Summary
A latest approach in bioethanol generation is the usage of yeast and bacteria that uses C5 sugars for fermentation and the valorization of bio refinery by products. Here it is shown that a hemicellulose-like fermentation mash has a high content of the artificial sweetener xylitol and that its purification by HPLC can be accomplished using polymer based Eurokat columns. The product is soluble in water and can easily be used for further applications.
Introduction
The second generation of bio refinery uses biomass with lower contents of C6 glucose and higher contents of C5 sugars. Besides ethanol generation its goal is the full usage of biomass by valorizing by products. Fermentation of C5 sugars with microorganisms result in mash that could be used for further applications. Polymer based Eurokat columns were tested for their ability to separate fermentation mash and among them the Eurokat Ca column had the best separation profile. Analysis of the mash revealed high contents of xylitol. Purification of highly pure xylitol was established.
Results
The fermentation mash was analyzed on different columns (Eurokat Na, H, and Ca) to determine the optimal stationary phase. The Eurokat Ca column showed the best separation profile for xylitol Fig.1 even though it has the longest run with about 28 min compared to Eurokat Na with 18 min and Eurokat H with 12 min (not shown). A more detailed analysis of the fermentation mash identified five components: xylose, arabinose, glycerol, mannitol and xylitol Fig.1. Xylitol had the highest concentration with 80 mg/mL in the sample, followed by glycerol with 20 mg/mL. The other three components had concentrations of 7–8 mg/mL Fig.1. The baseline separation of xylitol indicated promising batch purification. Overload studies with a semi-preparative Eurokat Ca column were performed . This column has a three times higher volume (50 mL) than the analytical column (15 mL) and larger particle size (25–56 μm) enabling higher sample loading and faster flow rates with lower back pressure. The collected fraction of xylitol Fig.2 had a purity of 99 %, measured with RI Fig.3.
Materials and Method
Sample preparation Vogelbusch Biocommodities GmbH provided the fermentation mash that resulted from fermentation with yeasts of hemicellulose-like hydrolysate with high xylose content. The sample was filtered through 0.45 μm filter after centrifugation. A 1:10 dilution was prepared and analyzed. For calibration a mixture of xylose, arabinose, glycerol, mannitol and xylitol was prepared and six dilution steps from 15 mg/mL to 0.3 mg/mL prepared. Method parameters Analytical runs were performed with 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/min using H2Odd as eluent. The KNAUER AZURA analytical HPLC system comprising 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 used. The puritifcation of xylitol was performed with KNAUER Eurokat Ca columns (250 × 16 mm) with 25–56 μm μm particles at 75 °C running at flow rates of 2.5 mL/min using H2Odd as eluent. The KNAUER AZURA Preparative HPLC system comprising of 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 used. The refractive index detector‘s Extended Dynamic Range (EDR) feature was used for preparative experiments.
More information
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