Biocalorimetry as a process analytical technology process analyser; robust in-line monitoring and control of aerobic fed-batch cultures of crabtree-negative yeast cells

Control of bioprocesses requires reliable and robust on- or in-line monitoring tools providing real-time information on process dynamics. Heat generation related to metabolic activity of living systems is currently gaining importance in bioprocess industry due to its non-invasive and essentially instantaneous characteristics. This study deals with monitoring and control of pure aerobic fed-batch cultures of three Crabtree-negative yeast strains, Kluyveromyces marxianus, Candida utilis and Pichia pastoris, based on in-line measured, metabolic heat flow signals. A high resolution biocalorimeter (BioRC1) was developed from a standard bench-scale heat flow calorimeter (RC1). The BioRC1 was equipped with in-line (dielectric spectroscopy, pH probe and dissolved oxygen probe) and at-line (exit gas analyser) sensors to characterise the growth behaviour of the yeast cells. Both metabolic heat flow and biomass profiles exhibited similar behaviour proving the significance of employing heat flow signal as a key-parameter for the system under investigation. A simple estimator for biomass concentration and specific growth rate was formulated based on heat flow values. In order to evaluate the potential of calorimetry as a reliable and powerful process monitoring tool, the robustness, reliability as well as the broad applicability of the developed estimators was assessed through comparison with off-line measurement techniques and showed promising results for general applicability with a wide range of bioprocesses.     

High-sensitive heat-flow calorimetry

Heat production rates of chemical reactions or of biological cultures are a valuable signal, able to provide crucial information about the system under investigation. Commercially available bench-scale calorimeters reach a sensitivity of ≈100 mW/l. This sensitivity is usually sufficient for investigations in the field of reaction engineering or for safety studies. For the investigation of processes that exhibit only small exothermic or endothermic effects on the observation of the growth of biological material, the sensitivity of the calorimeter needs to be improved. This paper describes the modifications undertaken to reach a sensitivity of a few milliwatts per liter with an RC1 from Mettler-Toledo.     

Anaerobic Calorimetry of the Growth of Lactobacillus Helveticus Using a Highly Sensitive Bio-RCl

To meet the need for studies of anaerobic microbial and animal cell cultures involving much lower heat effects as compared to aerobic microbial cultures, a bench scale calorimeter, Bio-RCl, has been improved for achieving a higher long-term sensitivity. This newly improved Bio-RCl was used for heat measurement of anaerobic growth of Lactobacillus helveticus. The results showed that the bench-scale calorimetry has powerful potential for on-line monitoring and control of anaerobic bioprocesses as well as fundamental studies, such as stoichiometry, thermodynamics and kinetics of cellular growth. This revised version was published online in August 2006 with corrections to the Cover Date.     

On-line calorimetry as a technique for process monitoring and control in biotechnology

This contribution reviews laboratory-scale investigations carried out on the usefulness of biological heat release measurements, as a means for monitoring and controlling the metabolic state of microbial cultures. Such studies are carried out in high-quality bench-scale calorimeters, but measuring heat generation rates by establishing energy balances ought to be applicable to large-scale bioreactors without resorting to sophisticated instrumentation. The signal received can either be interpreted by more qualitative correlation with the evolution of the culture, or it may be quantitatively exploited - together with other on-line measurements - in order to assess the rates at which various types of metabolisms proceed in the culture. The work described shows how this can be used to keep a culture in a desired metabolic state during fed-batch and transient continuous cultures of the yeast, S. cerevisae, and how a bacterial fed-batch culture can be controlled in order to optimize biosynthesis of an antibiotic.     

Painleve analysis for a semi-linear parabolic equation arising in smectic liquid crystals

This article presents a systematic method, employing a Painlevéanalysis for obtaining static and travelling wave solutionsto a semi-linear parabolic equation which frequently arisesin the smectic liquid crystal literature. The equation consideredhas sinusoidal non-linearities and shares some of the featuresof the double sine-Gordon equation; a brief discussion of therelationship of this equation to liquid crystals is also given.     

Calorimetry and thermodynamic aspects of heterotrophic,mixotrophic, and phototrophic growth

A simple stoichiometric model is proposed linking the biomass yield to the enthalpy and Gibbs energy changes in chemo-heterotrophic, mixotrophic, and photo-autotrophic microbial growth. A comparison with calorimetric experiments on the algae Chlorella vulgaris and Chlorella sorokiniana confirmed the trends but revealed large calorimetric measurement inaccuracies. The calorimetric data on purely photo-autotrophic growth was, however, in fair agreement with calculations. The thermodynamic characteristics of photosynthetic growth, including an estimation of the Gibbs energy dissipation, are compared with similar data for chemotrophic microbes.     

Formation of microcapsules from polyelectrolyte and covalent interactions

A new approach combining electrostatic and covalent bonds was established for the formation of resistant capsules with long-term stability under physiological conditions. Three kinds of interactions were generated in the same membrane: (1) electrostatic bonds between alginate and poly-L-lysine (PLL), (2) covalent bonds (amides) between propylene-glycol-alginate (PGA) and PLL, and (3) covalent bonds (amides) between BSA and PGA. Down-scaling of the capsules size (< or =1 mm diameter) with a jet break-up technology was achieved by modifying the rheological properties of the polymer solution. Viscosity of the PGA solution was reduced by 95% with four successive pH stabilizations (pH 7), while filtration (0.2 microm) and sterilization was possible. Covalent bond formation was initiated by addition of NaOH (pH 11) using a transacylation reaction. Kinetics of the chemical reaction (pH 11) were simulated by two mathematical models and adapted in order to preserve immobilization of animal cells. It was demonstrated that diffusion of NaOH in the absence of BSA resulted in gelation of 94% of the bead and death of 94% of the cells after 10 s reaction. By addition of BSA only 46% of the cells were killed within the same reaction time (10 s). Mechanical resistance of this new type of capsule could be increased 5-fold over the standard polyelectrolytic system (PLL-alginate). Encapsulated CHO cells were successfully cultivated for 1 month in a repetitive batch mode, with the mechanical resistance of the capsules decreasing by only 10% during this period. The combination of a synthetic and natural protein resulted in enhanced stability toward culture medium and proteolytic enzymes (250%).     

Calorimetry of dual limitations in yeast cultures

Chemostat cultures of one aerobic fermenting yeast (Saccharomyces cerevisiae) and one aerobic respiring yeast (Kluyveromyces fragilis) have been grown under dual C + N limitation in an isothermal reaction calorimeter. The dual limitations resulted in uncoupled oxidation of part of the glucose, which enabled the culture to adapt the ratio of nitrogen to carbon consumption rates precisely to the N/C ratio that was fed into the calorimeter. This conclusion has been reached based on the calorimetric measurements, which reflect uncoupled respiration conspicuously as abnormal heat yields or ratios of heat release per biomass grown.     

Simplified Fourier-transform mid-infrared spectroscopy calibration based on a spectra library for the on-line monitoring of bioprocesses

In order to significantly reduce the time involved in mid-infrared spectroscopy calibrations, a novel approach based on a library of pure component spectra was developed and tested with an aerobic Saccharomyces cerevisiae fermentation. Instead of the 30-50 standards that would have been required to build a chemometric model, only five solutions were used to assemble the library, namely one for each compound (glucose, ethanol, glycerol, ammonium and acetate). Concentration profiles of glucose, ethanol and ammonium were monitored with a fair accuracy, leading to standard error of prediction (SEP) values of 0.86, 0.98 and 0.15 g L⁻¹. Prediction of the two minor metabolites, acetate and glycerol, was less accurate and presented a detection limit of around 0.5 g L⁻¹. The overall performance of the library-based method proved to be very similar to a 49-standard chemometrics model. The model was shown to be very robust and uncorrelated, since it was able to predict accurately the concentration changes during a spiking experiment. Even though simple, this method allows more advanced calculations, such as determination of the explained variance and detection of unexpected compounds using residuals analysis.