Continuous Sterilization of Media in Biochemical ' Processes

v. F. PFEIFER AND CHAS. VOJNOVICH Norfhern Regional Research Laboratory, Peoria, 111.

HE succea9 of most fermentation processes is entirely depend- ent on the preparation of a sterile medium without appreci- ably affecting the fermentable substrate or the accessory

factors required by the organism involved. Dwtruction of mi- croorganisms 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 ul- travioIet radiation with wave lengths shorter than 2900 -4. (17); and a third method is mechanical removal of bacteria by atrat ion 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, deter- gents, and ethylene oxide (24) ; a fifth is by irradiation of the me- dium 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 com- binations of chemical disinfection and relatively mild heat treat- ment (6).

Batch sterilization with steam may be carried out in the fer- mentor itself, or in B separate tank. If a separate tank is used, the sterilized medium is pumped or blown by air pressure to 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 ul- traviolet 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 com- pletely removing contaminating microorganisms from media.

This paper describes pilot plant equipment used for continuous sterilization at the Northern Regional Laboratory, and its ap- plication in the continuous sterilization of various commercial media. Design of continuous sterilizers is discussed, and some general data obtained from commercial installations are pre- sented.

T

A Modified Form of the Arrhenius Equation Is Used to Calculate Sterilization Conditions

It is generally assumed that microorganisms are destroyed when subjected to 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 mi- croorganisms and time as a straight line by plotting the logarithm of the number of survivors against time. This may be rcprc- sented by

where h: is the specific reaction rate or rate of destruction of micro- organisms, co is the concentration of microorganisms at thc he- ginning 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 to pasa through one logarithm cycle, or by giving the period of half-life, the length of time necessary for half of the microorganisms to be destroyed. The half-life period, t l l g , may be obtained from thr ratc-timc Equation 1

(2)

The classical Brrhenius equation is used by Johnson (11) to

(3)

t i l 2 = 0.693/k

correlate temperature of sterilization with sterilization rate

log k = -0.219E/T + C

where ki and kz are specific reaction rates a t the two absolute tem- peratures, TI 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 indus- tries for approximating sterilization requirements. Using Equa- tion 3, a straight line is produced by plotting the logarithm of the specific reaction rate against the Iecipiocal of the absolute tem- perature.

A modified form of the Arrhenius relationship has bcon derived and verified experimentally by Higuchi and B u s e (9 ) to relate the sterilization time to the absolute temperature of sterilization

0.219E log fd = - T 4x lvhere t d is the necessary sterilization time, E is the heat of activa- tion 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 neces- sary sterilization time against the reciprocal of the nbsolutc t tm- perature of sterilization will give a straight line.

1940

August 1952 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y 1941

Since E and K are substantially constant, Equation 5 may be used to 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 that 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 unprofit- able. This is true in riboflavin production by fermentation with the yeastlike organism, Ashbya gossypii (14, 19), in which yields from continuously sterilized media we 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 con- tinuous sterilization are uniform, since the operating conditions are readily and simply maintained and duplicated.

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 in- vestment,

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, i f designed properly, is quite easy to clean and keep free from con- tamination. The problem of fouled heat transfer surfaces is minimized, because the high velocity of liquid flowing through the jet heater and holding coils helps to keep the surfaces clean.

Floor space required by a continuous sterilization installation is smaller than would be required for a comparable batch installa- tion 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 to keep the viscous mixture in motion is eliminated.

A continuous sterilizer of proper design may be used for a vari- ety of purposes. In addition to being used to sterilize a variety of media under widely varying conditions, i t may be used for cooking grain mashes, acid saccharification of mashes, or hydroly- sis 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

Figure 1. Horizontal Continuous Sterilizer Installation

1942 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 44, No. 8

cooler a substantial portion of the heat supplied by the steam may be recovered.

Metal contamination from a large tank during batch steriliza- tion 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 fermen- tation 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 to 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 con- densate. This may be counteracted by omitting that amount of water from the medium before sterilization. Although it is pos- sible to remove a part of the steam bj- flashing, cooling in a coun- tercurrent heat exchanger and recovering the heated water is sim- pler and more foolproof.

If iron must be excluded from the medium, special precautions may be required to 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 saccharifica- tion and hydrolysis conducted a t very low pH.

The medium to be sterilized is mixed in one of two 500-gallon Herculoy premix tanks, usually with hot water a t 120" to 140" F. to 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 to 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 1.5 to 12 gallons per minute.

The medium from the pump passes to the liquid side of a 1- inch 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 to form five sets of U's, mounted one above the other, and are well insulated. Valves are installed in a manner that allows use of from one to 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 pres- sure above the pressure of steam at sterilizing temperature to eliminate the 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

the inlet and outlet of the sterilizer holding coils. Temperature drop through the sterilizer amounts to 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 to about 8 pounds pcr square inch gage and cools to about 235 F. The medium cools t o fermenting tempera- ture as i t passes through a stainless steel, spiral-type heat ex- changer 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 main- tained 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 the cooler is adjusted. When operating conditions have become adjusted properly and the system is operating smoothly, water to the heater is re- placed 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 pure- culture 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 ex- pressed 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 at the h'orthern Laboratory is shown in Figure 3. This sterilizer was constructed of &-inch inside diame- ter 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 to 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 steril- izer 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 at 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-cul- ture 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 to investigate the relationships between time, temperature, pH, and raw materials in so far as they effect sterilization. In these ex- periments 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.

August 1952 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y 1943

Medium -C

E

/9'

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 con- ditions were adjusted properly and the system was operating smoothly, water to the heater was replaced with medium. Sam- ples 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. In 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 ascer- tained 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 de- scribed in this paper is listed in Table I. The medium de- scribed for vitamin Bl9 production is that developed by Hall et al. (7) .

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 sam- ples were taken over a range of temperatures. Minimum sterili- zation temperatures were determined by plating samples on nu- trient agar or by incubating samples in liquid nutrient medium. Results were checked when possible by microscopic examination

Results.

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 20- mesh screen. Results of typical data that were obtained in the treatment of riboflavin medium a t pH 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 to steril- ize this medium. A critical temperature range, above which sterilization took place and below which sterilization was not ob- tained, was determined for each of the various media described in Table I.

In another experiment with riboflavin medium treated for varying lengths of time at pH 4.4 and temperature of 237" F., medium retained for 10 minutes in the holding coils wm 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 tempera- ture 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 to 255 ' F.

If the solids were not uniformly and finely ground, much higher temperatures were required to effect sterilization of the medium. It was necessary to heat media containing coarse particles of ground corn or soybean meal to above 275" F. with 4 minutes re- tention time to sterilize them.

McCulloch ( l a ) has compiled the results of a number of inves- tigations on the exposure to moist heat necessary to kill all life. A summary of his compilation is given in Table IV, Although

The results are shown in Table 111.

1944 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 44, No. 8

Table 1. Media Used in Sterilization Experiments and Pure-Culture Fermentations

Afedium Used for Producing Ingredients in Medium, 70 Riboflavin Dextrose (anhydrous) 2 . 0

Corn steep liquor 1 . 8 Animal stick liquor 1 . 0 Soybean oil 0 .1

Vitamin Bln Diktiller's solubles plus soybean meal 4 0 Dextrose (anhydrous) 1 0 Calcium carbonate 0 5 Soybean oil 0 1

Butanol, acetone, and ethanol Xylose Ground corn (SHa)zSO4

3 . 8 1 . 8 0 . 1

Itaconic acid

Fungal amylase

Dextrose (anhydrous) (NHa)zSOd Corn steep liquor T\l,oS01 7H90

Distiller's solubles Ground corn

6 . 0 0 3 0 . 2 0 os 4 0 3 0

Table I t . Effect of Temperature in Continuous Heat Treatment of Riboflavin Medium

(Retention time, 4 minutes; pH, 4.4) Plate Count, Sterility Tube

Temp., O F. Colonies /Ml. Incubation No treatment 10,000 Contaminated

200 120 Contaminatnd 215 230 235 237 245

40 Contaminated 20 Contaminated

1 Contaminated 0 Contaminated 0 Sterilr

260 0 Sterile 275 0 Sterile

he claims that no living thing can survive 10 minutes direct CY-

posure to saturated steam at 250" F , other workers ( 4 ) have re- ported the existence of therniophilic spores which required 23 minutes of direct exposure to 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 me- dia 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 va- ried to conform to the types of raw materials used and that mini- mum conditions for a particular medium must be determined by expeiinient.

Operating Conditions Must Ensure Sterilization and Preclude Damage to Nutrients

The aotual operating conditions that are employed a t this lab- oratory 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 sterili- zation 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 par- ticle size of solids is often quite variable. Even though care is taken to 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

yields obtained in pilot plant fermentations of this sterile medium with Ashbya gossypii were 500 to 600 y per ml. compared to yields not exceeding 100 y from sterile medium that was batch sterilized a t either high or low pH. Results were most consistent and sat- isfactory t? hen sterilization was carried out continuously at 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 described (Table V). When batch sterilization R T ~ employed, it nas necessary to hold the medium for 11/2 to 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 TIL

Continuous sterilization was particularly useful in the prepara- t 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 wm used, very little furfural mas produced.

Medium for the pilot plant production of sodium gluconate which was sterilized continuously at low temperaturw was pccu- liarly 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.

In 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

Critical C m p . hLedium C'sed for Producing PH Range, F. Riboflavin 4.4 235-245 Vitamin BIZ, no soybean meal 6 7 230-240 Vitamin BIZ. with half soybean meal 6 7 240-250 Sodium gluconate 6 0 215-225 Itaconic acid 6 . 7 220-230 Fungal amylase 6.5 240-250

Table IV. Time-Temperature Relationships for Wet Sterilization

(Data of Novy, Jordan, hfuir and Ritcbie, l lacfarland. Erne, Besson and Sternberg, compiled by McCulloch. 1945)

Minutes for Thermal ,Death of -411 Bacterial Life Temp., O F.

265 248-250 239-246 10 230-244 15 221-242 20

Table V. Actual Operating Conditions Employed at NRRL for Continuous Sterilization of Media

Retention Time, Medium Used for Producing p H Temp., O F. Minutes

4 . 5 4 . 5 6 . 5 4.5 6 . 1 5 . 0

278 325 275 275 300 325

4 13 3 5 5

13

August 1952 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 1945

Table VI. Effect of Medium Sterilization on Yields of Riboflavin Produced by A. gossypii, NRRL Y-1056

Corn Animal Max. Steep Stick Riboflavin

Glucose, Liquora, Liquor", Yield, % % % Sterilization Method y/hfl.

2 . 0 1 . 9 0 . 8 Batchwise 250' F 45 min. 6 6 pl i 5 2 . 1 1 . 9 1 . 0 Batchwise: 250" F:: 25 min.: 4:4 p H 88 2 . 0 1 9 0 9 Continuous. 275' F.. 5 rnin.. 6.5 n H 360

stick sterilized separately a t 275°F. 2 . 0 1 . 9 0 . 9 Continuous. 275' F.. 5 min.. 6.5 UH 158

As is basis.

Table VII. Effect of Medium Sterilization on Yields of Vitamin BIZ Produced by S. oliveaceus, NRRL B-1125

Extd. AIaximuiii Distiller's Soybean Vitamin Solubles, Meal, Yield.

% % Sterilizlition Method ?/MI. 4 . 0 " . . . Batchwise, 2 hr., 250O F., p H 4.8 0 . 1

2 . 0 " 2 . 0 Batchwise, l l / a hr., 250' E'., pH 5.7 1 . 3 4 . 0 " . . . Continuous, 13 rnin., 325O F., pH 4.8 1 . 2

2 . 0 " 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.

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 that 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 to the heater, cent'rifugals predominate. The output rate is meas- ured 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 to 8 hours.

Media are heated to sterilization temperature by direct injec- tion of steam in all cases reported. The devices used for this pur- pose 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 classifi- cations: (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 to 12 inches in diameter.

Average liquid velocities in the holding pipes vary from 0.1 to 2.1 feet per second, with 0.2 to 0.4 foot per second the preferred range. The Reynolds numbers for these installations vary from 36,000 to 273,000, with 38,000 to 80,000 the preferred range. This indicates that medium passes through the retaining or hold- ing 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 to 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 oppo- site end of the cooker.

Sterile medium leaving the holding section is discharged through some device which retains the prcssure in the sterilizer itself. For this purpose throttling-type globc valves, instru- ment-operated back pressure valves, and fixed orifices are used. Manually adjusted throttling valves are preferred in most plants for this purposc.

Figure 3. Vertical Continuous Sterilizer Installation

Sterile inedia arc usually cooled to fermenting temperature by passing them through a double-pipe heat exchanger countercur- rent to flow of cold water. Special precautions are taken in in- stalling some typcs of indirect coolers t o ensure that both mash and water sides drain completely, so that t,he cooler may be ster- ilized properly before use. Precautions are also taken in desigii- ing and installing coolers to prevent nonsterile water from enter- ing sterilo medium in case of leakage. Coolers of the double- head type are also used for this purpose, since containination of the mash v i th cooling water is minimized in this type of construc- tion. In s o ~ c e plants, however, the medium passes tJo the fep mentor without cooling, flashes in the fernlentor to about 220" F., and is then cooled t o fermenting temperature by passing cold wa- ter through thc jacket or coils.

Hot water leaves the cooler a t 150" to 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.

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

Onc company (3 ) employs

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 Vol. 44, No. 8

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 tein- peratures reported on media used for pure-culture fermentations vary from 250' to 325' F. and holding times from 4 to 14 min- utes. Preferred conditions for sterilizing media containing rela- tively small amounts of rather finely divided suspended solids are in the range of 275 to 28.5' F. with a holding time of 3 to 5 min- utes and pH 4.0 to 5.0.

Brown et al. (,2) describc another type of direct steam injection heater which eliminates the possibility of burned material eollect- ing on the steam heated walls or pipes of the heater. The heater is constructed with tangential steam inlet to 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 ef- fecting 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 160 O F., to facili- tate pumping and passage through the cooker and cooler.

Slurry heated to 140" to 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 to 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 mix- ing 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-inch iron pipe, extra heavy, is a preferred construction material for the holding section. Some difficulties have been reported with erosion and subsequent failure in thr holding section, particularly in return bends.

Average liquid velocities in the holding pipes vary from 0.4 to 1.5 feetper second. Unger el al. (22) have reportedthat ataveloc- ity of 1.5 feet per second in the holding pipes, there was mb- stantially no deposit on t,he walls of the pipe, and it was not nec- essary t o clean the cooker of caramelized mash a t weekly inter- vals.

The patent literature also describes the UEC of one or more vei- tical 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" to 250" F. in a flash tank, and the regenerated steam a t 10 to 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 tempera- ture 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 to 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.

Continuous Sterilization Wil l Be Installed in Many New 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 pure- culture 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 apprcci- ably 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 ncc- c'ssary for profitable operation of the procees. Equipment is sim- ple, 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 proc- CSR, the possibilities of higher yields, snioot,her operations, and lowered operating costs should certainly be investigated for exist- ing fermentation processes. Continuous sterilization equipment will be installed i n many ~icw plants for the production of vita- mins, antibiotics, enzyme concentrates, and industrial organic chemicals.

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IIamre, D., J . Bact., 57, 279 (1949). Higuchi, T., and Busse, L. K., J . Am. P / ( U T / / [ , jlssoc., 39, .I i 1

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,

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 , ,

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Stumpf, P. K., Green, D. E., and Smith, F. W., J . Bucl., 51,487

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599'(1948). Unger, E. D., Willkie, H. F., and JSlaiikmoynr, Ii. C., Y m n s .

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