Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors.

S J Wewetzer,T Ladner,R Kloß,J Büchs,L Regestein,M Kunze,A Costa E Silva,N Rahmen,B Luchterhand,S Roth

doi:10.1186/s13036-015-0005-0

Abstract

BackgroundConventional experiments in small scale are often performed in a ‘Black Box’ fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device.ResultsThe same ‘mastermix’ (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects).ConclusionsThe parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0005-0) contains supplementary material, which is available to authorized users.

Highlights

Conventional experiments in small scale are often performed in a ‘Black Box’ fashion, analyzing only the product concentration in the final sample
Correct absolute values are provided which can for example be used to calculate material balances and stoichiometries. The aim of this current study is to present a new efficient experimental approach (Fig. 2) combining a Respiration Activity MOnitoring System (RAMOS) (Fig. 2b) and a BioLector device (Fig. 2c) coupled with an automated liquid handling system (Fig. 2d) or separate shake flasks (Fig. 2e) for offline sampling to reduce the necessary number of experiments in laboratory-scale stirred tank bioreactor (Fig. 2a)
Using Escherichia coli BL21 E. coli modified Flavin mononucleotide (FMN)-based fluorescence protein (FbFP) (EcFbFP) in modified WilmsMOPS mineral medium, Maximum oxygen transfer capacity (OTRmax) values were determined as described in the Methods section

Summary

Introduction

Conventional experiments in small scale are often performed in a ‘Black Box’ fashion, analyzing only the product concentration in the final sample. Fully equipped laboratoryscale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems They are too spacious, laborious and expensive to be operated in larger number in parallel. The aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device. The sequence, starting at strain construction and ending with pilot-scale trials (compare Fig. 1), results in long development times, termed as a “resource burden” by Bareither and Pollard in 2011 [1] This whole process may lead to false selection of strains and media due to insufficient data and overlooked unsuitable operating conditions in MTPs and shake flasks. Small-scale online monitoring systems such as the Respiration Activity MOnitoring System (RAMOS) and the BioLector devices have been developed [10,11,12,13,14,15,16]

Objectives

Methods

Results

Conclusion