Real-time data reconciliation of concentration estimates of process analytes and biomass in microbial fermentations is investigated. A Fourier-transform mid-infrared spectrometer predicting the concentrations of process metabolites is used in parallel with a dielectric spectrometer predicting the biomass concentration during a batch fermentation of the yeast Saccharomyces cerevisiae. Calibration models developed off-line for both spectrometers suffer from poor predictive capability due to instrumental and process drifts unseen during calibration. To address this problem, the predicted metabolite and biomass concentrations, along with off-gas analysis and base addition measurements, are reconciled in real-time based on the closure of mass and elemental balances. A statistical test is used to confirm the integrity of the balances, and a non-negativity constraint is used to guide the data reconciliation algorithm toward positive concentrations. It is verified experimentally that the proposed approach reduces the standard error of prediction without the need for additional off-line analysis. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 578-588, 2009