Biotechnology Colloquium: Dr. Ivan Schlembach (HKI Jena)
Guest: Dr. Ivan Schlembach (HKI Jena)
Title:
Online 2D-fluorescence spectroscopy coupled to PARAFAC decomposition as versatile multiparameter sensor for bioprocess analysis
Abstract:
Optical process analytical technology is a vital tool for non-invasive real-time characterization and control of bioprocesses. Among these, optical density and backscattered light measurements are widely used for assessing biomass growth. However, these technologies are strongly influenced by strain morphology, insoluble media components and air bubbles. In-situ fluorescence spectroscopy is a powerful technology capable of measuring a variety of variables, including biomass growth, substrate consumption and product formation, which is much less affected by these interferences. Furthermore, the possibility to acquire high-resolution 2D excitation-emission matrices enables precise decomposition and identification of fluorophores in complex systems, even with significant spectral overlap. This can be done by parallel factor analysis (PARAFAC), a tensor decomposition method capable of decomposing mixed trilinear spectra into its single components, without any prior knowledge. PARAFAC provides excitation and emission spectra of the components along with their concentration profiles and considerably increases the sensitivity. This enables detailed investigation of autofluorescence dynamics, which can be exploited for example to characterize cellular redox state, metabolic switches, amino acid uptake, and protein production. Additionally, spectral analysis of backscattered light can yield complementary information.
Despite these advantages, fluorescence spectroscopy is rarely applied in routine bioprocess analysis, primarily due to a lack of standardized equipment for stirred tank fermenters. Here, a novel and flexible setup for online fluorescence spectroscopy in stirred tank fermenters was developed, which allows to connect a standard benchtop fluorescence spectrometer with various bioreactors from lab to production scale.
To demonstrate the capabilities of this setup, a process for cellulase production using the industrially widely used fungus Trichoderma reesei was chosen. The process was conducted in a turbid mineral medium with solid cellulose particles as the substrate, highlighting the robustness against turbidity. To demonstrate the analytic performance in a complex mixture containing many fluorophores and compare recombinant vs. autofluorescence, an mCherry tagged strain was used.
Tryptophan autofluorescence served as excellent signal to follow biomass formation while simultaneously many other known and unknown autofluorescence compounds could be traced. The system also proved excellent comparability to small-scale Biolector fluorescence measurements. The work highlights the potential of online fluorescence spectroscopy as a powerful and yet underrepresented PAT-technology, which allows to evaluate a multitude of factors using a single sensor.