Laboratory Fer entor fo Aerobic Fermentations d
R. W. RIVETT, RI. J. JOHNSON,
AND
W. H. PETERSON
L'nizersity of Wisconsin, Madison, Wis.
Laboratory fermentors of 15-liter working capacity are described. Experience over the past 4 years with a number of aerobic fermentations has demonstrated the versatility and adaptability of the equipment. The fermentors are useful for studies of fermentation variables and for the preparation of small lots of fermentation products.
D
URING the past 5 or 6years the fermentation industry has experienced a great expansion, largely due to the development of antibiotic fermentations, all of which are aerobic in character. This development has required the study of many factors, such as cultures, media, aeration, and numerous physical factors that effect growth of the organism and formation of the antibiotic. In the research laboratory the primary means for studying these variables has been fermentation in a shake-flask. By this technique many problems have been solved, but results obtained in shake-flasks are usually not comparable to those secured in large tank fermentations or even in pilot plant operations. Hence there is need for laboratory equipment of adequate design to bridge the gap between shake-flasks and large containers. The 30-liter fermentor (15-liter working capacity) described in this paper allows investigation of many variables and a t the same time gives results that are more readily transposable to large scale operations. Another use for a laboratory fermentor of larger size is in the production of a sufficient quantity of a new antibiotic for isolation and characterization of the compound. The 30-liter fermentor described in this paper was developed largely for use in the isolation of a new antibiotic, streptolin. As a further test of its usefulness several other types of aerobic fermentations have been run, and the results of these experiments are reported along with the description of the apparatus. A number of these fermentors have been in constant use for about 4 years and have proved a most valuable tool for the study of penicillin, yeast, and other fermentations ( 3 , 7', 10). APPAR4TUS
A photograph of the fermentor is reproduced in Figure 1. A cylindrical Pyrex jar, A , 12 X 18 inches (Corning S o . 850), having a capacity of 30 liters, mas fitted mith a cover, B , of 0.38inch carbon steel plate 14 inches in diameter, into which were built various devices for aeration, agitation, and sampling. All metal parts inside the fermentor were made of stainless steel except the plate in the first lot of fermentors. I t was found that the plate rusted badly and in later models the plate also was made of stainless steel. The cover was held in place by four 0.38-inch tie rods, C, fastened to a similar plate on which the bottom of the jar rested. Rubber gaskets (0.13-inch) were inserted a t all metalglass contacts; these served as shock absorbers as well as an airtight, contaminationproof seal for the cover. Encircling the four tie rods, 5 inches below the cover, was a 0.19 X 1 inch round steel band on which handles were welded to facilitate lifting of the fermentors. The cover was removable and the tops of the tie rods were threaded and supplied with wing nuts so that the rover could be firmly held to the glass. The cover was fitted in the center nith a
hearing for a 0.5-inch shaft. Another idcntical beaiing vat; placed 5 inches directly above the cover bearing in a steel yoke, L). The bearings consisted of porous bronze pressed into steel housings which were threaded to screw into position in either the yoke or the cover. The lower bearing and housing were covered ~ i t i~h skirtlike shield, E, having a 0.25-inch overhang which rotated with the shaft and prevented air-borne contaminants from settling on the bearing and working their waj- down through it into thc feimentor. The cover was also provided v,ith a hole tapped for 1.5-inch pipe fitting. A pipe nipple covered with a pipe cap, E', was screwed into the hole, and this served as a means of inoculating and manually adding defoaming agents. Three pipe couplings 0.13-, 0.25-, and 0.5-inch nere welded into holes in the cover to serve as fittings for aerator, air exhaust, and sampling tubc, iespectively. The agitator consisted of a foul-bladrd paddle-typc piopeller, G, on the bottom end of a 24 X 0 5 inch shaft. The shaft passed through the bearings in the cover and the yoke. Abovc thc yoke was a pulley driven by a 17-belt running from a pulley on a horizontal line shaft. The propeller itself was made by inserting 1 X 2 inch pieces of 0.06-inch metal sheet into slots in 0.25-inch rods which in turn were screwed into a hub, 2 inches in diamctcr and 0.5 inch thick, which was fastened t o the bottom of the shaft by a set screw. The diameter of the piopeller, tip of blade to tip of blade, was approximately 8 inches and the pitch of the blades could be adjusted to any desired angle. I n this work a pitch of 30" to the horizontal was used. The shaft ivas rotated clockwise and the propeller blades were set in such a manner as t o forcc the liquid upward. The weight of the agitator x a s supported b j the pulley riding on the yoke bearing. The aerator, H , consisting of 0.13-inch pipe bent into a 6-inch circle and containing six 0.03-inch holes on the top side, !*as placed a t the bottom of the jar directly beneath the propellcr The aerator mas connected by a 90" elbow to a piece of 0.13-inch pipe threaded into the bottom of one of the 0.13-inch couplings welded into the top plate. A close nipple to Lvhich a 90" elborv had been fitted was screwed into the top of this coupling. An air filter, T, consisting of a 1 X 5 inch pipe packed with cotton and fitted with pipe caps containing suitably drilled and tapped holes for pipe fittings was screwed into the 90" elbo~v. The frce end of the air filter was provided with a rubber tubing connection so that the rubber tubing from the flowmeter could be readily rcmoved. The air exhaust, J , consisted of an inverted U-bhapcd pipc made of 0.25-inch pipe fittings screwed into one of thc 0 25-inch couplings in the cover. The open end of the exhaust was fitted with a 0.25-inch globe valve. For sampling, a piece of 0.25-inch pipc, IC, !\as fitted to extend from a coupling in the cover nearly to the bottom of the jar. Connected to the top of the coupling was ~tgate valve, L , and fitting, .I[, for collecting the sample without contaminating the broth in the fermentor. The essential feature of the fitting was a bulblilie enlargement that prevented sample from the end of the delivery tube being sucked back into the fermentor when the pressure was released. T o admit sterile air into the fitting, a hole was drilledin the pipe cap and into it was threaded a guard tube, AT, packed with cotton.
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To collect a sample the cap of the delivery tube was removed and a sterile flask was firmLy attached to the rubber stopper on the delivery tube. Then the gate valve was opened and the exhaust valve was closed. Pressure built up in the fermentor forced- the broth up the sampling tube and into the collecting flask. When sufficient sample had been collected, the exhaust valve was opened, releasing the pressure, and the sampling valve was closed. The collecting flask was removed and the delivery tube flamed and capped. A baffle plate, 0, to prevent coning of the liquid during agitation was fastened to the aerator pipe leading from the cover to the sparger ring. The baffle, made of 16-gage metal, extended 3.5 inches from the pipe toward the agitator shaft and was 9 inches in length. Ten such fermentors were constructed and incubated in two 8foot long water baths, each holding five fermentors in a row. The two water baths were placed parallel to each other, 24 inches apart with a 1-inch horizontal line shaft on brackets between them. The line shaft was rotated at 600 r.p.m. by an electric motor. By suitable combinations of pulleys on the line shaft and the agitator shafts connected by V-belts, the rate of agitation of fermentors could be varied from 100 to 600 or more r.p.rn. The temperature of the water bath was thermostatically controlled. Circulation through the baths was accomplished by means of a pump and a return line. Aeration was controlled by means of needle valves, and the air was metered from a constant pressure air line through rotameters. OPERATION
Fifteen liters of medium made to volume with hot tap water were placed in each fermentor. The open air-exhaust pipe was plugged with cotton and the fermentor was sterilized in a large autoclave a t 18 pounds per square inch steam. Ten fermentors could be sterilized a t one time. In general the time of sterilization was 30 minutes for synthetic media, 45 minutes for corn steep liquor media or other media of low pH, and 1 hour or more for natural media at neutral pH. As soon as possible, the hot sterilized fermentors were placed in the cold water bath and aeration and agitation were begun in order to hasten cooling. It required about 90 minutes before the fermentors were cool enough to inoculate. T o prevent and control foaming, a sterile solution of 2.5% octadecanol in lard oil was added in 10-ml. portions during the fermentation. One 10-ml. addition of antifoam agent was sufficient for synthetic media, whereas four to ten times that amount was required for natural media. Sterile samples were taken at various intervals and assayed for the fermentation product. EXPER1,MENTAL FERMENTATIONS
Penicillin. Two different media were tried in successive runs with two fermentors in each run. The culture used was Penicillium chrysogenum Q176. The fermentors were aerated a t 15 liters of air per minute and agitated a t 385 r.p.m. The inoculum consisted of 500 to 1000 ml., per fermentor, of 50-hour shakeflask culture grown from spores on 2% corn steep solids and 4% commercial glucose for run P-Iand 2% corn steep solids plus 6% dextrin for run P-11. Water bath temperature was held a t 24 to O
TABLE I.
Figure 1.
Assembled 30-Liter Fermentor
25" C. Penicillin assays were performed by a modified cylinder plate method (8). The results in Table I show that good yields of penicillin were obtained. Increasing the corn steep solids in the medium, run P-11, gave proportionately higher yields of penicillin. Duplicate fermentors checked very well. Several hundred other penicillin ferpentations have been run in these fermentors in the last 3 years (3). Yeast Production. A beet molasses medium (8) was used in comparing two yeast cultures in 30-liter fermentors. The cultures were Saccharomyces cerevisiae and Torula utilis 2. Twelve liters of the medium given in Table I1 were sterilized by autoclaving 30 minutes at 15 pounds per square inch steam. The sterile solutions of urea and monobasic potassium phosphate xvere added aseptically prior to inoculation of each fermentor with 300 ml. of culture from shake-flasks. The inoculated medium was aerated with 24 liters of air per minute and agitated at the rate of 385 r.p.m. The water bath was held a t 30" to 32" C. and Vegifat Y was used as a defoaming agent. Samples taken a t 0, 12, and 18 hours were analyzed for sugar and dry yeast by previously described methods ( 2 ) . Table I1 is a summary of analyses and calculations on 12-hour samples. Both
PENICILLIN FERMENTATION
Fer17 Hours 30 Hours 50 Hours 71 Hours 84 Hours mentor NO. No. Mediuma p H Units/ml. p H Units/ml. p H Units/ml. p H Units/ml. p H Units/ml. P-I 1 A 6.6 35 6.4 237 6.2 652 6.4 718 6.6 765 P-I 2 A 7.1 34 6.4 280 6.3 628 6.5 777 6.6 795 P-I1 1 B 7.3 124 6.5 322 6.5 760 6.7 1210 6.8 1240 P-I1 2 B 7.5 128 6.6 289 6.7 690 6.7 1040 6.8 1240 A = 2% corn steep solids and 4% lactose plus 1% calcium carbonate (sterilized separately a n d added a t time of inoculation); B .? 3% corn steep solids and 4% lactqse adjusted t o p H 4.85 with sodium hydroxide plus 1%calcium carbonate (sterilized separately and added a t time of inoculation).
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TABLE 11. PRODUCTION OF YEAST Fermentor No.
1
2 3 4
Sugara Added, Culture M g . / l I l . S.ce?euisiae 11.40 11.20 T. utilis 11.20 11.20
Sugar Utilized, llg./AIl. 10.20 10.07 10.04 10.02
Dry Yeast, iUg./AIl. 5.05 4.83 7.41 7.26
Yield of Dry Yeast 70 of %,of added utilized sugar sugar 44.3 49.5 43.1 48.1 66.2 73.7 64.8 72.3
l l e d i u i n contained clarified beet molasses a t a level of about 1% sugar, 0.025% corn steep solids, 0.1% urea, a n d 0.1% monobasic potassium phosphate; pH 4.55. a
cultures used about 90% of the sugar supplied in the 12-hour period. With T.utilis the weight of dry cells amounted to 72 to 74% of the n-eight of the sugar utilized, whereas with S.cerevisiae the figure was only 48 to 50%. The higher yield obtained with T . utzlis 2 is attributable to lower alcohol production and greater utilization of nonsugar carbon compounds by that yeast (1). About 50 other fermentations with four different yeasts have been run and the yields of yeast and its vitamin content have been reported elsewhere (10). Butylene Glycol. Two fermentors containing 15 liters of 10% commercial glucose, 0.025% magnesium sulfate, and 0. 18ycmonobasic potassium phosphate were autoclaved under steam at 15 pounds per square inch for 35 minutes. T o the sterilized medium, 0.2% urea and 0.57, calcium carbonate were added aseptically. Each fermentor was then inoculated with 600 ml. of shake-flask culture of Aerobacter aerogenes XRRL 199. Air was metered in a t the rate of 3 liters per minute for the first 24 hours and then cut back to 1 liter per minute. The agitators were run a t 200 r.p.m. The temperature of the mater bath was 30" to 32" C. and 5 ml. of 3% octadecanol in lard oil were added during the third hour to control foaming. Samples were taken every 2 01 3 hours beginning a t 22 hours. Reducing sugar, butylene glycol, and acetoin determinations were run on each sample by suitable methods ( 4 j 5, 9 ) . Table I11 summarizes the results obtained a t 29 hours. These yields were considered fair.
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TABLE 111. BUTYLEKE GLYCOL FERMENTATION Yerinentor NO.
Initial Sugar, Mg./hLl. 91.5 91.0
TABLE
ITr~
Sugar Used, lIg./l11. 83.5 76.9
2,3-Butylene Glycol, Mg./&II. 30.9 25.8
STREPTOMYCIX
Ferinentor 19 Hours 42 Hours No. pH U./ml. pH U./ml. 144 1 7.3 11 7 . 4 7.2 124 2 7.3 10 3 7.3 12 7.3 108
0.5 0.5
FERUEXTATION
67 Hours p H U./ml. 7.0 208
7.1 7.0
Acetoin, hIg./Ml.
160 130
91 Hours pH U./ml.' 7.9 220 7.4 185 7.7 165
114 Hours p H TJ./ml. 7.2 230 7 . 4 245 7.6 240
Streptomycin. Twelve liters of 1.5% soybean meal, 1.5% glucose, 0.5y0sodium chloride, and 0.1% calcium carbonate in each of three 30-liter fermentors were sterilized 2 hours at 17 pounds per square inch steam. After cooling, the fermentors were inoculated with 1000 ml. each of 48-hour submerged culture of Streptomyces griseus Waksman No. 4. Agitation was 360 r.p.m., aeration 6 liters per minute, temperature 27" C., and Vegifat Y was the defoaming agent. Streptomycin assays were made by the method of Loo et al. (6). The results are given in Table IV. Streptolin. Fifteen liters of medium containing 27, soybean meal, 2% glucose, 1% sodium chloride, ok5y0 Curbay BG, and 0.1% calcium carbonate were sterilized in the fermentors 2 hours
Vol. 42, No. 1
a t 18 pound< per square inch stemi. After cooling to 25" C. each fermentor was inoculated ii-ith 300 ml. of a 52-hour shake-flask culture of Streptomyces N o . 11. The aeration and agitation rates were varied as shown in Table I'. The results show clearly that better yields mere obtained in less time when the lower agitation rate was used. Subsequent evperiments have confirmed this observation. DISCUSSION
The experiments givkn above illustrate the application of these fermentors to the investigation of such variables as media, culture, agitation, and aeration. The 30-liter fermentors have been useful in studying other factors. Raffles, made of various structural materials, have been inserted in place of the stainless steel ones to investigate the effect of the material on a fermentation. In evaluation of defoamers, the fermentors have given some indication of the relative amounts that might be required in larger equipment. The internal structure of the fermentors is of such flexible design that it can be readily altered to permit investigation of various types of agitators and aerators.
TABLE v.
EFFECT O F AGITATIOX AXD L%ERATIOX O N STREPTOLIX
mentor Fer-&ir, No. L./Afin. 27 4 28 10 29 4 30 10
FERMESTATIOK -kgitation,
R.P.RI. 375 375 195 195
39 Hours U./ml. 5,300 4,000 19.800 13,300
49 Hours U./ml. 11,100 10,300 22,500 21,700
60 Hours U./ml. 19,400 13,600 26,400 23,900
83 Hours
U./ml. 23,400 15,400 25,200 23,800
I t was found that during sterilization of a charged fermentor 111 the autoclave there was a lag in temperature rise of the liquid medium. It required about 50 to 60 minutes for theliquid to reach 115" C. when the steam pressure in the autoclave was 18 pounds per square inch. This was due to the insulating effect of the thick glass walls of the fermentor. Another manifestation of this effect was that, with high agitation rates, the tempei ature of the medium inside the fermentor rose 3 to 4 degrees above that of the surrounding water bath during a run. During the last 2 years the glass jars have been replaced for most work with stainless steel containers of similar dimensions. This container gives not only better temperature control but also avoids the excessive breakage incurred with the Pyrex jars. ACKNOWLEDGMENT
The aut,hors are indebted to J. C. Sylvester, Abbott Laboratories, for suggestions regarding design and construction of the fermentors and other equipment. This work was aided by an industrial fellowship from Abbott Laboratories. LITERATURE CITED
Agarwal, P. hT.,and Petelson, W. H., Arch. Biochem., 20, 59 (1949). ilgarwal, P. N., Singh, K., King, P. 9 , and P e t e r s o n , IT'. H., Ibid., 14, 105 (1947). Brown, IT. E., a n d Peterson, W. H., IND. ENG.CHEM.,in press. J o h n s o n , M.J., IND. ENG.CHEM.,A i i a ~ ED., . 16, 626 (1944). Langlykke, A. F., and Peterson, W. H., I b i d . , 9, 163 (1937). Loo, Y . H., Skell, P. S., Thornberry, H. H., Ehrlich. J o h n , McGuire. J. M., Savage, G. hI., and Sylvester, J. C., J . Bad , 50,701 (1945). Rivett, R. W., a n d Peterson, \Ir, H., J.Am. Chem. SOC.,69, 3008 (1947). Schmidt, W. H., and Moyel, A J., J.Bact , 47, 199 (1944). Shaffer, P. A,, and Somogyi, M.,J . Biol. Chem., 100, 695
(1933). Singh, K., Agarwal, P. N., and P e t e r s o n , W. H.. Arch. Biochem 18,181 (1948).
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RECEIVED October 3, 1949. Published with the approval of the director of the Wisconsin Agricultural Experiment Station.
