( Part I...)Fundamentals of Fermentation: Techniques For Benchtop Fermentors

Part I - E. col

Prepared by the R & D Lab, New Brunswick Scientific Co., Inc.

The following document outlines step-by-step procedures for carrying out a benchtop fermentation, particularly when using an NBS fermentor. Provided in a question and answer format, this article covers such topics as what media formulation, tubing size, and concentration of various additives should be used, as well as the preparation, autoclaving and clean up procedures for the vessel and accessories. Unless otherwise indicated, our example refers to an E. coli fermentation in a benchtop BioFlo 3000 fermentor. However, in general, the information should be valid for any fermentation.

Question: What kind of MEDIA should be used, and does it differ from media used in shake flasks?

Answer: The media used in shake flasks does differ from the standard media used in a fermentation vessel. Shake flask media is generally of a much simpler composition. LB Broth (Difco catalog number 0446-17-3 ) and Tryptic Soy Broth (Difco catalog number 0370-07-5) are standard shake flask media.

An example of a more complex media used in a recombinant E. coli fermentation follows:

For fermentation, the glucose solution is usually sterilized in a separate flask. It is then added aseptically to the other (heat labile) components that can not be subjected to autoclaving, such as Ampicillin and the trace metal solution. These are prepared in advance by sterile filtration so that they are available as stock solutions. The magnesium sulfate is sometimes sterilized separately. Most materials are available from a variety of vendors. Note that Sigma and Difco are often the best sources for the more unusual biologic
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