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

Flitsch, David; Ladner, Tobias; Lukacs, Mihaly; Büchs, Jochen

doi:10.1186/s12934-016-0444-4

Cited by 21 publications

(31 citation statements)

References 42 publications

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“…The applied fluorescence dye is immobilized in fluorescence sensor spots (OXSP5, Pyro Science GmbH, Aachen, Germany) [25, 26]. The red light excitation and infrared light emission is advantageous for avoiding biogenic fluorescences [27, 28]. For well-resolved oxygen partial pressure measurements during stop phases, every well is equipped with a respective optical fiber and a fluorescence sensor spot (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For E. coli BL21 (DE3) pRotHi-YFP, two pre-cultivations were conducted according to the literature [17, 27, 28]. The first pre-cultivation on terrific broth (TB) medium was inoculated with 1 ml terrific broth (TB) cryocultures (200 g L −1 glycerol stocks, stock OD 600 = 1).…”

Section: Methodsmentioning

confidence: 99%

“…For the pre-cultivation of H. polymorpha RB11 pC10- FMD (P FMD - GFP ), synthetic Syn-6-MES medium was prepared according to the literature [27, 40]. The pre-cultivation was inoculated with 1 mL Syn-6-MES medium cryoculture (200 g L −1 glycerol stocks) and cultivated at T = 30 °C.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

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“…3e–h, q–t. Due to the fact that the k L a estimation is fraught with high systematic errors at low respiration activities [36], the estimated k L a is very noisy at OTR values below 5 mmol L −1 h −1 . Accordingly, the most reliable k L a estimation is conducted during oxygen-limited conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Both values were considered to be constants during cultivation. To test the sensitivity of the k L a determination, a Gaussian error propagation was performed as described in the literature [36]. The results are given in Eq.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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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.

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Parallel online multi‐wavelength (2D) fluorescence spectroscopy in each well of a continuously shaken microtiter plate

Ladner

Beckers

Hitzmann

et al. 2016

Biotechnology Journal

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Small-scale high-throughput screening devices are becoming increasingly important in bioprocess development. Conventional dipping probes for process monitoring are often too large to be used in these devices. Thus, optical measurements are often the method of choice. Even some parameters that cannot directly be measured by fluorescence become accessible via sensitive fluorescence dyes. However, not all compounds of interest are measurable by this technique. Recent studies applying multi-wavelength (2D) fluorescence spectroscopy in combination with chemometrics have shown that information on numerous analytes is obscured by the fluorescence data. Hitherto, this measurement technique has only been available on the scale of stirred tank fermenters. This work introduces a new device for multi-wavelength (2D) fluorescence spectroscopy in each well of a continuously shaken microtiter plate. Using a combination of spectrograph and CCD detector, the required time per measurement cycle in a 48-well microtiter plate was 0.5 h. Cultures of Hansenula polymorpha and Escherichia coli are monitored. The concentrations of glycerol, glucose and acetate as well as pH are determined using partial least square (PLS) models. Because a pH-sensitive fluorescence dye was not required, no dependency of the pK of a fluorescence dye exists, and measurements in the low pH range can be obtained.

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

Cited by 21 publications

References 42 publications

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

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

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

Parallel online multi‐wavelength (2D) fluorescence spectroscopy in each well of a continuously shaken microtiter plate

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