Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures

Abstract

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calorimeter (BioRC1), improved to a sensitivity range of 8 mW l(-1) in order to facilitate the monitoring of even weakly exothermic biochemical reactions. A proportional-integral feedback control strategy based on these estimators was designed and implemented to control the growth rate of Candida utilis, Kluyveromyces marxianus and Pichia pastoris by regulating an exponential substrate feed. Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore a key sequence for the step from quality by analysis to quality by design. The potential of biocalorimetry as a reliable biomass monitoring tool and as a key part of a robust control strategy for aerobic fed-batch cultures of Crabtree-negative yeast cells in defined growth medium was investigated. Presenting controller errors of less than 4% in the best cases, the approach paves the way for the development of a generally applicable process analytical technology platform for monitoring and control of microbial fed-batch cultures.

Real-time monitoring and control of microbial bioprocesses with focus on the specific growth rate: current state and perspectives

“…Of particular interest are the important oscillations in the values of μ that are observed by several authors Farza et al 1997;Claes and Van Impe 1999;Levisauskas 2001;Ferreira et al 2005;Kaiser et al 2008;Dabros et al 2010;Schuler et al 2012), even during the exponential phase in batch cultures (Fig. 1).…”

“…3 Excerpt of a fed-batch culture of E. coli where the specific growth rate was maintained at a sufficiently low level to avoid overflow metabolite production. (Reprinted from Biener et al 2010, with permission from Elsevier) as well as Schuler et al (2012) compared feed-forward control as given by Eq. 2 with closed-loop control.…”

“…FTIR and NIR spectroscopy could, as mentioned in Section "Substrates", be an alternative to at-line HPLC or gas chromatography for the quantification of overflow metabolites such as acetate, ethanol, or citric acid but might also be considered for determining the amount of recombinant protein produced in real-time (Sellick et al 2010). Reaction heat evolution rates can be determined by microcalorimetric (Maskow et al 2004(Maskow et al , 2006Ma et al 2007) and bench-scale calorimetric devices (van Kleeff et al 1998;Marison et al 1998;Schill et al 1999;Schuler et al 2012) and also by using simple instrumentation such as temperature probes and mass flow meters (Biener et al 2010), particularly at large-scale (Voisard et al 2002).…”

The Application of Dielectric Spectroscopy and Biocalorimetry for the Monitoring of Biomass in Immobilized Mammalian Cell Cultures

“…Biocalorimetry has been used in many different applications, including to monitor culture metabolism and control the feed of nutrients and hence the growth rate. By doing so, respiro-fermentative metabolism caused by overflow metabolism in Crabtree positive organisms could be avoided [34][35][36][37][38]. In addition, a study was performed in the RC1 to determine the existence of endothermic bacterial strains [39].…”

“…Once the cells have no longer any free space to replicate, the cell growth should become inhibited. As the heat flow rate is dependent upon the metabolic rate, it is forecast for the heat signal to fall and stabilise at an inferior heat flow rate, Figure 9B, [36,37].…”