Chemical Changes during Chloramphenicol (Chloromycetin) Fermentation JULIAN E. OYAAS, JOHN EHRLICH, AND ROBERT M. SMITH' Parkc, Dash 6. Company, Detroit 32, Mich. Biosynthesis of Chloromycetin by Streptomyces veneruelae occurs during the period of rapid mycelium formation in shaken flask cultures in a medium containing glycerol, tryptone, fermentation solubles extract, and sodium chloride. Glycerol utilization accompanies mycelium formation while both total and ammonia nitrogen are utilized in the early stage of the fermentation and released to the medium in the last phase due to observed autolysis.
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TUDIES of Streptomyces venezuelae have been reported by Smith et al. ( 4 )and Ehrlich et al. (I), but few data were given on the chemical changes which occur in the medium during biosynthesis of Chloromycetin. Chloromycetin is Parke, Davis & Company's registered trade-mark for D-threo-N-( 1,1'dhydroxy1-p-nitrophenylisopropyl)dichloroacetamide, the generic name of which is chloramphenicol. The purpose of this paper in to record results of an exploratory investigation of such changes in a single undefined medium.
CHLOROMYCETIN. A 30- to 40-ml. portion was centrifuged and assayed, in duplicate, turbidimetrically for Chloromycetin. MYCELIALDRYWEIQHT. The mycelium from three 25-ml. aliquots was collected on separate Seita filter pads which had been dried a t 105' C. overnight and tared. Each pad and m celium was washed three times with 25-ml. portions of distilledYwater, dried at 105" C. overnight, and weiahed to obtain drv weiaht of mycelium, The filtrate h o m I this determination was removed from the flasks before the washing procedure, pooled, and used to determine reducing substance, ammonia nitrogen, and 5'300 7 total nitrogen. 3 REDUCINQ S U B S T ~ C ERe. ducing substance was determined on Seitz filtered beer by the method of Hagedorn and x I I Jensen (2). In one experiment 0 the values were checked by the method of Sumner (6). NITR o Q E N DET E R M INATIONS. Ammonia nitrogen and total nitrogen in the clarified beer were determined by the method of Ma and Zuazaga (3). GLYCEROL.Glycerol in the filtered beer wm analyzed by oxidation with metaperiodic acid following an ether extrac1 I tion and barium precipitation, as outlined by Voris and Maynard (6). Glycerol determinations were performed on only two of the three runs.
EXPERIMENTAL
The medium used in this study was one described by Smith et al. ( 4 ) and modified to obtain a clear solution after autoclaving. It contained per liter: glycerol, U.S.P., 10 grams; BactoI 1 tryptone (Difco), 5 grams; 100B-Y fermentation solubles 500 MYCELIUM (C.S.C.) extract, 5 grams; sodium chloride, c.P., 5 grams; and was adjuRted with sodium hydroxide to pH 7.5 before sterilization.
A 5.0% suspension of B-Y fermentation solubles waa prepared in 0.570 sodium chloride solution in tap water. The suspension was incubated for 18 hours a t 37" C. and then boiled for 45 minutes. After coolin water was added t o give t t e original volume; the BUS ension was then centrifuged a n f clarified by Seita filtration. A volume of the filtrate equivalent to 0.5% fermentation solubles was taken for the medium. Aliquots of 100 ml. of the medium were placed in 500ml., wide-mouthed Erlenmeyer flasks. Three thicknesses of cotton milk filter disks (Johnson and Johnson) secured by a spring wire clip were used to cover the mouth of the flask. The flask and medium were autoclaved 25 minutes at 127 a to 129.5" C. Present address, University of Winoonsin, Mrdiaon 6, Wis. 1
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Each flask was inoculated with 1 ml. of a spore suspension of S. venezuelae. The spores were obtained from cultures, derived from Parke-Davis culture No. 04745, that had been grown on maltose-tryptone-mineral salts agar at 28' C. for I3 to 10 days until well sporulated. The spores were suspended in 0.01% castile soap solution. The inoculated flasks were pleced on a rotary shaker in a room maintained at 23" to 24" C. and swirled in a 4-inch circle a t 155 or 170 r.p.m. Initial samples were taken from autoclaved but uninoculated and unshaken flaska. Later samples were taken at %-hour intervals for 4 or 5 days after inoculation. Four flasks were removed from the shaker a t each sampling. The pH of each flask was determined, immediately after removal from the shaker, by means of a glass electrode. Smears were made from the individual flasks and observed for characteristics of growth and evidence of contctmination. The contents of the flaska were then pooled, thoroughly mixed, and divided for the following determinations.
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Figure 1. Chemical Changes during Chloromycetin Biosynthesis 1775
Three experimental runs were carried out and the values averaged for this report because results of the various individual determinations for the three runs were in good agreement.
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
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RESULTS AND DISCUSSION
The data obtained are summarized in Figure 1. The most rapid biosynthesis of Chloromycetin occurs during the time of most rapid mycelial formation, suggesting that Chloromycetin is a product of relatively young cells. The same period is characterized by a rapid decrease in assayable glycerol and ammonia nitrogen. The explanation of the concomitant sharp but transitory increase in reducing substance is not clear, but possibly may reflect a temporary excess of metabolizable material derived from glycerol. The changes in total nitrogen in the filtered beer are reflected in the corresponding changes in mycelial dry weight. Autolysis of mycelium during the last 2 days of incubation doubtless accounts for much of the observed increase in ammonia nitrogen which in turn is presumably responsible for the rise in pH. SUMMARY
1. An exploratory study has been made of chemical changes occurring in the medium during growth of Streptomyces venezuelae in shaken flask cultures.
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2. Data on nitrogen and glycerol utilization, reducing substance, mycelial dry weight, pH, and Chloromycetin content are reported. ACKNOWLEDGMENT
The authors are indebted to Gertrude Rodney for analyses for nitrogen and reducing substance, to Margaret Galbraith for Chloromycetin determinations and to Charles Childs for assistance with the glycerol analysis. LITERATURE CITED (1) Ehrlich, J., Gottlieb, D., Burkholder, P. R., Anderson, L. E., and Pridham, T. G., J . Bad., 56,467 (1946). (2) Hagedorn, H. C., and Jensen, B. N., Biochem. Z.,135, 46 (1923). (3) Ma, T. S., and Zurtzaga, G., IND.ENG.CIIEM.,ANAL.ED., 14, 260 (1942). (4) Smith, R. M., Joslyn, D. A., Gruhzit, 0. M., McLean, I. W,, Jr. Penner, M. A , , Ehrlich, J., J . B a d . , 55, 425 (1946). ( 5 ) Sumner, J. B., J . Bid. Chem., 65, 393 (1925). (6) Voris, L.,Ellis, G., and Maynard, L. A,, Ibid., 133, 491 (1940).
RECEIVED January 6,1950.
Riboflavin by Fermentation with Ashbya gossypii V. F. PFEIFER, F. W. TANNER, JR.1, CHARLES VOJNOVICH, AND D. H. TRAUFLER Northern Regional Research Laboratory, Peoria, Ill.
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IBOFLAVIN, vitamin B2, may be produced in commercial quantities by biosynthesis when nutritive mediums of proper compositions are subjected to pure culture fermentation by one of several yeastlike organisms. Currently most of the commercial riboflavin produced by aerobic fermentation is probably obtained by biosynthesis with Eremothecium ashbyiz in submerged culture
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capacity of 30 liters. They are 39 inches tall, 10 inches in diameter, and each is equipped with a top-entering mixer. The propeller blade is 5 inches from the dishedbottom of the vessel. An air sparger consisting of one Aloxite cylinder (No. 0 porosity) is mounted horizontally 1 inch from the bottom of the vat and 1 inch below the agitator blsde. Air from t,he service line is sterilized by passage through a column packed with glass wool. The inoculum, 1%by volume of a 24-hour culture of Ashbya gossypii NRRL Y-1056,was developed in accordance with the suggestions of Tanner, Vojnovich, and Van Lanen (9). Its production is discussed in detail in the description of the pilot plant work. Liquid inoculum was transferred aseptically to the small tanks from flasks equipped with siphons that were actuated by sterile air. Previous work showed that the complete medium was easily overcooked by batch sterilization in the fermentor with subsequent impairment of riboflavin yields. To minimize this, the corn steep liquor and the animal stick liquor were sterilized separately. After adjusting the pH of a water slurry containing the corn steep liquor and the glucose to 6.5 with sodium hydroxide, it was cooked in the vat fermentors a t 121" C. for 30 minutes by means of direct injection of steam. Cooling of the liquor was accomplished by circulating cold water through the jackets of the tanks while internal pressure was maintained with sterile air. The animal stick liquor, thoroughly dispersed in 3 to 4 volumes of tap water, was autoclaved for 30 minutes a t 121' C., cooled, and
Lperiments were conducted on semipilot plant and pilot plant scales to supply engineering data on the production of riboflavin by submerged culture of Ashbya gossypii. The following factors affecting the yield of riboflavin were investigated: sterilization methods, medium composition, aeration and agitation, fermentation temperature, and strain variation. In satisfactory fermentations, riboflavin yields of 500 to M8-y per ml. were obtained. One 1250-gallon fermentation was conducted, and the fermented liquor was processed to dried concentrate with a 969'' recovery of contained riboflavin. Operating and investment wsts for the process were approximated. Estimates indicate that riboflavin in the form of dried concentrate may be produced commercia!ly by this process at a total plant production w s t as low as 3.75 cents per gram.
Wickerham et al. (10)found that a related species, Ashbya gOSS@i, also produced large quantities of this vitamin when propagated under proper conditions. Tanner, Vojnovich, and Van Lanen (9) investigated most of the important factors influencing the biosynthesis of riboflavin in submerged aerobic cultivation by Ashbya gossypii on a laboratory scale, and established suitable mediums for commercial production employing inexpensive and readily obtainable raw materials. This paper described semipilot plant and pilot plant experiments that were conducted to obtain engineering data on the process and information on which to base approximate cost calculations. PRELIMINARY INVESTIGATIONS
Paralleling the pilot plant studies, experimental fermentations were conducted in two aluminum tanks to determine the effect of medium composition on the production of riboflavin. Each of these jacketed vats has a total capacity of 50 liters and a working 1 Present address, Chas. Pfizer C Company, Brooklyn, N. Y.
