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

Abstract

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.

Less Sacrifice, More Insight: Repeated Low‐Volume Sampling of Microbioreactor Cultivations Enables Accelerated Deep Phenotyping of Microbial Strain Libraries

“…Current MBR technologies enable data acquisition at high temporal resolution by means of non‐invasive measurements of various culture parameters . In addition, substrate consumption or product formation can be efficiently determined using spectrophotometric MTP‐based assays .…”

“…[18][19][20] Current MBR technologies enable data acquisition at high temporal resolution by means of non-invasive measurements of various culture parameters. [21][22][23] In addition, substrate consumption or product formation can be efficiently determined using spectrophotometric MTP-based assays. [24] Here, the integration of MBR systems with liquid handling robots provides the possibility to sample microbial cultivations in an automated way.…”

Prediction of Escherichia coli expression performance in microtiter plates by analyzing only the temporal development of scattered light during culture

“…Technical efforts might be reduced by applying more advanced mathematical methods. Recently, a system for online multi-wavelength (2D) fluorescence spectroscopy in each well of a MTP was presented [21]. By applying chemometrics based on the acquired multi-wavelength (2D) fluorescence spectra, models were developed to predict the concentration also of non-fluorescent compounds such as carbon sources and overflow metabolites (glucose, acetate and glycerin) during the cultures [21].…”

Microbioreactor Systems for Accelerated Bioprocess Development

“…For bioprocess development, measurements are needed that capture both the microbial phenotype and the process characteristics. Next to state‐of‐the‐art monitoring capabilities for biomass and other culture parameters, the incorporation of methods for individual determination of respiration activities from sub‐mL cultivations and metabolite quantification by multi‐wavelength fluorometric techniques or Fourier transformed infrared spectroscopy (FTIR) were reported to be valuable extensions for MBR systems. Especially, the high information content from 2D fluorescence spectra and FTIR requires chemometric models that rely on training and validation datasets, again highlighting the need for novel data evaluation procedures.…”