Respiration activity monitoring system for any individual well of a 48-well microtiter plate

Abstract

BackgroundSmall-scale micro-bioreactors have become the cultivation vessel of choice during the first steps of bioprocess development. They combine high cultivation throughput with enhanced cost efficiency per cultivation. To gain the most possible information in the early phases of process development, online monitoring of important process parameters is highly advantageous. One of these important process parameters is the oxygen transfer rate (OTR). Measurement of the OTR, however, is only available for small-scale fermentations in shake flasks via the established RAMOS technology until now. A microtiter plate-based (MTP) μRAMOS device would enable significantly increased cultivation throughput and reduced resource consumption. Still, the requirements of miniaturization for valve and sensor solutions have prevented this transfer so far. This study reports the successful transfer of the established RAMOS technology from shake flasks to 48-well microtiter plates. The introduced μRAMOS device was validated by means of one bacterial, one plant cell suspension culture and two yeast cultures.ResultsA technical solution for the required miniaturized valve and sensor implementation for an MTP-based μRAMOS device is presented. A microfluidic cover contains in total 96 pneumatic valves and 48 optical fibers, providing two valves and one optical fiber for each well. To reduce costs, an optical multiplexer for eight oxygen measuring instruments and 48 optical fibers is introduced. This configuration still provides a reasonable number of measurements per time and well. The well-to-well deviation is investigated by 48 identical Escherichia coli cultivations showing standard deviations comparable to those of the shake flask RAMOS system. The yeast Hansenula polymorpha and parsley suspension culture were also investigated.ConclusionsThe introduced MTP-based μRAMOS device enables a sound and well resolved OTR monitoring for fast- and slow-growing organisms. It offers a quality similar to standard RAMOS in OTR determination combined with an easier handling. The experimental throughput is increased 6-fold and the media consumption per cultivation is decreased roughly 12.5-fold compared to the established eight shake flask RAMOS device.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-016-0034-3) contains supplementary material, which is available to authorized users.

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

“…Therefore, oxygen transfer rates (OTRs) were calculated from the decrease in the oxygen partial pressure in the headspace of the reaction vessel. For determination of the OTR in 48‐round‐well plates, the µRAMOS technique was applied . By integration of OTRs over time, the total oxygen transferred (OT) was calculated.…”

Quasi-continuous parallel online scattered light, fluorescence and dissolved oxygen tension measurement combined with monitoring of the oxygen transfer rate in each well of a shaken microtiter plate

“…In this work, a new device for online monitoring in MTPs is presented. The system combines the benefits of BioLector [9] and µRAMOS technology [15]. Because all measurements are based on optical signals, a special measuring sequence had to be developed to avoid interference among various measurement systems.…”

“…A 48 round deep-well MTP (MTP-R48-B, m2p-labs GmbH, Baesweiler, Germany) is placed on the shaker table of an in-house constructed orbital shaking machine (shaking diameter d 0  = 3 mm). For OTR measurements in each well, the µRAMOS cover was fixed on top of the MTP [15]. A sterile barrier (900371-T, HJ-Bioanalytik, Erkelenz, Germany) is clamped between µRAMOS cover and MTP to avoid contaminations (Fig.…”

“…Based on the phase shift between excitation and emission, the oxygen partial pressure is calculated according to the Stern–Volmer equation. To allow simultaneous measurements for one whole row of wells (eight wells), different excitation light modulation frequencies (3550–4000 Hz) are applied in the optical multiplexer of the µRAMOS [15]. Thus, no interferences between neighboring oxygen sensors occur.…”

“…Nevertheless, to get also access to qualitative data in MTPs, parallel cultures in shake flasks were conducted and the results were considered as data received by one hypothetical bioreactor [14]. In a recent study, 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].…”