APPLIED MICROBIOLOGY, May 1967, p. 597-602Copyright © 1967 American Society for Microbiology

Vol. 15, No. 3Printed in U.S.A.

Ergotamine Production in Submerged Culture andPhysiology of Claviceps purpurea

ALBA M. AMICI, A. MINGHETrI, T. SCOTTI, C. SPALLA, AND L. TOGNOLI

Istituto Ricerche, Farmitalia, Milano, Italy

Received for publication 21 December 1966

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptidealkaloids in submerged culture, and strains V, C, and W, spontaneously obtainedfrom 275 FI and practicaUy unable to produce alkaloids, were compared. Strain275 Fl differs from the other strains in its capacity to accumulate lipids and sterols,as well as in its capacity to produce alkaloids. Strain 275 FI utilizes large quantitiesof sucrose and citric acid simultaneously; strain V utilizes large amounts of sucrose

but little citric acid; strain C utilizes large quantities of citric acid but only smaUamounts of sucrose; strain W consumes only small amounts of both substances. Weconclude that the production of large quantities of alkaloids, as wel as the accumula-tion of lipids and sterols, is correlated with the simultaneous utilization of largeamounts of sucrose and citric acid.

The production of ergotamine in submergedculture by strains of Claviceps purpurea has beenreported (1, 19). The physiology of ergot alka-loid-producing strains of Claviceps was studiedby Arcamone et al. (2), Taber (16, 17), Taber andVining (18), Pacifici et al. (13), Kobel et al. (11),and Castagnoli and Tonolo (7).

This paper reports the results of a comparisonbetween an ergotamine-producing strain andthree strains spontaneously obtained from itwhich are practically unable to produce alkaloids.

MATERIALS AND METHODS

Microorganisms. Strain 275 Fl has been described(1); it produces 1,100 to 1,500 jAg (per ml) of a mixtureof peptide alkaloids, chiefly ergotamine (80%), insubmerged culture. Strains V, C, and W were ob-tained by isolation from sectors of giant colonies ofstrain 275 FL on medium T2. Strain V producesamounts of alkaloids varying from 0 to 100 jug/ml;strains C and W do not show any alkaloid production.

Culture media. Medium T2 contained (grams perliter): sucrose, 100; L-asparagine, 10; Ca(N03)2.4H20,1; KH2PO4, 0.25; MgSO4-7H20, 0.25; yeast extract,0.1; KCI, 0.12; FeSO4-7H20, 0.02; ZnSO4-7H20,0.015; agar, 20; tap water to 1,000 ml; pH adjusted to5.2 with NaOH; sterilized by heating at 110 C for 20min.Medium TG contained glucose, 100; citric acid,

10; KH2PO4, 0.5; MgSO4 7H20, 0.3; yeast extract,0.1; FeSO4-7H20, 0.007; ZnSO4-7H20, 0.006; tapwater to 1,000 ml; pH adjusted to 5.2 with aqueousammonia; sterilized by heating at 110 C for 20 min.Medium T25 contained sucrose, 300; citric acid,

15; KH2PO4, 0.5; MgSO4-7H20, 0.25; yeast extract,0.1; KCl, 0.12; FeSO4-7H20, 0.007; ZnSO4-7H20,

0.006; tap water to 1,000 ml; pH adjusted to 5.2 withaqueous ammonia; sterilized by heating at 110 C for20 min.

Methods of culture. A 1-cm2 portion of the mycelialmat of the above strains, grown on slants of mediumT2 and cultivated at 28 C for 8 days, was mashed witha sterile spatula and placed into a 300-ml Erlenmeyerflask containing 60 ml of medium TG; flasks soprepared were incubated for 6 days at 24 C on a rotaryshaker, operating at 220 rev/min and describing acircle 8 cm in diameter. Samples (5-ml) of the culturethus obtained were employed as inoculum for 300-miflasks containing 50 ml of medium T25; these wereincubated for 14 days as described for the cultures onmedium TG.

Methods of analysis. Each analysis was performedon pooled culture broths from three flasks.The determinations of alkaloids, Qo0, protein

nitrogen, pH, and dry weight were made immediatelyafter the sampling, and each analysis was repeated.For the other analyses (which had to be delayed forvarious reasons), the mycelia were separated from theifitrates and kept at -20 C. For determination oftotal lipids, the mycelium alone was thoroughlyground with 5 parts of Na2SO4 (anhydrous) andextracted in a Soxhlet extractor for 24 hr with petro-leum ether and for 12 hr with a mixture of ether-ethyl alcohol (3:1); the extracts were pooled, evapo-rated, and weighed. Sterols were titrated in the lipids bythe Liebermann-Buchard reaction (9). Total nitrogenwas determined by the Kjeldahl method, with aK2SO4HgO catalyst (14). Ammonium nitrogen wasdetermined according to Russel (15). Citric acid wasdetermined according to Chambon (8). Sugars weredetermined by the anthrone method (20). The oxygenuptake of the mycelium was determined in Warburgvessels, by use of 3 ml of suspension, suitably diluted

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3 ^-- <ox sFIG. 1-4. Claviceps purpurea mycelia (8 days old), strai/is 275 FI (1), V (2), C (3), anid W (4), in submerged

culture. X 500.

with its own filtered culture broth. To determine dryweight, a portion of the mycelium suspension wasfiltered, washed thoroughly with 3 volumes of water,and dried at 85 C for 24 hr. Inorganic phosphorus inthe filtrate was determined by the method of Beren-blum and Chain (3).

Protein nitrogen was determined by using 5 ml ofculture broth mixed with 0.48 ml of 50% trichloro-

acetic acid; this mixture was held 1 hr at 0 C. Aftercentrifugation for 20 min at 5,000 X g the precip-itate was twice washed with 5 ml of 5% trichloro-acetic acid in the cold and once more at 100 C (within3 min). Then it was washed again twice with a mixtureof ethyl alcohol-ether (1:1) and once again withether. Nitrogen was determined in the residue by theKjeldahl method (14).

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VOL. 15, 1967 ERGOTAMINE PRODUCTION IN SUBMERGED CULTURE

0 3 5 j 10 1-2 14 Days

FIG. 5. Course of fermentation on medium T25 insubmerged culture of strain 275 FI.

FIG. 6. Course of fermentation on medium T25 insubmerged culture of strainz V.

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Alkaloids. Since about 90% of the alkaloid pro-duced is found in the mycelium, the extraction wascarried out by adding 10 ml of an aqueous solution of4% tartaric acid to 5 ml of culture broth. The mixturewas diluted to 40 ml with acetone and homogenizedfor 1 min. To 4 ml of the ifitrate, 1 ml of a 1: 2 mixtureof borate buffer (0.4 M) and NaOH (1 N) was added,to obtain a pH value of about 10. This preparationwas then extracted three times with 8 ml of CHC13(3 ml + 3 ml + 2 ml), and the pooled extracts werediluted to 10 ml with CHCl3. Of this solution, 1 mlwas dried under vacuum and the residue was dis-solved in 1 ml of 1%o aqueous phosphoric acid. Thecolorimetric determination was made by the methodof Voigt (21), based on the appearance of a blue colorwith the van Urk reagent. The reading was madeagainst a standard of ergotamine base. The exhaustedaqueous extracts still reacted positively with the vanUrk reagent after extraction with CHCl3 because ofsmall amounts of lysergic acid (which, however, werenot taken into consideration in determining the titerof alkaloids).

RESULTS

Morphological and cultural characteristics ofstrains 275 Fl, V, C, and W. Strain 275 FI (Fig. 1)grows on medium T2, forming bulky colonies,compact and rather sharp at the edge, pinkish-white, with darker hues in older cultures. Sub-merged cultures in medium T25 are first whitishand then, after 6 to 7 days, pink-brownish. Themycelium, thin and filamentous at the beginning,

0 3 5 7 10 12 14 Days

FIG. 7. Course of fermentation on medium T25 insubmerged culture of strain C.

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15

12.5

10

7.5

Days

TABLE 1. Principal metabolic characteristics ofstrains 275 FI, V, C, and W as observed

in submerged culture in mediumT25 after 14 days

StrainCharacteristic -

275 FI V C W

Alkaloids (ug/ml) 1,150 80 -

Dry weight (mg/ml)............... .38.5 28 13.7 22.5

Lipids (mg/ml) 1 15 4.5 1.5 3.8Dry weight without

lipids (mg/ml) 3 23.5 23.5 12.2 18.7Protein N (mg/100ml).9 92 95 98 97

Sterols (,ug/ml) 316 136 51 145Sucrose utilized (g/

liter)... .. 160 120 75 50Citric acid utilized

(g/liter).14 7.5 15 8Ammonium N (mg/

100 m).246 250 228 212Total N of filtrate

(mg/100 ml) 87 80 82 95

FIG. 8. Course offermentation on medium T25 insubmerged culture of strain W.

later grows thicker and partially fragments. Noconidia were observed at any time.

Strain V (Fig. 2) shows on medium T2 the samecharacteristics as 275 Fl, but its color is deeppurplish-violet. In submerged culture in mediumT25, the strain is dark brownish-violet at 6 to 7days. Microscopically, the young mycelium showsnumerous branching buds. Upon ageing, it breaks(more often than does 275 Fl) into irregular cells,some of which are dark purplish-violet. No coni-dia are present.

Strain C (Fig. 3) produces on medium T2 flatcolonies, cream-white in color and with creamyconsistency. Submerged cultures in medium T25are also cream-white, and the mycelium appearsto be very much fragmented from the beginning.No conidia are present.

Strain W (Fig. 4) produces on medium T2bulky compact white colonies with sharp edges.When grown in submerged culture in mediumT25, the mycelium appears to be formed by longthin hyphae that remain practically unchangedtill the end of the fermentation. No conidia areseen.

Course of fermentation. The course of typicalfermentations on medium T25, carried out withthese four strains, is reported in Fig. 5-8. Somedata on the metabolic characteristics of thesestrains are summarized in Table 1.

DIscussioN

From these data, it appears that strain 275 FI,besides its ability to produce alkaloids, differsfrom the others in accumulating lipids and sterolsthroughout the fermentation. The higher dryweight of 275 Fl is partially ascribable to lipidaccumulation. The amounts of ammonium nitro-gen utilized, of nitrogen incorporated into pro-teins, and of total nitrogen released into thebroth were the same for the four strains.

In all of the broths, there was a rapid disappear-ance of phosphates, due to a marked capacity ofthese strains to absorb and store them as poly-phosphates. Analogous behavior in strains ofClaviceps has been also reported by Arcamoneet al. (2) and by Taber and Vining (18).

In terms of sucrose and citric acid utilization,each of the four strains is different. Strain 275 Flconsumes large amounts of sucrose and citricacid; strain V utilizes large quantities of sucrosebut small amounts of citric acid; strain C useslarge amounts of citric acid but small amounts ofsucrose; and strain W utilizes only limited quan-tities of both substances.

Unlike the strain of C. purpurea producingclavines (16, 17), our strains did not show evidentcompetition between alkaloid production andlipid formation. We observed, on the contrary,that alkaloid synthesis occurs only in the strainable to store large amounts of lipids. With thisstrain, the two processes run together during

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VOL. 15,1967 ERGOTAMINE PRODUCTION IN SUBMERGED CULTURE

fermentation; together with alkaloids and lipids,sterols are accumulated as well. An unusuallylarge lipid content in the mycelium of a strain ofC. paspali, which produces lysergic acid deriva-tives, was reported by Arcamone et al. (2).

It is known that significant consumption of thecarbon source of the medium is favorable toaccumulation of metabolic products other thancell material and CO2 (10). In our case, theaccumulation of lipids, sterols, and alkaloids is,however, conditioned not only by a high con-sumption of the carbon sources but also by thesimultaneous high utilization of two differentcarbon sources: sucrose and citric acid.The correlation among alkaloid, sterol, and

lipid production allows us to think that the "primeprecursors" (5) of the synthesis of lipids andsterols are equal or biogenetically close to thoselimiting the synthesis of alkaloids, as for instanceacetyl coenzyme A and phosphoenolpyruvic acid(6). It is thus supposed that the considerable andsimultaneous utilization of sucrose and citric acidis favorable to the accumulation of these sub-stances.

If we assume that the utilization of the sugarstakes place chiefly through the glycolysis-Krebscycle route (12), the utilization of citric acid atthe same time as that of sugar could determine asaturation of the enzymes of the Kreb's cycle,with the consequence that accumulations occurat the entry of the cycle itself (in particular, ofacetyl-coenzyme A and phosphoenolpyruvic acid)if there is no adequate functioning of the controlmechanisms.

Strain 275 Fl, which readily utilizes sucrose andcitric acid, seems indeed to be lacking those con-trol mechanisms related to the utilization of thesetwo substances. In strain W, the regulating mecha-nisms could work normally, with the consequentutilization of an amount of sucrose and of citricacid proportional to that required for the growthof the microorganism. Accumulations of meta-bolic products are thus avoided. This strain canbe considered as a "normal" form; its morpho-logical characteristics (filamentous, regular andslightly fragmented mycelium) also strengthenthis consideration. Strains V and C have charac-teristics intermediate between those of 275 Fland W, and behave complementarily in utiliza-tion of sucrose and citric acid.

ACKNOWLEDGMENTS

We gratefully acknowledge the support andinterest shown by B. Camerino throughout theseinvestigations. We also thank A. Giangulano fortechnical assistance.

LiTERATiuRE CITED

1. AMICI, A. M., A. MINGHETrI, T. ScoTTI, C.SPALLA, AND L. ToGNOLI. 1966. Production ofergotamine by a strain of Claviceps purpurea(Fr.) Tul. Experientia 22:415-416.

2. ARCAMONE, F., E. B. CHAIN, A. FERRETTI, A.MINGHETTI, P. PENNELLA, A. ToNoLo, AND L.VERO. 1961. Production of a new lysergic acidderivative in submerged culture by a strain ofClaviceps paspali Stevens and Hall. Proc. Roy.Soc. Ser. B 155:26-54.

3. BERENBLUM, J., AND E. B. CHAIN. 1938. An im-proved method for the colorimetric determi-nation of phosphate. Biochem. J. 32:295.

4. BRADY, L. R., Am V. E. TYLER, JR. 1960. Alka-loid accumulation in two clavine-producingstrains of Claviceps. Lloydia 23:8-20.

5. BU'LOCK, J. D. 1961. Intermediary metabolismand antibiotic synthesis. Advan. Appl. Micro-biol. 3:293-342.

6. BU'LOCK, J. D. 1965. The biosynthesis of naturalproducts, p. 4. McGraw-Hill Publishing Co.Ltd., London.

7. CASTAGNOLI, N., JR., AmD A. ToNoLo. 1966.Ergot alkaloids in submerged culture. Symp.IX Intern. Congr. Microbiol., Moscow, p. 31-40.

8. CHAMBON, P. 1963. Dosage de l'acide citrique dansle sang et les autres milieux biologiques. Ann.Pharm. Frang. 21:613-617.

9. COOK, R. 1961. Reactions of steroids with aceticanhydride and sulphuric acid (the Liebermann-Burchard test). Analyst 86:373-381.

10. FOSTER, J. W. 1949. Chemical activities of fungi,p. 164. Academic Press, Inc., New York.

11. KOBEL, H., E. SCHREIERAND, AND J. RUTSCHMANN.1964. 6-Methyl-A'.'-ergolen-8-carbonsalure, einneues Ergolin-derivat aus Kulturen einesStammes von Claviceps paspali Stevens et Hall.Helv. Chim. Acta 47:1052-1064.

12. MCDONALD, J. K., V. H. CHELDELIN, AND T. E.KING. 1960. Glucose catabolism in the ergotfungus, Claviceps purpurea. J. Bacteriol. 80:61-71.

13. PACIFICI, R. L., W. J. KELLEHER, AND A. E.SCHWARTING. 1962. Production of lysergic acidderivatives in submerged culture. I. Fermenta-tion studies. Lloydia 25:37-45.

14. PERRIN, C. H. 1953. Rapid modified procedure fordetermination of Kjeldahl nitrogen. Anal.Chem. 25:968-971.

15. RUSSELL, J. A. 1944. The colorimetric estimationof small amounts of ammonia by the phenol-hypochlorite reaction. J. Biol. Chem. 156:457-461.

16. TABER, W. A. 1963. Ergot alkaloid productionand physiology of Claviceps purpurea (Fr.)Tul. Develop. Ind. Microbiol. 4:295-305.

17. TABER, W. A. 1964. Sequential formation andaccumulation of primary and secondary shuntmetabolic products in Claviceps purpurea.Appl. Microbiol. 12:321-326.

18. TABER, W. A., AND L. C. VINING. 1963. Physiology

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of alkaloid production by Claviceps purpurea(Fr.) Tul. Correlation with changes in mycelialpolyol, carbohydrate, lipid, and phosphorus-containing compounds. Can. J. Microbiol.9:1-14.

19. ToNoLo, A. 1966. Production of peptide alkaloids

in submerged culture by a strain of Clavicepspurpurea. Nature 209:1134.

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20. TREVELYAN, W. E., AND J. S. HARRISON. 1952.Studies on yeast metabolism. 1. Fractionationand microdetermination of cell carbohydrates.Biochem. J. 50:298-303.

21. VOIGT, R. 1959. Zur Bestimmung der Mutterkorn-alkaloide mit p-dimethylamino-benzaldehyd.Mikrochim. Acta, p. 619-630.

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