Integrated Optical Sensing of Dissolved Oxygen in Microtiter Plates: A Novel Tool for Microbial Cultivation Gernot T. John, 1 * Ingo Klimant, 2† Christoph Wittmann, 1 Elmar Heinzle 1 1 Biochemical Engineering, Saarland University, P.O. Box 151150, 66123 Saarbru ¨ cken, Germany; fax: +49-681-302-4572; e-mail: e.heinzle@mx.uni-saarland.de 2 Institute of Analytical Chemistry, Chemo- and Biosensorics, University of Regensburg, Regensburg, Germany Received 12 December 2001; accepted 27 August 2002 DOI: 10.1002/bit.10534 Abstract: Microtiter plates with integrated optical sens- ing of dissolved oxygen were developed by immobiliza- tion of two fluorophores at the bottom of 96-well poly- styrene microtiter plates. The oxygen-sensitive fluoro- phore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an inter- nal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen trans- fer coefficients, k L a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxy- gen was initially depleted by the addition of sodium di- thionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k L a values in one commercial reader were about 10 to 40 h −1 . k L a val- ues were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebac- terium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those ob- served in shaking flasks and in a stirred fermentor. Oxy- gen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during culti- vation in the microtiter plate were used to estimate k L a values in a 96-well microtiter plate. The resulting values were about 130 h −1 , which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h −1 . Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing condi- tions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 829– 836, 2003. Keywords: microtiter plate; oxygen sensor; oxygen transfer; dynamic k L a determination; Corynebacterium glutamicum; growth INTRODUCTION It is of general interest to develop tools for the cultivation of cells in large numbers both for screening purposes and for enhanced development of cultivation processes. Shake flasks are used intensively in industry, and there is an on- going discussion about the usefulness of data obtained from flasks with regard to scale-up and large-scale production (Henzler and Schedler, 1991; Humphrey, 1998; Maier and Bu ¨chs, 2001). Measurement of dissolved oxygen in microtiter plates is of potential interest for the screening of oxygen-consuming enzymes (e.g., oxidases), aerobic cell activities, and biologi- cal degradation of pollutants, and for toxicity tests. Conven- tional dissolved oxygen measurement uses amperometric electrodes (Hitchman, 1978). Optical sensors for the mea- surement of dissolved oxygen concentration have been stud- ied for more than a decade and have been receiving in- creased interest (Bacon and Demas, 1987; Klimant and Wolfbeis, 1995; Wolfbeis and Caroline, 1984) and commer- cial systems have been introduced. One commercial system uses an immobilized fluorescence sensor with a fluores- cence signal intensity that is related to dissolved oxygen concentration (Pitner et al., 1999). Fluorescence methods are often superior to other optical and electrochemical methods because of their high sensitiv- ity and their ability to measure fluorescence intensity as well as fluorescence decay time (Bambot et al., 1994; Huber et al., 2000). General problems with the measurement of fluorescence intensity include fluctuations in light intensity and detector sensitivity, irregular sensor spots, light scatter- ing, and intrinsic fluorescence of the sample. Therefore, the use of a second internal standard fluorophore with emission at a different wavelength is promising. The internal standard is insensitive to oxygen and serves as reference; the fluo- rescence intensity of the actual sensing fluorophore is re- duced by increasing oxygen concentration. Ideally, both fluorophores are excited at the same wavelength and emit light at two different wavelengths. Standard commercially available readers permit measurement of fluorescence in- tensity. Instruments measuring fluorescence decay time in Correspondence to: E. Heinzle * Present affiliation: Presens GmbH, Regensburg, Germany † Present affiliation: Institute of Analytical, Micro- and Radiochemistry, Technical University, Graz, Austria Contract grant sponsor: Deutsche Bundesstiftung Umwelt Contract grant number: AZ 13040/15 © 2003 Wiley Periodicals, Inc.