Development of a large-scale biocalorimeter to monitor and control bioprocesses.

Calorimetry has shown real potential at bench-scale for chemical and biochemical processes. The aim of this work was therefore to scale-up the system by adaptation of a standard commercially available 300-L pilot-scale bioreactor. To achieve this, all heat flows entering or leaving the bioreactor were identified and the necessary instrumentation implemented to enable on-line monitoring and dynamic heat balance estimation. Providing that the signals are sufficiently precise, such a heat balance would enable calculation of the heat released or taken up during an operational (bio)process. Two electrical Wattmeters were developed, the first for determination of the power consumption by the stirrer motor and the second for determination of the power released by an internal calibration heater. Experiments were designed to optimize the temperature controller of the bioreactor such that it was sufficiently rapid so as to enable the heat accumulation terms to be neglected. Further calibration experiments were designed to correlate the measured stirring power to frictional heat losses of the stirrer into the reaction mass. This allows the quantitative measurement of all background heat flows and the on-line quantitative calculation of the (bio)process power. Three test fermentations were then performed with B. sphaericus 1593M, a spore-forming bacterium pathogenic to mosquitoes. A first batch culture was performed on a complex medium, to enable optimization of the calorimeter system. A second batch culture, on defined medium containing three carbon sources, was used to show the fast, accurate response of the heat signal and the ability to perfectly monitor the different growth phases associated with growth on mixed substrates, in particular when carbon sources became depleted. A maximum heat output of 1100 W was measured at the end of the log-phase. A fed-batch culture on the same defined medium was then carried out with the feed rate controlled as a function of the calorimeter signal. A maximum heat output of 2250 W was measured at the end of the first log-phase. This work demonstrates that real-time quantitative calorimetry is not only possible at pilot-scale, but could be readily applied at even larger scales. The technique requires simple, readily available devices for determination of the few necessary heat flows, making it a robust, cost-effective technique for process development and routine monitoring and control of production processes.

On-line determination of animal cell concentration.

A new approach for the indirect determination of cell concentration in the case of nonconstant metabolic rates has been developed. The specific glucose-uptake rate was shown to be nonconstant in batch cultures of free suspended and immobilized CHO SSF3 cells. Time-independent models correlating the specific rate to the limiting substrate concentration were established, thus providing a continuous determination of the specific rate through on-line measurement of the limiting substrate. The method could be applied to determine on-line cell concentration in both free suspended and immobilized cell cultures. Results were verified off-line by crystal violet nuclei counting. The predicted cell concentration was in very good agreement with the off-line reference during the whole exponential-growth phase, until the specific glucose-uptake rate tended to zero.

A simple HPLC technique for accurate monitoring of mammalian cell metabolism.

We have developed a simple and accurate isocratic HPLC method, without any prederivatisation, for the determination of glucose, lactate, glutamine, glutamate, pyrrolidone carboxylic acid and alanine in samples from mammalian cell cultures. The method has been successfully validated with enzyme analysis for each of the compounds. Quantification of pyrrolidone carboxylic acid makes the correction for glutamine decrease due to chemical decomposition very simple and accurate, and avoids some possibly erroneous calculations.

Automated HPLC monitoring of broth components on bioreactors.

Under proper operating conditions, a low dead volume continuous filtration module operated on biological broths (yeast and bacteria suspensions in stirred reactors) still fulfills the flow-rate requirements of an analytical apparatus (for example HPLC or FIA) without membrane regeneration. The filtrate stream has been successfully connected to a bioreactor in order to perform the automated HPLC analysis of broth components. The monitoring of the carbon source (lactose), and minor products (glycerol, acetate and succinate) during a yeast culture (Kluyveromyces marxianus) is shown.