A near-infrared (NIR) spectroscopy technique for the control of lactic acid fermentation process has been proposed. Lactic acid, glucose, and biomass concentrations were determined by the NIR spectroscopy method. The three parameters examined were closely correlated to the results obtained with classical laboratory procedures. Moreover, the conditions for the on-line utilization of the NIR spectroscopy measurement system were pointed out. The great versatility of the NIR spectroscopy should permit its use for other fermentation processes. (c) 1994 John Wiley & Sons, Inc.

Near-infrared (NIR) spectroscopy has been developed as a noninvasive tool for the direct, real-time monitoring of glucose, lactic acid, acetic acid, and biomass in liquid cultures of microrganisms of the genera Lactobacillus and Staphylococcus. This was achieved employing a steam-sterilizable optical-fiber probe immersed in the culture (In-line Interactance System). Second-derivative spectra obtained were subjected to partial least-squares (PLS) regression and the results were used to build predictive models for each analyte of interest. Multivariate regression was carried out on two different sets of spectra, namely whole broth minus the spectral subtraction of water, and raw spectra. A comparison of the two models showed that the first cannot be properly applied to real-time monitoring, so this work suggests calibration based on non-difference spectra, demonstrating it to be sufficiently reliable to allow the selective determination of the analytes with satisfactory levels of prediction (standard error of prediction (SEP) < 10%). Direct interfacing of the NIR system to the bioreactor control system allowed the implementation of completely automated monitoring of different cultivation strategies (continuous, repeated batch). The validity of the in-line analyses carried out was found to depend crucially on maintaining constant hydrodynamic conditions of the stirred cultures because both gas flow and stirring speed variations were found to markedly influence the spectral signal.

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.

The utilization of mono-, di-, and oligosaccharides by Bifidobacterium adolescentis MB 239 was investigated. Raffinose, fructooligosaccharides (FOS), lactose, and the monomeric moieties glucose and fructose were used. To establish a hierarchy of sugars preference, the kinetics of growth and sugar consumption were determined on individual and mixed carbohydrates. On single carbon sources, higher specific growth rates and cell yields were attained on di- and oligosaccharides compared to monosaccharides. Analysis of the carbohydrates in steady-state chemostat cultures, growing at the same dilution rate on FOS, lactose, or raffinose, showed that monomeric units and hydrolysis products were present. In chemostat cultures on individual carbohydrates, B. adolescentis MB 239 simultaneously displayed alpha-galactosidase, beta-galactosidase, and beta-fructofuranosidase activities on all the sugars, including monosaccharides. Glycosyl hydrolytic activities were found in cytosol, cell surface, and growth medium. Batch experiments on mixtures of carbohydrates showed that they were co-metabolized by B. adolescentis MB 239, even if different disappearance kinetics were registered. When mono-, di-, and oligosaccharides were simultaneously present in the medium, no precedence for monosaccharides utilization was observed, and di- and oligosaccharides were consumed before their constitutive moieties.

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