigmentation starts to appear in the submerged hyphae. The color changes to reddish-brown with traces of violet, and the internal section turns yellow. Some of the pigment diffuses into the substrate and varies in its growth, often with a small area of a different color intensity. As the culture becomes older, brightly colored drops of exudate are sometimes formed. No sporodochial fructifications were observed on the media used. The old hyphae were deep red and thickened, but were sometimes colorless and developed apical swellings. The hyaline hyphae were usually,um in diameter, and the red hyphae were shorter, more branched, and larger (4,um in diameter; Fig. 1). Inoculum preparation for submerged culture. During the preliminary experiments, it was noted that growth and pigmentation of E. nigrum in submerged culture was similar to that of the surface culture, but that the growth in surface culture required approximately 5 days to develop, in contrast to the submerged culture in which it FIG. 1. Five-day aerial mycelium grown on agar medium 11. Differences in thickness and structure between hyaline and dark hyphae together with the formation of globose vesicles are noted. developed in 3 days. The latter method was chosen for our investigations. The culture was grown for 5 to 7 days at 4 C on medium 11 with agar and preserved under mineral oil. From this stock, transfers were made to new slants; they were incubated for 5 to 7 days at 4 C and inoculated into a 500-ml Erlenmeyer flask containing 100 ml of medium. After 3 to 4 days of incubation on a rotary shaker, the mycelium became dark red and formed fluffy, loose pellets, 0.5 to 0.7 cm in diameter, and some irregularly shaped flakes of Downloaded from on July 7, 018 by guest

3 VOL. 17, 1969 E. NIGRUM GROWTH IN SUBMERGED CULTURE 849 different size. When this heterogeneous material was used as inoculum, it gave extremely varied pigmentation. To eliminate this variation, 3- to 4-day-old culture was filtered without suction through a stainless-steel filter (1.5-mm pore diameter), and the filtrate containing homogeneous material was used as inoculum. Since the experiments with this material gave only slight differences in qualitative and quantitative production of carotenoids, this procedure was used throughout this study. Growth in shaken flasks. Mycelial growth in flasks was very poor during the first 40 hr and appeared as fluffy, loose pellets which were whitish or lightly pigmented. At 48 hr, they measured 0.5 to 0.8 cm in diameter and also formed smaller, starry flakes (0.1 to 0. cm in diameter). These agglomerates consisted mainly of long, pliable, and fine hyphae (.0 to.5 jsm in diameter) with short, numerous branches and, to a lesser extent, of thickened hyphae (4.0 to 5.0 um in diameter) without apical swellings. After 3 days, the mycelial broth became a wine-red color. The pellets consisted of hyphae of two different sizes: the peripheral ones, generally pliable, fine, and without pigment, and the central ones, intensely colored, with swollen hyphal portions between the septa and frequent apical or intercalary swellings without internal structure (Fig. ). From the apical swellings, in some cases, new germ tubes consisting of very fine extensions occurred (Fig. 3). As the culture aged, there was an increase in vesiculation and in autolysis of the individual cells. The color of the mycelial broth turned rapidly from wine-red to dark-brown. Growth in fermentors. No pellets were formed in the fermentors during growth, but the mycelial broth became increasingly viscous. During the first 4 hr, small, whitish centers with a prevalence of fine, long hyphae occurred (Fig. 4). After 30 hr, the culture produced red pigment; this pigmentation coincided with maximal acidity of the fermentation broth and with an almost complete utilization of glucose. At this time, the mycelial flakes were wine-red in color, with pliable and hyaline peripheral hyphae without the small branches characteristic of the growth in the shaken flasks. The hyphae were fine (1 to,um in diameter) and usually without swelling. FIG. 3. Fresh, red, nodose hyphae with germ tubes from vescicolized septa of 3-day mycelium from submergedflask culture. Downloaded from on July 7, 018 by guest FIG.. Three-day pellets from submergedflask culture. Central, thick, nodose hyphae becoming colored and peripheric, subtile hyphae, longer and hyaline, with characteristic vesicles (arrow). FIG. 4. Blue-cotton colored, 4-hr mycelium grown in fermentor. Fine andflexible pellets with hyphae.

4 850 TUTTOBELLO, FOPPEN, AND CARILLI APPL. MICROBIOL. TABLE 3. Fermentation in shaken flasks with Epicoccum nigrum Link in medium 11 Total amt of carotenoids Age of H Dry Glucose culture ph wt content Total amt of ergosterol Dry Broth Dry Broth Wt Dry hr mg/10 % lg/100 mg/1g0gioo ml ml mgg ml FIG. 5. More subtle and lessflexible, partially autolyzed hyphae, blue-cotton colored mycelium from 6-hr fermentor. After 44 hr, many crushed flakes were observed; at this stage, gradual lysis set in. Depending on the intensity of agitation, the hyphae were shorter, fragmented, more irregular, and without swelling. In the few swollen hyphae, some vesicular swelling could be observed. At this stage, the mycelial broth had its maximal wine-red color, which began to fade after 6 hr. The peripheral mycelium was smaller in diameter, less pliable, and highly fragmented, and autolysis was advanced (Fig. 5). At 86 hr, the mycelium became reddish-brown and was completely fragmented; the mycelial broth was viscous and difficult to filter. Chemical observations. Since only media 1, 4, 8, 9, and 11 gave red pigmentation (Table ), they were investigated further. Incubation was terminated when the carbohydrates were completely exhausted. After extraction of the fatsoluble carotenoids and subsequent separation by column chromatography, no qualitative differences were observed between the carotenoid pigments obtained from the mycelia grown in these five media. There was, however, a great difference in the gross pigmentation of the mycelial broths and of the acetone extracts of the mycelia after extraction with hexane. Only media 1 and 11 produced red filtrates. Amounts of the carotenoids, ergosterol, and mycelium in the five media that produced red mycelia were determined, and the results were correlated with the utilization of glucose. The data show that the carotenoid content was highest at the time of complete carbohydrate uptake (Table 3 and 4). Medium 11 was found to be most desirable because of the high production of carotenoids, good mycelial growth, and the ease of preparation (Table ). It is interesting to note that the quantitative ratio of p-carotene plus rhodoxanthin to -y-carotene plus torularhodin (4:1) was essentially TABLE. Effect of medium upon pigmentation, carotenoid synthesis, and ergosterol synthesis in shaker flasks by Epicoccum nigrum Link Medium Color of g. Mycelial dry Total amt of carotenoids Total amt of ergosterol no. fermentation broth Cooofmciu wtm/lo- D.bromlof broth) Broth Dry wt Broth Dry wt Downloaded from on July 7, 018 by guest Ag/100 ml pg/g mg/100 ml mg/g 1 Orange Red Brown Dark brown 1, Yellow Brown Pink Light red Light yellow Pink Colorless Pink Colorless Pink Orange Dark red Yellow-orange Light red Colorless Pink Wine-red Dark red

5 VOL. 17, 1969 E. NIGRUM GROWTH IN SUBMERGED CULTURE 851 constant in all media, with small fluctuations in the ratio between (-carotene and rhodoxanthin; the exception was medium 10, in which only f3-carotene was produced. Mycelial growth and pigmentation were studied in shaken flasks in medium 11 with glucose concentrations of 0.1, 0., 0.5, 1.0, 1.5,.0, and 3.0%. Qualitative observations showed that, with 0.1, 0., and 0.5% of glucose, growth was delayed; with.0 and 3.0%, the mycelial growth was abundant but the amount of carotenoids per gram of dry mycelium or per 100 ml of broth was very low. There was a slight but constant difference between the amount of carotenoids produced in this medium with 1.0 and 1.5% glucose: 35,Ag/g (dry weight) and 3,ug/g (dry weight), respectively. Inoculations with 0.1 to 10% (v/v) were compared, and an 0.5 % volume was preferred. An inoculum size of 1% or more yielded growth with intense red pigmentation. This pigmentation, however, was not caused by the carotenoids but by red, water-soluble pigments. The optimal inoculum for pigmentation is, therefore, the same percentage as that required for mycelial growth. Fermentations in two types of Erlenmeyer flasks, with and without baffles, were studied TABLE 4. Fermentation with Epicoccum nigrum Link in fermentors under different conditions of aeration and agitation Age of culture hr w 0 a a,^4 a 1 3 PHaI Dry wt mg/100 ml Glucose contenta Total carotenoids Dry wt jhg/g Broth jsg/100 ml Dry wt mg/g Broth mg/100 ml a At zero hour the ph was and the glucose content was 1.0%. with culture medium 11. In the baffled flasks, in which turbulence and aeration rates are considerably increased, approximately the same amount of mycelium per unit of broth was produced as in the unbaffled flasks, but the production of carotenoids was lower (30 Ag/g versus 35 Ag/g, dry weight). The same effect was observed] in stirred fermentors: higher aeration caused a reduction in the carotenoid production. These data indicate that an 0.5% inoculum of 100 ml of culture medium 11 in an unbaffled 500-ml Erlenmeyer flask and incubation at 4 C were optimal conditions for growth, red pigmentation, and carotenoid synthesis in E. nigrum. For the preparation of large amounts of the red pigments, growth and pigmentation were studied in 90-liter fermentors under three different conditions of agitation and aeration. With an illumination of 10 lux at 4 C with a small inoculum (1:100, v/v) of 50 liters of medium 11, growth and pigmentation (Table 4) paralleled the glucose utilization and the ph changes of the medium, which was similar to the findings previously described for shaken flasks. When glucose was exhausted, mycelial growth and carotenoid production ceased, and a rapid decrease in carotenoid content ensued (Fig. 6). This situation was not observed in shaken flasks in which the carotenoid contents decreased slowly after total utilization of glucose from the fermentation broth. Although greater aeration in shaken flasks reduced the production of carotenoids, the quantitative production of mycelium and carotenoids in the fermentors was higher under optimal conditions (vortex at 400 rev/ min) than in the shaken flasks. The relative ratios among the four carotenoids was not affected by the different mechanical conditions of the fermentors. Production of the red, water- TIME in hours FIG. 6. Fermentation characteristics of Epicoccum nigrum in fermentor. Downloaded from on July 7, 018 by guest

6 85 TUITOBELLO, FOPPEN, AND CARILLI APPL. MICROBIOL. soluble pigment was the same in fermentors as in shaken flasks. ACKNOWLEDGMENT We are grateful to V. Adriani and G. Bocci for their competent technical assistance. One of the authors (F. H. F.) is the recipient of a fellowship from the Istituto Superiore di Saniti. LITERATURE CITED 1. Bamford, P. C., G. L. F. Norris, and G. Ward Flavipin production by Epicoccum spp. Trans. Brit. Mycol. Soc. 44: Brian, P. W., P. J. Curtis, and H. G. Hemming A substance causing abnormal development of fungal hyphae produced by Penicillium janczewskii Zal. Trans. Brit. Mycol. Soc. 9: Brian, P. W., P. J. Curtis, and H. G. Hemming Glutinosin: a fungistatic metabolic product of the mould Metarrhizium glutinosum S. Pope. Proc. Roy. Soc. Ser. B, Biol. Sc. 135: Chain, E. B., S. Paladino, D. S. Callow, F. Ugolini, and J. Van Der Sluis Studies on aeration I. Bull. World Health Organ. 6: Chain, E. B., and G. Gualandi Aeration studies IL Rend. Ist. Super. Sanita (English ed.) 17: Clutterbuck, P. W., R. Lovell, and H. Raistrick Studies in the biochemistry of micro-organism. XXVL The formation from glucose by members of the Penicillium chrysogenum series of a pigment, an alkali-soluble protein and penicillin-the antibacterial substance of Fleming. Biochem. J. 6: Foppen, F. H., and 0. Gribanovski-Sassu lipids produced by Epicoccum nigrum in submerged culture. Biochem. J. 106: Moreau, C., and M. Moreau Sur quelches Hyphomycetes. Bull. Soc. Linn. Normandie 6: Naumann, C. W Epicoccum purpurascens und die Bedingungen flir seine Pigmentbildung. Hedwigia 51: Paladino, S A simple rotary shaker. Rend. Ist. Super. Sanith (English ed.) 17: Schol-Schwarz, M. B The genus Epicoccum Link. Trans. Brit. Mycol. Soc. 4: Somogyi, M Notes on sugar determination. J. Biol. Chem. 195:19-3. Downloaded from on July 7, 018 by guest