On-line determination of the intracellular poly(β-hydroxybutyric acid) content in transformed Escherichia coli and glucose during PHB production using stopped-flow attenuated total reflection FT-IR Spectrometry

An automated approach for rapid on-line monitoring of the solid and liquid phases present in bioprocesses based on mid-IR Fourier transform spectrometry is introduced. The principles of this new approach are presented using the example of the quantitative and qualitative analysis of poly(beta-hydroxybutyric acid) (PHB) accumulated in living bacterial cells as well as dissolved glucose during two 27-h fermentation processes. Fr-IR spectra were recorded on-line using a diamond-attenuated total reflection (ATR) cell connected to the fermentation broth by means of a computer-controlled flow system. For calibration of the Fr-IR method, standard reference analysis procedures for PHB (gas chromatography) and glucose (HPLC) were used. While pumping the fermentation broth through the flow cell, the recorded spectra corresponded to the fermentation solution, which allowed the determination of glucose in a range from 3.8 to -10.3 g/L. Upon stopping the flow, the cells settled on the ATR surface, and the thus recorded spectra enabled the determination of the intracellular PHB content of Escherichia coli in a range from 0.005 to 0.766 g/L. Errors of cross-validation of 0.264 g/L for glucose and 0.037 g/L for PHB were obtained. Application of one PLS calibration model to another fermentation was possible with prediction errors of 0.493 g/L for glucose and 0.105 g/L for PHB. Furthermore, from the position and shape of the PHB carbonyl band, it could be concluded that the PHB granules inside the E. coli are predominantly amorphous.