ARTICLE Non-invasive Online Detection of Microbial Lysine Formation in Stirred Tank Bioreactors by Using Calorespirometry Lars Regestein, 1 Thomas Maskow, 2 Andreas Tack, 1 Ingo Knabben, 1 Martin Wunderlich, 1 Johannes Lerchner, 3 Jochen Bu¨ chs 1 1 RWTH Aachen, AVT—Biochemical Engineering, Worringer Weg, 52074 Aachen, Germany; telephone: þ49-2418024633; fax: þ49-418022570; e-mail: jochen.buechs@avt.rwth-aachen.de 2 Department of Environmental Microbiology, Helmholtz Centre for Environmental Research—UFZ, Leipzig, Germany 3 TU Bergakademie Freiberg, Inst. Physical Chemistry, Freiberg, Germany ABSTRACT: Non-invasive methods for online monitoring of biotechnological processes without compromising the integrity of the reactor system are very important to generate continuous data. Even though calorimetry has been used in conventional biochemical analysis for decades, it has not yet been specifically applied for online detection of product formation at technical scale. Thus, this article demonstrates a calorespirometric method for online detection of microbial lysine formation in stirred tank bioreactors. The respective heat generation of two bacterial strains, Corynebacterium glutamicum ATCC 13032 (wild-type) and C. glutamicum DM1730 (lysine producer), was com- pared with the O 2 -consumption in order to determine whether lysine was formed. As validation of the proposed calorespirometric method, the online results agreed well with the offline measured data. This study has proven that calorespirometry is a viable non-invasive technique to detect product formation at any time point. Biotechnol. Bioeng. 2013;110: 1386–1395. ß 2012 Wiley Periodicals, Inc. KEYWORDS: calorespirometry; lysine; Corynebacterium glutamicum; stirred tank reactor Introduction It is crucial for development and control of bioprocesses to monitor biochemical parameters. Therefore, new non- invasive online measuring techniques are required (Locher et al., 1992; von Stockar et al., 2003). There are already classical ways for measuring parameters such as pH and dissolved oxygen that provide essential and very general information about the running process. Other techniques have been developed to provide more detailed information on the bioprocess, for example exhaust gas analysis, which allows conclusions about the kind of metabolic activity (e.g., respiratory quotient) (Anderlei et al., 2004; Erickson et al., 1978; Gjerkes et al., 2011). Impedance measurement can be used for online determination of viable biomass (Knabben et al., 2010a, 2011; Maskow et al., 2008). For online detection of product formation, however, the particular measuring strategy depends on the specific product. Therefore, special sensors for single products, like ethanol (Cavinato et al., 1990), as well as optical methods for fluorescing products (Hess et al., 2002; Kensy et al., 2009) were developed. The infrared-technique in general allows the simultaneous online monitoring of concentrations of several compounds in the fermentation broth at the same time (Rhiel et al., 2002; Tosi et al., 2003). However, the application of near and mid infrared-technique requires a lot of calibrations to obtain quantitative information (Ducommun et al., 2001). Additionally, all available online signals can be combined and compared with each other to predict and conclude yields, substance concentrations and metabolic pathways (Dietzsch et al., 2012; Herwig et al., 2001; Wang and Stephanopoulos, 1983). Calorimetry represents an additional method for online monitoring and controlling of bioprocesses (Larsson et al., 1991; Marison and von Stockar, 1988). Regarding the general measuring concept, there are two ways to realize non-invasive calorimetric measurements in pilot-scale and large-scale bioreactors (Regestein et al., 2012). The first one is based on the determination of the heat transfer coefficient Correspondence to: J. Bu¨ chs Received 7 September 2012; Revision received 26 November 2012; Accepted 11 December 2012 Accepted manuscript online 27 December 2012; Article first published online 17 January 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.24815/abstract) DOI 10.1002/bit.24815 1386 Biotechnology and Bioengineering, Vol. 110, No. 5, May, 2013 ß 2012 Wiley Periodicals, Inc.