dry the filter. Finally the steam supply to the steam jacket is stopped. Valve D is closed and valve E opened, thus introducing sterile air into the fermenter to achieve a slight positive pressure in the vessel.
Sterilization of the exhaust gas can be achieved by 0.2-fim filters on the outlet pipe (Fig. 5.21). Under normal operation aerosol formation may occur in the fermenter and moisture and solid matter may then plug the filter. To ensure satisfactory operation a cyclone separator (for solids) and a coalescer (for liquids) would be included upstream of two filters in series. The filters should be checked regularly to ensure that no viable cells are escaping. A test procedure to ensure integrity has been described by Hesselink et al. (1990).
The addition of inoculum, nutrients and other supplements
To prevent contamination when operating a fermenter requiring GILSP, it is essential that both the addition vessel and the fermenter should be maintained at a positive pressure and that the addition port is equipped with a steam supply.
At Containment levels 1 and B2, the addition of inoculum, nutrients, etc. 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 levels 2 and B3/4, 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).
Further details of the aseptic inoculation of laboratory, pilot-plant and production fermenters are described in Chapter 6.
The sampling points fitted to larger fermenters also illustrate the principles for maintaining sterility. A sterile barrier must be maintained between the fermenter contents and the exterior when the sample port is not being used and it must be sterilizable after use. A simple design (Fig. 7.20) is described by Parker (1958). In normal operation valves A, B and C are closed and a barrier is formed by submerging the end of the sampling port in 40% formalin or a suitable substitute. A sample is obtained by removing the container of formalin and closing valve A. Valves B and C are then opened until the piping has been sterilized by steam. Valves B and C are then partially closed to allow a slow stream of steam and condensate out of the sampling port. Valve A is then opened slightly to cool the piping. The broth is discarded. Valve C is then closed and a sample is collected. Valve A is then closed and the piping is resterilized and left in the out of use arrangement.
An alternative arrangement for a sampling port is illustrated in Fig. 7.21 where the sterile barrier between
Steam and -condensate
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