Introduction

It is essential that the culture used to inoculate a fermentation satisfies the following criteria:

1. It must be in a healthy, active state thus minimizing the length of the lag phase in the subsequent fermentation.

2. It must be available in sufficiently large volumes to provide an inoculum of optimum size.

3. It must be in a suitable morphological form.

4. It must be free of contamination.

5. It must retain its product-forming capabilities.

The process adopted to produce an inoculum meeting these criteria is called inoculum development. Hocken-hull is credited with the quotation ''once a fermentation has been started it can be made worse but not better" (Calam, 1976). Whereas this is an over-statement it does illustrate the importance of inoculum development. Much of the variation observed in small-scale laboratory fermentations is due to poor inocula being used and, thus, it is essential to appreciate that the establishment of an effective inoculum development programme is equally important regardless of the scale of the fermentation. Such a programme not only aids consistency on a small scale but is invaluable in scaling up the fermentation and forms an essential part in progressing a new process.

A critical factor in obtaining a suitable inoculum is the choice of the culture medium. It must be stressed that the suitability of an inoculum medium is determined by the subsequent performance of the inocu lum in the production stage. As discussed elsewhere (Chapter 4), the design of a production medium is determined not only by the nutritional requirements of the organism, but also by the requirements for maximum product formation. The formation of product in the seed culture is not an objective during inoculum development so that the seed medium may be of a different composition from the production medium. However, Lincoln (1960) stated that the lag time in a fermentation is minimized by growing the culture in the 'final -type' medium. Lincoln's argument is an important one, so the inoculum development medium should be sufficiently similar to the production medium to minimize any period of adaptation of the culture to the production medium, thus reducing the lag phase and the fermentation time. Furthermore, Hockenhull (1980) pointed out the dangers of using very different media in consecutive stages. Major differences in pH, osmotic pressure and anion composition may result in very sudden changes in uptake rates which, in turn, may affect viability. Hockenhull also emphasized that for antibiotic fermentations the inoculum medium should contain sufficient carbon and nitrogen to support maximum growth until transfer, so that secondary metabolism remains repressed during growth of the inoculum. If secondary metabolism is derepressed in the seed fermentation, then selection may enrich the culture with non-producing variants having a growth advantage over high-producing types. Hockenhull drew attention to Righelato's (1976) work in which it was shown that chemostat culture of Pénicillium chryso-genum under carbohydrate-limited conditions led to a loss of penicillin synthesizing ability and an increase in the proportion of non-conidiated variants whereas this did not occur in ammonia-, phosphate- or sulphate-limited conditions. The relevance of this phenomenon is supported by Hockenhull's observation that P. chrysogenum inocula produced under non-limiting conditions are remarkably free from variants whereas variants arise relatively frequently during the carbon-limited production phase. Examples of inoculum and production media are given in Table 6.1, from which it may be seen that inoculum media are, generally, less nutritious than production media and contain a lower level of carbon.

The quantity of inoculum normally used is between 3 and 10% of the medium volume (Lincoln, 1960; Meyrath and Suchanek, 1972; Hunt and Stieber, 1986). A relatively large inoculum volume is used to minimize the length of the lag phase and to generate the maximum biomass in the production fermenter in as short a time as possible, thus increasing vessel productivity. Thus, starting from a stock culture, the inoculum must be built up in a number of stages to produce sufficient biomass to inoculate the production-stage fermenter This may involve two or three stages in shake flasks and one to three stages in fermenters, depending on the size of the ultimate vessel. Throughout this procedure there is a risk of contamination and strain degeneration necessitating stringent quality-control procedures. The greater the number of stages between the master culture and the production fermenter the greater the risk of contamination and strain degeneration. Therefore, a compromise may be reached regarding the size of the inoculum to be used and the risk of contamination and strain degeneration. Another factor to be considered in the determination of the inoculum volume is the economics of the process. A seed fermenter 10% of the size of the production fermenter represents a considerable financial investment and must be justified in terms of productivity. A large-scale continuous fermentation for the production of biomass

Table 6.1. Inoculum development and production media for a range of processes

Process

Griseofulvin

Inoculum development medium

Whey powder \to Lactose / give:

Lactose Nitrogen Corn-steep liquor solids (to give approx. 0.04% N)

kh2po4

Production medium

Lactose Corn-steep liquor solids to give: Nitrogen Limestone kh2po4

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