Development of Inoculum for Industrial Fermentations

Development of Inoculum for Industrial Fermentations

The culture used to inoculate a fermentation should meet the following criteria and the process adopted to produce an inoculum meeting these criteria is called inoculum development.

1. It must be in healthy and active state to minimize the lag phase during fermentation.

2. It must be available in sufficiently large volumes to provide an optimum size inoculum.

3. It must be in a suitable morphological form.

4. It must be free of contamination.

5. It must retain its product-forming capabilities.

To obtain a suitable inoculum culture medium is important. The design of a production medium is determined by the nutritional requirements of the organism and also based on the requirements for maximum product formation. The seed medium may be of a different composition from the production medium since formation of product is not required during inoculum development. Still, in order to minimize the lag time in a fermentation the inoculum development medium should be sufficiently similar to the production medium.

The quantity of inoculum normally used is between 3 and 10% of the medium volume.  A relatively large inoculum volume is used to minimize the length of the lag phase and to generate the maximum biomass in the production fermenter for achieving maximum productivity.

Starting from a stock culture, the inoculum is built up in a number of stages to produce sufficient biomass to inoculate the production-stage fermenter.  This may involve two or three stages in shake flask and one to three stages in fermenters, depending on the size of the ultimate vessel. Throughout this procedure there is a risk of contamination and strain degeneration and thus stringent quality-control procedures have to be adopted. The greater the number of stages between the master culture and the production fermenter the greater is the risk of contamination and strain degeneration.

A typical inoculum-development programme

·         The master culture is reconstituted and plated on to solid medium

·         Approximately ten colonies of typical morphology of high producers are selected and inoculated on to slopes as the sub-master cultures.  Using the sub master culture, shake flasks will be inoculated to check the productivity of these cultures

·         A sub-master culture is used to inoculate a shake flask (250 or 500 ml flask containing 50 or 100 ml medium)

·         Shake flask culture is used as inoculum for a larger flask, or a laboratory fermenter

·         The larger flask or laboratory fermenter culture is then used to inoculate a pilot-scale fermenter.

Culture purity checks are carried out at each stage to detect contamination as early as possible.

References

1.      Principles of fermentation technology, PF Stanbury, A Whittakker, SJ Hall, 1995, Butterworth-Heinemann publications