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Research Division, Abbott laboratories, North Chicagc
Fermentor Design for Small Scal Submeraed Fermentation A versatile 30-liter fermentor makes possible study of a wide range of experimental conditions ,
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Figure 1 is a photograph of a fermentor unit without the stainless steel can. The frame consists of a round ‘/, X 14l/, inch base plate, A, with six vertical tie rods, B. Near the top of the tie rods is a supporting ring, C, which has two handles welded to it for lifting the frame or complete unit. The tie rods a r e s/e X 19”d inches, with the tops threaded so that the head of the fermentor can be bolted tightly to the can. Standard
,The fermentor described gives resulk at least as qood as thore obtained in larger equipment with fermentations that require high aeration rates. Maintenance cask are law, and the mik are easily assembled, disaswmbled, and cleaned. Operating volumes are large enough to provide samples for analyses during a fermentation withwt an appreciable change in vdume. Variables such as temperotvre, inoculum, or medium far a series of fermentem can be mode very uniform.
wpven asbestos gasket material is used to ensure an airtight seal between the can and head. The gasket is cemented in a grwve cut in the underside of the head. The head of the fermentor, D, is a ’/*-inch stainless steel plate 14 inches in diameter. The handles, E, can be used for lifting the head or the complete fermentor unit. A lS//,-inch port with a threaded cap, F, is provided for adding medium, inoculum, special supplements,
F,-N,oN v e s d ranging h m shaken flazb to equipment of pmduction sue are employed for r a w r c h on industrial fermentations. One of the mmt versatile pieces of intermediate equipment is the 30-liter fermentor or stirred jar described by Rivet$ Johnson, and Pevrson (2) in 1950. A number of modifications have been made in these units and this article d d b e s the design and operation of p e 30-liter fermentors currently being used by the Microbiological Research Department of Abbott Laboratories.
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Design and Conshuetion The basic design for these unirg was developed by the Engineering and Microbiological Departments of Abbott Laboratories in the early days of penidllin during World War 11. Each fermentor unit consists of three basic parts: a fermentation vessel, a head to which the necessary accessories are attached, and a frame to hold the head and vessel together. When the iirst units were built, stainless steel was unavailable, so 30-liter borosilicate glass battery jars were selected for fermentation vessels and most nf the construction was of hiehcarbon steel. The glass jars were replaced as soan as possible after the war with stainless steel cans with a capacity of about 27 liters. These cans are made of 16-e Type 304 stainleas steel and are about 11 inches wide and 18 inches high. Au construction now is of Type 304 stainless steel, except for scveral special parts.
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-1 Figure 1.
A 30-liter fermentor head and frame S u p M n g ring, 0. F-ntor head; E. Hondlm; F. Port
A. Base plahi 0. Tie rod; C. cop; G. Foam d o t u b r ; H. Agitatw shaft) 1. Impellu blode; 1. Sparwen I(. Sompk line; Baffle; M. Air exhauslti N. Antifom inlet; 0. Seorhg housing> P. Spacers Q. boring E tridgq Y. Grease fllting
Figure 2.
Thirty-liter fermentor bearing assembly plus the~ovcnizedimpeller reduce8 whipping ofthe impeller and m a r on the mal to a minimum. The drive pulley, T,is a t the top ot .the s l ~ c .Coupling bctwcu~the heating and ahaft is p m vided by a pin extending through the stiaft, which fita into a slot in the bottom of the bearing sleeve. A, drawing 6f the imp+er shaft sed (Figure show8 & @arm in the bearing houDiog and the pin, U,tbrough the impeller shaft that engaga the bearing d-. %,rotating V, of the d is a stainlea# steel'dislZ'/. inches in diaqeter and p d htopoaitiou on the im+r shaft. ,' The stationary part, W,of the seal is a matching disk of
~.F/,X9inchplate,L,bpltedtoa .'/*inch md crtendiug down, Fmm the hcad. The aha&, M,and antilengths of '(r for+? ink$, N, are
MwliabearingbronzewithanOilite bushing insert, X,a m d the shaft. A radial sl0t.k cut in the face of the bmm disk and connected to a drive-rype greax fitting, Y,for lubricatin. Thisarrangement maLes it poaaible to lubricate the positively at i n y time. It is .im-
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inch tubing projecting , ihrough the head.
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on top, in the center-of the head, is a
h r a ~lxaring housing, 6,to hold the ~cmovabkbearing assembly. A brass
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P,is kept in the bearing housing : spacer, to hold the agitator ahaff stationary wheneyer the%* d d g q g, is not
'e.,$ace..
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This : p " e n e a d v e wear . on the a@atmuhaft roal when the head ishamnedforeleaningor~bling. ' T h e m m b k bearing cartridge is put place only during actual Operation of the fekentor. Because the bcarina.is ~, m i rquired to w i t h s h skr&hn . icmpCraturea, its a p r u c t i o n is iimple. Adrmingoftheesamtialpartsof.the.
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shaft
etc. The f& detector, G,is a '/rinch copper d held in place.by an insulating phenolic.: pkstie nut (Synthane, S k The agitator shaf6 H, stainless,steeldwith a four-bladed propeller attacbed to the The 1 X Z'inch blade+ I, bwer set at b y an& and am held in pipti? b y a lo& nut. A &-type . sparger, J, ,of.'/rinch tubing is used with five 0.WS-inch h$ca spaced at , equal intinab -ad the top of the ring. The 'Sampk. K, iS tubing extending d&G about Z'inchts from the boof the can. This tube . has a sligbx bcql to allow dearaqce for the &npeklcr.. Bafaing is pmvided by
".btarihgisahowninFigu~2: Thelong central t u b R , - t o which the ball bear-
S,are,anacheda c w a s a . + d sleeve aroid the impeller ahaft. This sleeve
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portant that the common surlaces of the two didu be properly lubricated, be- , c a w in addition to serving as the scal for the impeller shaft, these di& carry the Wei#ht .of the shaft and impeller. It iwtandd ploeedure to lubricate the d with a high tempvature silicone ~ M K (Dow Corning 44 grease) before sterillsation. This ensum adequate and d e lubrication when the fcrmentwisisatartcd. Wheninuse, thescalsam lubricated -3aiIy.with.silicone or.petroleum grraae.. No attrmpt tisterilize
theselubricants~harbamlnadeorappears to be nmssary. The %%crews, 2,kthe bearing housing to hold the bearingcamidgepr'apacerinplace. F i i 4 shows a gmup of six fennen-
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tors in operation. A constant temperature water bath is used to maintain the fermentors at the dca-&d temperam. The temperabnc&ting unit ( A d term, Powers Regulating Co.) is shown in the lower a n t a of the tank and the hot and cold water control valvw (plowrite valve, Powers Regulating Co.) are to the left of the fermentors. A pamp circulata the water in the bath and an overflow pipe in the bath keep the water at a constant level. A continuous tempera t u n ncader (MinneapobHoncywcll Co.) is located on the left side of the control panel o w the fermentom. Agitation spceds of 240, 360, 480, and 600 r.p.m. are available from four step cone pulleys mounted on the Boston spud gear unita (Boston Gear Works, Inc.) located between pairs of water baths. Standard V-belts are used to drive the agitamrs. Power for each Bet of 12 fermentors is a 2-hp. motor located between the water baths. The foam detector is connected to a relay behind the control panelbyna lead wire and an insulated alligator clip. W h e n foam touches the detector, a circuit is completed which dom the relay. This, in turn, opens a soknoid whicb pasvs low p r m air (2 to 4 pounds per square iqch gage) to the antifoam r-r. T h e m e v o i r s a r e modified d u m i m q paint spray cans (Spray Engineering Co.) h o l d i i about 400 ml. Each ca'n ha8 a smdl cotton plug to filter the incoming air. The flow of anagent (lad ~it-octadecanol, Vegifat Y,or CLRS) is controlled by a stopcock. The gksa drip tube is for Viribly adjusting the flow to the d d rate. Air of 15 to 20 pounds pa cquamiach
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tors are as good as or better than in larger equipment with fermentations that require high aeration rates. Two factors are believed to be responsible for this fermentation efficiency. First, the diameter of the impeller and size of the impeller blades are proportionately larger in the 30-liter fermentors. Secondly, the power input to the medium is high enough to maintain good aeration even with very heavy mycelial growth. Power input as high as 0.045 hp. per gallon has been measured during fermentations. Discussion
Group of six 30-liter fermentors in operation
Figure 4.
gage for each set of six fermentors is filtered through a large filterijust below the control panel and then enters a manifold behind the control panel. ,4ir for each fermentor passes through a rotameter (Flowrator. Fischer & Porter Co.) on the control panel and then through a regulating needle valve to a small cottonpacked filter on the fermentor. The individual filters are 8-inch lengths of 1inch iron pipe fitted with nipples at each end. From this filter the air goes to the sparger. On the right side of the fermentor is the sample line. There is a connecting link between this line and the sparger for flushing the sample line with sterile air before and after sampling. The line on the left side of the fermentor is the air exhaust. All connections on the fermentor are of rubber tubing R.P.M.
BLADE ANGLE
480 360
240
90" 90" 90"
480 360 0---02 4 0
45" 45O 45"
X-X X---X X---X
0-0
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5
5.0
t
4.0
-
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ON VI YI
2.0
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9 1.0 0
YX
E./x/:/
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0 x,. X 'H
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w 0.5 0.8 1.5 AIR RATE(v/v/MIN.)
Figure 5. Sulfite oxidation values for 30-liter fermentors
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(pure gum rubber tubing, Compound 4830, United States Rubber Co.). Operation and Performance The fermentors are assembled as complete units when needed. A charge of 10 to 15 liters of medium is added and the units are sterilized in a large horizontal autoclave, which has a capacity for 12 fermentors. Sterilization timing begins when the autoclave reaches 121' C. The maximum temperature obtainable is about 125" C. After autoclaving, the fermentors, are placed in water baths and connected to the air supply. When cooled to the proper temperature, the bearing cartridges are put in place and the desired agitator and air flow rates are set. The fermentors may be inoculated by two general methods. The inoculum can be poured in through the port in the head of the fermentor, or a connection can be made to the sample line and the inoculum blown in with sterile air. With either method some precautions are necessary to prevent contamination of the fermentor during the inoculating operation. Sulfite oxidation values for the normal operating range of the fermentors are shown in Figure 5. These data were measured a t 30" C. with a volume of 12 liters by the method of Cooper, Ferstrom, and Miller (7). As may be seen from the curves, the aeration efficiency increased as the speed of agitation was increased and also as the pitch of the impeller blades was increased from 45" to 90". For simplicity the data are not shown here, but the aeration efficiency increased slightly as the volume of liquid in the fermentor was increased. Most fermentations are run at sulfite oxidation values of 2.0 to 2.5, which are lower than those for the standard operating conditions of larger experimental fermentors. However, the results in 30-liter fermen-
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Some fermentors of this design have been in use for about 3 years with very little trouble during operation and generally low maintenance costs. All parts except the bearing cartridges are interchangeable. The units are easily assembled, disassembled, and cleaned. The operating volumes are large enough to provide the necessary samples for analyses during a fermentation without an appreciable change in volume. The fermentors provide convenient quantities of beers for laboratory processing of products or cells, yet medial costs are negligible. I t is possible to study a wide range of experimental conditions with this equipment. But when necessary, such variables as temperature, inoculum, or medium for a series of fermentors can be made very uniform. The principal disadvantage of this type of fermentor is the necessity for stationary batch sterilization of media. With very heavy media or with heat-sensitive ingredients this can present a problem. The use of rubber tubing limits the operating air pressures, but this has not been a serious handicap. The antifoam system has a tendency to deliver too much antifoam, a situation which can be corrected by putting a timer in the circuit to add defoamer intermittently when demanded. The dissimilarity in general design between the 30-liter fermentors and larger experimental and production fermentors is a factor which is undergoing continuous evaluation. In many instances the scale-up of results has been very encouraging. Acknowledgment The authors wish to acknowledge the assistance of many members of the Engineering Department, Abbott Laboratories, in developing these 30-liter fermentors. literature Cited (1) Cooper, C. M., Ferstrom, G. A., Miller, S. A., IND.ENG. CHEM.36, 504-9 (1944). (2) Rivett, R. W., Johnson, M. J., Peterson, W. H., Zbid.,42, 188-90 (1950). RECEIVED for review April 2, 1956 ACCEPTEDJuly 2, 1956
