Arabinose

Cultivations were performed as described in [13 (link)]. Briefly, bioreactors (Multifors, Infors HT, Bottmingen, Switzerland) with 500 mL working volume were used for growing R. toruloides CBS14 containing either CGHH (50% CG, 10% HH) or only CG (50% CG) as carbon sources as well as 0.75 g/L yeast extract (BactoTM Yeast Extract, BD, France), 1 g/L MgCl₂ 6xH₂O (Merck KGaA, Germany), 2 g/L (NH₄)₂HPO₄ (≥ 98%, Sigma-Aldrich, USA) and 1.7 g/L YNB without amino acids and ammonium sulphate (DifcoTM, Becton Dickinson, France). To inoculate the bioreactors, cells from a −80 °C stock culture were streaked on YPD-agar (glucose 20 g/L, yeast extract 10 g/L, peptone 20 g/L) and incubated for 3 days. From the plates, cells were inoculated into 100 mL YPD in 500 mL baffled Erlenmeyer flasks. After incubation on an orbital shaker at 125 rpm and 25 °C for 72 h, the cells were transferred to 50 mL-Falcon tubes, washed twice with NaCl-solution (9 g/L) and re-suspended with NaCl-solution. Cultures were inoculated with the cell suspension, to reach an initial OD of 1 in the cultivation. Cultivations were performed in triplicate at 25 °C, pH 6, and an oxygen tension of 21%.
CG was obtained from Perstorp Holding AB, Sweden. The glycerol concentration was specified as 80%; however, there were differences from batch to batch. For the cultivations described here, the same batch was used as in the bioreactor experiments in [13 (link)]. HH was generated from wheat straw, after steam explosion and enzymatic digestion. Pretreatment was performed at Lund University, Sweden, as described previously [16 (link)]. Briefly, the straw was soaked with 1% acetic acid overnight, and then steam exploded at 190 °C. The liquid fraction, representing the solubilised hemicellulose, was removed from the solid fraction by pressing and used in the experiments. HH contained 26.2 g/L xylose, 7 g/L glucose, 6.6 g/L acetic acid and small amounts of arabinose (< 0.5 g/L). The nitrogen content was 0.6 g/l [27 (link)]. The pH was set to 6 by addition of appropriate amounts of 5 M NaOH [13 (link)]. The C/N-ratios were 32.5 for CGHH and 30.7 for CG.
Samples for RNA-isolation and determination of the concentrations of biomass, carbon sources and lipids were isolated from R. toruloides CBS14 cultures grown at different growth conditions: they were taken from CG cultures after 10 h, 30 h, and 60 h and from CGHH cultures after 10 h, 36 h, and 60 h. Cell dry weight was determined gravimetrically, and glucose, xylose, acetic acid and arabinose were determined by HPLC [17 (link)]. Lipid content was measured using FT-NIR, as described previously [28 (link)]. Cell samples for RNA isolation (3 mL) were rapidly collected in Falcon tubes and placed in ice to decrease sample temperature.

Molecular weight distributions of lyophilized crude EPS were determined by size exclusion chromatography. In brief, crude EPS powder was suspended in 0.1 M NaNO₃ (0.5 mg/mL) and then filtered through a 0.45 μm pore diameter polyvinylidene fluoride membrane (Millipore Corporation, USA). The average molecular weight (MW) was determined by high-performance molecular exclusion chromatography (HPLC-SEC, Agilent 1,100 Series System, Hewlett-Packard, Germany) associated with a refractive index (IR) detector (Ibarburu et al., 2015 (link)). 50 μL of the samples were injected and eluted at a flow rate of 0.95 mL/min (pressure: 120:130 psi) at room temperature using 0.1 M NaNO₃ as mobile phase. Dextrans (0.5 mg/mL) with a molecular weight between 10³ and 2.10⁶ Da (Sigma-Aldrich, USA) were used as standards.
Once the molecular weight distributions were determined, low and high molecular weight fractions that composed the crude EPS obtained at 20°C were separated. For this purpose, EPS solutions (0.2% w/v) were centrifuged through a Vivaspin™ ultrafiltration spin column 100 KDa MWCO, (Sartorious, Goettingen, Germany) for 20 min at 6000 g, eluting only the low MW fraction. Subsequently, high MW fraction retained in the column was eluted using hot distilled water. The eluted fractions were passed through a Vivaspin column (cut-off 30KDa) in order to separate the middle and low MW fraction of EPS.
Monosaccharide composition of crude EPS and their fractions were determined by gas chromatography as previously described (Notararigo et al., 2013 (link)). Briefly, 1–2 mg of EPS were hydrolyzed in 1 mL of 3 M trifluoroacetic acid (1 h at 120°C). The monosaccharides obtained were converted into alditol acetates by reduction with NaBH₄ and subsequent acetylation. The samples were analyzed by gas chromatography in an Agilent 7890A coupled to a 5975C mass detector, using an HP5-MS column with helium as carrier gas at a flow rate of 1 mL/min. For each run, 1 μL of sample was injected (with a Split 1:50) and the following temperature program was performed: the oven was heat to 175°C for 1 min; the temperature was increased to 215°C at a rate of 2.5°C/min and then increased to 225°C at 10°C/min, keeping it constant at this temperature for 1.5 min. Monosaccharides were identified by comparison of retention times with standards (arabinose, xylose, rhamnose, galactose, glucose, mannose, glucosamine and galactosamine) analyzed under the same conditions. Calibration curves were also processed for monosaccharide quantification. Myo-inositol was added to each sample as internal standard.