UBTILIN JOHN A. GARIBALDI AMD ROBERT E. FEESEY United States D e p a r t m e n t of , l g r i c u l t u r e , Albany, Caltf. T h e antibiotic subtilin was routinely produced by the submerged culturing of Bacillus subtilis in a fermentor operating with a charge of 190 to 200 liters of medium. High levels of antibiotic activity and heavy growth were obtained after only 11 to 12 hours of fermentation on asparagus juice, supplemented asparagus juice, and a partially synthetic medium. Inorganic nitrogen supplied as ammonia adequately served as the main source of nitrogen. Purified water was found essential for production in the partially synthetic medium. The fermentor employed and operating procedures and problems involved are described.
BCTERIX of the genus Bacillus have been studied for the production of enzymes (16, %), indust,rially important solvents ( 2 ) , and a variety of antibioiics (10, 14, 26, 17, 24). These reports, however, largely concern work perfornicd cither on a laboratory scale or under conditions which did not alloa. for the heavy growth obtainable from many members of this group on simple media ( I I ? 22). The present work was undertaken when substantial quantities of subt'ilin (3, 13, I S ) , an antibiotic produced by a particular strain of Bacillus subtilis, became necessary for animal and clinical evaluation studies ( 1 , 4 , 23). It concerned the stdaptat,ion of surface and submcrged culturing studies (18, $5) and nutritional investigat'ions (5-7') to the routine, large scale, submerged production of cultures from which the antibiotic could be isolated wit>hrelative ease (9). The culture of I?. sztbfilis used was originally obtained from N. R. Smith, Bureau of Plant Industry, Soils, and Agricultural Engineering of the United States Department of Agriculture, as No. 231 of his collection. It, is now deposited in t'he hmcriran Type Culture Collection as No. 6633, and in the culture collect,ion of the Korthern Regi0na.L Research Laboratory as KO.B-543. FERMENTATION EQUIPMEXT
arid serve as the main agitators of the culture medium (Figure 4) They are 8 inches and 3 inches in diameter at, the open ends and 6 inches long. The foam breaker is 16 inches in diameter and consists of bvo blades 1.5 X 7 inches set a t a pitch to impel tho foam downward. The two baffles, 2.6 inches wide and 34 inches long, are set opposite each other vertically along the fermentor wall with the lower end 12 inches from the bottom of the fcrinentor. These baffles eliminate swirling and increase the efficiency 01 aeration. All of the mixing and stirring devices are movable on the shaft in order t o meet widely different stirring requirements t'hat may arise. A stainless steel temperature bulb extends into the interior of the fermentor and i s in direct contact wit,h the culture medium. It activates the throttling valve, which controls the amount of steani adnlixed Tvith cold water flowing through the jacket of the fermentor and thus provides automatic temperature regulation during fermentation. Three pairs of sight glasses are set opposite each othcr so that the bottom, central, and top portions of the fermentor can be observed a t any time during the process. Air is passed from a service line through a 3-inch pipe 20 inches long packed mith absorbent cotton l o separate any liquid or solid particles froin the incoming air. The service-line pressure of approximately 80 pounds per square inch then is reduccd to 15 pounds per square inch and the air passed through flowmeters. The air then is passed into the air sterilizer, which is a jacketed %inch pipe 6 feet' long packed mith 5.5 leet o l glass .rlrool. After passing through t'he filters, the air is introduced into the fcrmentor through the sparger. OPERATING PROCEDURES
Inoculum. The bacteria were washed from an agar slant into t w o Fernbach flasks containing 500 ml. each of sterile medium. Aft>erincubating at, 35" C. for 36 to 48 hours, these cultiiros were blended in a sterile Waring Biendor, and 50-ml. aliquots of the
resulting suspension were used to inoculate the niedium in each of fifteen other Fernbach flasks. After 16 to 20 hours of growth a t 35" C., the cultures were blended and used to i~ioculate initial runs. For consecutive batch runs, 7 . 5 liters of cult,ure of the preceding run were drained from. the fermentor a t the time of harvest and used as promptly as possible for inorulation of the succeeding run. Sterilization of Fermentor and Accessories. The fermentor, air sterilizer, and air lines were kept under a steam pressure of 15 pounds per square inch for at least 1hour. The st,eani was turned off and air was passed through the air sterilizer arid air lines to dry them. The fermentor was brought t o atmospheric pressure and the medium, which had been prepared previously, was pumped in. Preparation and Sterilization of the Medium. While the fcrmentor was being sterilized, 150 or 200 liters of medium were prepared in s, stainless steel tank and the pM was adjusted t o 7.0.
The equipment consists of a fermentor with a capacity- of 75 gallons and accessory equipment (Figures 1, 2 , and 3). The fermentor is pat'terned after the Feustel-Humfeld laboratory scale fermentor (8),which permits thorough agitation of the medium. The body of the fermentor (Figure 2 ) is a etainless steel cylinder 18 inches in diameter and 68 inches long; the lower 30 inches are surrounded by a jacket. h propeller, two truncat,ed cones, and a foam breaker are mounted on ri stainless steel shaft (1-inch diameter) which can be rotated at speeds of 50 to 650r.p.m. by a 1.5-hp. motor. The ring sparger, G inches in diameter, is made of 0.5-inch stainless steel pipe with holes 0.094-inch in diameter on its upper surface. The air-dispersing propeller is 7 inches in diameter and consists of four blades set a t a n angle to impel the medium downward on to the entering air. It is mounted 4 inches from t,he bot'torn of the fermentor and 2 inches above the sparger. The truncated cones are open a t both ends 432
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Figure 1.
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Fermentor and Accessories
The medium was pumped into the fermentor and sterilized a t a pressure of 15 pounds per square inch for 30 minutes. The steam was turned off and cold water was run through the jacket,. Air pressure of 15 pounds per square inch was maintained in the fermentor during cooling and standing overnight. The following morning the air-outlet valve was opened and the stirrer was set t o rotate a t 360 r.p.m. Water was run through the fermentor jacket, and steam and the temperahure regulator were turned on. When the medium had reached the operating temperature (35 C.), the p H was readjusted and the inoculum was introduced. Aeration was started immediately after inoculation. For consecutive batch runs, the medium was adjusted to p H 5.0 and sterilized by steam passed directly to the stainless steel tank. The medium was kept at 98 to 100" C. for 30 minutes. I t then was cooled by cold water running through coils inserted i n the tank pnor to steaming, The p H was adjusted to 7.0 and the medium pumped into the fermentor immediately after the previous run was harvested. Addition of Defoam Agent. Excessive foaming during the fermentation was prevented by addition of a defoam agent. Initially, the agent was added manually through the inoculum inlet (Figure 2) as needed. Later an adjustable mechanical oiler, operated from a pulley on the main shaft and connected to the inoculum inlet, was used to give a continuous and constant addition, which was usually 12 to 15 drops per minute. To prevent excessive addition of defoam agent during the early stages of the fermentation, a well just large enough to intercept that added during the first 2 hours was placed between the oiler and the inorulum inlct. Sampling. Hourly samples were taken from the outlet a t the bottom of the fermentor (Figure 2). The pH wm determined immediately. Cell production was followed by measurements of
volume of bacterial cells as previously described (25). A 50-m1. sample of culture medium was added to 150 ml. of 95% ethyl alcohol and stored in thc refrigerator pending bioassay with Micrococcus conglomeratus (19). AntiFEED INLET biotic activities are AIR O U T L E T expressed in mg. INOCULUM per liter of medium INLET as determined SIGHT G L A S S against the subtilin standard used in FOAM BREAKER this laboratory, Samples of purified subtilin now availW A T E R OUTLET, STEAM I N L E T ablein this laboratory are approxiTRUNCATED CONE mately twice as acSTIRRERS tive as the standard employed in this work (9). Dry JACKET weights were deterBAFFLE mined by ccntrifugWATER INLET, STEAM O U T L E T ing 50 ml. of culTEMPERATURE ture, washing the BULB centrifuged cells P R O P E L L E R AGITATOR twice with distilled SPARGER RING SAMPLING OUTLET water, and drying PRODUCT DISCHARGE in forced air draft AIR I N L E T at 50 to 55" for Figure 2. Schematic Outline of 24 hours. Fermentor
c.
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moderate levels of antibiotic activity on simple media composed of a n appropriate source of carbohydrate, in& GLOBE I VALVE organic sources of nitrogen, phos6 N E E D L E VALVE phorus, and sulfur, and mineral salts PI CHECK VALVE and citrate (to control salt solubilities FEED I N L E T %4 S A F E T Y VALVE and ionizations), Antibiotic activity dl was maximal after 12 hours of fermenTRAP tation (3 to 5 hours after maximum P R E S S U R E REGULATING VALVE cell volume was attained). I n Figure 5 are presented comparisons of the @ 3 - W A Y PORT F E RME NTO P L U G COCK utilizations of ammonia nitrogen, nonVESSEL HOSE TO ammonia nitrogen, and sugar with the FEED MIXING cell volumes, dry cell weight, and antibiotic activities attained in a product,ion run on this medium, Disagreements betweon cell volumes and dry weights similar to those shown in Figure 5 m-ere found in other runs. Although maximum antibiotic activity did not appear to have been reached AIR S T E R I L I Z E R in this experiment,, the activity ob-. A T E D ABOVE trained (980 mg. per liter) approximated the maximum values obtained in nine other runs whosc activities ranged from 850 to 1100 mg. per liter. Rjeldahl-nitrogen and phosphorus determinations on the dried, washed, centrifuged cells showed, respectively, 9.5 to 1 0 . O ~ oand 2.1 to !2.3y0;these \Tere similar to analyses of pelliclee produced on synthetic media in surTO CONDENSATE c. RETURN O I L SEPARAT face cultures ( 6 ) . This medium purposely contained a large excess of ammonia and citrate. However, its conFigure 3. Diagram of Fermentor and Accessories stitution was not changed, because it was employed periodically for suppleRESULTS A N D DISCUSSIOh tiieritation studies wherein more nitrogen might be necessary and because fermentations could be completed without adjustments of Production on Different Media. Initial runs on beet molasses pH, etc. and asparagus juice media (1944 season) showed (Table I) that After ten weekly runs on the ammonia-yeast-citrate medium, conditions found optimal for the production of subtilin in subthe average maximum activity dropped from 950 mg. per liter to merged culture on a laboratory scale (26) were largely applicable 300 mg. per liter for the next six runs. The rates and extents of to production in the pilot plant. The runs on beet molasses were growth in theso six runs were similar to those of the first ten runs. completed essentially in 12 to 13 hours, but the antibiotic activiLaboratory experiments indicated that the water supply was a t ties were approximately 50% of those obtained on a laboratory fault, possibly because of changes in the calcium content. When scale. The use of beet-molasses medium also was undesirable production was resumed with distilled water in place of tap water, because the isolation and purification of the antibiotic thereactivities of 900 to 1200 mg. per liter were obtained and remained from were difficult with the methods employed at that time ( 3 ) . in that range until the studies were discontinued. Although i t Much more satisfactory results \\-ere obtained on the asparagusappeared that complex media, such as asparagus juice, were less juice (1944 season) medium. The maximum antibiotic activity was reached after 11 hours of fermentaAkSTIBIOTIC A4CTIVITIES OBTAIXED WITH TABLE I. TYPICAL tion and was approximately 757” of VARIOUSMEDIA f / that obtained on a laboratory scale. Maximum Activity, Mg./L. With a second batch of asparagus Media Laboratory scale Pilot plant scale juice (1946 season), it was necessary 600 1100 B e e t molassesa 880 1200 Aaparagus juice 1944 aeason) b to add various supplements t o ob300 Asparagus juice {I946 season) b As aragus juice (1946 season)b 4 tain appreciable antibiotic activi1 8.6% Steffen’s waste liquor Y 450 550 ties. Highebt activities were obconcentrate 600 ... Asparagus juice (!946 season)&-!- “so tained by supplementation with am1000 1400 Ammonia-seast-citrated monia. Supplementation with Beet molasses 20% solids: (NHa)zHPOd, 0.8%; M n , 60 p.p.m. b Asparapus-bu$t-juice concentrate diluted to 10% solids. Media conSteffen’s waste liquor concentrate sisting of, or containing, asparagus juice were sterilized by steaming at (21) also was satisfactory. pH 5-0 C Gufficient concentrated N H 4 0 H (3.4 t o 3.6 ml. per liter of medium) to The high-yielding ammonia-yeast adjuet pH t o 7.0. d Sucrose 100 g.; citric acid, 11.7 9 . ; NazSOa, 4.0 g . ; yeast extract, 5.0 citrate medium was a direct outg.; (NHn)zHPOa, 4.2 g.; asparagus-Juice concentrate, 5.0 g. (dry bas&; salts to give 400 p.p.m, K, 50 p.p.m, M g , 18 p.p.m. M n , 10 p.p.m. Fe growth of nutritional stuxies in surand 10 pap.m. Zn; sufficient concentrated ”4OH t o adjust the pH t o 6.8 Figure 4. Diagram t o 6.9 (approximately 13 ml.); a n d water t o ylve 1 liter. face cultures (5-71, wherein it was of Agitating Meoham nism shown t h a t the organism produced
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