FERMENTATION RESEARCH & ENGINEERING Sterilization of Filled Fermentors
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HENRY R. BUNGAY 111 and PETER HOSLER Antibiotics Manufacturing and Development Division, Eli Lilly and Co., Indianapolis, Ind.
In terms of scale-up of sterilization conditions, simple operation, and flexibility of medium changes, the autoclave venting technique has definite advantages over alternate procedures for la b-scale fermentors O
N
E
OF
THE
COMMON
TYPES
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equipment in a fermentation laboratory is the 30-liter stirred fermentor. This equipment is large enough to have most of the operational characteristics of a larger fermentor but small enough to be moved easily, so that the expense of pressure coding, jacketing, and permanent piping is avoided. The obvious way to sterilize such small fermentors is to fill them with nutrient media, place them in an autoclave, and perform a sterilization heating cycle. Unfortunately, there are several drawbacks to such an operation. Although dilute media have adequate heat transmission for relatively short sterilization
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times, media with solid ingredients require much longer a t 121' C. to ensure sterility. The time is dependent on the amount of solids which settle to the bottom to form a layer resistant to heat transfer. Another serious drawback is familiar to anyone xvho has operated an autoclave; superheated liquid may suddenly boil out of the vessel when pressure is released. The larger the fermentor, the more serious the problems. Throughout the years, attempts have been made to improve autoclave operations, Exhausting air from the autoclave with vacuum allows steam to penetrate into the load more rapidly to shorten the heating period. Newer auto-
POSITIONS FOR OPERATIONS
In sterilization position, sample lines leading t o spargers and exhaust hoses connect t o headers Individual air filters are plugged. Three-way valve i s in by-pass or sample position, so that air filter is isolated from sparger line. W h e n outoclave i s opened, main valve on exhaust header i s closed and union broken t o permit removing fermentors. Sample valves ore closed, and air filters are unplugged so that pressure equalization can occur. Before removing headers, exhaust hoses a r e pinched off b y doubling and binding with rubber bands. For comparison, valve positions for normal operation a r e also shown. Advantage of three-way valve system over a separate sample line i s that little broth stands in a dead line, so a representative sample of broth may be obtained o n a small quantity without having i o flush out stagnant portions
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INDUSTRIAL AND ENGINEERING CHEMISTRY
clave designs avoid reducing valves by connecting full line pressure to the chambe1 through a solenoid valve controlled by a temperature sensing element This reduces the time to reach sterilization temperature by half, but a good veniing system is needed to rcmove air which would add its partidl pressure to that of the steam. Another recent improkement uses steam jets during cooling ( I ) . The expansion of the jet into the chamber allows this steam to absorb heat from the load in the autoclave. None of these ideas, however, attack the principal defects of autoclave sterilization because heat
Figure 1. Solids in bottom of fermentor lag in temperature during typical sterilization
iThermocouple
On cooling, thermocouples read together because temperature i s primarily a function of staturated steam pressure
in solids at bottom
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transfer to solids and boiling out are not affected. The heating and cooling of one of eight 30-liter fermentors is shown (Figure 1). With this conventional procedure, several hours were needed to ensure sterility, presumably because of the quiescent nature of the operation. Since even 1 hour at sterilization temperature usually causes undesired changes, a previous alternate procedure was to sterilize the medium separately in agitated batch cookers. The venting technique described here has a 2-year sterility record at least as good as that of the alternate procedures. Special sterility runs, with bacterial counts, have shown adequate sterilization in 20 minutes, whereas much longer rimes were required by the conventional method.
and one or more fermentors would lag behind the pressure release of the autoclave itself. This reversed the flow through the sparger line, and liquid poured out of the fermentor. Equal flow was effectively obtained by connecting the jars to an inlet header and outlet header within the autoclave. The flow resistance for each is only the pipe and liquid head; thus the flow rate is almost identical for each fermentor. T h e maximum deviation in final volume of the fermentors has been
200 ml. or 1.3%. Furthermore, no fermentor can lag during pressure release: and loss of liquid by reversed flow in the sparger line is prevented. Both headers are protected with filters to prevent entry of foreign organisms at the end of the sterilization cycle. Positions of the valves on the jar fermentor are shown (p. 746). These fermentors are piped with a three-way valve so that a sample can be taken by backing media up through the sparger line.
Principle of Operation
By connecting the fermentor exhaust lines to a vent in the wall of the autoclave, steam is permitted to enter the fermentors through the sparger lines, pass through the medium, and exhaust to the atmosphere. The mixing advantage of this technique is shown (right), The autoclave is exhausted during termination of the cycle by continuing the passage of steam through the fermentors. This continued agitation and equilibration with steam effectively prevents boiling out. The principal engineering problem in developing the venting technique was obtaining equal flow through the fermentors. Several preliminary designs failed because of this factor. With unequal flow, the amount of vaporization loss would cause the volume to vary as much as 1 liter from the desired amount. Furthermore, the pressures within the fermentors would differ,
Steam flow disperses solids during autoclave sterilization with the proposed technique VOL. 53, NO. 9
SEPTEMBER 1961
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Comparison w i t h Production
The heating curve for the autoclave venting technique is roughly similar to that for a 12,000-gallon fermentor (Figure 2). The different shape results from a difference in driving force. Fermentors sterilized in place are connected to a 45-p.s.i.g. steam supply and utilize the higher temperature steam to get
increased heating rates. The steam valve is closed just before attainment of sterilization temperature, and the temperature coasts u p slightly. This is indicated by a dotted line because it can vary slightly, depending upon such factors as insulation, positon along the steam header, operator judgment, and the like. The autoclave uses only 16p.s.i.g. steam; thus the driving force
1200 GAL.
12,000 G A L
I20 Figure 2. Practicality of any steriliI IO zation technique depends upon time required to reach ster- u' ilization temperature O and then cool back to W'IOO nondestructive tem- 3 perature. As tank size increases, sparger steam dominates over that of jacket or coils k
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AUTOCLAVE OPENED-
WATER BATH 90 I O V'
Figure 3. Critical cooling period i s the same as that in a large tank
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a
Acknowledgment
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The authors received help and suggestions from R. B. Brigham, Herman DeValeria, D. G. Farr, R . J. Davis, C. F. Simons, and R. JV. Squires.
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AUTOCLAVE METHOD
12 GAL.
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MINUTES 748
decreases rapidly as the Sterilization temperature is approached and the heating curve decreases in slope. Typical cooling curves are shown in Figure 3. Vaporization cooling is the predominant mechanism for the fermentors in the autoclave and may contribute to the cooling of the large production tanks when pressure is reduced. The relatively rapid temperature drop for the 12-gallon and 1200-gallon fermentors results from the greater ratio of heat transfer surface to volume. T h e effect is much more pronounced as the temperature difference between the contents and jacket water decreases. Again, the curve for the autoclave method resembles that for a big tank, at the higher temperatures. The question of proper sterilization heating and cooling rates for pilot plant equipment has not been resolved. Large fermentors have rates imposed by the design characteristics of the equipment; it would be possible to simulate these rates in pilot plant equipment by design modifications or by using automatic controllers. The principal objection would be the long delays in the pilot plant while cooling fermentors at a rate copied from production equipment. I n some cases: the sterilization cycle seems to have little effect on the character of the fermentation, while in others a substantial effect is noted. Relative amount of browning ofthemediumisoften an indicator of differences in operations. The autoclave venting technique seems a good compromise for resolving sterilization differences. because the times at temperatures exceeding 100' C. are much the same as in production equipment. but cooling to operating temperature is complete in about 45 minutes because the hot fermentors are placed in a water bath. -4n analogous cycle seems logical if other pilot plant equipment is scaled more closely to production. The temperatures above 100" C. could be set on the production time schedule, and the better heat transfer could be utilized below 100' C. to speed operations. Since most heat damage probably occurs above looo C., the media should resemble production media closely, and another scale-up variable would be under control.
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literature Cited (1) Wilmot Castle Co., Rochester, N. Y., "Orthomatic Sterilizer" bulletin, 1959.
KECEIVED for review December 30, 1960 ACCEPTED March 9, 1961 Division of .4gricultural and Food Chemistry, 138th Meeting. ACS, New York, September 1960.
