Continuous Sterilization of Media in Biochemical' Processes v.
F. PFEIFER AND CHAS. VOJNOVICH
Norfhern Regional Research Laboratory, Peoria, 111.
T
HE succea9 of most fermentation processes is entirely dependent on t h e preparation of a sterile medium without appreciably affecting the fermentable substrate or the accessory factors required by the organism involved. Dwtruction of microorganisms in a medium or their removal from it may be carried out in a variety of ways: One is the application of heat, usually supplied in the form of steam; a second is the application of ultravioIet radiation with wave lengths shorter than 2900 -4.(17); and a third method is mechanical removal of bacteria by a t r a t i o n of the medium before inoculation with the desired microorganism. A fourth is by treatment of the medium with chemical sterilizing agents OP disinfectants such as phenols, heavy metal salts, detergents, and ethylene oxide ( 2 4 ) ; a fifth is by irradiation of the medium with high energy Roentgen rays, high energy cathode rays, or u l t r d o r t electrical impulses ( 1 , 6, d l ); a sixth is by treatment of the medium with high frequency sound waves above about 9000 cycles per second ( 8 , d O ) ; and a seventh method consists of combinations of chemical disinfection and relatively mild heat treatment (6). Batch sterilization with steam may be carried out in the fermentor itself, or in B separate tank. If a separate tank is used, the sterilized medium is pumped or blown by air pressure t o a presterilized fermentor containing sterile air under moderate pressure (IO, 23). Batch sterilization with steam is the method of choice in most fermentation processes. However, the many disadvantages of batch sterilization have caused a gradual trend toward installation of equipment for continuous sterilization, especially in new plants. Continuous processes capable of sterilizing the media for use in commercial fermentations may possibly be developed with ultraviolet radiation, chemical sterilizing agents, or irradiation with Roentgen rays, cathode rays, or ultrashort electrical impulses. At present, however, continuous sterilization with steam is the simplest, most economical, and most efficient method of completely removing contaminating microorganisms from media. This paper describes pilot plant equipment used for continuous sterilization at the Northern Regional Laboratory, and its application in the continuous sterilization of various commercial media. Design of continuous sterilizers is discussed, and some general data obtained from commercial installations are presented.
A
Modified Form of the Arrhenius Equation
Is Used to Calculate Sterilization Conditions It is generally assumed that microorganisms are destroyed when subjected t o moist heat because of denaturation of proteins and inactivation of essential enzymes. Since these reactions seem to
be of the unimolecular type (167,it is usually possible to represont the relationship between survival of a uniform population of microorganisms and time as a straight line by plotting the logarithm of t h e number of survivors against time. This may be rcprcsented by
where h: is the specific reaction rate or rate of destruction of microorganisms, co is the concentration of microorganisms at thc heginning of the sterilization, and c is the concentration after timt, t, has elapsed. Sterilization rates may be described conveniently by givlng thc numerical value of k , by giving the time required t o pasa through one logarithm cycle, or by giving the period of half-life, the length of time necessary for half of the microorganisms t o be destroyed. The half-life period, t l l g , may be obtained from t h r ratc-timc Equation 1 t i l 2 = 0.693/k
(2)
The classical Brrhenius equation is used by Johnson (11) to correlate temperature of sterilization with sterilization rate log k = -0.219E/T
+C
(3)
where kiand kz are specific reaction rates a t the two absolute temperatures, T I and Ta; C is a constant; and E is a constant, thc energy of activation, which is usually in the range of 50,000 to 100,000 calories for the most resistant microorganisms and spores. A value for E of 65,000 calories is used in the food industries for approximating sterilization requirements. Using Equation 3, a straight line is produced by plotting the logarithm of the specific reaction rate against the Iecipiocal of the absolute temperature. A modified form of t h e Arrhenius relationship has bcon derived and verified experimentally by Higuchi and B u s e ( 9 ) t o relate the sterilization time to the absolute temperature of sterilization log fd
=
0.219E T
4x
lvhere t d is the necessary sterilization time, E is the heat of activation characteristic of killing the most thermally resistant species present, T is the absolute temperature of sterilization, and K is tl constant depending on the number and kind of the most thormally resistant species present, A plot of the logarithm of the necessary sterilization time against the reciprocal of the nbsolutc t t m perature of sterilization will give a straight line. 1940
August 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
Since E and K are substantially constant, Equation 5 may be used t o calculate any set of suitable sterilizing conditions. The equation is also extremely useful for comparing sterilization rates with decomposition rates of nutrients in a medium undergoing sterilization. Fortunately, the rate of sterilization increases much more with temperature than the rate of decomposition of nutrients, RO t h a t high temperature sterilization at a short time does have an advantage in preserving the nutritive value of the medium.
Continuous Sterilization Yields Uniform Products at a High Rate; Processing Can Be Automatic and Economical The principal advantage of continuous over batch sterilization with steam is the retention of the nutritive value of the sterile medium, The total heating time usually is well under 10 minutes. Consequently, a fermentation process incorporating continuous sterilization may be successful and profitable, whereas the same process carried out with batch sterilization might give product yield 80 much lower that the operation would be unprofitable. This is true in riboflavin production by fermentation with the yeastlike organism, Ashbya gossypii (14, 19),in which yields from continuously sterilized media w e from three to ten times the yields from batch sterilized media. Carnarius ( 8 ) has described the savings in labor and materials that are possible with a continuous sterilization installation. With suitable instrumentation, the process becomes automatic and may be conducted by remote control. The products of continuous sterilization are uniform, since the operating conditions are readily and simply maintained and duplicated.
Figure
1.
1941
The sterilizer itself may be fabricated from standard sizes of pipe, often readily available in a variety of materials. The cost of the complete installation is a small part of the total plant investment, Since a continuous sterilizer operates a t constant capacity a t all times, each component may be constructed of the optimum size for its particular function. The sterilizer itself is mall and, if designed properly, is quite easy t o clean and keep free from contamination. The problem of fouled heat transfer surfaces is minimized, because the high velocity of liquid flowing through the jet heater and holding coils helps t o keep the surfaces clean. Floor space required by a continuous sterilization installation is smaller than would be required for a comparable batch installation with its separate batch tanks. The holding coils or pipes may be arranged in such a way that no floor space is taken up by them. In continuous sterilization, the steam load is conshnt, so that the plant boilers may operate at or near their designed capacity for maximum efficiency and economy. When grain mashes are processed, considerable reductions in electrical power result when the continuous procesa is used. The need for large agitators t o keep the viscous mixture in motion is eliminated. A continuous sterilizer of proper design may be used for a variety of purposes. In addition t o being used t o sterilize a variety of media under widely varying conditions, i t may be used for cooking grain mashes, acid saccharification of mashes, or hydrolysis of starch slurries by the variation of operating conditions. If the continuously sterilized medium is cooled to fermenting temperature by passing it through an efficient countercurrent
Horizontal Continuous Sterilizer Installation
1942
INDUSTRIAL AND ENGINEERING CHEMISTRY
cooler a substantial portion of the heat supplied by the steam may be recovered. Metal contamination from a large tank during batch sterilization may be a serious problem. Use of a continuous sterilizer with retaining coils constructed of corrosion-resisting material will minimize this metal contamination and may allow fermentation to proceed satisfactorily in tanks built of metal in which the medium cannot be batch sterilized. Continuous sterilization eliminates the possibilities of cold pockets or steam flow stoppage due t o high liquid heads, since the fermentor is sterilized while it is empty. d point to be considered in the use of continuous sterilization with steam is the 10 to 20% dilution of the medium with the condensate. This may be counteracted by omitting that amount of water from the medium before sterilization. Although it is possible t o remove a part of the steam bj- flashing, cooling in a countercurrent heat exchanger and recovering the heated water is simpler and more foolproof. If iron must be excluded from the medium, special precautions may be required t o remove iron rust from injection steam, or it may be necessary to use an indirect heat exchanger. Continuously sterilized mediuni may have a greater foaming tendency than batch-sterilized medium, and it may be necessary to raise the sterilizing temperature, or increase the retention time considerably in order to eliminate this difficulty.
The Pilot Plant Sterilizer Is Constructed of %Inch Herculoy Pipe with Bronze Fittings Nearly all the media used a t this laboratory for pure-culture fermentations on a pilot plant scale are sterilized continuously. A photograph of the complete continuous sterilizer used for this purpose is ahown in Figure 1. Herculoy is used extensively in this equipment so that it may be used also for acid saccharification and hydrolysis conducted a t very low pH. The medium t o be sterilized is mixed in one of two 500-gallon Herculoy premix tanks, usually with hot water a t 120" to 140" F. t o hasten solution of nutrients and afford smoother cooker operation. With water at that temperature a pasteurizing effect is obtained, thus the number of microorganisms is reduced before the medium passes t o the sterilizer proper. Medium from the premix tanks is pumped t o the sterilizer by a duplex piston pump driven by an electric motor with variable speed transmission. The pump is of stainless steel construction and has a capacity of from 1.5to 12gallons per minute. The medium from the pump passes t o the liquid side of a 1inch Schutte and Koerting Co. steam jet heater for liquids, where it is mixed with high-pressure steam and heated instantaneously to sterilizing temperature. Pressure drop through the jet heater ranges from 6 to 10 pounds per square inch gage. The hot slurry leaves the heater through the Venturi tailpiece and passes to the holding section. The holding section of the sterilizer is shown in detail in Figure 2. The sterilizer is constructed of binch Herculoy pipe, with bronze tees, ells, and valves. The pipes are arranged t o form five sets of U's, mounted one above the other, and are well insulated. Valves are installed in a manner t h a t allows use of from one t o five sets. In this manner, the holding capacity of the sterilizer may be varied from 19 to 62 gallons. By varying the pumping rate and the holding capacity, the retention time in the sterilizer may be varied from l l / z to 41 minutes. With steam a t 125 pounds per square inch gage, a top temperature of about 335 O F. may be obtained. Medium in the sterilizer is maintained at 5 to 10 pounds pressure above the pressure of steam at sterilizing temperature t o eliminate t h e possibility of the sterilizer containing vapor. The pressure is maintained by adjusting the discharge valves, and once set they require no further adjustment. Dial temperature indicators and pressure gages are installed a t
Vol. 44, No. 8
the inlet and outlet of the sterilizer holding coils. Temperature drop through the sterilizer amounts t o between 10" and 20" F., depending on the inlet temperature and the retention time. The sterile medium, after passing through the discharge valve, drops in pressure t o about 8 pounds pcr square inch gage and cools t o about 235 F. The medium cools t o fermenting temperature as i t passes through a stainless steel, spiral-type heat exchanger operated in countercurrent fashion with 60' F. water as cooling medium.
Medium Passes through Sterilizer at about 0.2 Foot per Second Operation of the continuous sterilization system is quite simple. The holding coils, cooler, fermentor, and connecting piping are steamed for a minimum of 2 hours with steam a t 15 pounds per square inch gage. A valve past the cooler is closed, and the fermentor is filled with sterile air and maintained a t about 5 pounds per square inch gage. Water is pumped to the jet heater, the steam rate is adjusted downward t,o give the proper temperature, and mater to t h e cooler is adjusted. When operating conditions have become adjusted properly and the system is operating smoothly, water to the heater is replaced with medium. After the water in the sterilizer has been displaced with medium, the valve to the fermentor is opened and the sterile medium passes into it. Liquid passes through the st'erilizer with an average velocity of about 0.2 foot per second. With most media used for pureculture fermentation, the Reynolds number, D V p / p , is between 20,000 and 25,000, where D is the inside diameter, V is the average velocity, p is the density, and p is the viscosity, all expressed in any consistent set of units. Liquid passing through a pipe a t a Reynolds number above 3000 is considered to be in a condit'ion of turbulent flow, and the maximum velocity of any particle is approximately 1.25 times the average velocity.
A photograph of a vertical continuous sterilizer and cooker which was originally used a t the h'orthern Laboratory is shown in Figure 3. This sterilizer was constructed of &-inchinside diameter glass-lined iron pipe and was made up of four 5-foot and two 15-inch sections, a total volume of 34 gallons. The medium to be sterilized or cooked was pumped t o the sterilizcr by a steam-driven duplex piston pump. The discharge from the pump entered the liquid side of a 1-inch steam jet heater for liquids, was mixed with high pressure steam in the heater, and was heated instantaneously to sterilizing or cooking temperature. Discharge from the sterilizer was through a manually operated globe valve, adjusted to keep a constant level in the gage glass. Sterile medium passed through a cooler on its way to the fermentor. This unit was used principally for acid saccharification of grain mashes with 1% sulfuric acid. The liquid level was maintained a t 30 gallons and the slurry containing 15 t o 20% ground grain was pumped a t the rate of 6 gallons per minute, giving an actual retention time of about 4 minutes, with a temperature of 325" F. Underthese conditions the average liquid velocity in the vessel waa about 0.08 foot per second. With most media used for pure-culture fermentations, the Reynolds number was 16,000 to 18,000. Because of difficulties in controlling the level and in keeping the gage glass clean, this vertical sterilizer wag abandoned in favor of the horizont.al-pipe, continuous sterilizer previously described. Temperature, Time, pH, Medium Composition, and Grind of Solids Affect Sterilization Methods. The continuous sterilizer shown in Figures 1 and 2 has been used chiefly for the preparation of sterile media for pure-culture fermentations carried out on B pilot plant scale. Some experiments have been conducted with this equipment t o investigate the relationships between time, temperature, pH, and raw materials in so far as they effect sterilization. In these experiments 250-gallon batches of medium were made up in the premix tank, with warm water a t about 120' F. The p H of the medium was adjusted when necessary with sodium hydroxide.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
1943
Medium - C
/9' E
3' Pipe- Hercdoy 42/ 3. Valve* Tees. El/s Bronze. F/anged A l l Gote Vo/ves. Except as Shown Flonges Welded on Ptpc
-
Figure 2.
Horizontal Continuous Sterilizer Details
The continuous sterilizer, cooler, and connecting piping were steamed for 2 hours and started on water. When operating conditions were adjusted properly and the system was operating smoothly, water to the heater was replaced with medium. Samples were taken aseptically from a sampling valve installed past the cooler and fitted with a protecting hood to minimize danger of accidental contamination from dust. The sample valve and its nipple were protected between samples by gentle flaming with a gas burner. I n any experiment the most stringent sterilizing conditions were employed first, and these were then progressively reduced until the mildest conditions were reached. Survival of microorganisms in the treated medium was ascertained by three methods: plating in nutrient agar, incubating in sterility tubes (M), and incubating processed samples for 5 days. Plating of contaminating microorganisms was the least sensitive method employed for their detection and was practically useless for samples containing coarse suspended solids. Composition of the media used in the various experiments described in this paper is listed in Table I. The medium described for vitamin Bl9 production is that developed by Hall et al. (7). Results. The following factors affecting sterilization in this equipment were investigated: temperature, time, pH, medium composition, and grind of solids. In one set of experiments, the various media listed (Table I ) were retained in the sterilizer for a period of 4 minutes, and samples were taken over a range of temperatures. Minimum sterilization temperatures were determined by plating samples on nutrient agar or by incubating samples in liquid nutrient medium. Results were checked when possible by microscopic examination
of incubated samples, although this method was ineffective when the p H was below about 5.0. In these experiments the dried distiller's solubles, soybean meal, and corn were finely ground, with less than 1% retained on a 20mesh screen. Results of typical data that were obtained in the treatment of riboflavin medium a t p H of 4.4 for 4 minutes are given in Table 11. Examination of Table I1 indicates that a temperature in the range of 235 O to 245 O F. was required t o sterilize this medium. A critical temperature range, above which sterilization took place and below which sterilization was not obtained, was determined for each of the various media described in Table I. The results are shown in Table 111. In another experiment with riboflavin medium treated for varying lengths of time a t pH 4.4 and temperature of 237" F., medium retained for 10 minutes in the holding coils w m sterilized, whereas medium held for 6 minutes or less still contained viable microorganisms. Sterilization of riboflavin medium at a low pH waa somewhat easier to accomplish than a t a neutral pH. The critical temperature range for sterilizing this medium at p H 4.4 with 4 minutes holding time was 235' to 245" F., whereas a t p H 7.2 the range was 245 O t o 255 ' F. If the solids were not uniformly and finely ground, much higher temperatures were required t o effect sterilization of the medium. It was necessary t o heat media containing coarse particles of ground corn or soybean meal to above 275" F. with 4 minutes retention time to sterilize them. McCulloch ( l a ) has compiled the results of a number of investigations on the exposure t o moist heat necessary to kill all life. A summary of his compilation is given in Table IV, Although
INDUSTRIAL AND ENGINEERING CHEMISTRY
1944 Table 1.
Media Used in Sterilization Experiments and Pure-Culture Fermentations Ingredients in Medium, 70 Used f o r Producing
Afedium Riboflavin
Dextrose (anhydrous) Corn steep liquor Animal stick liquor Soybean oil
2.0 1.8
Vitamin Bln
Diktiller's solubles plus soybean meal Dextrose (anhydrous) Calcium carbonate Soybean oil
4 0
Butanol, acetone, and ethanol
Xylose Ground corn (SHa)zSO4
3.8 1.8 0.1
Itaconic acid
Dextrose (anhydrous) (NHa)zSOd Corn steep liquor T\l,oS01 7H90
6.0 0 3 0.2 0 os
Fungal amylase
Distiller's solubles Ground corn
4 0 3 0
Effect of Temperature in Continuous Treatment of Riboflavin Medium
Heat
Table
It.
1.0 0.1
1 0 0 5 0 1
(Retention time, 4 minutes; p H , 4.4) Plate Count, Sterility Tube Temp., O F. Colonies /Ml. Incubation N o treatment 10,000 Contaminated 200 120 Contaminatnd 215 40 Contaminated 230 20 Contaminated 1 Contaminated 235 237 0 Contaminated 245 0 Sterilr 260 0 Sterile 275 0 Sterile
he claims t h a t no living thing can survive 10 minutes direct CYposure t o saturated steam at 250" F , other workers ( 4 )have reported the existence of therniophilic spores which required 23 minutes of direct exposure t o satuiated steam a t 248" F. for their destruction. Results of our pilot plant experiments conform in general t o the data presented in Table IV, with the exception of the simple media for producing itaconic acid and sodium gluconate. These media contained larger amounts of sugar and were quite easy to sterilize. It is evident that sterilization conditions must be varied to conform to the types of raw materials used and that minimum conditions for a particular medium must be determined by expeiinient.
yields obtained in pilot plant fermentations of this sterile medium with Ashbya gossypii were 500 to 600 y per ml. compared t o yields not exceeding 100 y from sterile medium t h a t was batch sterilized a t either high or low pH. Results were most consistent and satisfactory t? hen sterilization was carried out continuously a t low pH. Typical results of pilot plant fermentations with riboflavin medium are shown in Table VI. Yields of vitamin Biz from some nutrients were much higher in copper tanks when the media were continuously sterilized as ~ it described (Table V). When batch sterilization R T employed, nas necessary t o hold the medium for 11/2 t o 2 hours a t 250" F. in order t o eliminate contaminating organisms, so that the total heating time was well over 3 hours. Results of actual pilot plant fermentations are shown in Table T I L Continuous sterilization was particularly useful in the preparat on of medium containing xylose from corncob6 to be used for production of butanol, acetone, and ethanol by fermentation with a C l o s t d i u m species, Wisconsin A-14, on a pilot plant scale. When batch sterilization of the medium was employed, excessive amounts of furfural were produced, and these interfered with the fermentation, but when the continuous process w m used, very little furfural mas produced. Medium for the pilot plant production of sodium gluconate which was sterilized continuously a t low temperaturw was pcculiarly susceptible to foaming. Medium which had been sterilized continuously a t a temperature of 275" F. and 5 minutes retention time exhibited much more foaming than medium which had been hatch sterilized. I n Commercial Installations Using Various Types of Equipment Sterilization Temperatures Vary from 250" to 325" F.
Through the cooperation of a number of companies engaged in a variety of industrial fermentations, data were collected on the operation of commercial processes of continuous sterilization and cooking. These processcp include continuous sterilization of me-
Table 111. Critical Temperature Ranges for Sterilization of Various Media for Pure-Culture Fermentations hLedium C'sed for Producing Riboflavin Vitamin BIZ,no soybean meal Vitamin BIZ. with half soybean meal Sodium gluconate Itaconic acid Fungal amylase
Table IV. Operating Conditions M u s t Ensure Sterilization and Preclude Damage t o Nutrients
The aotual operating conditions that are employed a t this laboratory in using the continuous sterilizer are considerably more drastic than would be necessary on the basis of the experiments listed. A reasonable factor of safety is necessary so that sterilization is enmred, but overheating must be avoided to minimize damage t o the nutrients. TVith common materials such as are usually incorporated into commercial media formulas, the particle size of solids is often quite variable. Even though care is taken t o grind the solid constituents, some large particles usually get through, and these require harsher treatment. Table V lists the actual operating conditions used a t this laboratory for a variety of pure-culture fermentations. The actual operating conditions used for sterilizing riboflavin medium (Table V) are such that sterilization of the medium is ensured and damage t o the nutrients is minimized. Riboflavin
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PH
Critical C m p . Range, F.
4.4 6 7 6 7 6 0 6.7 6.5
235-245 230-240 240-250 215-225 220-230 240-250
Time-Temperature Relationships for W e t Sterilization
(Data of Novy, Jordan, hfuir a n d Ritcbie, llacfarland. Erne, Besson and Sternberg, compiled b y McCulloch. 1945) Minutes for Thermal ,Death Temp., O F. of -411 Bacterial Life 265 248-250 239-246 230-244 221-242
Table
V.
Actual
10 15 20
Operating
Conditions
Employed
at
NRRL for Continuous Sterilization of Media Medium Used for Producing
pH 4.5 4.5 6.5 4.5 6.1 5.0
Temp., 278 325 275 275 300 325
O
F.
Retention Time, Minutes 4 13 3 5 5 13
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
August 1952
Table VI. Effect of Medium Sterilization on Yields of Riboflavin Produced by A. gossypii, NRRL Y-1056 Corn Animal Steep Stick Glucose, Liquora, Liquor",
%
%
%
2.0 2.1 2.0
1.9 1.9 1 9
0.8 1.0 0 9
2.0
1.9
0.9
Sterilization Method Batchwise 250' F 45 min. 6 6 p l i Batchwise: 250" F:: 25 min.: 4:4 p H Continuous. 275' F.. 5 rnin.. 6.5 n H stick sterilized separately a t 2 7 5 ° F . Continuous. 275' F.. 5 min.. 6.5 U H
Max. Riboflavin Yield, y/hfl.
1945
Sterile medium leaving the holding section is discharged through some device which retains the prcssure in t h e sterilizer itself. For this purpose throttling-type globc valves, instrument-operated back pressure valves, and fixed orifices are used. Manually adjusted throttling valves are preferred in most plants for this purposc.
5 88 360 158
As is basis.
Table VII. Effect of Medium Sterilization on Yields of Vitamin BIZ Produced by S. oliveaceus, NRRL B-1125 Distiller's Solubles,
%
Extd. Soybean Meal,
% ... ...
AIaximuiii Vitamin Yield.
Sterilizlition Method ?/MI. Batchwise, 2 hr., 250O F., p H 4.8 0.1 Continuous, 13 rnin., 325O F., pH 4.8 1.2 2.0 Batchwise, l l / a hr., 250' E'., pH 5.7 1.3 2.0 Continuous, 13 rnin., 330° F., p H 5.7 2.0 Each medium supplemented with 1.0% glucose, 0.5% CaCOa, and 2 p.p.111. COCIZ. 4.0" 4.0" 2.0" 2.0"
dia for pure-culture fermentations and continuous cooking of grain mashes for alcohol production. Pure-Culture Fermentation Processes. Media are mixed Ilatchwise in large tanks (rather than continuously in small tanks) using hot water up t o 180" F. In the most concentrated mixturct reported, the concentration of nutrients in the mix tank was three times t h a t desired in the fermentor. Mixing tanks are equipped with mechanical agit,ators or air spargers. Although a varietg of pumps are used for pumping mixed media t o the heater, cent'rifugals predominate. The output rate is measured and controlled. Reciprocating piston pumps and positive displacement rotary pumps are also used, with controlled output rates. Pumping rates vary from 20 t o 200 gallons per minute, and fermentors are filled in 11/2 t o 8 hours. Media are heated to sterilization temperature by direct injection of steam in all cases reported. The devices used for this purpose are mixing tees or commercial steam jet heaters for liquids. Construction of the retaining or holding section of the sterilizer is extremely variable but usually falls into rather general classifications: ( a ) series of vertical pipes connected with 180" return bends; ( b ) series of horizontal pipes mounted one above the other and connected with 180" return bends; (c) long straight runs of pipe; and (d) combinations of vcrtical and horizontal pipes, Generally there is a moderate slope to the horizontal pipes, preferably with the low end at the point of entrance of medium, so that the sterilizer may be drained easily and also vented of noncondensable gases. In some instances the holding pipes are arranged around the building so that they require no floor space whatsoever. Pipc sizes vary from 4 t o 12 inches in diameter. Average liquid velocities in the holding pipes vary from 0.1 t o 2.1 feet per second, with 0.2 t o 0.4 foot per second the preferred range. The Reynolds numbers for these installations vary from 36,000 t o 273,000, with 38,000 t o 80,000 the preferred range. This indicates that medium passes through the retaining or holding coils in a condition of turbulent flow. Conventional, horizontal, rake-agitated batch cookers used in the alcohol industry are also used as continuous sterilizers. For this purpose the medium t o be sterilized is fed to the bottom end of the cooker, sterilized as i t passes through the baffled cooker in a tortuous path, and leaves through an overflow pipe in the opposite end of the cooker.
Figure 3.
Vertical Continuous Sterilizer Installation
Sterile inedia arc usually cooled t o fermenting temperature by passing them through a double-pipe heat exchanger countercurrent to flow of cold water. Special precautions are taken in installing some typcs of indirect coolers t o ensure t h a t both mash and water sides drain completely, so t h a t t,he cooler may be sterilized properly before use. Precautions are also taken in desigiiing and installing coolers t o prevent nonsterile water from entering sterilo medium in case of leakage. Coolers of the doublehead type are also used for this purpose, since containination of the mash v i t h cooling water is minimized in this type of construction. In s o ~ c eplants, however, the medium passes tJo the f e p mentor without cooling, flashes in the fernlentor to about 220" F., and is then cooled t o fermenting temperature by passing cold water through thc jacket or coils. Hot water leaves the cooler a t 150" t o 200" F., and is used in some plants for medium make-up, boiler iced water, and other hot watcr rcquiremcnts. When pII adjustment of t,lie iitcrilc medium is necessary, it is accomplished in most plants by following the medium through with a ciustic solution until the dcsired pH in the fermcntor is reached. Onc company ( 3 ) employs series of largo v w t i d tanks as liolding vessels for retitining the heated medium for t,he required time. The tanks are four to fivc times as high as they are widc, and channelling is minimized by pumping the heated niediuni
1946
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
tangentially into the top of the tank and passing i t through the tank in spiral fashion. For the various types of cquiprnent employed, sterilizing teinperatures reported on media used for pure-culture fermentations vary from 250' t o 325' F. and holding times from 4 t o 14 minutes. Preferred conditions for sterilizing media containing relatively small amounts of rather finely divided suspended solids are in the range of 275 t o 28.5' F. with a holding time of 3 to 5 minutes and pH 4.0 t o 5.0. Brown et al. (,2) describc another type of direct steam injection heater which eliminates the possibility of burned material eollecting on the steam heated walls or pipes of the heater. The heater is constructed with tangential steam inlet t o effect instantaneous heating of the medium, a very smnll holding chamber, and a iixed orifice discharge. Very short rctention times are employed. Although this equipment mas not designed for the purpose of effecting sterilization, some media are sterilized when treated in it a t 245 O F. for 1 second.
Temperatures far Continuous Cooking of Grain Mashes Vary from 320" to 360" F. Because of the large scale of operation involved, ground gi alii IS usually slurried continuously-that is, the ground grain and hot water are mixed continuously in a rather small tank. The slurry IS preferably treated with malt in a subsequent tank, using about 1 % of the weight of grain processcd, a t 145 O t o 160O F., t o facilitate pumping and passage through the cooker and cooler. Slurry heated to 140" t o 1SO" F. is pumped t o the cooker by means of duplex and triplex reciprocating piston pumps and b\ centrifugal pumps. ST'illkie et al. ($3)found reciprocating triple\ pumps most serviceable for pumping abrasive grain slurries at high pressures. Pumping rates vary from 100 t o 300 gallons per minute, and fermentors are usually filled in from 4 t o 8 hours. Mash is heated to cooking temperature by direct injection 01 steam with steam jets, steam mixing tees, and simple steam mixing nozzles. Heated mash is retained a t cooking temperature in the same types of equipment as described for use in pure-culture fermentations. Tm-elve-inchiron pipe, extra heavy, is a preferred construction material for the holding section. Some difficulties have been reported with erosion and subsequent failure in t h r holding section, particularly in return bends. Average liquid velocities in the holding pipes vary from 0.4 t o 1.5 feetper second. Unger el al. (22)have reportedthat atavelocity of 1.5 feet per second in the holding pipes, there was mbstantially no deposit on t,he walls of the pipe, and it was not necessary t o clean the cooker of caramelized mash a t weekly intervals. The patent literature also describes the UEC of one or more veitical tanks, with top feed and bottom discharge, as holding tanks for continuous cooking of grain mashes (3,18). Cooked mash leavea the cooker through a manually operated throttling valve, a fixed orifice, or an air-operated diaphragm valve actuated by a pressure controlling instrument. The cooked mash is usually cooled t o 240" t o 250" F. in a flash tank, and the regenerated steam a t 10 t o 20 pounds per square inch gage is used in the beer still, evaporators, or for heating process water. The regenerated steam contains noncondensable gases formed by decomposition of grain or nutrients at the high cooking temperature and should be disposed of directly rather than by passing t o an indirect heater for reclamation of its heat content. Cooling the cooked mash t o the malting temperature of 145' t o 150' F. takes place in conventional heat exchangers or by flashing in a vacuum tank. Cooking temperaturcv reported for corn and sorghum m a s h vary from 320 O t o 360 O F., with holding times from 2 t o 7 minutes. Most steps in the cooking and malting processes are conbrolled automatically, and very little attention is necessary after they are started and brought into synchronization.
Vol. 44, No. 8
Continuous Sterilization Will Be Installed in M a n y N e w Biochemical Processes A useful and flexible pilot plant installation designed primarily for continuous sterilization is described, and its application in thc sterilization of various commercial media to be used for pureculture fermentations is outlined. Continuous sterilization is a practical method of prepa.riny media for industrial pure-culture fermentations and is the only method presently available for sterilizing media without apprcciably affecting the fermentable substrate or the accessory factors required by the organism involved. W t h some fermcntations the use of continuous sterilization is one of the main factors nccc'ssary for profitable operation of the procees. Equipment is simple, requires but moderate amounts of floor space, and is readily adaptable to instrumentation, so that the entire sterilization and cooling processes m8.y be carried out automatically. Although in s o m instances it may be uneconomical to modify installed batch Sterilization equipment t o allovv opcmtion of the continuous procCSR, the possibilities of higher yields, snioot,her operations, and lowered operating costs should certainly be investigated for existing fermentation processes. Continuous sterilization equipment will be installed i n many ~ i c wplants for the production of vitamins, antibiotics, enzyme concentrates, and industrial organic chemicals. Literature Cited Brasch, h.,arid U'oligang, ll., S c i e / ~ c c 105, , 112 (1947). 13mwn. A . ET., Lazar, hl. E., \T-asserman. T., Smith, G. S.,:LJICI Cole, M. JV., I z n . ENG.CIix,i.. 43, 2949 (1961). Carnarius, E. €I., E.S. Pat,erit 2,423,580 (July 8. 194;). Davis, F. L., Jr., and JVilliams. 0. 13.. .I. Btrct.. 56, 55.5 ( 1 9 & > , Ilunii, C. G., Campbell. \T'. L.. Pram, I € . . and Hutchins, >l,, .I. 9ppZied Pitys., 19, 605 (1948).
Krne, II., presented before the Division of Agricultural a n t i od Chemistry, 119th Xeeting of t h c Aincricm C1lernic:il ciety, Boston, Mass. IIamre, D., J . Bact., 57, 279 (1949). Higuchi, T., and Busse, L. K., J . Am. P / ( U T / /jlssoc., [, 39, .I i 1 (1950).
Inskeep, G. C., Bennett, R. E., Dudley, J . F., arid Shepard, M. W., Ian. EXG.CHERI., 43, 1488 (1951). Johnson, M. J., "Factors Involved in Rapid Sterilization of Fermentation Media," U. of \Vis. Lecture Notes (October 1949). hIcCulloch, E. C., "Disinfection and Sterilization," 2nd cd., pp. 69-104, Philadelphia, Lea and Uebiger (1945). Olive, T. R., Chem. Eng., 56, 107 (1949). Pfeifer, V. I?., Tanner, F. W., Jr., Vojnovich, Chas., and Trmiiw, D. H., IND. E m . CHEM.,42, 1776 (1950). Pittman, M., J. Bacl., 51, 19 (1946). Porter, J. R., "Bacterial Chemistry and Physiology," p. 174, Yew York, John Wiley & Sons, Rentschler, IT. C., and Nagy, R., 20, 1949). Saltzman, €3. E., U. S. Patent 2,309,989 (Feb. 2 , 1943). Smiley, IC. I,., Sobolov, X., Austin, F. L., Rasmussen, It. A , , Smith, 31. B., Van Lanen, J. M., Stone, L.,and Boruff, C. S., 1x0. E N G . CHEM., 43, 1380 (1951). Stumpf, P. K., Green, D. E., and Smith, F. W., J . Bucl., 51,487 (1946). Trumo. J. G.. and Van de Graafl. R. J.. .J. Aaalied I'/L?Is..19. _599'(1948). Unger, E. D., Willkie, H. F., and JSlaiikmoynr, Ii. C., Y m n s . Am. I u t . Chem. Engrrs., 40, 421 (1844). Willkie, H. F., and Kolachov, P. J . , "Food for Thought," JJ. 136, Indianapolis, Indiana Farm Bureau, Inc.,(1942). Wilson, A. T., and Bruno, P., J . Ezpll. X e d . , 91, 449 (1960). i i c a m r m June 2,,1933. I~ECEIVED for review April 21, 1952. One of t h o laboratories of the Bureau OS Agricultural and Industrial C h e n istry, Agricultiiral Research Administration, 5. S. Department of Apricultmre. Report of a study in which certain phasea were carried on under t h e Research and Marketing Aot of 1946.
