The Achievement And Maintenance Of Aseptic Conditions

Once the design problems of aeration and agitation have been solved, it is essential that the design meets the requirements of the degree of asepsis and containment demanded by the particular process being considered. It will be necessary to be able to sterilize, and keep sterile, a fermenter and its contents throughout a complete growth cycle. There may be also a need to protect workers and the environment from exposure to hazardous micro-organisms or animal cells (Van Houten, 1992). As has been mentioned earlier, the containment requirements depend on the size of the fermentation vessel.

The following operations may have to be performed according to certain specifications to achieve and maintain aseptic conditions and containment during a fermentation:

1. Sterilization of the fermenter.

2. Sterilization of the air supply and the exhaust gas.

3. Aeration and agitation.

4. The addition of inoculum, nutrients and other supplements.

5. Sampling.

6. Foam control.

7. Monitoring and control of various parameters.

On a small scale, below 10 dm3, the biohazard risk can be controlled by a combination of containment cabinets and work practices (Van Houten, 1992). When the volume of culture exceeds 10 dm3, GILSP is required for those non-pathogenic non-toxigenic agents which have an extended history of large scale use. For this category there should be prevention of contamination of the product, control of aerosols and minimization of the release of micro-organisms during sampling, addition of material, transfer of cells and removal of materials, products and effluents. It should be appreciated that the majority of fermentations fall into this category.

At level 1, B2 or at higher containment levels, the following points need to be considered when designing a fermenter or other vessel, so that it can operate as a contained system (Tubito, 1991):

1. All vessels containing live organisms should be suitable for steam sterilization and have sterile vent filters. This is discussed in Chapter 5.

2. Exhaust gases from vessels should pass through sterile filters. This is discussed in Chapter 5.

3. Seals on flange joints should be fitted with a single 'O'-ring at the lower levels of containment. Flange joints on vessels for Containment levels 3 and B3/4 need double 'O'-rings or double 'O'-rings plus a steam barrier. This has been discussed in an earlier section of this chapter.

4. Appropriate seals should be provided for entry ports for sensor probes, inoculum, sampling, medium addition, acid, alkali and antifoam. This will be discussed later in this chapter.

5. Rotating shafts into a closed system should be sealed with a double acting mechanical seal with steam or condensate between the seals. This has been discussed in an earlier section in this chapter.

6. During operation a steam barrier should be maintained in all fixed piping leading to the 'contained' vessels.

7. Provision of appropriate pressure relief facilities will be discussed later in this chapter.

Further details for containment are given by Giorgio and Wu (1986), Hesselink et al. (1990), Kennedy et al. (1990), Janssen et al. (1990), Hambleton et al. (1991), Tubito (1991), Leaver and Hambleton (1992), Van Houten (1992), Vranch (1992) and Werner (1992).

Some of the issues discussed in points 1, 3, 4, 5 and 7

will need to be considered for aseptic operation of GILSP processes.

Sterilization of the fermenter

The fermenter should be so designed that it may be steam sterilized under pressure. The medium may be sterilized in the vessel or separately, and subsequently added aseptically. If the medium is sterilized in situ its temperature should be raised prior to the injection of live steam to prevent the formation of large amounts of condensate. This may be achieved by steam being introduced into the fermenter coils or jacket. As every point of entry to and exit from the fermenter is a potential source of contamination, steam should be introduced through all the entry and exit points except the air outlet from which steam should be allowed to leave.

All pipes should be constructed as simply as possible and slope towards drainage points to make sure that steam reaches all parts of the equipment and is not excluded by siphons or pockets of condensate or mash. Each drainage point in the pipework should be fitted with a steam trap. This will be described in the section on valves and steam traps. Parker (1958), Chain et al. (1954) and Muller and Kieslich (1966) and others have all stressed the need to eliminate fine fissures or gaps such as flange seals which might be filled with nutrient solutions and micro-organisms. Hambleton et al. (1991) described a high specification pilot scale fermenter with surfaces free of crevices greater than 0.05-mm depth, which is needed if the vessel was to be used for animal cells in suspension culture or on micro-carriers. For long-term aseptic operation welded joints should be used wherever possible, even though sections may have to be cut out and re-welded during maintenance and repair (Smith, 1980).

Sterilization of the air supply

Sterile air will be required in very large volumes in many aerobic fermentation processes. Although there are a number of ways of sterilizing air, only two have found permanent application. These are heat and filtration. Heat is generally too costly for full-scale operation (see also Chapter 5).

Historically, glass wool, glass fibre or mineral slag wool have been used as filter material, but currently most fermenters are fitted with cartridge-type filters as discussed in Chapter 5. However, before the filter may



Steam Trap Fermenter
Fig. 7.18. An arrangement of packed air filter and fermenter (Richards, 1968).

be used it, too, must be sterilized in association with the fermenter. Two procedures are commonly followed depending on the construction of the filter unit.

Figure 7.18 shows the simple unit described by Richards (1968). During sterilization the main nonsterile air-inlet valve A is shut, and initially the sterile air valve B is closed. Steam is applied at valve C and air is purged downwards through the filter to a bleed valve at the base. When the steam is issuing freely through the bleed valve, the valve B is opened to allow steam to pass into the fermenter as well as the filter. It is essential to adjust the bleed valve to ensure that the correct sterilization pressure is maintained in the fermenter and filter for the remainder of the sterilization cycle.

An alternative approach is to use a steam-jacketed air filter (Fig. 7.19). At the beginning of a sterilization cycle the valve A will be closed and steam passed through valves B and C, and bled out of D. Simultaneously steam will be passed into the steam jacket through valve F and out of G. When steam is issuing freely from valve D, valve F, may be opened and steam circulated into the fermenter. The bleed valve D will have to be adjusted to ensure that the correct pressure is maintained. Once the sterilization cycle is complete, valves B and E are closed and A is opened to allow air to pass through the heated filter and out of valve D to

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  • Elias
    How are aseptic conditions maintained in a fermenter?
    8 years ago
  • Anne Kost
    How are aseptic conditions in fermenter achieved?
    8 years ago
  • Elsa
    How do you maintain aseptic condition during fermentation?
    8 years ago
  • Uwe
    Why do you have to maintain aseptic conditions in a fermenter?
    8 years ago
  • Dionisio
    What are aseptic conditions in fermentation?
    8 years ago
  • pierino
    How to maintain aseptic conditions during fermentation?
    8 years ago
  • Teemu S
    Why maintenance of aseptic environment required?
    2 years ago
  • leon
    Why fermentation tanks must kept sterile?
    1 year ago
  • amanuel
    How to aseptic condition are maintained?
    1 year ago

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