INDUSTRIAL AND ENGINEERING CHEMISTRY
January 1952
A P = pressure drop, inches of water = viscosity, pounds per hour foot = density, pounds per cubic foot
p p
Subscripts refers tu conditions a t the bottom of column 1 refers to conditions a t the base of partial condenser sec2 tion refers to the product leaving top of partial condenser d refer to values of H~and N based on individual gas and G, L liquid film resistances, respectively of H~ and N on an over-all basis, as deOG, 01, refer to fined by gas and liquid compositions, respectively. ACKNOWLEDGMENT
The authors are grateful to H. J. Garber and R. M. for valuable criticisms and suggestions and to c. c. Furnas for Buggesting the idea originally. LITERATURE CITED
(1) Badger, w. L., and McCabe, W. L.. "Elements of ~ h e m i c dEngineering," p. 351,New York, McGraw-Hill Book Co., 1936. (2) Chilton, T. H., private communication, quoted in reference 16. (3) Chilton, T.H., and Colburn, A. P., IND. ENQ.CHEM.,26, 1183 (1934).
231
(4) Ibid., 27, 255 (1935). (5) Colburn, A. P.,Trans. Am. Inst. Chem. Engrs., 35,211 (1939). (6) Duncan, D.W., Koffolt, J. H., and Withrow, J . R., Ibid., 38, 259-71 (1942). (7) Fenske, M. R.,U.S. Patent 2,037,316(April 14, 1936). (8) Furnas, C. C., U. 5. Bur. Mines, Bull. 307 (1929). (9) Furnas, C. C., and Taylor, M. L., Trans. Am. Znst. Chem. Ens?..., 36,135 (1940). (10) Goldstein,B. J., B.S. thesis, University of Tennessee, 1947. (11) Jackson, M. L., and Cea&.ke, N. H.3 I N D - ENO. CHEM., 42, 1188-97 (1950). (12) Johnstone, H. F.3 and pigford, L.3 Trans. Am. Inst. Chem. Engrs., 38,25 (1942). (13) Minard, J. H., Koffolt, J. H., and Withrow, J. R., Ibid., 39, 813 (1943). (14) Peck, R.E.,and Wagner, E. F., Ibid., 41,737 (1946). (15) Perry, J. 1% Ed., "Chemical Engineers' Handbook," 3rd ed., pp. 164,187,369,538,574, NewYork, McGraw-Hill BookCo., 1941. (16) Schoenborn,E. M.. Jr., Dissertation, The Ohio State University, 191 (1941). (17) Schoenborn, E. M.,Koffolt, J. H . , and Withrow, J. R., Trans. Am. Inat. Chem. E w r s . , 37, 1000 (1941). (18) Suroweic, A.J.,and Furnas,C. C., Ibid., 38,53 (1942). cHEM,, 34, 126 (1942). (19) Westhaver, J. w., (20) Willingham, C. B., Sedlak, V. A., Rossini, F. D., and Westhaver, J. W.,Ibid., 39,796812(1947). RECEIVED December 26, 1950.
EngFinTring
Submerged Production of Hydroxystreptomycin
pocess development I
MERLIN H. PETERSON, MARJORIE E. HANES,
AND
JOHN C. SYLVESTER
ABBOTT LABORATORIES, RESEARCH DIVISION, NORTH CHICAGO, ILL.
M
ICROBIOLOGICAL
and chemical studies of hydroxystreptomycin and taxonomic studies of productive actinomycetes have been Previous~Yreported ( 1 , 8,4 , 8). ~ h u far, s two cultures have been shown to produce the antibiotic under the conditions of submerged fermentation. The first of these, isolated by Benedict et al. (I), was designated as Streptomyces gn'seocarneus new Species. The second culture, designated 88 NA232-M1 in the stock culture collection of Abbott Laboratories, North Chicago, Ill., is believed t o be a strain of S. g+eocar7teUB (4), The following presentation deals with the submerged Production of hYdroxYstrePtomYcin by StWJtoW c e s NA232-M1.
shake flask inoculum grown in medium A-4b (see Table I), which had itself been previously inoculated with 4% of first-transfer, 4&hour, veqetative shake flask inoculum grown in the same medium. First-transfer, shake flask inoculum cultures were inoculated with a suspension of Streptomyces NA232-M1 spores grown on test tube of a medium COmPoSd of: tWPtone, Oa3%'0; beef extract, Oeo3%; glucose, O.l%; yeast extract, agar, 1.5%; and t a p water. Ten milliliters of spore suspension were repared from each test tube slant and three flasks were inocuEted from each suspension. All seed and production shake flasks were incubated a t 24' $0 26" c, on a rotary Shakin machine operating at 240 r a m . with an eccentric radius of2.25 inches. Samples from production shake flasks for hydroxystreptomycin assays were withdrawn from the third through the seventh day. os1%;
I n Table I are shown the yields of antibiotic obtained with EXPERIMENTAL seven different media. The figures are averages for triplicate METHODOF HYDROXYSTREPTOMYCIN ASSAY. The streptomyflwks. In addition to the results given it was also found that cin assay method of LOOet al. ( 6 ) was used for a]] assays of hydefinitely lower yields were obtained when soybean 'meal was droxystreptomycin described in this paper. The standard used waa Dure StreDtomvcin sulfate: FERMENTATIONS IN SHAKEN __ FLASKS.The s h a k e n flask TABLEI. SHAKEN FLASKYIELDSOF HYDROXYSTF~EPTOMYC~N ON VARIOUS MEDIA technique was employed in exMedium Composition periments designed t o find a Calcium Soybean Sodium Antibiotic Yields ,in good medium for hydroxyGlucose catbonate, meal, chloride, Corn steep Peptone, Glycerol, StreptomYcln Units s t r e p t o m y c i n pmduction. Medium grams/' g:am/ grams/ grams/ (wet baa@ grams/ grams/ Run 305 Run 307, Cotton-Dlugaed 500-ml. ErlenNo. liter liter liter liter grams/liter' liter liter units/ml: units/ml. a e y e r flask%>ontaining125 ml. A-4 10 1 10 5 a3 119 A-4b" 15 1 16 5 166 140 of the production medium t o 20 1 15 5 177 1.83 A-49 be tested were autoclaved 30 10 1 5 10 72 60 t o 40 minutes a t 15 pounds of 15 1 15 5 2.5 197 230 A-6 10 1 10 5 a4 100 steam pressure. After ooolA-Ba 15 1 20 10 90 104 ing, each flask was inoculated a Also used for production of all aeed culture inoculum. with 4 % (by volume) of secondtransfer, 48-hour, vegetative ~
ki!~
232
INDUSTRIAL AND ENGINEERING CHEMISTRY
replaced by Brewers’ yeast, inorganic nitrogen salts, or Curbay B.G. The soybean meal yield effect could be partially compensated for, however, by a corn steeppeptone combination (medium A-6). A further enhancement of antibiotic yield over that obtained with the best simple soybean meal medium (A-4c) was Sugar, Mg. X 10 per MI. Antibiotic, Streptomycin Units per MI.
pH X 100 Irjitrogen, Micrograms per M I .
Vol. 44, No. 1
rapid mycelium formation. The period of maximum antibiotic production is also characterized by rapid sugar utilization and marked increases in total soluble nitrogen, the latter indicating autolysis of mycelium. The p H rose only slightly but steadily throughout the fermentation. These findings are practically identical with those described by Dulaney and Perlman ( a ) for the production of streptomycin by S. griseus on a glucosepeptone-meat extract medium. FERMENTATION IN ~OO-GALLON FERMENTERS. Fermentations similar to those carried out in the 30-liter stirred jars were studied in 400-gallon stainless steel tanks. Two hundred and eighty Eallons of medium A-4c were sterilized in such a fermenter a t 120 6.for 45 minutes. After cooling, each fermenter waB inoculated with 3 liters of 72-hour vegetative inoculum of Streptomyces ”32-M1 grown in an aerated bottle on medium A-4b. The fermenters were held for 114 hours under the following conditions: a itation, 200 r.p.m.; aeratim, 30 cubic feet per minute or 0.8 vo7ume of air per volume of medium per minute; temperature, 26‘ C.; and antifoam, Vegifat-Y, added automatically as needed. Although the hydroxystreptomycin yields in these runs were 25 to 50% lower than those obtained on the same medium in shake flasks and 30-liter stirred jars, the culture grew well and the fermentation appeared identical to that in 30-liter stirred jars. The poorer yields obtained were probably due to the use of Vegifat-Y as the fermenter defoamer in place of 2.5% octadecanol in lard oil used for the 30-liter fermenters.
Figure 1. Chemical Changes Occurring in Streptomycin NA232-M1 Fermentations in 30-Liter Stirred Fermenters
obtained by supplementing with glycerol. Yield increases of 15 to 20% were obtained with the resulting medium (A-4h). From these results i t was decided t o use medium A-4c for purposes of hydroxystreptomycin production in large equipment, particularly in view of its simple and inexpensive composition.
For fermentation FERMENTATION IN ~O-LITERFERMENTERS. studies in small equipment closely resembling that used in large scale submerged fermentation production, 80-liter stirred jars of the type described by Peterson (7‘) were used. Studies on chemical changes during the fermentation were also carried out on runs made in this type of equipment. Media A-4c and A-6 (see Table I) were used for production and metabolism studies, respectively. Twelve liters of the production medium used were placed in a fermenter which was then autoclaved a t 15 pounds of steam pressure for 45 minutes. After coolin ,each fermenter was inoculated with 250 ml. of vegetative inocuyum of Streptomyces NA232M 1 which had been grown in medium A-4b in shake flasks under the same conditions as described for shake flask fermentations. The fermenters were held for 114 hours under the following conditions: agitation, 480 r.p.m.; aeration, 10 liters per minute or 0.8 volume of air per volume of medium per minute; temperature, 26’ C.; and antifoam, 2.570 octadecanol in lard oil added automatically as needed. Hydroxystreptomycin yields with medium A-4c were similar to those obtained in shake flasks on this medium-ranging from 100 to 150 streptomycin units per ml. Extensive studies to improve this yield in the 30-liter fermenters were not made, as material of this potency was found adequate for recovery and isolation of purified concentrates to be used in microbiological, pharmacological, and characterization studies. Medium A-6, which was used in 30-liter fermenter runs for purposes of following chemical changea in the medium during the fermentation, gave slightly better antibiotic yields in this equipment than in shake flasks. Hydroxystreptomycin levels obtained ranged from 100 to 150 streptomycin units per ml. Other conditions for the A-6 runs in 30-liter fermenters were identical with those for the A-4c runs. The chemical changes studied in these 30-liter fermenter runs with medium A-6 are summarized in Figure 1. Total nitrogen of sample filtrates was determined by the micro-Kjeldahl method of Johnson (6). From these values the mycelial nitrogen at any particular time may be calculated approximately by difference from the zero-time sample. Sugar analyses were carried out by the method of Shaffer and Somogyi (9). Biosynthesis of hydroxyStreptomycin occurs late in the fermentation, after the period of
SUMMARY
The production of hydroxystreptomycin by submerged fermentation with Streptomyces NA232-M1 has been studied in shake flasks, 30-liter stirred jars, and 400-gallon tanks. I n shake flask studies it was found that media containing soybean meal consistently gave the best antibiotic yields, ranging from 150 to 200 streptomycin units per ml. Trials in 30-liter and 400-gallon stirred fermenters indicated that the antibiotic yields obtained on these media in shake flasks are easily duplicated in larger scale deep culture fermentation equipment. A study of the chemical changes taking place during the fermentation in 30-liter stirred, fermenters showed that biosynthesis of hydroxystreptomycin occurs after the period of rapid mycelium formation. The time of biosynthesis is also characterized by rapid sugar utilization and marked increases in total soluble nitrogen, the latter indicating autolysis of mycelium. ACKNOWLEDGMENT
The authors wish to express their appreciation to Austin Alm a,nd William White for carrying out the 30-liter and 400-gallon fermenter runs, and to Reece Cantrell for performing the Gitrogen and sugar analyses. LITERATURE CITED
( I ) Benediot, R. G., Stodola, F. H., Shotwell, 0. L., Borud, A. M., (2)
and Lindenfelser, L. A,, Science, 112, 77-8 (1950). Dulaney, E. L., and Perlman, D., Bull. Torrey Botan. Club, 74, 504-11 (1947).
(3) Grundy, W. E., Schenck, J. R., Clark, R. K., Hargie, M. P., Richards, R. IC., and Sylvester, J. C., Arch. Biochem., 28,150-2
(1950). (4) Grundy, W.E.,Whitman, A. L., Hanes, M. E., and Sylvester, J. C., J. Antibiotics and Chemotherapy (in press). (5) Johnson, M. J., J. Biol. Chem., 137,576-86 (1941). (6) Loo, Y.H., Skell, P. S., Thornbeny, H. H., Ehrlich, J., McGuire, J. M., Savage, (3. M., and Sylvester, J. C., J. Bact., 50, 701-9 (1945). (7)Peterson, W.H.,Harvey Lectures Ser., 42, 276-302 (1946-47). (8) Schenck, J. R.,“Hydroxystreptomycin,” paper presented at the 117th meeting, Am.Assoc. Advancement Sci., Cleveland, Ohio (1950). (9) Shaffer, P. A., and Somogyi. NI., J . Bid. Chem., 100, 695-713 (1933). (r
RECEIVED May 23, 1951. Presented before the Division of Agricultural and Food Chemistry a t the 119th Meeting of the AMERICAN CHEMICAL SOCIETY, Cleveland, Ohio.
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