Biomass with higher contents of C5 sugars and lower contents of C6 glucose is used by the second generation of bio refinery. In addition to ethanol generation, the goal of the second generation biorefinery is the complete usage of biomass through the valorization of by-products. Fermentation of C5 sugars with microorganisms yields mash that could be used in further applications.
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Among the polymer-based Eurokat columns that were assessed for their ability to separate fermentation mash, the Eurokat Ca column showed the best separation profile. High contents of xylitol were observed in the analysis of the mash. Purification of highly pure xylitol was established.
Analysis of the fermentation mash was carried out on different columns (Eurokat Na, H, and Ca) to determine the optimal stationary phase.
The Eurokat Ca column exhibited the best separation profile for xylitol (Figure 1) despite having the longest run with about 28 minutes compared to Eurokat H with 12 minutes (not presented in this article) and Eurokat Na with 18 minutes. An in-depth analysis of the fermentation mash revealed five components: xylose, arabinose, glycerol, mannitol and xylitol (Figure 1).
The concentration of xylitol in the sample was the highest with 80 mg/mL, followed by glycerol with 20 mg/mL. The remaining three components had concentrations of 7–8 mg/mL (Figure 1).
Figure 1. Chromatogram of 1:10 dilution of fermentation mash 10 µL injection on Eurokat Ca – 1 xylose (8.2 mg/mL), 2 arabinose (8.3 mg/mL), 3 glycerol (21.0 mg/mL), 4 mannitol (7.0 mg/mL), 5 xylitol (80.6 mg/mL).
The baseline separation of xylitol signified promising batch purification. Overload studies were carried out on a semi-preparative Eurokat Ca column which has a volume (50 mL) three times higher than the analytical column (15 mL) and larger particle size (25–56 μm), thus allowing for higher sample loading and faster flow rates with lower back pressure.
The collected fraction of xylitol (Figure 2) showed a purity of 99%, measured with RI (Figure 3).
Figure 2. Fractionation of xylitol from 1000 μL injection; yellow fraction area (9.5 mL).
Figure 3. Comparison of sample and fraction chromatograms; blue = sample, red = fraction from batch purification.
Materials and Method
Vogelbusch Biocommodities GmbH supplied the fermentation mash that was obtained by fermentation with yeasts of hemicellulose-like hydrolysate with high xylose content. The sample was subjected to filtration through 0.45 μm filter following centrifugation.
A 1:10 dilution was prepared and analyzed. For calibration, a mixture of xylose, arabinose, glycerol, mannitol and xylitol and six dilution steps from 15 mg/mL to 0.3 mg/mL were prepared.
Analytical runs were performed using KNAUER analytical Eurokat columns (300 × 8 mm) with integrated pre-columns (30 × 8 mm) with 10 µm particles at 75 °C running at flow rates of 0.5 mL/minutes using H2Odd as eluent.
The KNAUER AZURA analytical HPLC system consisting of the AZURA P 6.1L HPG 10 mL pump, 3950 autosampler, AZURA DAD 2.1L diode array detector with high sensitivity KNAUER LightGuide cartridge flow cell, AZURA RID 2.1L refractive index detector, AZURA CT 2.1 column thermostat controlled by the OpenLAB® EZChrom Edition software was employed.
Xylitol was purified using KNAUER Eurokat Ca columns (250 × 16 mm) with 25–56 µm particles at 75 °C running at flow rates of 2.5 mL/minutes using H2 Odd as eluent. The KNAUER AZURA Preparative HPLC system featuring the AZURA P 6.1L HPG 50 mL pump, 3950 autosampler (preparative version), AZURA RID 2.1L refractive index detector, AZURA CT 2.1 column thermostat controlled by the OpenLAB® EZChrom Edition software was employed. Preparative experiments were conducted using the extended dynamic range (EDR) feature of the refractive index detector.
The results demonstrate that among the the Eurokat columns tested, the Eurokat Ca column was the best column for analysis of fermentation mash.
A high content of xylitol (80 mg/ml) was present in the fermentation mash analyzed. A semi-preparative batch purification of the xylitol led to high recovery (95%) of xylitol with a purity of 99%. Upscaling of the batch process or application of SMB (simulated moving bed) chromatography holds potential for xylitol production from fermentation mash.
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This information has been sourced, reviewed and adapted from materials provided by KNAUER Wissenschaftliche Geräte GmbH.
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