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treated wool, and that the resistance increases as the content of unchanged cystine decreases. Table V indicates results obtained when wool samples were subjected t o the action of moth larvae, and photographs of the actual samples are shown in Figure 2. The modified materials having cystine contents of 6 per cent or less were only slightly attacked, and this attack was mainly around the edges, while numerous holes were eaten into the untreated samples. It also appears that reducing the cystine content t o 6 per cent provides about as much protection as the more extensive treatments. It should be pointed out that the moth-proofness produced by the present method is not removed by laundering or dry cleaning, since it is “built into” the molecule as an integral part of its chemical structure. Carpet beetles were found to damage both the untreated and the modified wools more extensively than did moth larvae (Table V and Figure 2). Nevertheless, the modified wools were decidedly more resistant than untreated wool t o such attack.
Acknowledgment The miters wish to thank John A. Levering and J. W. Creely of Eavenson & Levering Company, Camden, N. J.,
Vol. 34, No. 11
for the tests with moth larvae, and A. G. Ashcroft and R. C. Allison of Alexander Smith & Sons Carpet Company, Yonkers, N.Y., for the tests with carpet beetles.
Literature Cited Crowder, J. A., and Harris, M., J. Research Natl. B u r . Standards, 16, 475 (1936). Ibid., 17, 577 (1936). Elsworth, F. F., and Phillips, H., Biochem. J.,35, 135 (1941). Geiger, W. B., Patterson, W. I., Mizell, L. R., and Harris, LM., J. Research Natl. B u r . StandarLs, 27, 459 (1941). Harris, M., Mizell, L. R., and Fourt, L.. IND.ENG. CREM., 34, 833 (1942); J . Research Natl. B u r . Standards. 29. 885 (1942).
LinderstrZm-Lang, K., and Duspiva, F., 2. phusiol. Chem., 237, 131 (1935).
Lowe, C . S., Lloyd, A. C., and Smith, A. C., Am. Duestuff Reptr., 30. 81 - 11941). Mease, R. T., 2. Research Natl. Bur. Standards, 13, 617 (1934). Natl. Bur. of Standards, Commercial Standard C S K - 3 6 (1936). Patterson, W. I., Geiger, W.B., Misell, L. R., and Harris, M., J . Research N a t l . B u r . Standards, 27, 89 (1941). Reimann, G. B., and Mease, R. T., Natl. Bur. Standards, C ~ T C617 . (Nov. 8 , 1940). Rutherford, H. A., and Harris, M., J . Research Natl. B u r . Standards, 20, 559 (1938). Smith, A. L., and Harris, M., Ibid., 16, 301 (1936). Sullivan, M. X., Public Health Rept. 86 (1930). I
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Continuous Aerobic rocess for Distiller’s Yeast ENGINEERING AND DESIGN FACTORS E. D. UNGER, W. H. STARK, R. E. SCALF, A”D P A U L J. KQLACHQV Joseph E Seagram RE Sons, Inc., Louisville, Ky. HE present practice usually employed for the production of distillery yeast involves: (a) The preparation of mash consisting of equal parts of rye and barley malt meals. (a) Souring this mash under conditions of active growth for lactic bacteria. This lactic bacteria can be produced spontaneously either from the mash or from a pure culture medium. Souring is carried out until the pH is lowered to the range 3.54.0. (c) Pasteurizing the mash under conditions R-hichdestroy substantially all of the lactic bacteria. (d) Cooling the mash to the yeast inoculating temperature. (e) Inoculation with a yeast culture and propagation of the yeast under anaerobic conditions a t temperatures suitable for yeast growth. In the above technique the yeast inoculum to the fermenter amounts to 3.54.0 per cent, based upon the weights of grain used. Therefore, equipment for mashing and propagation is large in volume. Over-all processing time from mashing to final yeast is long. In the case of a lactic-soured yeast mash 40 hours are required; for a sweet yeast mash or where sulfuric acid is used for pH adjustment, this time can be reduced t o 20-22 hours. As a result of the above factors nine yeast tubs, each having a capacity of 3000 gallons, are required for a distillery fermenting 10,000 bushels of grain per
T
day in the case of lactic-soured yeast. Although the anaerobic yeast process has been employed with success in distillery operations by the application of thorough sanitation conditions to the yeast room and yeasting equipment, this yeast is not pure culture and may be nonuniform with regard to quality and fermentability. For these reasons work was initiated on an entirely new concept of distillery yeast production, having the following objectives: the production of a pure culture yeast; the production of a yeast having high quality, uniformity, and fermentability ; production under conditions which would result in a very high concentration of yeast--500,000,000 cells per ml.; production of yeast in a process that would be continuous in operation. The obvious method of attack on this problem was to utilize the aerobic method for the propagation of the yeast. The effect of aeration of the yeast medium is twofold: Oxygen in the air promotcs respiration of the yeast cell, and therefore a greater concentration of yeast cells is produced; and agitation caused by the sparged air results in a more rapid growth of the yeast. It is common practice in the production of baker’s yeast to employ the aeration process because of the above advantages.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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produced in comparison with the usual 150,000,000 cells per ml. obtained by the sour-mash anaerobic process. Fermentation data (2) proved that this yeast compared favorably with the old process in fermentability. Thus a means of solving three of the initial objectives was accomplished in the laboratory. I n order to obtain data comparable to a production basis, a pilot plant was constructed (Figure 1). The operation of this unit is outlined in Figure 2.
Present yeasting practice in distilleries is undesirable because the resultant yeast is not pure culture and lacks uniformity, and because long processing time requires large equipment. The advantages of a completely aseptic technique for yeast production, combining the principle of an aerobic process and a continuous system for propagation of distiller’s yeast, are discussed. Problems involved in the development of a continuous process of yeast production are mentioned. The design of a pilot plant for carrying out the experimentalwork on such a process is presented. Factors involved, such as effect of media, filtration of mash, sterilization, sparger porosity, air consumption, and rate of propagation, are discussed. The design is given of a proposed system for carrying out this process on a production scale.
FIGURE 1. PILOT YEASTPLANT
However, no data are available in the literature on the quality of yeast produced by such a process for distillery purposes. Furthermore, none of these commercial yeast processes are completely continuous in operation, a fact which was one of the primary objectives of this investigation. Laboratory data have already been presented by Stark et al. (I,$) on factors which influence yeast propagation. The results of these studies have led to the development of a continuous process for distiller’s yeast production as described below.
Experimental Procedure
It was found possible in laboratory experiments to produce a good quality yeast by the use of a wort prepared from a mash bill1 containing 40 per cent corn, 30 per cent distiller’s barley malt, and 30 per cent long-fiber malt sprouts. Five hundred milliliters of sterile 10” Balling wort a t a pH of 4.5 were inoculated and were aerated with 0.4 cubic foot of sterile air per minute through two spherical Aloxite (electrically fused alumina) spargers. By this means a pure culture yeast at a concentration of 500,000,000 cells per ml. was 1 In distillery terminology the “mash bill” means a listing of the quantities of grain constituents used in the preparation of the mash; these constituents can either be calculated on a percentage basis totaling 100% or on a weight basis adding up to the total weight of the grain in the mash. “Dona” refers to the stock culture used to inoculate the yeast media, which in turn is required to inoculate thefermenter. “Diamalt” is a sugary sirup prepared by the infusion of barley malt in water, the filtration of mash slurry to give a clear liquid, and the evaporation of this liquid t o yield a sirup. The sirup contains maltose, dextrins, and amylase enzymes.
(I
M A S H I N G VESSEL
9
I
yyzER
OLIVER FILTER
STRAINED WORT
STORAGE
E ; : ; : STORAGE
1
FIUUREI 2.
BOWSER FILTER
FERMENTER
YEAST
P R O PA G A T 0 R -----STERILE
-
AtR YEAST
STORAGE
SCHElbiATIC LAYOUT OB PILOT YEA8T PLANT
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Simultaneously with the operation of the pilot plant, laboratory experiments were continued to determine the following optimum conditions:
W+SH
WATER
PfiOTEiN COAGULATION INFUSION
,
CLARIFIED iTEAM
COdLIhG
WATER
TRANSFER PUMPS
MASHING &
FII T R A T I O N
WORT
i
SYSTEM
A M M O N I A
FOAM
DONA VESSEL
I I
WORT STERILIZATION YEAST STORAGE
C O N T I N UOU 5
AERATION
P R OCE 5 5
FIGURE 3. PROPOSED YBASTPLANT
A previously prepared mash consisting of 40 per cent corn, 30 per cent barley malt, and 30 per cent malt sprouts a t a concentration of 30 gallons per bushel is strained on a rotary Oliver filter to remove most of the grain husks. The strained wort is dropped into a tank to which is added 1-2 per cent of filter aid. This mixture is then atered through a Bowser filter to yield a 10' Balling clarified wort, which is transferred to storage vessels and sterilized. Meanwhile a yeast inoculum is prepared in the dona' vessel using an 18" Balling medium prepared from diamalt'. This inoculum is added to a portion of the clarified wort in the propagator, and the sterile air is sparged into the vessel. As the yeast count increases, more wort is sprayed into the propagator until the volume has increased to 300 gallons and the count reaches 400,000,000 cells per ml. At this point operation becomes continuous with tt wort stream to the propagator of 75 gallons per hour and an equal drawoff rate of yeast. In this process it is necessary to add ammonium sulfate and ammonia to the wort to maintain an amino nitrogen level of 20-25 mg. per 100 ml. and OCcasionally to add an antifoam agent of Vegefat and lard.
TO
ST0 RAGE
STERILE WOR?
ANTI
Vol. 34, No. 11
SOURCE OF MEDIA. The two most satisfactory media were obtained from a mash bill prepared from a mixture of (a) 40 per cent corn, 30 per cent barley malt, 30 per cent lon -fiber malt sprouts, or ( b ) 85 er cent barfey malt and 15 per cent long-figer malt sprouts. CONCENTRATION OF WORT. A 10' Balling wort was capable of producing 500,000,000 cella per ml. STERILIZATIONCONDITIONS. Twentyminute sterilization at 121" C. (250' F.), followed by immediate cooling, will produce complete sterility. AIR SPARGERSURFACE AND POROSITY. Three square inches of sparger area per gallon of workin capacity are required. A No. 60 teardrop-sf aped carbon sparger manufactured by the National Carbon Company was found capable of giving good air dispersion. AIR CONSUMPTION. One fourth cubic foot of air per minute per gallon of working capacity is required. EFFECTOF YEAST~VUTRIENTS. No nutrients other than ammonia and ammonium sulfate are required. CONCENTRATION OF AMINONITROGEN. The amount of ammonia and ammonium sulfate required is enough to maintain the amino nitrogen level at 40-45 mg. per 100 ml. in the propagator. TEMPERATURE OF PROPAGATION. A temperature of 28.3" C. (83" F.) is maintained. pH OF MEDIUM.The pH is maintained at 4.0-4.2 by addition of ammonia.
From an engineering standpoint the problem of filtration was of considerable importance. No one-stage system has yet YERST T O been found capable of producing a clariFER M E N T E RS fied wort from the mash, nor would such a system be desirable. The best method developed to date involves first the use of an Oliver filter for straining the wort and washing the grain husks. This unit is capable of straining 20-25 gallons of mash per hour per square foot. The recovery of sugar from the mash is 82 per cent complete from an 85-15 mash bill. The strained wort contains 1-2 per cent suspended solids. To filter out this protein material is difficult. Regardless of the type of filter used, the cycle yields only 1-2 gallons per square foot before cleaning is required. Using filter aid, this capacity per cycle can be increased to 3 gallons per square foot, but even this is inadequate for production purposes. Therefore, either a selfcleaning precoat filter or a centrifuge is required. The use of a centrifuge works out satisfactori1y"and is being adapted for final clarification. Data obtained with a centrifuge are shown in Table I. Previous to final clarification, it is desirable to preheat the strained wort to 100-121" C. (212-250' F.) and hold i t for a period of 5 minutes. This causes coagulation of colloidal protein and greatly facilitates the centrifuging process.
Proposed Yeast Plant The problems involved in a completely continuous system for processing grain into yeast are complex, and to date i t cannot be stated that they are completely solved nor can
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INDUSTRIAL AND ENGINEERING CHEMISTRY
they be solved until experience has been gained under actual plant operating conditions. These problems can be summarized as follows: It is essential that all steps in the process be completely synchronized with each other and automatically controlled; it is essential that the equipment and the wort be completely sterile in the yeastpropagating section of the process; sterilization of the wort must be carried out in a continuous system and must be 100 per cent complete. I n addition, the usual problems of efficient process design are encountered. The method proposed for carrying out the yeasting process on a production basis and using a continuous processing system is shown in Figure 3. This system has sufficient capacity to supply a distillery having a capacity of 5000 bushels per day. For this size plant the yeast grain requirements are approximately 50 bushels per day in contrast to 175 bushels by the anaerobic process.
TABLE I.
CLARIFICATION OF STRAINED YEAST WORT' BY
CENTRIFUGATION^
Suspended Solids in Supernatant Liquorc. % Speed, R. P. M. 0.18 1600 0.08 2000 0.06 2500 0 Strained wort 1.5 per cent suspended solids. b In a solid bo41 oentrifuge; basket dimensions are 11-inoh diameter and 4-inch height. Rate of wort feed, 0.2 gallon per minute.
For this process a wort prepared from a mash bill consisting of 85 per cent barley malt and 15 per cent malt sprouts is preferred. The barley malt is obtained in the form of a stream of mash at a concentration of 35 gallons per bushel and 60" C. (140" F.) from the continuous cooker system (3)used for the preparation of the main mash for fermentation. A quantity of malt sprouts (7.5 bushels) sufficient for 24 hours operation is charged into the scale hopper from where it is fed continuously by a feeder device into the malt mixing chamber having a working capacity of 10 gallons. Mixing and agitation are effected by a centrifugal pump which circulates the combined mixture. The mash is simultaneously heated to 63" C. (145' F.) by steam sparged into this vessel. From here a continuous stream of the mash is pumped into the infusion tube where it is maintained a t this temperature for 30 minutes to permit solubilization of malt starch by enzymes in the malt and infusion of the yeast nutrients from the barley malt sprouts into the water phase. This tube is 8 inches in diameter and 25 feet long. The mash is then transferred to the rotary Oliver filter 3 feet in diameter and 1 foot wide, where i t is strained through a 40-mesh screen and the cake is washed to recover all available sugar. This cake is then repulped with water and pumped back to the continuous cooker system. The strained wort flowing down to the vacuum receiver vessel is pumped to a Paraflow plate-type heat exchanger where its temperature is raised to 100-121" C. (212-250" F.). The heated wort is maintained at this temperature for 5 minutes in the protein coagulation tube which is 8 inches in diameter and 6 feet long. The coagulated wort is then cooled in this heat interchanger to 49-65.5" C. (120-150" F.) and is centrifuged. The clarified wort is pumped to the yeasting system while the suspended solids are returned to the continuous cooker system. The over-all recovery of sugar by this process is 75-77 per cent complete. The clarified wort is transferred to a small storage vessel preceding the yeasting process. This vessel has a working capacity of 60 gallons, sufficient for 0.5 to 1hour of operation.
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It is jacketed, and refrigerated brine is circulated through it to maintain the temperature a t 7" C. (45" F.) to prevent bacterial growth. The vessel is maintained under air pressure, which serves to transfer the wort to the sterilizer and propagator. The wort is sterilized continuously by first passing i t at a measured rate into the preheating section of a heat exchanger, then into a jet heater where its temperature is raised to 121" C., holding it a t this temperature for 20-30 minutes in B tube to effect sterilization, and finally cooling it in the heat exchanger before transferring i t to the propagator. The propagator is a stainless steel vessel with a volumetric capacity of 530 gallons and a working capacity of 225-250 gallons. This allows for foaming of the aerated wort. Its dimensions are 3 feet in diameter and 10 feet in height. Into the vessel air is continuously sparged. This air at a pressure of 40 pounds per square inch in the compressed air tank is first filtered to remove all suspended particles and is then passed into a chamber fitted with Westinghouse Sterilamps where all remaining bacteria are destroyed. This type of sterilization for air is claimed to be 100 per cent effective. As yet, however, no tests have been made on this system. I n pilot plant experimentation a series of cotton pad filters proved to be entirely satisfactory for air sterilization purposes. The air requirements for this propagator are 60 cubic feet per minute, equivalent to 0.25 cubic foot per minute per gallon of wort. The sparger surface required is 875 square inches, equivalent to 10 cubic feet per minute per square foot of surface. As pointed out previously, the teardrop-shaped carbon sparger is the preferred equipment. Good dispersion is attainable, and tests indicate a satisfactory life cycle. An air pressure of 10 pounds per square inch is maintained within the vessel by means of a back-pressure control valve; this prevents any bacteria from entering the propagator from the outside. The level is maintained in the propagator at 240 gallons, and the sterile wort is sprayed into it at the rate of 60 gallons per hour. This allows an average propagation time of 4 hours to grow the yeast and deplete the medium of sugar. The finished yeast wort contains approximately 1.75 per bent alcohol. This yeast is drawn off continuously at the bottom of the vessel and passes to a centrifuge where the yeast cell concentration is further increased from 500,000,000 to 2,000,000,000 per ml. The supernatant liquor is then returned to the distillery, and the yeast cream is transferred to refrigerated storage where i t is kept until required for use. With this type of continuous propagation, sterility is extremely important. It is known that this operation can continue for one week without developing any contamination and will produce a good quality yeast. It is believed that this period of operation can be extended to 30 days without difficulty. Conclusions With this process as outlined, all of the initial objectives have been accomplished. Furthermore, we believe that it can readily be adapted to the production of baker's and brewer's yeast and to the construction of central yeast plants for distillery and industrial alcohol plants.
Literature Citations (1) Stark, W. H., Kolachov, P. J., and Willkie, H. F., Am. SOC. Brewing Chem., PTOC. 4th Ann. Meeting, 1941,49-56. (2) Stark. W. H.. Scalf, ,R.E., and Kolachov, Paul, Soc. of Am. Bacteriologists, Baltimore, 1941. (3) Unger, E. D., Theais, Case School of Applied Scieneb. 1941. PR~SH~NTBD before the Division of Industrial and Engineering Chemistry at the 103rd Meeting of the AMBBIOAN CHEMICAL SOCIETY, Memphis, Tenn.
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