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Spalla et al. (1989)

Nakayama (1972)

would be expected to operate at steady state in excess 0f 100 days. Thus, the number of times that the fermenter is inoculated should be very few compared with batch or fed-batch systems. In such circumstances it may be more economic to compromise on the size of the inoculum and to tolerate a relatively lengthy period of growth up to maximum biomass than to invest a large seed vessel which would be used on very few occasions. This is particularly relevant for biomass continuous processes where one very large fermenter may be used and, thus, any seed vessel would only be servicing the one production vessel.

A typical inoculum-development programme will now be described in detail. The master culture (see Chapter 3 for an account of master-culture maintenance) is reconstituted and plated on to solid medium; approximately ten colonies of typical morphology of high producers are selected and inoculated on to slopes as the sub-master cultures, each sub-master culture being used for a new production run. At this stage, shake flasks may be inoculated to check the productivity of these cultures, the results of such tests being known before the developing inoculum eventually reaches the production plant. A sub-master culture is used to inoculate a shake flask (250 or 500 cm3 containing 50 or 100 cm3 medium) which, in turn, is used as inoculum for a larger flask, or a laboratory fermenter, which may then be used to inoculate a pilot-scale fermenter. Culture purity checks are carried out at each stage to detect contamination as early as possible. Although the results of these tests may not be available before the culture has reached the production plant, at least it is known at which stage in the procedure contamination occurred. For a sporulating organism the process may be modified to facilitate the use of a spore suspension as inoculum and this will be discussed in more detail later in this chapter.

Lincoln (1960) suggested a more elaborate procedure for the development of inoculum for bacterial fermentations which, with minor modifications, is applicable to any type of culture. The procedure involved the use of one sub-master culture to develop a bulk inoculum which was subdivided, stored in a frozen state and used as inocula for several months. A single colony, derived from a sub-master culture, was inoculated into liquid medium and grown to maximum log phase. This culture was then transferred into nineteen times its volume of medium and incubated again to the maximum log phase, at which point it was dispensed in 20 -cm- volumes, plug frozen and stored at below — 20°. At least 3% of the samples were tested for purity and productivity in subsequent fermentation and, provided these were suitable, the remaining samples could be used as initial inocula for subsequent fermentations. To use one of the stored samples as inoculum it was thawed and used as a 5% inoculum for a seed culture which, in turn, was used as a 5% inoculum for the next stage in the programme. This procedure ensured that a proven inoculum was used for the penultimate stage in inoculum development.

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