Assessing the capabilities of 2D fluorescence monitoring in microtiter plates with data-driven modeling for secondary substrate limitation experiments of Hansenula polymorpha

Abstract

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.

Advancing 2D fluorescence online monitoring in microtiter plates by separating scattered light and fluorescence measurement, using a tunable emission monochromator

“…The sampling broth was analyzed for pH value (HI21; HANNA instruments Inc., electrode: InLab Solids; Mettler Toledo GmbH), OD 600 , and carbon source concentrations. Except for a temperature of 60°C used during HPLC measurement, the procedure for the offline measurements was identical to the previous studies (Berg et al, 2022(Berg et al, , 2023. The linear correlation between OD 600 and CDW is given in Supporting Information: Figure 3.…”

“…The subsequent data processing, and MVDA were performed using MATLAB 9.10.0.1710957 (R2021a) Update 4 and the opensource toolbox mdatools (Kucheryavskiy, 2019). The post-experimental spectral processing steps were comparable to those described in Berg et al (2022Berg et al ( , 2023. The detected spectral range was reduced to include only intensities of λ em between −20 and +270 nm relative to the λ ex .…”

“…The MVDA was conducted identically to the descriptions in Berg et al (2022Berg et al ( , 2023. PCA was performed using singular value decomposition (Wall et al, 2005).…”

“…The models were generated using the SIMPLS algorithm (de Jong, 1993). In contrast to the approach presented in Berg et al (2022Berg et al ( , 2023, the number of latent variables (LVs) used to generate the PLS models of this study was arbitrarily chosen to five. This facilitates the comparison and interpretation of the models, generated for different measurement For the sorbitol and glucose concentration, before PLS model generation, the residual carbon source concentrations were converted to the total consumed carbon source concentrations.…”

“…Only by combining a grating‐based emission monochromator with a CCD camera, comprehensive online 2D fluorescence monitoring of 48 parallel cultures with up to 40,000 single intensities per spectrum was enabled (Ladner, Beckers, et al, 2016). This groundbreaking advancement of the measurement concept was applied in multiple studies to investigate the robustness of PLS modeling for screening experiments (Berg et al, 2023) and the possibility to reduce the required sampling for model calibration (Babor et al, 2023; Berg et al, 2022; Paquet‐Durand et al, 2017).…”