LANTS Production of Circulin HARRISON A. NELSON, CLARENCE DEBOER, AND WILLIAM H. DEVRIES The Upjohn Company, Kolomamo, Mich.
A process for the production of circulin, an antibiotic described by Murray et 02. (2), was developed from preliminary bench experiments to a pilot plant scale. A medium was developed which produced yields of approximately 1 mg. (or 6OOO units) per ml. of beer. In 100gallon pilot fermentors, conditions of pH, agitation, and aeration were found to be critical. Two extraction pmcedures were developed in the pilot laboratory, using sulfonated alkyl aryl compounds aa specific precipitants. The circulin complex dissolves in acid aqueous organic solvent-that is, n-butanol or acetone. The complex breaks when the concentration of the organic solvent is increased, and the acid circulin salt is removed as a precipitant. The final line products averaged 65% of theoretical purity at an over-all recovery of 44%.
c
IRCULIN, described by Murray and Tetrault (W), is an antibiotia produced by an organism resembling BaciUue Cirnckzns. The pharmacology of circuliin has been reported by VanderBrook (4,and preliminary testing reports by Blias and Todd ( 1 ) indicate interesting possibilitiae for this compound. Preliminq fermentation, m a y , and extraction studies on circulin were published by Murray et al. (3). This paper describes the development of fermentation and extraction processes for circulin from the preliminary bench studies to pilot production in a 2000.gallon fermentation tank.
a t the rate of 2% to inoculate 6oo-ml. shake flasks for fermentation studies. Shake-flask seed was also used a t the rate of 0.2% to inoculate &gallon agitated bottles containing 6 liters of medium for use in seeding 100-gallon fermentation tanks. The &gallon seed bottles were agitated a t 220 r.p.m. a t 24' C. with aeration at the rate of 6 liters per minute for 3 days. The bottle seed was used to inoculate 100-gallon fermentation tanks a t the rate of 6%. In order to facilitate microscopic examination in early shakeflask studies, a clear medium was used. This medium, called BPLY, has the following composition: beef extract, 3 g r a m ; peptone, 10 grams; soluble liver, 1 gram; yeast extract, 1 gram; sodium chloride, 5 grams; and tap water to make 1 liter. Development of larger scale seed preparation, however, indicated the desirability of a heavier inoculum and consequently of a richer seed medium. Investigation of other types of media in shake flash indicated that heavier seed growth and roughly equivalent fermentation yields were produced when a dextrinyeast medium (DYI) was substituted for the BPLY seed medium. Table I indicates the yields obtained in three fermentation media inooulated with seed grown in four different seed media. The composition of the media used in these studies is presented in Table 11. Trial seed preparation runs in Bgallon bottles produced heavier growth in DYI than BPLY, but the resulta were not
FERMENTATION PROCEDURES
TABLEI. EFFECTOF SEEDMEDIUM VARIATION ON FERMENTATION YIELDS
Preliminary fermentation studies were made in 6Wml. Erlenmeyer flasks containing 100 ml. of medium. The flasks were stoppered with cotton and agitated on a reciprocal shaker a t ninety Pinch strokes per minute. Inoculation was with vegetetive seed a t the rate of 2%. In the 100-gallon fermentor studies, glass- or plsstic-lined tanks were used; these were equipped with automatic temperature control a t 24' C. and with automatic antifoam droppers; aeration was a t the rate of 140 liters per minute and agitation a t 270 r.p.m. The volume of medium in the tank was 240 liters and the medium was inooulated a t the rate of 5% with seed grown in &gallon agitated bottles.
(Averages of eight runs eaoh)
Fermentation Medium
Seed Medium BPLY
DSI
Oat I
for Seed Medium
1766 1194 939 1276
2686 2769 1819 1914
2241 2405 1964 2780
2231 2163 1674 1990
DYI D8I Oat I
TABLE 11. COMPOSITION OF MEDIA DYI DYIII
SEED PREPARATION
The circulin oulture, 0 1 9 , was maintained on
Over-All Av.
DYI
agar slante- -
incubated for 6 day at 24' C. and stored at 4' C. until used. Inooulum for shakeflask studies was prepared in 500-ml. Erlenmeyer flasks containing 100 ml. of medium, inoculated with a loop of Q-19 culture from a stock agar slant and inoubated 48 hours at 24' C. on a reciprocating shaker. This seed was used
20 25
4 16 0.2
4 16 0.2
20
CaCOs
Soybean meal
%8? 1259
20 10
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20
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Medium, Grams per Liter D8I Oat I Oat I1 20 20 20 10 10 10
.. 4
16 0.2
... .
26'
.. 4 5
4
20
20
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6
8.4
TOpH'6.5-7.0
YG
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 42, No. 6
I,vtassiuni e l i l w i d e reduced tho yicld to 1100 units per ml., tis did the omission of h d f the calcium carbonate. A study of protein substilutcs for y w t in the DYI medium was run, w i t h tlic results shown in Table III. l i d l e d oats appeared tn be an enaellerrt protein source, hili the standpoint of yirld and economy, and BS a result an oatmeel medime (hi,I ) w~ doveloped and used io furthw ihske-fiash stedirs.
The de"ri"pm
