Aseptic transferspore suspension

Test-tube working slant culture inoculated from lyophile stock

Roux bottle

Aseptic transferspore suspension 100 ml

Aseptic transfervegetative seed

Production stage 2000 liter fermenter 1500 liter medium 90 hr 26-C

Vegetative seed 100 liter fermenter 75 liter medium 72 hr 26°C

Fig. 6.4. The inoculum development programme for the production of clavulanic acid from Streptomyces clauuligerus (Butterworth, 1984).

may be harvested. Substrates such as barley, hard wheat bran, ground maize and rice are all suitable for the sporulation of a wide range of fungi. The sporulation of a given fungus is particularly affected by the amount of water added to the cereal before sterilization and the relative humidity of the atmosphere, which should be as high as possible during sporulation (Vezina and Singh, 1975). Singh et al. (1968) have described a system for the sporulation of Aspergillus ochraces in which a 2.8-dm3 Fernbach flask containing 200 grams of 'pot' barley or 100 grams of moistened wheat bran produced 5 X 1011 conidia after six days at 28° and 98% relative humidity. This was 5 times the number obtainable from a Roux bottle batched with Sabouraud agar and 50 times the number obtainable from such a vessel batched with Difco Nutrient Agar, incubated for the same time period. Vezina et al. (1968) have published a list of fungi which are capable of sporulating heavily on cereal grains. El-Sayed (1992) quoted the use of cooked rice for the production of spores of Penicillium and Cephalosporium in penicillin and cephalosporin inoculum development. Sansing and Cieglem (1973) described the mass production of spores of several As-

pergillus and Penicillium species on whole loaves of white bread and Podojil et al. (1984) quoted the use of millet for the sporulation of Streptomyces aureofaciens in the development of inoculum for the Chlortetracycline fermentation (see Fig. 6.5).

Sporulation in submerged culture

Many fungi will sporulate in submerged culture provided a suitable medium is employed (Vezina et al., 1965). This technique is more convenient than the use of solid or solidified media because it is easier to operate aseptically and it may be applied on a large scale. The technique was first adopted by Foster et al. (1945) who induced submerged sporulation in Penicillium notatum by including 2.5% calcium chloride in a defined nitrate-sucrose medium. An example of the use of this technique for the production of inoculum for an industrial fermentation is provided by the griseo-fulvin process. Rhodes et al. (1957) described the conditions necessary for the submerged sporulation of the griseofulvin-producing fungus, Penicillium patulum, and

Table 6.3. Solidified media suitable for the sporulation of some representative streptomycetes



S. aureofaciens



Malt extract (Difco) Yeast extract (Difco) Glucose


Williams et al. (1974)

S. erythreus

S. vmaceus



Beef extract (Difco) Yeast extract (Difco) Casamino acids (Difco) Glucose

Corn-steep liquor

(50% dry matter) Starch (NH4)2S04 NaCl CaCO,

Williams et al. (1974)

Williams et al. (1974)

S. clavuligerus

Clavulanic* acid

Soluble starch k2hpo4






1.0% 0.1% 0.1% 0.1% 0.2% 0.2% 0.001% 0.001 % 0.001%

Butterworth (1984)

S. hygroscopicus


Soluble starch


Meat extract

Yeast extract


Miyagawa et al. (1979)

"Agar content not quoted.

Table 6.4. Solidified media suitable for the sporulation of some representative fungi



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