The Development Of Inocula For Mycelial Processes

The preparation of inocula for fermentations employing mycelial (filamentous) organisms is more involved than that for unicellular bacterial and yeast processes. The majority of industrially important fungi and streptomycetes are capable of asexual sporulation, so it is common practice to use a spore suspension as seed during an inoculum development programme. A major advantage of a spore inoculum is that is contains far more 'propagules' than a vegetative culture. Three basic techniques have been developed to produce a high concentration of spores for use as an inoculum.

employed to produce sufficient spores. Parker (1950) described the 'roll-bottle' technique for the production of spores of Penicillium chtysogenum. Quantities of medium (300 cm3) containing 3% agar were sterilized in 1 dm3 cylindrical bottles, which were then cooled to 45° and rotated on a roller mill so that the agar set as a cylindrical shell inside the bottle. The bottles were inoculated with a spore suspension from a sub-master slope and incubated at 24° for 6 to 7 days. Parker claimed that although the use of the 'roll-bottle' involved some sacrifice in ease of visual examination, it provided a large surface area for cultivation of spores in a vessel of a convenient size for handling in the laboratory.

Hockenhull (1980) described the production of 1010 spores of Penicillium chrysogenum on a 300-cm2 agar layer in a Roux bottle and El Sayed (1992) quoted the use of spore suspensions derived from agar media containing between 107 and 108 cm-3. Butterworth (1984) described the use of a Roux bottle for the production of a spore inoculum of Streptomyces clavuligerus for the production of clavulanic acid. The spores produced from one bottle containing 200-cm2 agar surface could be used to inoculate a 75-dm3 seed fermenter which, in turn, was used to inoculate a 1500-dm3 fermenter. The clavulanic acid inoculum development programme is illustrated in Fig. 6.4. Some representative solidified media for the production of streptomycete and fungal spores are given in Tables 6.3 and 6.4 respectively.

Sporulation on solidified media

Most fungi and streptomycetes will sporulate on suitable agar media but a large surface area must be

Sporulation on solid media

Many filamentous organisms will sporulate profusely on the surface of cereal grains from which the spores

Table 6.2. The inoculum development programme for the clostridial acetone-butanol fermentation (Spivey, 1978)


Cultural conditions

Incubation time (hours)

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