Pilot Plant Equipment for SCTBMERGEID PRODUCTION OF
T
HE
tit'vc.lol)iiitbiit of corii stt.ep liquor-lurtosc. nirdia by the, Sorthtmi Itegional Research 1,aboratory (,5) and thv isolation of improved penicillin-producing cultures at t,hat laboratory (6) and elsewhere made possible large scale penicillin production by the suhrnrrged culture procedure. When tank frrmentations were first tried, penicillin yields were often very low. I t \vas neccssary to determine the proper medium, aerat,ion level, and operating prorcdure. Aeration and agitation were often found t o be critical. I n this laboratory experimental fermentations were carried out in 100-gallon t,anks. Some d a t a on the chemical changes during fermentation in these tanks were given by Kofflcr et nl. (4). The effect of variations in steep liquor concentration \vas reported by Bowden and Peterson (1). The present paper dcnls with t,he design of equipment, the operating procedure, and t h r effect of variations in aeration and agitation. Somr experiments on metal toxicity are also reported. Another p:ipcr ( 2 ) describes the rcsults ohtained b y the use of various pcnicillinproducing cultures. The cultures for the experiments here reported are the following: Pert 1 2 i / t n ~tiotot/tm KRRL 832, P . chrysogptium XRRL 1951-B25, P . chrynogrwztm X-1612 (Carnegie), and P . chrysogcnirm Q170 (Kisconsin). Data 0 1 1 thcw cultures and on the mrthods usrd for thrir maintcnnnw :itid ~ I Y I T I ag:rtiori givcw rlsc~\vhc~rt~ (2).
J . J . STEFANIAK, F. B. GA41LEY, C. S. BROWS, ~ N DM. J. JOHNSON I'nicersity of
rP iscnnain. Cladison, Wis.
p i a t i ~ ti, iiir*ticxh uidc i i i i i l 22 iiiches long, is we1dc.d t o the thernio.;t tit well. This b:Lffle aids :it:ration :ind minimizes coning. A jacket for temprrnturc~,coritrolcover6 tht, lower half of the tank. The air for aerating the medium is supplied by a service line :it 85 pounds per square inch gage. T o eliminate line fluctuations, the, service line pressure is reduced t o 50 pounds per square inrh gagc before the air is sterilized by pnssage through a cotton filter. Air sterilizing filter 10 (Figure 3) consist,s of an 11-inch irngtli of 3-inch pipe (packed length, 9 inchcs) and is packed with 9 ounces of nonabsorbent cotton. During sterilization-steam i* i*irculatedthrough the jacket of filter 10, t,hroughthe cotton filter, :uid finally through the pipe lines connected to the sparger. Conrltmsnte formed i ~ ]the jacket is drained before the steam ir 1):issrdthrough the cotton. A steam pressure differential bet'ween ,jacket and filter prevents wetting of the cotton. bntifoam agent is added aut,omatically b y the arrangement sliriwn i n Figure 3. Jacketed vessel 12, containing antifoam :igcmt, is mounted directly above the fermenter. This vessel is fillcd through standpipe 13 and two valves 15 and 16. T h e intiTriia1 construction of the vessel is shown in Figure 5 . T h e antifomi agent is sterilized by passing steam t,hrough the jacket at :I pressure of 25 pounds per square inch gage. T h e condensate is (1r:tined through steam cock 17. T h e automatic mechanism and thc fermenters are sterilized simultaneously. Throughout stei ilimtion, stcam from the fermenter, at 15 pounds per square inch. i j 1msc.d through l o m r magnetic valve l 4 b , through chamber 19. through upper magnetic valve 14a, and finally out of valve 18 at the lowcr end of the standpipe. During fermentation, the vessel and the chamber are connected t o thc sterile air line a t a point betxeen the cheek valve and t h e rotameter. The pressure in the jackctcti vessel and the chamber, therefore, always exceeds the pres.sure in the fermenter by an amount equal to the pressurr' drop through the sparger. Thc antifoam 'agent is added ab iiwded through the chamber and magnetic valves which arc' rwntrollrd by a relay connected t o a foam-detecting electrode. \Then tlic foam level is below the elcctrode level, upper magnetic valve 14a is open, and the antifoam agent fills chamber 19. Lower magnetic valve 14h is closed. \Then the foam level reacheb thc electrode, lower magnetic valve l4b is opened and upper valvc 140 is closed by a relay. The air under pressure in t h e line lecqding to the chamber blows the antifoam agent, into the fermenter. The foam-detecting electrode consists of a Pyrex tube extending about 6 inches dt)wn into the tank. The lower end of the tube bear?; :in e!ectrode madv from a standard '/,-inch pipe cap sealed to tht, fl:wed ends of the. tube h y means of habbitt metal. A copper v-irc p i w tliriiugh ~ the tiihe nnd i s r.mhedded in the babhitt metal.
FERMENTATION EQUIPMENT
The equipmriit ineludcs a tank for producing inoculum (Figuw
I ) , two fermenters (Figure a), and accessory equipmcnt. Figurtx
3 is a f l o diagram ~ of the apparatus. The inoculum tank (Figure l), which has a total volume of 115 liters, is 16 inches in inside diameter and 36 inches long, with a Full lrrigth jacket. T h e working capacity of the tank is 68 liters of medium. It is stirred at 360 revolutions per minute by means of a two-blade stirrer 12 inches in diameter, mounted on a central shaft 2 t o 3 inches above the sparger which is located 2 inches above the bottom of the tank. A baffle plate minimizes coning. The aerator is a 9-inch square sparger and has sixteen holes, inch in diameter. These holes are countersunk in ordcr t o shorten the bore of the hole. One of the accessory pieces of equipment (3, Figure 3) is :I small tank for measuring the inoculum. This cylindrical tank has three outlets located at levels such t h a t 5, 10, 6r 20 liters caii he measured. Tht, two 100-gallon fermenters (Figures 2 and 3) were not designed for penicillin fermentations, b u t accessory eqiiipmrnt, such as antifoam vessels and air filter, was designed and ronstructed t o meet the conditions of this fermentation. Tho two fermcntcrs a,re identical in construction; Figure 4 shows otic 01' them in detail. Inside the fermenter are a sparger, a n agit:ttor. B baffle, and a n electrode. The sparger, a 12-inch square, i a made of S/s-inch stainless steel pipe and standard pipe fittings. I t contains 54 holes 1/32 inch in diameter on its upper side. These, holes are also countersunk. The agitator, which is 18 inches ill diameter, runs a t 270 r.p.m. and consists of two flat blades, 2.5 X 5 inches in size, set a t a pitch 30" from the horizontal. Th(3 pitch is such t h a t rotation of these blades imp& the rnrtiium upward. The agitator is located 6 inches above the bottom of the tank. and the sparger i s 2 inchrs bclow thc agitator. This h;ifflr 666
PENICILLIN EquipmeiiL for production of penicillin ill 100-ga11orl tanks is described. Accessory equipment includean inoculum tank, air filters, and agitators. With this equipment penicillin yields could be reproducibly obtained of more than 200 units per ml. with culture 195l-B25 (NRRL) and more t h a n 400 unit. per ml. with culture X-1612 (Carnegie). .in aeration rate of one \olume of air per minute per \olume of medium was found optimal. Agitation wa. essential. Xletal tosicity experiments w i t h Perticillium notatum 832 showed t h a t aluminum and Alleghenj metal were nontoxic whereas iron exhibited slight toxicity. A tot'al iron content of 500 micrograms per ml. or more i n the fermentation medium lowered penicillin rields in khalte flaqkq.
f
Figure 1. Inoculum Tank and hcessory Equipment
+Figure 2. HundredGallon Iron Fermenters
The jacket of tlie i'ermentcr is used for. cooling the medium after sterilization. During fermentation, flow of cooling water through the jacket is controlled by magnetic vdve 14c, activated through a relay by a mercury thermostat in t h e t,hermometer ne11 of the tank. There is no provision for supplying hcat t o the tanks; this has been found unnecessary except in experimental high temperature fermentations. K h e n high operating temperatures are desircd, steam is circulated through the jacket of the air filter in order to heat t h ( * nir supplied t o t,hc fcrmentw. OPERATING PROCEDURES
The operation of the fermenters is planned so t h a t two runs are made in each fermenter
rvery week. 667
Length of fermentation is set
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
668
E-
245 64 hours 200 P , chrysogrnum S - I 6 1 2 270 ,470 72 hours t o g i w peak carbon dioxide pro200 I270 200 737 f'. rhryqogenum Q176 66 hours tluctiori rate of 8 t o 10 volumes 55; inoculum used. carbon dioxide per 1000 volumes h 1Oyo inoculum used. 93 units per mi. a t 50 culture 1)w mitiuttx \vas csscntial to good periiciHiii prcidurtion on the mcdium uscd. I milliliters of snmpltx A u c*q)vriiiit~iit is summarized iri Figure 7 in which ii tank w n UJ were digested for t:tiriiiig culturib P . chrysogcnum SRltT, 105l-B23 in regular 3 16 minutes with 3 nicdiiim (medium 3) was subjected tu various aeration r:itc>s at inl. of concentratid 'LO Iiours. As the, :terntioil rat? increased, t h i s rnc~t:iholismrate, nitric acid and 1 ml. r n t ~ : i ~ r eas d r:itc, of cn~~iciri ciiosidc protiuctiuri, tt,ritIi%tIto lipo f concentrated sul1)ro:tcIi ii niaximiim. .4t arbnition rates in excess of I volumt: per furic. acid. Aft.r.1. i t i i t i i i t c , , :tv~iil:tblc~ air was nu longer the chief fartor limiting oxidadigestion ttits samt i o n r:itc. (':irboii dioside production at : i n aeratioii r:itcb of 200 liter? pr.1. minute w:is ahout 10 volumw per 1000 volumes of 1)Iv \vas atljustcd tci :I p H h e t ~ ~ e 2.0 ~ri n i d i u m . I t may he c.oncludrd that optim:tl penicilliii yic,lds are 0 I i arid 5.0 by thc,:itldio t ) t : t i r i c d n.ht,n tho amount of osygen avnilahle to tht. orgmir;m is VOLS. AIK PER MIN PER VOL. CULTURE 7 , Effect of Le\-el t i m of : t m n i ~ ) ~ i i u n ~ r i i r t i t h a t iiir siiplily is not t h v rhicf factor limitiiip mc*t:iholiPm on RIetabolisni Rate h y d r u x i d t ' . Alir:iti,. quots of ttit. clilutrtl material tve~ri~a r i a I>.zcd. Calcium carbonate i l l the medium intrrfercd Ivitti tlic, i*omplcss. rate 0 1 color production hy the a,a'-dipyridyl-iron Si>vw:il hours werr often required for full color dt.vi*lopnii~iit
TABLEIT. E:FF.WT
OF
AERATION AND AGITATIOS ON PRODKCTION OF PEXICILLIS
'1
EXPERIMENTAL DATA
ISYLUEXCE OF AERATIONRATES. During the c,arly 1it.rIod ol w r experimental studies the equipment, stirring, rat? of' :ter:tt i o i t , m d inoculum were varied. Table I1 gives yield data for somc. ot
.\letal Sone Steel
Zletiil Area. Sq. Cm./ 100 hII. 2 5
Experiment 1 Age a t Penimax. cillin yield, yield, days uiiits/ml. 65 5 52 6
5 5
Experiment 26 PeniAge a t cillin max. yield, yield, dal-s units/ml. 60 6 45
0
6
10 3 5 0 the conditions employed. Later d a t a on improved c,ulturc*r:irv included for comparison. Only with high aeration ratrs : i I i i l :I 4ll~~~heu> 2 63 5 45 6 5 61 5 54 5 propeller of large diameter were t h e best yields obtained. 10 27 5 30 6 After installation of a n inoculum tank so t h a t a 10% i i i o i ~ u l u ~ ~ ~ \Iuiiiiiciini 2 61 5 50 6 cvuld be used, and after standardization of the aerating arid stir5 51 5 54 6 10 75 5 50 5 ring equipment, a series of experiments were carried out to dc~t(~1~mine the effect of variation in aeration rate. The culturt: uscd P. r i u l u l u m 832 was growu 011 mediuiii of following coniposition: lactose 20 g,, steep liquor solids 20 g., RlgSOd.7H~O0.15 g.. KHiPOi 0.25 g . , NaNOz was P.chrysogenun X-1612. These experiments were peri'ornicd 1.5 g. and water to 1 liter. Each figure given represents the average of thrce kplicilte 500 ml. Erlenmeyer flasks, each containing 100 ml. medium. in duplicate and checked well, with the exception of O I I ~eoiit:inii6 T h e metal pieces in experiment 1 were used without cleaning in experinitted tank. Standardized procedures were folloa-cd. Pcriiriliiient 2. lin, pH, total sugar, ammonia nitrogen, and carbon dioxide prciduction were determined at intervals during the fermentation. T ~ R II\', X EFFECTOF FIXED ~ ~ E T APIECES" L O N PENICIILIS The d a t a for a typical tank fermentation are summarized i n TIEI.DS I N SHAKE FLASKS Figure 6.4. T h e rate of aeration, 200 liters pur minutv per t a n k , Metal Area, Penicillin Yieldb. lletal Sq. Cm./100 MI. Units/.\ll. n x s slightly less than 1 liter of air per liter of medium siiic'c, 200 53 2 liters of medium plus 20 liters of inoculum Tverc used. 45 6 45 10 An increase in the aeration rate to 300 liters per iniriutc 11t'r 42 20 tank (Figure 623) did not alter the yield or the time of tlic fermcsti39 2 4llegheny tation. T h e rate of caibon dioxide production was iricrc:iscd, 46 6 58 10 :tnd the p H plateau was rearhed in a shorter time. Therr \v:ic 51 20 considerable fluctuation in the observed values for ratch of V:II'39 2 Steal bon dioxide production. Because of bicarbonate formation, the, 34 6 37 10 solubility of carbon dioxide at pH 7.5 is fifteen times its solubility 37 20 a t low p H values; therefore, slight fluctuations in the tank pr?.-None 29 sure during sampling resulted in large fluctuations in the carbon (tioxide content of exhairst air. As a result of this and of sindl ' ' l l ~ einetala were fixed in position so t h a t no friction occurred between xlabs and metal strips. I n other respects experimental conditions were iden\&ations in the rate of aeration during sarnpiing, it WRS diftical with those of Table 111. b .\laximum yields were obtained a t 5.5 days in all cases. ficult to obtain accurate carbon diosidc mc;isurcmrntr. \Vhen the aeratii~iit ' : i t i * \I,:&*Iri\vered (k'igiiris 6. (' : I n t i D ) . t l i v 0
..
12
-
506
400
300
200
IO0
10
It,>
0
I N F L U E NOCF E
] 600
A
.O 8r
67 1
INDUSTRIAL AND ENGINEERING CHEMISTRY
July, 1946
500
EO
40
60
80 ,400
'-' 20
1'0
PEN.
. HOUR5 40
60
80
A E R A T I O XR A T E CHANGES.Two experiments were performed in which the aeration rate was changed after periods of low or high aeration. For the first of these experiments (E igure SA) the penicillin yield and rate of carbon dioxide production were lower than those for a typical fermentation (Figure 6 A ) . However, the ammonia nitrogen, sugar utilization, and p H curves were similar. I n a second experiment (Figure 8B) the fermentation was very slow during the initial 24-hour period of low aeration. When the aeration rate was increascd, fermentation recovered and a good penicillin yield was obtained. Low ammonia nitrogen content during fermentation may be correlatedowithlow pH values. I
EFFECTOF STIR-
Because of mechanical breakdown OF the stirring Carbon dioxide, volumes per m i n u t e per equipment, a fcrlo00 volumes of culture; sugar, grams per 100 m l . ; ammonia nitrogen, m g . mentation was run per 10 m l . ; penicillin, units per ml without. agitation. A . Effect of change i n aeration rate: 0 to 6 hours, 60 liters per m i n u t e : 6 to Figure 8C shows 24 hours, 200 liters; 24 to 72 hours, 50 litera the results obtained B . Effect of low aeration rate for first 42 where the agitator hours: 2 t o 24 hours, 50 liters per m i n u t e ; 24 to 12 hours, 200 liters was not in operaC. Effect of aeration without agitation tion after the second hour. With an aeration rate of 200 liters per minute, but without stirring, growth was exceedingly slow and the penicillin yield \vas low. Figure 8 .
Results of Aeration Studies
allomd to move freely in the shaking flasks, and friction undoubtedly increased the amount of metal dissolved. I n another experiment (Table IV), the metal strips were fixed in position. Ender these conditions even steel showed no toxicity. The yield on the control flasks was low in this experiment, but in no case did an increase in metal surface area result in a decreased yield. 1x1order t o determine in an actual tank fermentation whether the amount of iron dissolved from the tank was sufficient to affect penicillin yields, the increase in iron content of the culture during fermentation was determined and compared R ith the level of iron required t o decrease yields in shake flasks. The data given in Tables V and VI indicate t h a t the presence of 200 micrograms of iron did not decrease shake flask yields. Since the iron content of tank fermentations was much below this level, it was concluded t h a t iron toxicity was not a factor in our tank fermentations. TABLE v.
IRON
CONTENT
Day
TANK hIEDICA1'
OF
PER
IN > f I L L I G R A M S
LITER
Total Iron in AIediuni R u n 11 R u n 12
Day
Total Iron in XIR u n 11 R u n 12
Composition of t h e medium was as follows:' lactose 20 grams. steep liquor solids 20 grams, NaNOs 3.0 grams, AIgSOe. i"i0 0.125 g r a m , Z n S 0 4 . 7HzO 0.4 gram,, FHz,PO, 0.5 gram, and water to make 1 liter. b Belore sterrlizntion. After sterilization, p H 1.6.
TABLE VI. EFFECT Iron Content of
Iledium", lIg./L. in
Pen,-cillin Yield, Units/'Ill.
OF .iDDED SHAKE
Ace a t
IIaLinium Yield, Dayb
IRON
OS PEXICILLIN
YIELDS
IX
FL~SKS Iron Content of lledium, Ilg./L.
Penicillin Yield, Units/All. 43 22 4
4ee at
AIaximum Yield, Days 6
6
a Iron was added as FeCh t o t h e medium, whirh had th,e lollowing composition: lactose 2 0 grams steep liquor solids 20 grama NaNOs 1.5 grams. AIgS04 7H20 0.125 g r n m , 'l\H?PO( 0.23 gram. ZnSO4.7kzO 0.02 gram. a n d water t o 1 liter.
RING.
ACKNOWLEDGMENT
This a o r k is part of a cooperative project on penicillin cwricd out a t the Univcrsity of Wisconsin in cooperation with arid support by the Office of Production Research and Developmcnt, War Production Board Contract S o , 118. rldditionsl funds were furnished by the Heyden Chemical Corporation and Lcdcrle Laboratories, Inc. The authors are indebted t o C. D. Gutscliy, J. T. Park, and B. H. Olson for assistance in obtaining some of the d a t a for this paper. They are also indebted t o R.H. Petcrson for counsel in the planning and execution of the woSk. Credit is due Margaret Lnrson for the pcnicillin assays. This wwrk is published with the approval of the Director of the 'Kisconsiri Agricultural Experiment Station.
METAL TOXICITY
When experimental tank fermentations n e r e begun, it was necessary t o determine Lvhether iron was sufficiently toxic t o the organism t o make impractical the use of iron tanks. For the shake flask experiments, pieces of different metals were placed in the flask. It was then incubated a t 23" C. in a reciprocating shaker which had a 4-inch stroke and operated at 92 cycles per minute. T h e results obtained in two experiments are sumrrmized in Table 111. Aluminum did not decrease yields, but Allegheny metal was toxic a t the higher levels, and steel was very toxic. In these experiments, however, the pieces of metal were
LITERATURE CITED
Boxden, J. P.. and Peterson, W . H., A r c h . Biochem., 9 , 3YI (1946'. G a i l e y , F. B., Stefaniak, J. J., Olson, B. H., and Johnson, .\.I. J , ' J . B a c t . , 52 (in press). K i t a e s , G . , Elvehjem, C. A . , and Schuette, H. A., J. B i d . C'hent.. 155, No. 2 , 653 (1944). KoMer, H., Emerson, 1%.L . , Perlman, D., and Burris, I t . H . . J . Bact., 50, 517 (1945). l l o y e r , A . J., and C o g h i l l , R . D.. I b i d . , 51, 79 (1940). Raper, K. B . , A n n . S . Y . Acad. Sci. (in press). S t e f a n i a k , J. J.. Gailey, f.B., Jarvis, f.G . , and Johnson. 11. J , J . Bact., 52 (in press).
