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Aug 1, 2011 (Vol. 31, No. 14)

Fed-Batch Cultivation in Baffled Shake Flasks

Option Enables the Assessment of Bioprocesses in Early Development

  • While industrial bioprocesses have been operated in fed-batch mode for a long time, the screening of high-producer cell lines and a lot of other development work is still performed in batch mode for a variety of reasons. One downside to fed-batch fermentation is that it requires more instrumentation, e.g., pumps, to realize the substrate feeding.

    Another factor that favors batch mode in small scale is that the huge number of clones in screening projects requires researchers to utilize parallel and easy-to-handle cultivation vessels, including shake flasks, which have known limitations.

    m2p-labs recently developed a ready-to-use medium to address these shortcomings. The Feed-in-Time (FIT) media series offers enzyme-assisted glucose release to enable fed-batch mode in any fermentation scale. Fed-batch medium is especially useful for small-scale fermentations where simple and easy-to-use bioreactors are preferred. Currently, FIT fed-batch medium is available in synthetic and complex formulations for several cell types such as E. coli, Pichia pastoris, and Hansenula polymorpha.

    When performing an aerobic fermentation, the maximum substrate feeding rate must always be adjusted to the maximum oxygen-transfer capacity of the bioreactor, otherwise the culture cannot process the substrate over the aerobic pathway and thus, inevitably, the cells produce anaerobic byproducts such as organic acids.

    Shake flasks are generally known as bioreactors with low oxygen transfer capacities. Baffled shake flasks typically provide higher oxygen-transfer capacities but often also exhibit higher heterogeneity of results. Since the baffles are introduced manually into classical Erlenmeyer flasks by glassblowers, these baffled shake flasks are unique and reproducibility is missing.

    Duran® solved this problem by designing an automated, two-step industrial production process for a new, reproducible baffled shake flask. The baffled Duran flask possesses reproducible geometry with four bottom baffles, which ensures reproducible behavior in respect to oxygen-transfer capacity and culture growth.

  • Oxygen-Transfer Capacities

    Click Image To Enlarge +
    Figure 1. The baffled Duran flask and classical Erlenmeyer flask (left) and the corresponding kLa values determined by the sulfite oxidation method (right).

    We recently undertook a study to determine oxygen-transfer capacities in baffled and classical shake flasks. The oxygen-transfer capacity was determined by applying the sulfite oxidation method.

    The basic conditions for determining the oxygen-transfer capacity, here expressed as kLa values, were as follows: 250 mL nominal volume of the shake flasks, 15 mL filling volume, 25 mm shaking diameter, 300 rpm shaking frequency, temperature 28eC, membrane cap for Duran baffled flask, and pulp plug for Erlenmeyer flask.

    Under these conditions a kLa value of 160 h-1 ± 7% for the Duran baffled flask and a kLa value of 100 h-1 ± 3% for the Erlenmeyer flask were determined (Figure 1). We were able to show that the Duran baffled flask exceeds the classical Duran Erlenmeyer flask in oxygentransfer capacity by more than 50%, enabling a better aeration for aerobic cultures.



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