Spectrometers are enjoying increasing popularity in bioprocess monitoring due to their non-invasiveness and <i>in situ</i> sterilizability. Their on-line applicability and high measurement frequency create an interesting opportunity for process control and optimization tasks. However, building and maintaining a robust calibration model for the on-line estimation of key variables of interest (e.g., concentrations of selected metabolites) is time consuming and costly. One of the main drawbacks of using infrared (IR) spectrometers on-line is that IR spectra are compromised by both long-term drifts and short-term sudden shifts due to instrumental effects or process shifts that might be unseen during calibration. The effect of instrumental drifts can normally be reduced by referencing the measurements against a background solution, but this option is difficult to implement for single-beam instruments due to sterility issues. In this work, in order to maintain the robustness of calibration models for single-beam IR and to increase resistance to process and instrumental drifts, planned spikes of small amounts of analytes were injected periodically into the monitored medium. The corresponding measured difference spectra were scaled-up and used as reference measurements for updating the calibration model in real time based on dynamic orthogonal projection (DOP). Applying this technique led to a noticeable decrease in the standard error of prediction of metabolite concentrations monitored during an anaerobic fermentation of the yeast <i>Saccharomyces cerevisiae</i>.

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