Abstract
BackgroundMicrotiter plates (MTP) are often applied as culture vessels in high-throughput screening programs. If online measuring techniques are available, MTPs can also be applied in the first steps of process development. For such small-scale bioreactors dipping probes are usually too large; therefore, optical measurements are often used. For example, the BioLector technology allows for the online monitoring of scattered light and fluorescence in each well of a continuously orbitally shaken MTP. Although this system provides valuable data, these measurements are mainly of a semi-quantitative nature. Therefore, signal calibration is required to obtain absolute values. With the µRAMOS technology it became possible for the first time to quantify the oxygen transfer rate (OTR) separately in each well of an MTP. In this work, a device is presented that combines both techniques, to provide a hitherto unparalleled high amount of information from each single well.ResultsBecause both systems (BioLector and µRAMOS) are based on optical measurements, the measurements need to be synchronized to avoid interferences with the optical signals. The new experimental setup was applied for online monitoring in cultures of Escherichia coli and Hansenula polymorpha. It has been demonstrated that the well-to-well reproducibility is very high, and that the monitored signals provide reliable and valuable information about the process. With varying filling volumes, different maximum oxygen transfer capacities (OTRmax) were adjusted in oxygen-limited cultures. The different degrees of stress during the culture due to oxygen limitation affected microbial growth and also impacted reproducibility from culture to culture. Furthermore, it was demonstrated that this new device significantly simplifies the experimental efforts: instead of parallel cultures in a shake flask and MTP, just one single experiment in MTP needs to be conducted to measure the OTR, dissolved oxygen tension (DOT), scattered light and fluorescence.ConclusionsThe new device is a very suitable system for the online monitoring of cultures in continuously orbitally shaken MTPs. Due to the high number of parameters that can simultaneously be measured with this small-scale device, deeper insight into the investigated microbial system can be achieved. Furthermore, the experimental efforts to obtain OTR, DOT, scattered light and fluorescence signals during a culture are decreased. Ultimately, this new technology and the resulting high amount of collected data will eliminate the currently existing separation between screening and process development.Graphical abstractPicture of the combined μRAMOS and BioLector setup which allows for measurements of the oxygen transfer rate (OTR), dissolved oxygen tension (DOT), scattered light and fluorescence in each single well of an orbitally shaken microtiter plate. Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0608-2) contains supplementary material, which is available to authorized users.
Highlights
Microtiter plates (MTP) are often applied as culture vessels in high-throughput screening programs
A so-called μRAMOS system was presented that allows for the direct online quantification of oxygen transfer rate (OTR) in each well [15]. μRAMOS is a miniaturized device adopted from the established respiration activity monitoring system (RAMOS) (Respiration Activity MOnitoring System) technique which allows online monitoring of eight parallel cultures in shake flask [16, 17]
Well‐to‐well reproducibility of E. coli BL21 (DE3) pRhotHi yellow fluorescent proteins (YFP) cultures The greatest benefit of high-throughput screening devices is the high number of parallel cultures that can be conducted within a single experiment
Summary
Microtiter plates (MTP) are often applied as culture vessels in high-throughput screening programs. If online measuring techniques are available, MTPs can be applied in the first steps of process development For such small-scale bioreactors dipping probes are usually too large; optical measurements are often used. The BioLector technology allows for the online monitoring of scattered light and fluorescence in each well of a continuously orbitally shaken MTP. This system provides valuable data, these measurements are mainly of a semi-quantitative nature. An alternative DOT measurement system based on dispersed infrared oxygen-sensitive nanoparticles was presented It can be applied in any type of MTP without the need of optodes [13]. A so-called μRAMOS system was presented that allows for the direct online quantification of oxygen transfer rate (OTR) in each well [15]. μRAMOS is a miniaturized device adopted from the established RAMOS (Respiration Activity MOnitoring System) technique which allows online monitoring of eight parallel cultures in shake flask [16, 17]
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