Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes.

Martin Kunze,Clemens Lattermann,Wolfgang Kroutil,Jochen Büchs,Sylvia Diederichs

doi:10.1186/1754-1611-8-22

Martin Kunze, Clemens Lattermann + Show 3 more

Open Access

https://doi.org/10.1186/1754-1611-8-22

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Abstract

BackgroundBioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex.ResultsIn this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay.ConclusionMicrotiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

Highlights

Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones
The wells are jacketed by these radiators providing efficient heat transfer between the thermostating block and the microtiter plates (MTP) wells
Sufficient aeration can be assured as discussed for microbial cultivations. With all these presented features the system allows the investigation of a great variety of biocatalysts: psychro, meso- or thermophilic; oxygen dependent or not. Running bioprocesses at their temperature optima is essential for their economical operation

Summary

Introduction

Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Systems for rapid determination of temperature dependent reaction kinetics are rare. There is a need for a high-throughput screening procedure of temperature dependent process behavior. Bioprocesses, either fermentations or enzymatic catalysis, depend on a number of different operating parameters. Thereby, the temperature optimum can be different within one microbial or enzymatic system, either the focus is on growth or product formation [2], on enzyme activity or stability [3,4]. The analysis of temperature-specific activities and kinetic parameters of enzymes is traditionally performed in a spectrophotometric way by use of a temperaturecontrolled water jacketed single cuvettes [5]. Even though the external thermostat provides constant and accurate temperature control over a broad temperature range, the numbers of samples handled for simultaneous reading in such spectrophotometers are usually very limited

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