Glazyrina et al. Microbial Cell Factories 2010, 9:42 http://www.microbialcellfactories.com/content/9/1/42 Open Access RESEARCH © 2010 Glazyrina et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor Julia Glazyrina †1 , Eva-Maria Materne †1 , Thomas Dreher 2 , Dirk Storm 1 , Stefan Junne 1 , Thorsten Adams 2 , Gerhard Greller 2 and Peter Neubauer* 1 Abstract Background: Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT ® CultiBag RM system pave the way for applying advanced fed- batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase ® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production. Results: Cultivation of E. coli in the BIOSTAT ® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD 600 ) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD 600 of 60 (20 g L -1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase ® Flo, OD 600 of 30 (10 g L -1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase ® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor. Conclusions: Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT ® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion- type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase ® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities. * Correspondence: peter.neubauer@tu-berlin.de 1 Laboratory of Bioprocess Engineering, Department of Biotechnology, T echnische Universität Berlin, Ackerstraße 71-76, D-13355 Berlin, Germany † Contributed equally Full list of author information is available at the end of the article