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Integrated optical sensing of dissolved oxygen in microtiter plates: A novel tool for microbial cultivation
Author(s) -
John Gernot T.,
Klimant Ingo,
Wittmann Christoph,
Heinzle Elmar
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10534
Subject(s) - laboratory flask , microtiter plate , oxygen , chemistry , dithionite , limiting oxygen concentration , cuvette , chromatography , sodium dithionite , shaker , ph indicator , analytical chemistry (journal) , light intensity , corynebacterium glutamicum , dissolved organic carbon , fluorophore , environmental chemistry , fluorescence , inorganic chemistry , organic chemistry , biochemistry , optics , physics , quantum mechanics , vibration , gene , enzyme
Abstract Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96‐well polystyrene microtiter plates. The oxygen‐sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen‐insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well‐defined media. Oxygen transfer 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 oxygen was initially depleted by the addition of sodium dithionite 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 values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation 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 m M h −1 . Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 829–836, 2003.