Browsing by Subject "High-throughput"

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Assessing the capabilities of 2D fluorescence monitoring in microtiter plates with data-driven modeling for secondary substrate limitation experiments of Hansenula polymorpha
(2023) Berg, Christoph; Herbst, Laura; Gremm, Lisa; Ihling, Nina; Paquet-Durand, Olivier; Hitzmann, Bernd; Büchs, Jochen; Berg, Christoph; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany; Herbst, Laura; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany; Gremm, Lisa; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany; Ihling, Nina; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany; Paquet-Durand, Olivier; Department of Process Analytics & Cereal Science, Institute for Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Hitzmann, Bernd; Department of Process Analytics & Cereal Science, Institute for Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Büchs, Jochen; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany
Background: Non-invasive online fluorescence monitoring in high-throughput microbioreactors is a well-established method to accelerate early-stage bioprocess development. Recently, single-wavelength fluorescence monitoring in microtiter plates was extended to measurements of highly resolved 2D fluorescence spectra, by introducing charge-coupled device (CCD) detectors. Although introductory experiments demonstrated a high potential of the new monitoring technology, an assessment of the capabilities and limits for practical applications is yet to be provided. Results: In this study, three experimental sets introducing secondary substrate limitations of magnesium, potassium, and phosphate to cultivations of a GFP-expressing H. polymorpha strain were conducted. This increased the complexity of the spectral dynamics, which were determined by 2D fluorescence measurements. The metabolic responses upon growth limiting conditions were assessed by monitoring of the oxygen transfer rate and extensive offline sampling. Using only the spectral data, subsequently, partial least-square (PLS) regression models for the key parameters of glycerol, cell dry weight, and pH value were generated. For model calibration, spectral data of only two cultivation conditions were combined with sparse offline sampling data. Applying the models to spectral data of six cultures not used for calibration, resulted in an average relative root-mean-square error (RMSE) of prediction between 6.8 and 6.0%. Thus, while demanding only sparse offline data, the models allowed the estimation of biomass accumulation and glycerol consumption, even in the presence of more or less pronounced secondary substrate limitation. Conclusion: For the secondary substrate limitation experiments of this study, the generation of data-driven models allowed a considerable reduction in sampling efforts while also providing process information for unsampled cultures. Therefore, the practical experiments of this study strongly affirm the previously claimed advantages of 2D fluorescence spectroscopy in microtiter plates.
Online 2D fluorescence monitoring in microtiter plates allows prediction of cultivation parameters and considerable reduction in sampling efforts for parallel cultivations of Hansenula polymorpha
(2022) Berg, Christoph; Ihling, Nina; Finger, Maurice; Paquet-Durand, Olivier; Hitzmann, Bernd; Büchs, Jochen
Multi-wavelength (2D) fluorescence spectroscopy represents an important step towards exploiting the monitoring potential of microtiter plates (MTPs) during early-stage bioprocess development. In combination with multivariate data analysis (MVDA), important process information can be obtained, while repetitive, cost-intensive sample analytics can be reduced. This study provides a comprehensive experimental dataset of online and offline measurements for batch cultures of Hansenula polymorpha. In the first step, principal component analysis (PCA) was used to assess spectral data quality. Secondly, partial least-squares (PLS) regression models were generated, based on spectral data of two cultivation conditions and offline samples for glycerol, cell dry weight, and pH value. Thereby, the time-wise resolution increased 12-fold compared to the offline sampling interval of 6 h. The PLS models were validated using offline samples of a shorter sampling interval. Very good model transferability was shown during the PLS model application to the spectral data of cultures with six varying initial cultivation conditions. For all the predicted variables, a relative root-mean-square error (RMSE) below 6% was obtained. Based on the findings, the initial experimental strategy was re-evaluated and a more practical approach with minimised sampling effort and elevated experimental throughput was proposed. In conclusion, the study underlines the high potential of multi-wavelength (2D) fluorescence spectroscopy and provides an evaluation workflow for PLS modelling in microtiter plates.