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Mother stock, first sporulation second sporulation _^

freeze dried generation, generation, stationary „ , 3

agar slant culture on 1U ,p°res/m millet (flasks) inoculation broth

7-10 days 14 days first submerged generation, preinoculation tank

5% volume of preinoculation tank second submerged -

generation, 5-10%

inoculation volume of tank inoculation tank

Production culture, fermentation tank

24-26 hr 18-20 hr 100-200 hr

Fig. 6.5. The inoculum development programme for the production of Chlortetracycline by Streptomyces aureofaciens (Podojil, 1984).

the medium utilized is given in Table 6.5. These authors found that for prolific sporulation the nitrogen level had to be limited to between 0.05 and 0.1% w/v and that good aeration had to be maintained. Also, an interaction was demonstrated between the nitrogen level and aeration in that the lower the degree of aeration the lower the concentration of nitrogen needed to induce sporulation. Submerged sporulation was induced by inoculating 600 cm3 of the above medium, in a 2-dm3 shake flask, with spores from a well-sporulated Czapek-Dox agar culture and incubating at 25° for 7 days. The resulting suspension of spores was then used as a 10% inoculum for a vegetative seed stage in a stirred fermenter, the seed culture subsequently providing a 10% inoculum for the production fermentation. The vegetative seed and production media are given in Table 6.1.

Most actinomycetes do not sporulate in submerged culture (Whitaker, 1992) and, thus, solid or solidified media tend to be used for the production of spore inocula.

The use of the spore inoculum The stage in an inoculum development programme

Table 6.5. Media for the submerged sporulation of selected fungi

Rhodes et al. (1957): Pénicillium patulum

Whey powder, to give Lactose 3.5%

Nitrogen 0.05%

KH2P04 0.4%

Corn-steep liquor solids to give approx. 0.04% N 0.38%

Foster et al. (1945): Pénicillium notatum

Sucrose 2.0%

NaN03 0.6%

KH2P04 0.15%

MgS04-7H20 0.05%

CaCl2 2.5%

Vezina et al. (1965): Aspergillus ochraceus

Glucose 2.5%

Corn-steep liquor 0.5%

Molasses 5.0%

at which a large-scale spore inoculum is used varies according to the process; it appears to be common practice that the penultimate stage is so inoculated, but this will depend on the scale of the production fermentation. In the inoculum development programme for the early penicillin fermentation described by Parker (1950) the penultimate stage was inoculated with a spore suspension (from a 'roll-bottle') and this stage may have produced either a vegetative or a submerged spore inoculum for the final fermentation. For the griseofulvin process, Rhodes el al. (1957) stated that the spore suspension obtained from the submerged sporulation stage could either be used for direct inoculation of the production fermentation or it could be germinated in an inoculum development medium to yield a vegetative inoculum for the final fermentation. The latter course was preferred and an inoculum volume of 7-10% was used. From Figs 6.4 to 6.6 it can

One loopful spores

One loopful spores

Culture of producer organism on agar slant

30ml of 1st seed medium in 250 ml Erlenmeyer flask

30ml of 1st seed 30°C for 5 days culture

30ml of 1st seed 30°C for 5 days culture

30°C for 2 days on rotary shaker on rotary shaker

300ml of 2nd seed medium in 2 liters Erlenmeyer flask on rotary shaker

Culture of producer organism on agar slant

30ml of 1st seed medium in 250 ml Erlenmeyer flask

30°C for 2 days on rotary shaker

300ml of 2nd seed medium in 2 liters Erlenmeyer flask

1.5 liters of 2nd seed culture

1.5 liters of 2nd seed culture

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