Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

Wewetzer, Sandra J.; Kunze, Martin; Ladner, Tobias; Luchterhand, Bettina; Roth, Simon; Rahmen, Natalie; Kloß, Ramona; Silva, A. Costa e; Regestein, Lars; Büchs, Jochen

doi:10.1186/s13036-015-0005-0

Cited by 64 publications

(75 citation statements)

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“…In addition, previous works have demonstrated the efficiency of the BioLector in scaling up directly from microtiter plates to a bioreactor [21, 22]. We therefore used the BioLector to screen six strains of Y. lipolytica that differed in lipid accumulation in order to set up and validate a method to discriminate among strains based on lipid production traits.…”

Section: Resultsmentioning

confidence: 99%

“…To this, the BioLector adds the ability to perform fermentation in conditions close to those of bench-top bioreactors, and, in addition, enables online fluorescence readings [21–23, 46]. To our knowledge, microfermentation systems like the BioLector have never before been used in studies of the model oleaginous yeast Y. lipolytica , either for real-time measurements of lipid production or for growth monitoring.…”

Section: Resultsmentioning

confidence: 99%

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High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production

et al. 2016

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BackgroundBecause the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment. To address this issue, small cultivation systems have been developed that mimic the conditions in benchtop bioreactors. In this work, we used one such microbioreactor system, the BioLector, to develop high-throughput fermentation procedures that optimize growth and lipid accumulation in Y. lipolytica. Using this system, we were able to monitor lipid and biomass production in real time throughout the culture duration.ResultsThe BioLector can monitor the growth of Y. lipolytica in real time by evaluating scattered light; this produced accurate measurements until cultures reached an equivalent of OD600nm = 115 and a cell dry weight of 100 g L−1. In addition, a lipid-specific fluorescent probe was applied which reliably monitored lipid production up to a concentration of 12 g L−1. Through screening various growing conditions, we determined that a carbon/nitrogen ratio of 35 was the most efficient for lipid production. Further screening showed that ammonium chloride and glycerol were the most valuable nitrogen and carbon sources, respectively, for growth and lipid production. Moreover, a carbon concentration above 1 M appeared to impair growth and lipid accumulation. Finally, we used these optimized conditions to screen engineered strains of Y. lipolytica with high lipid-accumulation capability. The growth and lipid content of the strains cultivated in the BioLector were compared to those grown in benchtop bioreactors.ConclusionTo our knowledge, this is the first time that the BioLector has been used to track lipid production in real time and to monitor the growth of Y. lipolytica. The present study also showed the efficacy of the BioLector in screening growing conditions and engineered strains prior to scale-up. The method described here could be applied to other oleaginous microorganisms.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production

et al. 2016

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“…1 the well-to-well reproducibility of parallel E. coli cultures is investigated. In this model, the E. coli cells are cultivated on a synthetic Wilms-MOPS auto-induction medium that contains three initial carbon sources (0.5 g L −1 glucose, 2 g L −1 lactose and 5 g L −1 glycerol) [13, 14, 18]. All presented signals are averages of parallel cultures and the surrounding shaded areas indicate their corresponding standard deviations.…”

Section: Resultsmentioning

confidence: 99%

“…The signals are usually presented as arbitrary units or as relative data requiring calibration. 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].…”

Section: Introductionmentioning

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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“…10 The method has the advantage that it can create almost any complex shape or geometric feature; 3D printers have reached extremely high resolution within microns and are constantly being improved. 11,12 Thus, 3D printing provides an alternative method of mass-fabricating electrochemical devices, such as stainless steel electrodes, 13 or electrochemical chips for direct biological measurements. 14 However, the fabrication of electrochemical probes with micron diameters has not been reported with 3D printed devices.…”

Section: Introductionmentioning

confidence: 99%

Novel carbon-fiber microelectrode batch fabrication using a 3D-printed mold and polyimide resin

Trikantzopoulos

Yang

Ganesana

et al. 2016

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Glass insulated carbon-fiber microelectrodes (CFMEs) are standard tools for the measurement of neurotransmitters. However, electrodes are fabricated individually and the glass can shatter, limiting application in higher order mammals. Here, we developed a novel microelectrode batch fabrication method using a 3D-printed mold and polyimide resin insulating agent. The 3D-printed mold is low cost, customizable to change the electrode shape, and allows 40 electrodes to be made simultaneously. The polyimide resin is biocompatible, quick to cure, and does not adhere to the plastic mold. The electrodes were tested for the response to dopamine with fast-scan cyclic voltammetry both in vitro and in vivo and performed similarly to traditional glass-insulated electrodes, but with lower background currents. Thus, polyimide-insulated electrodes can be mass-produced using a 3D-printed mold and are an attractive alternative for making cheap, biocompatible microelectrodes.

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Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

Cited by 64 publications

References 80 publications

High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production

High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production

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

Novel carbon-fiber microelectrode batch fabrication using a 3D-printed mold and polyimide resin

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