Abstract
BackgroundDuring the past years, new high-throughput screening systems with capabilities of online monitoring turned out to be powerful tools for the characterization of microbial cell cultures. These systems are often easy to use, offer economic advantages compared to larger systems and allow to determine many important process parameters within short time. Fluorescent protein tags tremendously simplified the tracking and observation of cellular activity in vivo. Unfortunately, interferences between established fluorescence based dissolved oxygen tension (DOT) measurement techniques and fluorescence-based protein tags appeared. Therefore, the applicability of new oxygen-sensitive nanoparticles operated within the more suitable infrared wavelength region are introduced and validated for DOT measurement.ResultsThe biocompatibility of the used dispersed oxygen-sensitive nanoparticles was proven via RAMOS cultivations for Hansenula polymorpha, Gluconobacter oxydans, and Escherichia coli. The applicability of the introduced DOT measurement technique for online monitoring of cultivations was demonstrated and successfully validated. The nanoparticles showed no disturbing effect on the online measurement of the fluorescence intensities of the proteins GFP, mCherry and YFP measured by a BioLector prototype. Additionally, the DOT measurement was not influenced by changing concentrations of these proteins. The kLa values for the applied cultivation conditions were successfully determined based on the measured DOT.ConclusionsThe introduced technique appeared to be practically as well as economically advantageous for DOT online measuring in microtiter plates. The disadvantage of limited availability of microtiter plates with immobilized sensor spots (optodes) does not apply for this introduced technique. Due to the infrared wavelength range, used for the DOT measurement, no interferences with biogenic fluorescence or with expressed fluorescent proteins (e.g. YFP, GFP or mCherry) occur.
Highlights
During the past years, new high-throughput screening systems with capabilities of online monitoring turned out to be powerful tools for the characterization of microbial cell cultures
In the recent past, high-throughput screening systems such as the BioLector technology [1, 2] which is based on shaken microtiter plates (MTP), the μ24 system [3] as combination of bubble column and shaken bioreactor system or the stirred ambr bioreactors [4, 5] became true
The dissolved oxygen tension (DOT) becomes accessible in MTPs and in small scale stirred bioreactor systems via oxygen-sensitive fluorescence dyes [16, 17]
Summary
New high-throughput screening systems with capabilities of online monitoring turned out to be powerful tools for the characterization of microbial cell cultures. These systems are often easy to use, offer economic advantages compared to larger systems and allow to determine many important process parameters within short time. The BioLector system has been introduced and proven as powerful technology for quasi-continuous online monitoring of cultivations in MTPs. With the help of Ladner et al Microb Cell Fact (2015) 14:161 an optical fiber bundle and a fluorescence spectrometer the online measurement of scattered light as online biomass signal and biogenic fluorescences was made accessible (Fig. 1a) [1, 2]. These dyes change their fluorescence behavior in response to DOT and are usually immobilized as sensor spots (optodes) at the bottom of the cultivation vessel
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