ONLINE MONITORING WITH FEEDBACK-CONTROL OF BIOREACTORS USING A HIGH ETHANOL TOLERANCE YEAST BY MEMBRANE INTRODUCTION MASS-SPECTROMETRY

We have investigated the fermentation of glucose with a genetically en gineered yeast, strain 1400, which has a tolerance to high ethanol con centration. The concentration of the major product in the liquid phase , ethanol, was monitored on-line as a function of time using membrane introduction mass spectrometry (MIMS). Minor products produced during the fermentation and identified by MIMS include lactic acid and glycer ol. The concentration of the substrate, glucose, was monitored off-lin e using a glucose analyzer. A flow injection analysis (FIA) sampling s ystem was used to inject, in sequence, the sample, the standard, and t he flush solution (deionized water). Microfiltered broth plugs were in troduced into the mass spectrometer, through a direct insertion membra ne probe which uses a hydrophobic silicone membrane as the interface b etween the aqueous sample and the mass spectrometer. Isobutane chemica l ionization was used to produce the protonated molecular ion of ethan ol (m/z 47) in experiments with a quadrupole mass spectrometer and wat er chemical ionization was used in comparative experiments with an ion trap. All operations such as sampling, scanning, data acquisition, co ntrol of the FIA, calibration, and feedback control were carried out a utomatically, with the help of a control program written in C. The fee dback control system described in this paper was employed to automate substrate addition. This allowed the inhibition of ethanol formation d ue to high substrate (glucose) concentration to be avoided. Batch ferm entations with initial concentrations of glucose up to 150 g/l were ca rried out in this mode and an ethanol concentration of about 50 g/l wa s achieved after monitoring for 8 h. Fed-batch fermentations were also carried out, to reduce the inhibitory effect of the substrate. In a t ypical fed-batch fermentation, a final ethanol concentration of about 120 g/l was achieved, and the bioreactor was monitored for 50 h.