Vol. 57, No. 1, January 1968
plotted against time, and Fig. 6 was obtained. The slope of the straight line was calculated to be 0.0605 hr.-l, and D,/h to be 1.234 cm./hr. or 3.43 X lo-‘ cm./sec. This same value was also used under Appendix of the previous report (1). The Computation of Theoretical Values-The computations were executed by the help of the IBM 7090 digital computer. The simplified flow chart is shown in Fig. 7 . Results are in Tables I1 and I11 and shown in Figs. 4 and 5. Discussion-The excellent agreement of computed values and experimental results can be seen in Figs. 4 and 5. REFERENCES
Keyphrases Pyridine release from emulsions Boundary effect on pyridine release as function of pH-w/o emulsions Membrane effect on pyridine release-w/o emu1sions Diffusion equations, one-dimensional Computer flow chart-theoretical values of pyridine release
(1) Koizumi, T., and Higuchi, W. I., J . Phurm. Sci., 57, 87 (1968).
Chemically Defined Medium for the Production of By ROBERT D. IMHOLTE and L. C. SCHRAMM* A chemically defined medium for production of Fusariurn graminearurn has been developed and modified. A series of carbon and nitrogen sources was studied, resulting in the selection of glucose and ammonium succinate as primary substrates. Using the economic coefficient as a criterion for efficiency, the optimum concentrations for glucose and succinic acid were 3 and 0.5 percent, respectively. Growth weight and pH changes as well as glucose, nitrogen, and succinic acid utilization were determined during a typical fermentation period.
is the imperfect stage of Gibberella zea [G. saubinetti (Mont.) Sacc.], a plant pathogen causing stalk, root, and ear rot of corn and scab disease on wheat and barley. This organism has been classified in the order Hypocreales of the class ascomycetes. Ingestion of scabbed grains by domestic animals having simple stomachs elicits digestive system disturbances, vomiting, and in extreme cases, death (1-3). Bread made from scabbed grains has been called “intoxicating bread.” Recent work on the fungus has resulted in the isolation of an
Received Anril 14. 1967. from the Collesze of Pharmacv. University of Minnesota, Minneapolis, M N 55455 Accepted for publication September 11, 1967. Presented to the Pharmacognosy and Natural Products Section, APHA Academy of Pharmaceutical Sciences, Las Vegas meeting April 1967. The authors’ acknowledge support of this work by grant UI-00133-04 from the National Center for Urban and Industrial Health and a Mead-Tohnson undereraduate award (1965-1966). Present address: School of Pharmacy, University of Georgia, Athens, G A 30601
anabolic uterotrophic compound (4, 5). Most investigators concerned with F. graminearurn metabolites have utilized natural or incompletely defined substances as growth media. These have included potato infusion enriched with dextrose, scabbed barley, and cracked corn (1-5). The present report is concerned with the development of a completely defined chemical medium for the production of the fungus and a study of the growth habits on this medium. With such a medium it is possible to control the total environment of the fungus in order to facilitate extraction, metabolic, and animal toxicity studies. EXPERIMENTAL MATERIALS A N D METHODS A culture of F. graminearurn was maintained by serial transfer on potato dextrose agar (PDA) slants. After initial growth for 1week in an incubator a t 28’, the cultures were stored at 4,. The liquid medium
Journal of Pharmaceutical Sciences
used at the beginning of these experiments was derived from the PDA slant culture medium. I t consisted of 20y0 potato infusion and 37, glucose, and was designated potato-dextrose broth (PDB). Analytical determinations were accomplished in the following manner. A sufficient quantity of 500ml. conical flasks, each containing 100 ml. of media, were plugged with disposable foam enclosures and autoclaved for 15 min. Inoculum was prepared by transferring a small portion of mycelium from a stock PDA slant to a flask of the same medium to be used for each experiment. This inoculum flask was incubated on a rotary shaker (1 in. diameter rotation, 300 r.p.m.) at 28" for 5-7 days. At the end of the incubation period, the contents of the flask were homogenized in a sterile Waring blender for 30 sec. The experimental flasks were inoculated by aseptically transferring a small loop of the well-stirred homogenized inoculum t o each flask. The flasks were then incubated on the rotary shaker for a sufficient period of time. At arbitrarily specified intervals two flasks were harvested and treated as follows. The contents of each flask were filtered through a previously tared filter paper, the mycelium and filter paper frozen, then freeze-dried and weighed. The weight of the freeze-dried mycelium represented growth weight. The pH of the filtrate from the filtration described above was measured; a 15-nil. sample of the filtrate was preserved frozen. The quantity of glucose remaining in the filtrate was measured by the method of Shaffer and Soniogyi (6). Nitrogen was measured by the method of Kala (7) and also by the Kjeldahl method.
RESULTS AND DISCUSSION
In an early investigation of the toxic metabolite of F. graminearurn the PDB medium was devised to permit rapid production of large quantities of mycelium. When cultured in the PDB medium cither in 500-1111,conical flasks on a shaker or in a 5-1,. fermentor, the organisni produced abundant growth and profuse quantities of red colored niicroconidia. Growth weight, pH, glucose, and nitrogen changes were determined during a typical incubation period of F.graminearurn on this medium. The results are summarized in Fig. 1. From this figure i t can be ascertained that the iiiaximum dry weight of mycelium obtained w a s
I I, \I
L , I
Ftg. 1-Patterns of growth, p H rlzange~,und glutose uttlzzutzon of Fusarium graminearuin cn potutodextrose broth.
0.94 Gm./100 ml., occurring approximately 100 hr. after inoculation. The pH of the medium varied from 5.25 to 7.25. The pH a t the termination of the experiment (140 hr.) was 6.9. After an initial 15-hr. lag period, glucose was depleted in a logarithmic fashion during the logarithmic growth phase of the organisni. ( I t is interesting to note that the minimum pH was recorded at the beginning of this logarithmic growth phase.) Glucose was apparently completely utilized at 110 hr. Maximum growth weight was reached before the glucose was depleted, indicating that glucose was not limiting growth. Nitrogen did not appear to be utilized since the slope of the nitrogen analysis regression line was -0.001. Because of the abundant production of the colored microconidia (about 8% of total dry weight) which hindered extraction studies, and because the mycelium produced in this medium was not acceptable to experimental animals as a food substitute in toxicity studies, it was necessary to investigate other media in anticipation that a completely defined synthetic medium might result from such further experimentation. Semisynthetic media contain in part natural products and in part discrete chemical compounds. The absolute quantities of the chemical compounds may be varied between zero and the upper limits of solubility. Natural product constituents used in growth media are in many cases unknown. In addition, tlie reproducibility of the composition of a natural product is difficult due to the inherent variability of most biological systems. A synthetic niediuin would therefore be desirable if for 1110 other reason than absolute control of all ingredients in the environment of the organism. In order to evaluate and establish the limits of certain medium constituents capable of supporting growth, a number of semisynthetic media were constructed before attempting growth on a synthetic defined medinin. Glucose, mannitol, sucrose, and starch were screened singly and in combination, using neopeptone, yeast extract, and casein hydrolysatc also singly and in combination as nitrogen sources. In these early screening experiments dry weight of mycelium after a 120-hr. incubation period was used as the criterion for efficiency. Although noiie of the media werc as efficient as the original PDB medium, growth was supported by all media containing a nitrogen source; no growth w a s noted in media lacking nitrogen. Nitrogen was thus established as a requirement for growth. Since F. grurninearuin did not appear to be a fastidious fungus, a chemically defined synthetic iriediurn developed by I'acifici et nl. (8) for tlie productioii of C/noic.eps pzirpurea was employed in the hearcti for suitable substrates. This medium (see under u p p e n d i s ) riot only supported growth, but was superior to PDB using terminal growth weight as criterion for efficiency. The carbon and nitrogen rnodifications of Pacifici's niediurri are illustrated in Tables I and 11. Table I indicates that although Pacifici's original medium containing mannitol is capable of supporting growth of G. zeu, either glucose or sucrose is more efficient in the combination with succinic acid. From the modifications made, i t is apparent that a medium containing 3(]; glucose or sucrose arid 0.19; to 10; succinic acid is most cllicierit. Thc criterion
VOL.57, No. 1, January 1968
I-CARBON %URCE VARIATIONSPACIFICIMEDIUM
Mannitol, 3 Sucrose, 5 Sucrose, 3 Sucrose, 3 Sucrose, 1 Glucose, 5 Glucose, 3 Glucose, 3 Glucose, 3 Glucose, 3 Glucose, 3 Glucosc, 1 Glucose, 0.5
Succinic Acid, %
0.5 3 0.5 0.5 3 3 1 0.5 0.1 0.05 3 3 3
M yceli urn Dry Wt.,a Gm.
&con. Coeff. h
1.59 1.95 1.23 1.38 0.56 2.03 1.72 1.65 1.50 1.29 0.84 0.85 0.47 0.45
63 83.5 48 93 87.5 61 68 101 105 104 69 49.5 31 34
* Average of three replicate flasks; 168 hr. lei-mentation. ICconomic coefficient = mycelium dry wt./Gm. carbon consumed X 100.
11-NITROGEN SOURCE VARIATIONMEDIUM^ MODIFIEDPACIFICI Nitrogen Sourceb
Ammonium succinated Ammonium nitrate Ammonium chloride Ammonium formate Ammonium bromide Ammonium carbonate Ammonium dihydrogen phosphate Ammonium sulfate Potassium nitrate Calcium nitrate Sodium nitrate Neopeptone
Mycelium Dry Wt.,' Gm.
0 0 0.18 0.32 0 0 0.38 0.78 0.23 0.20
Variations in concentration of the major salts (magnesium sulfate and potassium hydrogen phosphate) indicated that the original quantities were in fact optimum. No modifications of the micro elements have been attempted. During the course of medium modification, a second analysis was accomplished. The medium used consisted of glucose 3%, succinic acid 37&, and salts as in Pacifici's original medium. Growth weight and pH changes as well as glucose utilization were determined. The results are summarized as Fig. 2. Glucose was utilized in a logarithmic fashion, being depleted 80 hr. after inoculation. Growth weight did not reach a maximum until after glucose had been completely used, indicating utilization of succinic acid after glucose depletion. After an initial lag phase, the pH rose throughout the incubation period. The beginning of the rise coincided with the end of glucose utilization. The increase in p H could be explained on the basis of nonutilization of nitrogen (or only slight utilization) and metabolism of succinic acid after glucose had been almost completely depleted. This would account for the observed predominant ammonia odor in the medium, and the high ending p H (8.3). Maximum growth weight was 1.4 Gm./100 ml. Decreasing the succinic acid to 5 Gm./L. (see under Appendix) resulted in still a different picture of metabolism. A fermentation similar to the previous ones was accomplished using the low succinic
OThe basic medium consisted of 3% glucose, 3% succinic acid, and salts as listed in the Pacifici medium under Appendiz. Nitrogen sources were supplied in an amount equivalent to the nitrogen content of 3% ammonium succinate. Average of three replicate flasks; 168 hr. fermentation. dSuccinic acid was deleted from this modification.
for efficiency in this experiment was the economic coefficient; the greater numbers indicate greater utilization of carbon substrate. Succinic acid was considered as part of the carbon source in calculating the economic coefficient. For purposes of calculation, it was assumed that all sugar and succinic acid added t o the medium was consumed. The quantity of succinic acid does not appear to be critical between 0.1 and 1%. The economic coefficient drops sharply below 0.1% and above 1%. Using optimum succinic acid concentration, glucose appears to be the growth limiting factor a t 3 % ; no further modifications of glucose concentration have been attempted. Table 11 indicates that succinic acid, partially neutralized with ammonium hydroxide, is the most efficient nitrogen source. Although the nitrogen variations were accomplished before the optimum concentration of ammonium succinate was known, it is probable that similar results would be obtained using a nitrogen content equivalent to 0.1 to 1% ammonium succinate. Starting pH of the media was adjusted to 5.1-5.5 in all cases.
Fig. 2-Patterns of growth, p H changes, and glucose utilization of Fusarium graminearum in the defined medium containing 3% succinic acid.
60 TIME, hr.
Fig. 3-Patterns of growth, p H changes, and glucose utilization of Fusarium graminearum in the dejined medium containing 0.5% succinic acid.
Journal of Pharmaceutical Sciences
100 acid concentration. The results are summarized in Fig. 3. As in the previous experiment, glucose was utilized in a logarithmic fashion, being depleted in this case 45 hr. after inoculation. Growth weight, reached a maximum at the same time, indicating glucose as the growth weight limiting factor. After dropping from an initial pH of 5.4, the pH of the medium began t o increase a t the time glucose was depleted from the medium. It is probable that the succinic acid again was metabolized after glucose was depleted. In this fermentation, because of the low concentration of succinic acid, ammonia was also present in a much lower concentration. The final pH (4.0) was much lower than that encountered in other media. Maximum growth weight was 1.4 Gm.
acid, 30 Gm.; KHzPO4, 1.0 Gm.; MgS04.7Hz0, 0.3 Gm.; FeS04.7Hz0, 14 mg.; MnS01.4H20, 6.7 mg.; ZnS04.7H~0,3.7 mg.; H3B03, 2.5 mg.; KI, 0.76 mg.; AlC13.6H~0, 0.054 mg.; CuS04.5Hz0, 0.03 mg.; NHIOH p.s. to pH 5.5; distilled water q. s. ad. 1000 ml. Defined Medium-Glucose, 30 Gm.; succinic acid, 5.0 Gm.; KHsPO4, 1.0 Gm.; MgSOa.7Hz0, 0.3 Gm.; FeS04.7H~0,14 mg.; MnS04.7Hz0, 6.7 mg.; ZnS04.7H~0, 3.7 mg.; H3B03, 2.5 mg.; K1,0.76 mg.; A1C13.6H~O,0.054mg.; CuS04.5Hz0, 0.03 mg.; NH40H q.s. to p H 5.4; distilled water q. s. ad. 1000 ml. REFERENCES
(4) Christenson C M. Nelson, G. H., and Mirocha, C. J.. A p p l . Microbial., i 3 , 653(1965). ( 5 ) Stob, M . , Baldwin, R. S., Tuite, J . . Andrews, P. N . , and Gillette K. G. Naluve 196 1318(1968). (6) Shaffir, P. k., and Somigyi, M., J . Btol. Chem., 100, 6!)5f 1933). (7) Kala, H., Die Phavmasie, 18, 29(1943). (8) Pacifici, L. R., Kelleher, W. J., and Schwarting, A. E., LloycEta, 25, 37(1962).
(1) Ashley, J. N., Hobbs, B. C., and Raistrick, H., Biochem. J.. 31,385(1937). (2) Hoyman, W. G., Phylopalhology, 31, 871(1941). (3) Mull, R. P., and Nord, F. I?., Arch. Biochem., 4, 419 (1941) \____,.
The experiments indicated above have resulted in the formulation of a completely defined medium for the production of F. graminearurn. This medium was derived from one used for the production of another hypocrealic ascomycete, Claviceps purpurea. It appears that the optimal carbon sources are glucose (3%) and succinic acid (0.57,). Nitrogen is supplied in the form of ammonium hydroxide t o adjust the pH of the medium to 5.15.5.
APPENDIX Potato -Dextrose Medium (PDB)-Freshly diced potato, 400 Gm.; glucose, 30.0 Gm.; tap water p. s. ad. 1000 ml. Pacifici Medium-Mannitol, 30 Gm. ; succinic
Keyphrases Chemical media for Fusarium graminearurn Potato-dextrose broth Glucose-succinic acid-ammonium hydroxide media Glucose consumption-analysis Nitrogen consumption-analysis