Respirometric 13C flux analysis, Part I: design, construction and validation of a novel multiple reactor system using on-line membrane inlet mass spectrometry

Abstract

A novel method for (13)C flux analysis based on on-line CO(2) labeling measurements is presented. This so-called respirometric (13)C flux analysis requires multiple parallel (13)C labeling experiments using differently labeled tracer substrates. In Part I of the work, a membrane-inlet mass spectrometry-based measurement system with 6 parallel reactors with each 12 ml liquid volume and associated experimental and computational methods for the respirometric (13)C data acquisition and evaluation are described. Signal dynamics after switching between membrane probes follow exactly first-order allowing extrapolation to steady state. Each measurement cycle involving 3 reactors takes about 2 min. After development of a dynamic calibration method, the suitability and reliability of the analysis was examined with a lysine-producing mutant of Corynebacterium glutamicum using [1-(13)C(1)], [6-(13)C(1)], [1,6-(13)C(2)] glucose. Specific rates of oxygen uptake and CO(2) production were estimated with an error less than +/-0.3 mmol g(-1) h(-1) and had +/-3% to +/-10% deviations between parallel reactors which is primarily caused by inaccuracies in initial biomass concentration. The respiratory quotient could be determined with an uncertainty less than +/-0.02 and varied only +/-3% between reactors. Fractional labeling of CO(2) was estimated with much higher precision of about +/-0.001 to +/-0.005. The detailed statistical analysis suggested that these data should be of sufficient quality to allow physiological interpretation and metabolic flux estimation. The obtained data were applied for the respirometric (13)C metabolic flux analysis in Part II.