SPectroscopies for the Evaluation and ConTrol in Real time of biological cells physiological statE – SPECTRE

At the heart of bioprocesses the activity and the physiological state of microorganisms are variables still difficult to assess. Most of the information is obtained from delayed off-line measurements and remains insufficient for the development of real time control strategies to optimize the potential of micro-organisms and design high performance processes. On-line quantification of the physiological state of cells is paramount for the understanding and improvement of cell metabolism and thus to control pathways of interest.
The main objective of SPECTRE is to develop an on-line system able to monitor the physiological state of microorganisms during fermentation or cell cultures.
SPECTRE is partly in continuity with the work developped during the previous ANR FASST program (Programme ANR-06-BIOE-003-01-FASST : Fermentation Alcoolique d'hydrolysats lingo-cellulosiques et obtention de Souches adaptées aux Stress Technologiques). During FASST, advanced methods for the determination of yeast strain viability state were developed. In association with off-line data, on-line dielectric spectroscopy was able to track variations of cell cytoplasm conductivity and microscopy image analysis showed that cell size distribution and cell optical properties were strongly correlated with yeast cell viability. The results of the program have been positively evaluated by the ANR and ADEME expert boards.

Dielectric spectroscopy (DS) has been operational for the last ten years. This technique is now routinely used in a number of cell culture and fermentation processes for the determination of biomass concentration. However, it can also give access to informations dependent on the biomass state, but has to be completed by additional techniques to access the value of biologically significant variables. The determination of total cell volume, viability, and cell size are required to calculate the membrane capacitance Cm, representative of the cell enveloppe state, and the cytoplasmic conductivity si, a marker of water and ion exchanges between cells and their environment. Off-line measurements, on samples taken during fermentation or cell cultures, give a differed access to the information provided by the DS and are not suitable for online control.

The proposed SPECTRE project is based primarily on :
- the study of a coupling of two innovative technologies - spatially resolved optical spectroscopy (SRS) and dielectric spectroscopy (DS) - for the online determination of cell physiological marker variables (size, membrane capacitance, intracellular conductivity...).
- the implementation of associated measurement (quantitative microscopy, flow cytometry, optical density, fluorescence, ...) which will allow
- the validation of the information collected by DS and SRS, and
- the selection of the most relevant additional physical variables (and their associated measurement techniques) eventually able to further improve the robustness of the physiological state evaluation of the cultivated populations.
The project will lead to the development of generic tools allowing the real-time control of the physiological state of microbial populations. SPECTRE connects six academic teams expert in Bioprocess Engineering and an SME, leader in the SRS domain and in the associated data analysis techniques. Each team will use cell models chosen both for their established academic and industrial interest.