I
H. G. MAISTER, E. N. HEGER‘, and W. M. BOGARTZ Northern Utilization Research and Development Division, U.S. Department of Agriculture, Peoria, 111.
Continuous Freeze-Drying of SerrufiCr murcescens This is the most complete development study of freeze-drying yet published
Tm
principles of vacuum sublimation of ice from frozen materials have been studied extensively (7, 3-6, 8, 9, 77), and in the past decade considerable interest has developed in their application to the preservation of biological substances. Freeze-drying techniques have made it possible to store living organisms over considerable periods (6,8,70, 72, 74). T o study the effects of various conditions on the ability of a representative bacterium, Serratia rnarcescens, strain 8 UK, to survive continuous freeze-drying and subsequent storage, it was necessary to dry the bacteria on a prepilot-plant scale and to ensure good recovery of viable cells. A continuous freeze-dryer was designed and assembled a t Fort Detrick, Md. [the Fort Detrick continuous dryer (73)]. I t was especially desired to determine the most suitable methods of processing fermented medium prior to drying, the best fortifying materials for protecting the viable cells during drying, and the best operating conditions for drying S. marcescens.
a cooling system, two vacuum pumps, gages, and thermometers. The main vacuum pump takes suction from the condenser, which consists of a third glass bel1 jar containing a copper coil with 8.5 square feet of heat transfer surface. A stream of mineral spirits (boiling point 300 to 410’ F.), cooled by dry ice, is circulated through the coil to freeze out moisture in the vapors passing to the vacuum pump. The condenser is connected to the main vacuum chamber by a pipe with a 4-inch outside diameter, in which is installea the ionization chamber of an Alphatron vacuum gage. This gage is coupled to a recording unit which produces a continuous record of the vacuum inside the main chamber. The screen belt is of 11mesh stainless steel, 11 inches wide, and approximately 10 feet long with an effective length, between rollers, of 4
feet. I t is connected through a system of pulleys and belts to a motor capable of varying the speed of the screen between l .5 and 10 feet per hour. The feed chamber is located about 2 feet above the center of the screen belt; it is jacketed with 24 feet of 3/s-inch copper tubing, through which a stream of mineral spirits cooled by dry ice is circulated to prevent melting of the pellets during feeding. Pellets pass from the feed hopper through a diaphragm valve onto the inclined fluted chute in the main drying chamber. A magnetic vibrator actuated by an external push button, mounted on the chute, shakes pellets down into a hopper just above the forward end of the belt. A vibrator attached to this hopper settles the pellets uniformly on the belt, which moves them under a roller designed to prevent “doubling” in the
Equipment and Procedures
Equipment. The continuous freezedryer is composed of a main vacuum chamber made up of two glass bell jars, 17l/2 inches in inside diameter, a screen belt driven by a variable-speed motor, a feed chamber, an inclined feeding chute, a hopper for distributing feed pellets on the screen, a Nichromewire heater, an inclined discharge chute, a collection bottle, a condenser,
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layer. The pellets then move into the heating zone, which begins 1 foot from the forward end of the belt and extends to about 5 inches from the discharge end. Radiant energy is supplied from electrically heated Nichrome wire stretched back and forth across the belt, and about 1 inch above it. The heater is divided into two parts for close control of temperatures. The heating elements are arranged so that the energy applied decreases along the length of the heating zone in logarithmic fashion. The front section consists of 14 feet of straight Nichrome wire, No. 24 American wire gage, distributed in 18 transverse lengths, with a resistance of approximately 25 ohms, and capable of dissipating up to 500 watts at 110 volts. The rear section consists of 16 feet of No. 25 A.W.G. coiled Nichrome wire distributed in four transverse lengths, with a resistance of approximately 34 ohms, and capable of dissipating up to 350 watts at 110 volts. During normal operation, most of the required heat is applied in the front section, and only about 15 to 30 watts are applied to the rear section. Voltage applied to each section is adjusted with a separate variable transformer. Three dial thermometers are mounted in the heating zone: one at each end, and the third 5 inches from the discharge end. An inclined fluted discharge chute, mounted below the screen discharge, is equipped with a vibrator to roll the dried product down the chute into the collection bottle. The collection bottle and the feed chamber can be isolated from the main chamber, and the two are connected to the second vacuum pump with a l/Z-inch manifold system. Air entering the system is dried by passage through concentrated sulfuric acid and then through Drierite. Operation. Before the continuous freeze-dryer is started, the mineral spirits bath is cooled to -63' C. by dry ice, and the cooled liquid is pumped through the condenser and feed chamber jacket. The entire system is closed and evacuated to 50 microns. The feed chamber is isolated from the drying chamber, and brought to atmospheric pressure with dried air. Frozen pellets are poured into the feed chamber, which is evacuated to at least 300 microns before the valve connecting it to the drying chamber is opened. Frozen pellets are fed through the diaphragm valve, down the inclined chute into the hopper, and are carried by the screen out of the hopper, under the roller, and into the heating section, where the two heating circuits are adjusted to give the desired temperatures. The dried product drops from the end of the belt to the inclined discharge chute, and passes to the collection bottle. When the collection bottle is full, it is isolated from the drying chamber, brought up to atmospheric pressure with dry air, and re-
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placed with an empty bottle. The feed chamber is filled with frozen pellets when necessary, the heating circuits are adjusted to give the desired conditions, and the collection bottles changed as required. Methods of Analysis. The solids content of fermented medium or fortified concentrate was determined by measuring the weight loss of a 5-ml. sample. The sample was frozen while spinning in a test tube, desiccated in high vacuum for 3 hours, and placed in a vacuum oven operating below 150 microns for 22 hours at 50 O C. The final moisture content of the dried pellets was determined by the method of Flosdorf and Webster (7), for dry biological substances. The dried pellets were placed for 22 hours in a vacuum oven operating at 50' C. and a vacuum below 150 microns, and dried to constant weight. Reproducible results were obtained. Viable cell counts were made by a "streak plate" method originated at Fort Detrick and modified by Benedict and others (2). Solids and final moisture content were determined in triplicate; viable cell counts were made in duplicate. Preparation of Biological iMaterial for Drying. Three types of cell suspensions were obtained from the following fermentation products: Fermented medium fortified with a protective material, having a solids content of 9 to 10 grams per 100 ml. (fortified fermented medium). Bacterial paste produced by centrifuging diluted fermented medium in a Sharples supercentrifuge, reconstituted in water or effluent from the centrifugation and fortified (reconstituted Sharples concentrate). Solids content was approximately 10 grams per 100 ml. Bacterial slurry produced from undiluted (solids content approximately 15 grams per 100 ml.) or diluted (solids content 16 to 17 grams Rer 100 ml.) fermented medium by continuous centrifugation in a Sharples DV-2 centrifuge, and fortified (undiluted and diluted DV-2 concentrates). Before any suspension was dried, it was first converted into solid, frozen pellets by feeding the liquor very slowly to electrically heated hypodermic needles which projected through the bottom of a steel tank containing a mixture of equal parts of Freon 11 and Freon 113 a t -50' C. The droplets froze immediately to form small spheres which floated to the surface, and were transferred by a wire-screen scoop to a wire basket surrounded by crushed dry ice, where they remained for several minutes to allow drainage. Finally, they were transferred to a cabinet kept a t -40' C. for storage and vaporization of the remaining traces .of Freon. Immediately prior to drying, the pellets were screened in U. S. Standard sieves, and only those passing No. 4, and retained on No. 6, were used. Preliminary studies showed that the
INDUSTRIAL AND ENGINEERING CHEMISTRY
formation of spheres, rather than particles of irregular shape, in the pelleting process was strongly favored by a high solids contenK of the liquor. Fermented mediums could be pelleted only with considerable difficulty; concentrated liquors produced very uniform, almost spherical, pellets. Operation of the dryer was most satisfactory with pellets from concentrates. Pellets prepared from fermented medium tended to stick together, especially at pressures over 100 microns, and formed uneven layers on the screen belt.
Results The following factorswere investigated : protective materials, variations in feed materials, harvest time in the fermentor, moisture content in the dried product, pH control, temperature in the heating section, and belt speed. I t was necessary to have fermented media of uniformly high quality, as satisfactory drying recoveries and storage stability of the dried products were always associated with satisfactory fermentations. Protective Materials. Naylor and Smith (70) found that addition of a mixture of ammonium chloride, thiourea, ascorbic acid, and dextrin to a fermented liquor greatly increased the ability of S. marcexens to survive drying. They proposed adding 0.5% each of the first three components, and 2.0% of dextrin, based on the weight of the liquor being fortified. In the present work nu dextrin was used, because large amounts of solids were present in the medium and concentrates; 0.5% each of ammonium chloride, thiourea, and ascorbic acid gave excellent results with fermented medium, and 1.0% of each was satisfactory for fortifying suspensions of concentrates. Benedict and others (Z), found that several other protective materials gave results comparable to or better than the Saylor-Smith combination in drying under very mild conditions. Experiments in which DV-2 concentrates were fortified with a variety of protective materials are reported in Table I. Only the Naylor-Smith mixture gave satisfactory results under the conditions in the continuous freeze-dryer. T o prevent damage to the microorganisms during fortification and drying of the fermented medium or concentrates, the pH was maintained at or near 7.0 by addition of sodium hydroxide. Feed Materials. The ability of living cells contained in the pellets produced from various types of fermentation products to survive drying is shown in Table 11. All the materials were fortified with the same protective material prior to pelleting. The survival of viable cells from concentrated materials is considerably higher than from fermented medium. Sharples concentrates reconstituted with effluent, and DV-2 concentrates from medium in which the
C O N T I N U O U S FREEZE-DRYING and drying recovery and storage stability of the viable cells. The drying recovery of viable cells increases with increasing moisture content of the product, but cells are better able to survive storage if dried to a lower moisture content. To determine the conditions for best balance between these two opposite effects, frozen pellets produced from one fermented medium were dried to various moisture contents, and stored a t 50' C. in sealed tubes evacuated to 40 microns of mercury pressure for up to 6 weeks. A final moisture content of O.51y0 resulted in the highest over-all recovery. Subsequent drying experiments with frozen pellets from fermented medium were therefore directed toward producing pellets having a moisture content of 0.5 to 1.O%. A similar experiment was carried out with pellets from DV-2 concentrate; highest over-all recovery was obtained from dried pellets containing 1.lo% moisture. In subsequent drying experi-
pH was controlled during fermentation, yielded the highest recoveries of viable cells in the dried product. Harvest Time of Fermentation. In several experiments fermenting media were collected after 16, 20, 24, and 28 hours of fermentation, to determine whether ability to survive drying was dependent on the length of growing time in the fermentor. Figure 1 shows that the percentage recovery of viable cells was low for material harvested before 16 hours, but much higher after 18 hours. The viable cell count in the fermented medium was a t or near a maximum after 22 hours of fermentation. As this point was a convenient one a t which to discontinue the propagation, most fermentations were halted a t age 22 hours, and the media were processed immediately. Optimum Moisture Content of Dried Product for Recovery and Storage. Figure 2 shows the relationship between moisture content of the dried pellets
ments with pellets from DV-2 concentrates, the moisture content was kept in the range of 0.8 to 1.5%. Influence of pH Control during Fermentation, During the first part of the fermentation cycle sugar in the medium is used rapidly in the formation of organic acids; pH is lowered to 5.5 or slightly less. Thereafter, proteins and amino acids are assimilated, liberating ammonia, which increases the pH to 8.0 or higher. Preliminary experiments indicated that the pellets dried and stored better if the pH during the acid formation cycle was controlled to 6.5 to 7.0 by addition of sodium hydroxide. Later experiments indicated that drying recovery and storage ability were further improved if pH was 6.8 to 7.2 during the entire fermentation. Experiments were carried out with pellets from DV-2 concentrates, in which sodium hydroxide was used to control the pH during the acid formation cycle, and a variety of inorganic acids was used during the ammonia formation cycle. Hydrochloric acid gave slightly
Table I.
Effect of Fortifying Materials on Recovery of Viable S. marcescens Product * Feed ProducV.C. Fortification Solids, tion rate, Moisture, recovery, % g./100 ml. V.C.C.'/g.D.S. g./hr. % % None 14.16 5512 X 109 30.3 0.55 None Urea 3.0 16.22 3661 46.0 1. 7gb 19.3 Guanidine sulfate 0 . 5 15.28 3256 53.6 1.52 None Maleic acid 0.5 Guanidine sulfate 0 . 5 15.72 3855 54.8 1.39 None Maleic acid 1.0 Guanidine sulfate 0 . 5 16.68 3115 57.0 1.27 0.6 Maleic acid 2.0 1967 1802 2883
Urea oxalate Maleic acid
62.2 57.7 55.0
0.66 5 . 04c 0.84
5.2 10.2 2.4
NHiCle 1.0 Thioureae 1.0 17.46 3444 55.3 1.00 86.8 Ascorbic acids 1.0 Viable cell count/gram dry solids. High result due to sublimation of urea during moisture determination, O Run at low temperatures to maintain viability of cells. Urea oxalate and maleic acid adjusted t o pH 8.3-6.9 before adding to concentrate. Naylor-Smith ingredients.
Product Feed Solids, V.C.CT g./100 ml. g. D.S.a
Fermented mediumb 9.73 Sharples concentrate reconstituted with HzOb 10.45 With eauent pH control (HaP04) 16.36 DV-2 concentrate0 no pH control 15.78 pH control (HaPO4) 17.46 Acid0 HCl 15.67 HzSO4 16.65 Hap04 17.46 a X lo9. Belt speed = 1.5 ft./hr.
O
18
20
22
24
26
28
H I R Y E S T TIME, HOURS
Figure 1. Correlation of recovery of viable cells with harvest time of fermented materials
r-
fi P
i--
70-
ui 60-
Recovery After 6 Weeks Storage, B a t e d On Dried Pellets
0
Production rate, g./hr.
Moisture, %
V.C. recovery, %
1361
21.6
1.38
73.1
5057
21.3
0.54
55.9
4566
61.3
2.32
91.5
3996 3444
35.1 55.3
1.28 1.00
79.1 86.8
3766 4182 3444
44.1 46.4 55.3
0.96 0.77 1.00
94.9 79.7 86.8
Belt speed = 2.0 ft./hr.
16
0'
Effect of Feed Material and Acid on Recovery of Viable S. marcescens
Type of Feed or Acid
90-
80-
~
Table II.
5
W
50-
5
8
40-
F I N A L MOISTURE CONTENT, PERCENT
Figure 2. Correlation of drying and storage recovery with moisture content, based on 6 weeks'. storage at 50' C. VOL. 50, NO. 4
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Acknowledgment Table Ill.
Effect of Dryer Belt Speed on Recovery of Viable S. marcescens
Feed
Belt Speed, Solids, g./ Feet/Hour 100 ml. 1.5a 2.0b 2.5b 3.0b a
9.73 17.46 16.90 16.89
Heat Product Watt-Hours/ Input, Production Moisture, V.C. re- Gram Ice Watts rate, g./hr. covery, % Sublimed %
V.C.C./ g. D.S. 1361 X IOQ 172.3 3444 2850 2921
21.6 55.3 71.9 84.5
232.6 345.1 389.6
1.38
73.1 86.8 88.2 82.6
1 .oo
1.30 4.22
0.87 0.85 0.93 0.93
Pellets from fermented medium. Pellets from DV-2 concentrate.
The authors are grateful to Geraldine B. Meyers, Carolyn S. Kemp, and Edward F. Baer, Fermentation Section, for making viable cell counts of the materials used in these experiments, and to Carl R. Martin and Albert Clark, Engineering and Development Section, for assistance in carrying out the experiments. Literature Cited
better protection against the loss of viable cells during drying than phosphoric or sulfuric acids (Table 11). Phosphoric acid afforded somewhat better protection in storage, and imparted superior pelleting qualities to the fortified suspensions. In most of the subsequent fermentations, therefore, sodium hydroxide and phosphoric acid were used to control the pH between 6.8 and 7.2. Determination of Optimum Drying Temperatures. I n several preliminary experiments the relationship among discharge vapor temperature, recovery of viable cells, and final moisture content was investigated. Results of experiments in which DV-2 concentrates were dried at different discharge temperatures are shown in Figure 3. The discharge vapor temperature should not be assumed to be the same as that of the pellets, but is of value in controlling final moisture content. Figure 3 shows that a t a belt speed of 2 feet per hour, both final moisture content and percentage recovery of viable cells drop of: rapidly as the temperature rises above 50’ C. I n subsequent drying operations a t this speed, therefore, the temperature was controlled as closely as possible at 49’ Similar experiments showed that for a belt speed of 2.5 feet per hour, the discharge temperature should be maintained a t 56’ C. Maximum Production Rate. After the dryer had been operated successfully a t belt speeds of 1.5 and 2 feet per hour, an attempt was made to find the maximum rate for producing pellets of satisfactory cell counts and moisture content. At 2.5 feet per hour, a satisfactory product was obtained a t an average production rate of 71.9 grams per hour (Table 111). At 3 feet per hour, however, high temperatures and pressures in the drying chamber produced sticky pellets and operation was unsatisfactory. Heat Requirements. The energy required to obtain a product of 1% final moisture content can be estimated from material and energy balances over the dryer. T o dry a suspension having a total solids content of 17% at a belt speed of 2 feet per hour, a production of about 55 grams per hour can be expected (Table 111). Figure 4, a plot of the energy expenditures involved in
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preparing dried pellets over a wide range of final moisture contents, was compiled from over 100 runs in the continuous freeze-dryer. Using a value of 0.88 watt-hour required per gram of ice sublimed, the following calculations can be made:
Antoine, L. D., Hargett, M. V., J . Bacterid. 46, 525 (1943). Benedict, R. G., Koepsell, J. H., Tsuchiva, H. M., Sharpe, E. S., Corman,.J., Meye&, G. B.; Remp; C. E., Jackson, R. W., A$$. Microb i d . 5 , 308 (1957). Bradish, C. J., Chem. Prod. 10, 60 (1947). Bradish, C. J., Brain, C. M., McFarlane, A. S., Nature 159, 28 (1947). Chizhov, G., Chem. Zentr. 1949, p. 1000. Flosdorf, E. W., “Freeze Drying,” Reinhold, New York, 1949. Flosdorf, E. W., Webster, G. W., J . Bid. Chem. 121, 353 (1937). Harris, R. J. C., “Biological Applications of Freezing and Drying,” pp. 87-127, 215-52, Academic Press, New York, 1954.. Harris, R. J. C., “Freezing and Drying,” pp. 41-9, Institute of Biology, London, 1951. Naylor, H. B., Smith, P. A., J . Bucteriol. 52, 565 (1946). Plank, R., Angew. Chem. 19, 36 (1947). Proom, H., Hemmons, L. M., J . Gen. Microbiol. 3, 7 (1949). Rhian, M. A , , Maister, H. G., Hutton, R. S., Appl. Microbiol. 5,323 (1957). Stamp, Lord, J . Gen. Microbiol. 1, 251 (1947).
Grams HzO/gram dry solids, initial = 83/17 = 4.88 Grams HlO/gram dry solids, final = 0.01/0.99 = 0.01 Grams ice sublimed/gram dry solids = 4.87 4.87 X 55 X 0.88 = 236 watts required. Summary
I n the apparatus described, S. marcescens, strain 8 U K , was dried successfully to a moisture content of 1%, suitable for satisfactory storage stability, without excessive loss of viable cells. With high quality fermented medium, recovery of viable cells was 80% or higher in the product. Suspensions of concentrates were processed more readily and gave better products than fermented medium. Fortification of medium or concentrates with a protective material was necessary for satisfactory survival of the viable cells during drying. Only the Xaylor and Smith mixture, minus dextrin, provided effective protection. Control of hydrogen ion concentration during fermentation increased the ability of the organisms to survive drying. The maximum rate of production of dried pellets of satisfactory moisture content and viable cell levels was 50 to 55 grams per hour at a belt speed of 2 feet per hour.
RECEIVED for review June 7, 1957 ACCEPTED September 7, 1957 Work supported by U. S. Chemical Corps, Fort Detrick, Frederick, Md., under contract with the Agricultural Research Service, U. S. Department of Agriculture.
loo i’O
““I 04
; z -
o n 0 2 Feet per H w r
1:’
0 6 1
on0
I
2
3 iN4L
4
MOISTURE
I 5
1
1
6
7
L--J 8
9
IO
COVTENT PERCENT
Figure 3. Correlation of final moisture content and recovery of viable cells with discharge vapor temperature
INDUSTRIAL AND ENGINEERING CHEMISTRY
40
45
50
DISCHARGE
55 VAPOR
MI
65
70
TEMPER4TURE.
75
’C
Figure 4. Correlation of final moisture content with electrical energy input
