Assessment of In-Line Near-Infrared Spectroscopy for Continuous Monitoring of Fermentation Processes Simona Tosi, † Maddalena Rossi, ‡ Elena Tamburini, † Giuseppe Vaccari, † Alberto Amaretti, ‡ and Diego Matteuzzi* ,‡ Chemistry Department, University of Ferrara, Via L. Borsari, 46, 44100 Ferrara, Italy, and Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna Italy The application of NIR in-line to monitor and control fermentation processes was investigated. Determination of biomass, glucose, and lactic and acetic acids during fermentations of Staphylococcus xylosus ES13 was performed by an interactance fiber optic probe immersed into the culture broth and connected to a NIR instrument. Partial least squares regression (PLSR) calibration models of second derivative NIR spectra in the 700-1800 nm region gave satisfactory predictive models for all parameters of interest: biomass, glucose, and lactic and acetic acids. Batch, repeated batch, and continuous fermentations were monitored and automatically controlled by interfacing the NIR to the bioreactor control unit. The high frequency of data collection permitted an accurate study of the kinetics, supplying lots of data that describe the cultural broth composition and strengthen statistical analysis. Comparison of spectra collected throughout fermentation runs of S. xylosus ES13, Lactobacillus fermentum ES15, and Streptococcus thermophylus ES17 demonstrated the successful extension of a unique calibration model, developed for S. xylosus ES13, to other strains that were differently shaped but growing in the same medium and fermentation conditions. NIR in-line was so versatile as to measure several biochemical parameters of different bacteria by means of slightly adapted models, avoiding a separate calibration for each strain. 1. Introduction Any fermentation takes advantage of quantitative analysis providing real-time information about the evolu- tion of the process. Despite the availability of powerful analytical tools, their application is limited by infrequent sampling and laborious techniques (1). The ideal ap- proach to monitor and control fermentations is to mea- sure the relevant parameters in-line and hopefully in- situ. Therefore, the implementation of sensors is essential in order to get direct access to the data and intervene with proper controls to optimize the final outcome of the process. In fact, the lack of suitable measurements hinders process control and can reduce the productivity through unidentified faults. This drawback can be overcome by the development of noninvasive techniques based on optical measurement principles, such as near-infrared spectroscopy (NIR). The near-infrared spectral region lies between the end of the visible absorption band at 700 nm and the beginning of the fundamental infrared (IR) absorption region at 2500 nm. The absorption by organic molecules of these radia- tions arises from transition in stretching and bending vibrations. The weak absorption of NIR energy enables direct analysis of samples that are strongly absorbing and highly light scattering, such as microbial cultures. Fur- thermore, it does not require the short optical path- lengths necessary with traditional spectroscopic tech- niques. A typical NIR spectrum encompasses information for all constituents of the sample matrix. Almost every analyte absorbs at more than one wavelength, and therefore the final absorbance at each wavelength results from all components of the broth. Deconvolution of spectral information to get meaningful data is obtained by multivariate statistical analysis (2). NIR spectroscopy has been applied for analytical purposes in the food, chemical, and pharmaceutical industries. The improvement in NIR technology, paral- leled by the development of powerful chemometric tools for spectral analysis, has made it possible to monitor dynamic complex mixtures, such as fermentation broths (3). This spectroscopic technique seems ideal to analyze broth components, because it ensures rapid responses (assay time is usually no longer than 2 min) and provides the opportunity to measure several constituents simul- taneously (4). However, the application of NIR to monitor a bioprocess presents challenges related to the spectro- scopically complex nature of microbial broths, the so- phisticated chemometrics required to translate raw spectra into useful correlation models, and the wide variations in the concentration of analytes over the time. These features made high demands on model robustness. Up to now, NIR has been applied to external monitor- ing of bioprocesses, supplying information close to real time by a prompt analysis of the withdrawn sample or by a flow-through cell. NIR at-line (i.e., rapid off-line) was used to follow submerged fermentations for antibiotic production (5, 6). NIR on-line, built up with the sample cell connected with the bioreactor through a loop, was * To whom correspondence should be addressed. Tel: +39-51- 2099733, Fax: +39-51-2099734. E-mail: diego.matteuzzi@unibo.it. † University of Ferrara. ‡ University of Bologna. 1816 Biotechnol. Prog. 2003, 19, 1816-1821 10.1021/bp034101n CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 09/17/2003