Design And Analysis Of Defined Minimal Growth Media

To grow and synthesize their own cell material, organisms must obtain all the required building blocks (or their precursors) and the necessary energy from their environment. Consequently, to cultivate microbial cells in the laboratory these nutrients must be supplied in a culture medium in adequate amounts and in a form accessible to the organism.

As a result of the physiological diversity of the microbial world, a myriad of media of different compositions have been published, for either selective enrichment or cultivation of particular microorganisms (consult, for example, LaPage et al, 1970; Balows et al, 1992; Atlas, 1997). All these media contain components whose nutritional function is obvious, particularly when considering their elemental or energetic function. Nevertheless, most nutritional studies made have been qualitative rather than quantitative and different nutrients have been added in more or less arbitrary amounts. Also, many of the media contain components whose reason for inclusion cannot be clearly identified because their inclusion is based more on experience or tradition than on a clear purpose.

The identification of the nutritional requirements of microbial cells usually calls for the use of defined synthetic media. The design of defined culture media is based on quantitative aspects of cell composition and it allows the influence of the growth of a microbial culture at three major levels. First, the choice of which nutrient is to limit the growth of the culture stoichio-metrically and kinetically is made. Second, for nutritionally flexible microbial strains, the choice of the type of metabolism that the organism is to perform is made by the selection of the compounds that are supplied to fulfill a particular nutritional requirement, including electron donors and acceptors. Third, and often linked with the second point, the choice of the maximum specific growth rate to be achieved during unrestricted growth in batch culture is set.

A. Setting fimax during unrestricted growth

In addition to physicochemical parameters such as temperature or pH, the maximum specific growth rate of a microorganism is influenced by the diversity of the nutrients supplied in the medium. This has been elegantly illustrated for the growth of Salmonella typhimurium by Schaechter et al. (1958), who used 22 media of different compositions to obtain growth of the culture at differing rates under nutrient excess conditions (a selection is given in Table 62.5). Although the four media supporting the highest specific growth rates are undefined, the other media consist of a minimal salt medium to which different carbon sources or amino acid mixtures are added. Hence, the selection of the quality of precursors supplied in the mineral medium allowed the adjustment of the growth rate of the culture in a defined and reproducible way.

B. Medium design and experimental verification of the limiting nutrient

1. Designing a growth medium

In the design of a defined growth medium, the initial decisions to be made are the choice of the maximum concentration of biomass the medium should allow to produce (Xmax), and the definition of the growth-limiting nutrient (according to Liebig's principle). Typically, defined growth media for heterotrophic microbes are designed with a single carbon-energy source restricting the amount of biomass that can be produced, whereas all other nutrients (each of them usually added in the form of a single compound) are supplied in excess. Having set Xmax, it is possible to

TABLE 62.5 Composition of a selection of media used to set the maximum specific growth rate of

Salmonella typhimurium in batch culture0

TABLE 62.5 Composition of a selection of media used to set the maximum specific growth rate of

Salmonella typhimurium in batch culture0

No.

Medium

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Mmax (h )

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