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.

BackgroundConventional experiments in small scale are often performed in a ‘Black Box’ fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device.ResultsThe same ‘mastermix’ (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects).ConclusionsThe parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0005-0) contains supplementary material, which is available to authorized users.

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.

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.

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.

Background Escherichia coli is often used for recombinant protein production. The expression of recombinant proteins negatively affects the microbial growth, thus, a balance between protein expression and biomass formation is preferable to reach high product- and space-time-yield. Already in screening experiments, suboptimal conditions causing too weak or too strong induction must be avoided. High-throughput screening devices such as the BioLector are often applied for screening experiments. The BioLector allows optical online monitoring of each well in a continuously orbitally shaken microtiter plate via scattered light and fluorescence measurements. This technique enables a fast identification of promising clones. However, to determine the expression performance of non-fluorescent products elaborated offline analysis is often required.MethodsA mathematical method is developed to distinguish between cultures, which are insufficiently, optimally or too strongly induced. Therefore, just the temporal development of the scattered light intensity signal is investigated. It is found that discrimination between the different intensities of induction is possible via principal component analysis. By fitting an extended sigmoidal function to the trajectory of the scattered light over time, two characteristic parameters are found. These are used in an empirical model to predict the expression performance.ResultsThe method was established for a wide range of culture conditions based on 625 E. coli cultures. Three E. coli host strains (Tuner(DE3), BL21(DE3), and BL21-Gold(DE3)) expressing either flavin-mononucleotide-based fluorescent protein (FbFP) or Cellulase celA2 were investigated. Cultures were conducted in two different types of microtiter plates (48- and 96-wells), in two online measurement devices at four temperatures (28 °C, 30 °C, 34 °C, and 37 °C). More than 95% of the predicted values are in agreement with the offline measured expression performances with a satisfying accuracy of ±30%.ConclusionsThe properties of cultures studied can be represented by only two characteristic parameters (slope at and time of the inflection point) received from fitting an extended sigmoidal function to the respective scattered light trajectory. Based on these two characteristic parameters, predictions of the standardized expression performance are possible and for a first screen elaborated offline analysis can be avoided. To the best of our knowledge, this is the first work presenting a method for the general prediction of expression performance of E. coli based solely on the temporal development of scattered light signals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-017-0064-5) contains supplementary material, which is available to authorized users.

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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