On-line monitoring with feedback control of bioreactors using a high ethanol tolerance yeast by membrane introduction mass spectrometry

Abstract

We have investigated the fermentation of glucose with a genetically engineered yeast, strain 1400, which has a tolerance to high ethanol concentration. 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 glycerol. The concentration of the substrate, glucose, was monitored off-line using a glucose analyzer. A flow injection analysis (FIA) sampling system was used to inject, in sequence, the sample, the standard, and the flush solution (deionized water). Microfiltered broth plugs were introduced into the mass spectrometer, through a direct insertion membrane probe which uses a hydrophobic silicone membrane as the interface between the aqueous sample and the mass spectrometer. Isobutane chemical ionization was used to produce the protonated molecular ion of ethanol (m/z 47) in experiments with a quadrupole mass spectrometer and water chemical ionization was used in comparative experiments with an ion trap. All operations such as sampling, scanning, data acquisition, control of the FIA, calibration, and feedback control were carried out automatically, with the help of a control program written in C. The feedback control system described in this paper was employed to automate substrate addition. This allowed the inhibition of ethanol formation due to high substrate (glucose) concentration to be avoided. Batch fermentations with initial concentrations of glucose up to 150 g/l were carried out in this mode and an ethanol concentration of about 50 g/l was achieved after monitoring for 8 h. Fed-batch fermentations were also carried out, to reduce the inhibitory effect of the substrate. In a typical fed-batch fermentation, a final ethanol concentration of about 120 g/l was achieved, and the bioreactor was monitored for 50 h.