Equalizing growth in high‐throughput small scale cultivations via precultures operated in fed‐batch mode

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.

Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics

“…During the lag phase of the microorganisms the released glucose accumulates in the medium [10]. When the biomass concentration increases during the cultivation the accumulated glucose is consumed until the substrate-limited fed-batch phase starts.…”

“…precultures for screening experiments), then all cultures enter the fed-batch phase at different points in time. When all cultures are in the fed-batch mode, their biomass concentrations and their growth rates are equalized dependent on the glucose release kinetics [10]. In contrast, the pH-control with Na 2 CO 3 containing polymer-based controlled-release discs presented in this paper is not self-regulating.…”

Recombinant protein expression in Escherichia coli: advances and challenges

“…Upon dilution into fresh media, cells will grow at different rates leading to irreproducible induction points (Huber et al, 2009). A proper preculture (cells in an active equalized growing phase) can be prepared by growing the overnight starter culture at 20–25°C or by using a slow-release system for glucose, among other methods (Busso et al, 2008; Huber et al, 2009; Sivashanmugam et al, 2009). After inoculation and further growth, the inducer is often added in mid-log phase because the culture is growing fast and protein translation is maximal.…”

Development of a modified Respiration Activity Monitoring System for accurate and highly resolved measurement of respiration activity in shake flask fermentations

“…Since its introduction, RAMOS has been used for multiple applications: e.g., for determining oxygen limitations in shake flask cultivations [18,25-30], screening of microorganisms [31-33], optimizing media [34-36], investigating secondary substrate limitations [37,38] and stress phenomena [39], process development and optimization [2,40-44] and for monitoring precultures for fermentations in stirred tank reactors [45,46]. The setup of a RAMOS device is illustrated in Figure 1.…”