The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.

An in-situ, mid-infrared sensor was used to monitor the major analyte concentrations involved in the cultivation of Gluconacetobacter xylinus and the production of gluconacetan, a food-grade exopolysaccharide. To predict the analyte concentrations, three different sets of standard spectra were used to develop calibration models, applying partial least-squares regression. It was possible to build a valid calibration model to predict the 700 spectra collected during the complete time course of the cultivation, using only 12 spectra collected every 10 h as standards. This model was used to reprocess the concentration profiles from 0 to 15 g/L of nine different analytes with a mean standard error of validation of 0.23 g/L. However, this calibration model was not suitable for real-time monitoring as it was probably based on non-specific spectral features, which were correlated only with the measured analyte concentrations. Valid calibration models capable of real-time monitoring could be established by supplementing the set of 12 fermentation spectra with 42 standards of measured analytes. A pulse of 5 g/L ethanol showed the robustness of the model to sudden disturbances. The prediction of the models drifted, however, toward the end of the fermentation. The most robust calibration model was finally obtained by the addition of 34 standard spectra of non-measured analytes. Although the spectra did not contain analyte-specific information, it was believed that this addition would increase the variability space of the calibration model. Therefore, an expanded calibration model containing 88 spectra was used to monitor, in real time, the concentration profiles of fructose, acetic acid, ethanol and gluconacetan and allowed standard errors of prediction of 1.11, 0.37, 0.22, and 0.79 g/L, respectively.

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A two-step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP-captured material in batch mode. Using multi-modal anion exchange in bind-elute mode, mAb was produced consistently within the purity specification. The benchmark was a state-of-the-art 3-step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2-step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase.