Purospher star nh2 column

Sugar content was determined by using high-performance liquid chromatography and a refractometric detection (La-Chrom, Merck, Hitachi, Japan) according to the method described by Bogdanov et al. [19] . The extraction was performed by treating the homogenized jams with distilled water in an ultrasonic cleaner Sonic 14 (Polsonic, Warsaw, Poland) at a temperature of 40 °C for 45 min. The obtained sample was clarified using Carrez reagents I and II, and filtered. The sample solution was purified by using a Millex-LCR-type filters (PTFE) with 0.45 μm pore size directly prior to HPLC analysis. A mixture of acetonitrile with water (80:20, v/v) was used as a mobile phase. A flow rate was 1 ml/min. Separation was carried out using a Purospher Star NH2 column (Merck, Darmstadt, Germany) (25 × 4.6 mm, particle size 5 μm) equipped with a precolumn and thermostated at 30 °C. Quantification of individual saccharides was based on a comparison with standards.

Absorbance was measured spectrophotometrically (Cintra 202 GBC DBUV instrument, Australia) at 590 nm. Nutrient medium was used as a blank. Absorbance values at 590 nm were converted into biomass concentration (g DW/l), using a standard curve. DW was determined by centrifuging the cultures (Hettrich Zentrifugen Universal 320), as described previously in Lima-Costa et al. [6] . Sugars and ethanol analyses were performed by a high-performance liquid chromatography (HPLC) using samples previously centrifuged at 13,400 ×g for 10 min. Analyses were performed on a Beckman System Gold HPLC (Beckman, USA) equipped with a Jasco 1530 refractive index detector (Jasco, Japan). To analyze sugar concentrations, a Purospher STAR NH 2 column (Merck KGaA, Germany) was used with an isocratic elution of acetonitrile:water (75:25) at 35ºC. Ethanol determinations were performed on an OH AY column (Merck KGaA, Germany), at room temperature, with an isocratic elution of 0.002 N H 2 SO 4 at 0.5 ml/min.