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120 420 2,500 15,000 100,000

Fig. 6.2. The development of inoculum for the commercial production of bakers' yeast. PC 1, 2 and 3 are pure culture batch fermentations. F1 and 2 are non-aseptic batch fermentations. F3 and 4 are fed-batch fermentations and F5 is the final fed-batch fermentation (Reed and Nagodawithana, 1991a).

120 420 2,500 15,000 100,000

Fig. 6.2. The development of inoculum for the commercial production of bakers' yeast. PC 1, 2 and 3 are pure culture batch fermentations. F1 and 2 are non-aseptic batch fermentations. F3 and 4 are fed-batch fermentations and F5 is the final fed-batch fermentation (Reed and Nagodawithana, 1991a).

to 2 days on a solid or liquid medium and then transferred to a seed vessel where the organism was allowed to grow for a further ten generations before transfer to the production stage. Priest and Sharp (1989) cited the use of a 5% inoculum, still in the exponential phase, for the commercial production of Bacillus «-amylase.' Underkofler (1976) emphasized that, in the production of bacterial enzymes, the lag phase in plant fermenters could be almost completely eliminated by using inoculum medium of the same composition as used in the production fermenter and employing large inocula of actively growing seed cultures. The inoculum development programme for a pilot-plant scale process for the production of vitamin Bu from Pseudomonas denitrifi-cans is shown in Fig. 6.3 (Spalla et al, 1989).

The necessity to use an inoculum in an active physiological state is taken to its extreme in the production of vinegar. The acetic-acid bacteria used in the vinegar process are extremely sensitive to oxygen starvation. Therefore, to avoid disturbing the system, the cells at the end of a fermentation are used as inoculum for the next batch by removing approximately 60% of the culture and restoring the original level with fresh medium (Conner and Allgeier, 1976). The advantage of a highly active inoculum apparently outweighs the disadvantages of possible strain degeneration and contaminant accumulation. However, strain stability is a major concern in inoculum development for fermenta tions employing recombinant bacteria. Sabatie et (1991) demonstrated that plasmid stability and produ tivity in an E. coli biotin fermentation was oTeJ~ improved if stationaiy, rather then exponential phas cells were used as inoculum. They postulated that tlT plasmid copy number may be higher in stationary eelk than in exponential ones, resulting in a lower plasmid loss in the subsequent fermentation when a stationary culture is used as inoculum. A stationary phase inocu lum would result in a lag phase, but this disadvantage" was more than compensated for by the considerable improvement in plasmid retention and biotin produc tion compared with that obtained using an exponential inoculum.

In the lactic-acid fermentation the producing organism may be inhibited by lactic acid. Thus, production of lactic acid in the seed fermentation may result in the generation of poor quality inoculum. Yamamoto et al. (1993) generated high quality inoculum of Lactococcus lactis IO-l on a laboratory scale using electrodialysis seed culture which reduced the lactate in the inoculum and reduced the length of the lag phase in the production fermentation.

An example of the development of inoculum for an anaerobic bacterial process is provided by the clostridial acetone-butanol fermentation. The process was out-competed by the petrochemical industry but there is still considerable interest in re-establishing the fermen-

STOCK CULTURE Lyophilised with skim milk

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