Fermenters

The inoculation of plant-scale fermenters may involve the transfer of culture from a laboratory fermenter, or spore suspension vessel, to a plant fermenter, or the transfer from one plant fermenter to another. Obviously, it is extremely important that the fermentation is not contaminated during inoculation but if the process is a contained one then it is equally important that the process organism does not escape. Thus, the nature of the inoculation system will be dictated by the containment category of the process (see Chapter 7).

At Containment levels 1 and B2, the addition of inoculum must be carried out in such a way that release of micro-organisms is restricted. This should be done by aseptic piercing of membranes or connections with steam locks. At Containment level 2 and B3/A, no micro-organisms must be released during inoculation or other additions. In order to meet these stringent requirements, all connections must be screwed or clamped and all pipelines must be steam sterilizable (Werner, 1992).

Inoculation from a laboratory fermenter or a spore suspension vessel

Several systems have been described in the literature which are suitable for inoculating fermentations requiring only Good Industrial Large Scale Practice (GILSP, see Chapter 7). To prevent contamination during the transfer process it is essential that both vessels be maintained under a positive pressure and the inoculation port be equipped with a steam supply. Meyrath and Suchanek (1972) described a system for the inoculation of a plant fermenter from a laboratory vessel. The apparatus is shown in Fig. 6.7. The connecting point A is normally covered with the blank plug a and prior to inoculation this plug is slightly loosened, valve E closed and valve F opened to allow steam to exit at A. Valve F is then closed and E opened so that when a is removed sterile air will be released from the vessel. After removal, plug a is placed in strong disinfectant. Blank plug b is then removed and a coupling made between B and A. Valve E is closed and an air ]m, attached to point C, establishing a pressure inside th*1 inoculum fermenter greater than in the plant vessel Valve E is then opened and the inoculum will be forced into the plant fermenter. After closing valve E the inoculum fermenter may be removed, plug a re placed, and the line steamed out by opening valve I This system may be modified by using the quick connection devices which are now available (see Chaptcr

Jackson (1958) has described a very similar system for the introduction of a spore suspension into a plant fermenter. The apparatus is shown in Fig. 6.8 and its operation is identical to that described by Meyrath and Suchanek.

Parker (1958) described a more complex system for inoculation of a plant fermenter from a spore-suspension vessel. The apparatus is shown in Fig. 6.9. The sterile spore-suspension vessel is batched with the spore suspension in the sterile room. The plant vessel, containing the medium and with blank plugs screwed on at A and B, is sterilized by steam injection. The plugs A and B are slightly loosened to allow steam to emit for 20 minutes when the whole system is under steam pressure. The blanks are then tightened, the valves E and G shut and sterile air is allowed into the plant fermenter by opening valves D, F and C. The spore suspension vessel is loosely connected at A and B and valves D, H, I and C closed and E, F and G opened. After 20 minutes steaming A and B are tightened and G and E are closed. D is then opened to establish a positive pressure in the pipework. When the pipework has cooled, the pressure in the plant vessel is reduced to approximately 5 psi, valve F is closed and H, I and C are opened. This procedure allows the spore suspension to be forced into the fermenter. Valves D, H, 1

Meyrath Suchanek Inoculation

Plant fermenter

Fig. 6.7. Inoculation of a plant fermenter from a laboratory fermenter (Meyrath and Suchanek, 1972).

Plant fermenter

Fig. 6.7. Inoculation of a plant fermenter from a laboratory fermenter (Meyrath and Suchanek, 1972).

Filter

Inoculum Vessel

6 « Inoculation of a plant fermenter from a spore suspension vessel (Jackson, 1958).

Filter

6 « Inoculation of a plant fermenter from a spore suspension vessel (Jackson, 1958).

nd C arc closed ami the suspension vessel replaced by blank plugs at A and B and the pipework steamed by opening valves H and G.

GILSP and category 1 fermentations may also be inoculated by aseptic piercing of a membrane. In this system the inoculum vessel is connected to an inoculating needle assembly (as shown in Fig. 6.10) and the sterile needle pierces a rubber septum set into a fermenter port. However, the use of a needle presents safety problems and many companies prohibit such systems. Also, aerosols may be created on the removal of a needle.

The inoculation of level 2 or B3/4 contained fermentations requires the use of modified systems. It must be possible to steam sterilize all the pipework

Sterile air

Sterile air

Spore suspension vessel

Fig. 6.9. The pension vessel inoculation of a plant fermenter from a spore sus-(Parker, 1958).

Spore suspension vessel

Membrane

Inoculation needle

Inoculation Device

Fig. 6.10. Needle inoculation device (Werner, 1992).

Opening for flame scarfing

Membrane

Inoculation needle

Fig. 6.9. The pension vessel inoculation of a plant fermenter from a spore sus-(Parker, 1958).

Fig. 6.10. Needle inoculation device (Werner, 1992).

after the inoculation and the condensate from the sterilization must be collected in a kill tank (see Chapters 5 and 7). The inoculation flask is then removed after inoculation and sterilized in an autoclave. One such system is shown in Fig. 6.11 (Vranch, 1990).

Inoculation from a plant fermenter

Figure 6.12 illustrates the system described by Parker (1958). The two vessels are connected by a flexible pipeline A B. The batched fermenter is sterilized by steam injection via valves G and J, valves D, I, A, B, H, E and F being open and valve C closed. Valves H and I lead to steam traps for the removal of condensate. After 20 minutes at the desired pressure the steam supply is switched off at J and G and the steam-trap valves I and H are closed. F, E and D are left open so that the connecting pipeline fills with sterile medium. The medium in the fermenter is sparged with sterile air and when it has cooled to the desired temperature the pressure in the seed tank is increased to at least 10 psi whilst the pressure in the fermenter is reduced to about 2 psi. Valve C is opened and the inoculum is forced into the production vessel. After inoculation is

Steam supply

Steam supply

Fermenter

To kill vessel

Fig. 6.11. Inoculum system suitable for contained fermentations (Vranch, 1990).

Fermenter

To kill vessel

Fig. 6.11. Inoculum system suitable for contained fermentations (Vranch, 1990).

complete the connecting pipeline is resterilized before it is removed. For a contained fermentation the condensate from the two steam traps attached to valves I and H would be directed to a kill tank.

Steam

Trap

Trap

Fig. 6.12. Inoculation of a plant fermenter from another plant fermenter (Parker, 1958).

Steam

Trap

Trap

Fig. 6.12. Inoculation of a plant fermenter from another plant fermenter (Parker, 1958).

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