Easy to use and reliable technique for online dissolved oxygen tension measurement in shake flasks using infrared fluorescent oxygen-sensitive nanoparticles.

David Flitsch,Mihaly Lukacs,Tobias Ladner,Jochen Büchs

doi:10.1186/s12934-016-0444-4

Abstract

BackgroundDespite the progressive miniaturization of bioreactors for screening purposes, shake flasks are still widespread in biotechnological laboratories and industry as cultivation vessels. Shake flasks are often applied as the first or second step in applications such as strain screening or media optimization. Thus, there are ongoing efforts to develop online measurement techniques for shake flasks, to gain as much information as possible about the cultured microbial system. Since dissolved oxygen tension (DOT) is a key experimental parameter, its accurate determination during the course of experiment is critical. Some of the available DOT measurement techniques can lead to erroneous measurements or are very difficult to handle. A reliable and easy to use DOT measurement system, based on suspended oxygen-sensitive nanoparticles, is presented in this work.ResultsIn a cultivation of Kluyveromyces lactis, a new DOT measurement technique via suspended oxygen-sensitive nanoparticles was compared with the conventional DOT measurement via fixed sensor spots. These experiments revealed the main disadvantage of applying sensor spots. With further cultivations of Escherichia coli and Hansenula polymorpha, the new measurement technique was successfully validated. In combination with a RAMOS device, kLa values were determined during the presented cultivations. The determined kLa values are in good agreement with a correlation recently found in the literature.ConclusionsThe presented DOT measurement technique via suspended oxygen-sensitive nanoparticles in shake flasks turned out to be easy to use, robust and reliable under all applied combinations of shaking frequencies and filling volumes. Its applicability as an online monitoring system for cultivations was shown by means of four examples. Additionally, in combination with a RAMOS device, the possibility of experimental kLa determination was successfully demonstrated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0444-4) contains supplementary material, which is available to authorized users.

Highlights

Despite the progressive miniaturization of bioreactors for screening purposes, shake flasks are still widespread in biotechnological laboratories and industry as cultivation vessels
Characterization of the dissolved oxygen tension (DOT) measurement system Ladner et al [29] demonstrated the biocompatibility of the applied oxygen-sensitive nanoparticles in cultivations of Gluconobacter oxydans, Hansenula polymorpha and Escherichia coli with concentrations up to 1 g L−1
By using dispersed oxygen-sensitive nanoparticles, an easy to use, robust and reliable DOT measurement was developed, which is applicable for almost all cultivation conditions in shake flasks and for both, soluble complex and synthetic media

Summary

Introduction

Despite the progressive miniaturization of bioreactors for screening purposes, shake flasks are still widespread in biotechnological laboratories and industry as cultivation vessels. There are ongoing efforts to develop online measurement techniques for shake flasks, to gain as much information as possible about the cultured microbial system. [11,12,13] This technology is based on oxygen and pressure measurements in the head space of modified 250 mL shake flasks during phases of air supply and phases of terminated air supply. In the latter phase the linear decrease of the oxygen partial pressure is evaluated and from this information the OTR is calculated. Online DOT measurement became possible by mounting a classical electrochemical Clark electrode into the shake flask [15, 16]. It has been shown that the baffling effect of these immersed electrodes can lead to significant hydrodynamic changes in shaken cultures [17, 18]

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