A novel milliliter-scale chemostat system for parallel cultivation of microorganisms in stirred-tank bioreactorsby Andreas Schmideder, Timm Steffen Severin, Johannes Heinrich Cremer, Dirk Weuster-Botz

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Accepted Manuscript

Title: A novel milliliter-scale chemostat system for parallel cultivation of microorganisms in stirred-tank bioreactors

Author: Andreas Schmideder Timm Steffen Severin Johannes

Heinrich Cremer Dirk Weuster-Botz

PII: S0168-1656(15)30028-6

DOI: http://dx.doi.org/doi:10.1016/j.jbiotec.2015.06.402

Reference: BIOTEC 7147

To appear in: Journal of Biotechnology

Received date: 27-3-2015

Revised date: 10-6-2015

Accepted date: 16-6-2015

Please cite this article as: Schmideder, Andreas, Severin, Timm Steffen, Cremer,

Johannes Heinrich, Weuster-Botz, Dirk, A novel milliliter-scale chemostat system for parallel cultivation of microorganisms in stirred-tank bioreactors.Journal of

Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2015.06.402

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A novel milliliter-scale chemostat system for parallel cultivation ofmicroorganisms in stirred-tank bioreactors

Andreas Schmideder, Timm Steffen Severin, Johannes Heinrich Cremer, Dirk


Institute ofBiochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching

E-Mail-Addresses: a.schmideder@lrz.tum.de t.severin@lrz.tum.de johannescremer@web.de d.weuster-botz@lrz.tum.de *Corresponding footnote:

Postal address: Institute of Biochemical Engineering, TechnischeUniversitätMünchen,

Boltzmannstr. 15, 85748 Garching, Germany. Phone: + 49 (089) 28915712; Fax: + 49 (089) 28915714; E-mail: d.weuster-botz@lrz.tum.de 1

Running title: milliliter-scale chemostat system 2


ApH-controlled parallel stirred-tank bioreactor system was modified for parallel continuous cultivation on a 10 milliliter-scale by connecting multichannel peristaltic pumpsfor feeding and medium removalwith micro-pipes (250 µm inner diameter).Parallel chemostat processes with E. colias an example showed high reproducibility with regard to culture volume and flow rates as well as dry cell weight, dissolved oxygen concentration and pH control at steady states (n = 8, coefficient of variation< 5 %). Reliable estimation of kinetic growth parametersof E. coliwas easily achieved within one parallel experiment by preselecting ten different steady states.

Scalability of milliliter-scale steady state results was demonstrated bychemostat studies with a stirred-tank bioreactor on a liter-scale. Thus, parallel and continuously operated stirred-tank bioreactors on a milliliter-scale facilitate timesaving and cost reducing steady state studies with microorganisms. The applied continuous bioreactor system overcomes the drawbacks of existing miniaturized bioreactors, like poor mass transfer and insufficient process control.

Key Words

Miniaturized stirred-tank bioreactors, chemostat, Escherichia coli, growth kinetics, scale-up 3


The applicationofchemostats (continuously operated ideal stirred-tank bioreactors) enablephysiological studies of cells at defined and controlled reaction conditions at steady states since the 1950s (Monod, 1950; Nocick& Szilard, 1950). The growth rate of cells can easily be controlled by the residence time of the medium in a steady state.

Nowadays, in the post-genomic era, a fundamental knowledge of microbial genomes and technologies for studies of the intracellular protein, mRNA, metabolite profiles and intracellular fluxes are available.Hence, the “omic” technologies provide the opportunity to characterize physiology of microorganisms at a molecular level. To gain a maximum of reliable data, the growth of cells under a defined, constant and highly controllable set of physico-chemical reaction conditions is essential. Therefore, the chemostat is the ideal experimental system for such investigations (e.g. Hoskisson and

Hobbs,2005). Parameters like medium composition, pH, temperature and oxygen supply can be controlled. Furthermore, cells are kept at a steady state with a constant growth rate and metabolic activity.That provides the possibility of detailed analysis of microbial metabolism under single substrate limitation (Hoskisson and Hobbs, 2005).

The major drawbacks of chemostatcultivations are the time-consuming experimental setup and the high substrate consumption, which can be a problem if expensive substratesare used, e.g. isotopic labelled substrates for 13C metabolic flux analysis (Niklas et al., 2010). A general strategy to avoid these disadvantages is the miniaturization and parallelization of bioreactor systems (Akgun et al., 2008; WeusterBotz, 2005). However, only few miniaturized chemostat bioreactor systems have been 4 developed in recent years. Nanchen et al. (2006) reported the design of parallel bioreactors for continuous cultivation of Escherichia coli(E. coli) in Hungate tubes with a working volume of 10mL. Water saturated air was sucked into the bioreactors by applying negative pressure, and small stirrer bars inside the culture vessels allowed sufficient mixing and oxygen transfer. The system was equippedwith online measurement of dissolved oxygen concentration (DO) and online analysis of exhaust gas (Klein et al., 2013). Seletzky et al. (2007) introduced special shake flasks for continuous cultivation of Corynebacteriumglutamicum.Furthermore, various systems for continuous operation on amicroscale (<1mL) have been reviewed (Zhang et al., 2006; Lee et al., 2011). Most of the developed miniaturized approaches show low oxygen transfer rates. Thatlimits maximal biomass concentrations and feeding rates (Kirk and Szita, 2013). Further drawbacks are insufficientprocess control andthe restricted probe volume for further analysis of cells and fermentation broth.