Abstract

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non-parallel and non-equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag-phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed-batch mode in precultures in microtiter plates and shake flasks. Fed-batch operation in precultures is realized through a slow-release system for glucose. After differently growing cultures turn to glucose-limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow-release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high-throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha.

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