Micro guard cation h guard column

Free monosaccharides were extracted according to the method presented by Bell et al. (2017a) (link), with the exception that 0.2 g of lyophilized leaf powder was extracted. Extracts were analyzed on an Agilent 1100 series HPLC system equipped with a binary pump, degasser, and auto-sampler, with an external column heater (50°C). A Bio-Rad Aminex HPX-87H (300 × 7.8 mm, 9 μm particle size) column with a Micro-Guard Cation H guard column (Bio-Rad, Watford, United Kingdom) was used to achieve separation with an isocratic gradient of 5 mM sulfuric acid, and a flow rate of 0.6 mL per min. A Polymer Laboratories ERC-7515 refractive index detector (Church Stretton, United Kingdom) was used to detect monosaccharides. Compounds were quantified using authentic standards and analyzed with Agilent ChemStation software (Santa Clara, CA, United States).

Initial spore concentration was measured using a Bürker counting chamber (with a depth of 0.1 mm) under a light microscope (Carl Zeiss Axiostar plus, Oberkochen, Germany). The spore solution was diluted ten times before the measurement, and the spores were counted in a volume of 1/250 μl each. HPLC (Waters 2695, Waters Corporation, Milford, USA) was used to analyze all liquid fractions. A hydrogen-based ion-exchange column (Aminex HPX-87H, Bio-Rad Hercules, CA, USA) at 60 °C with a Micro-Guard cation-H guard column (Bio-Rad) and 0.6 mL/min 5 mM H₂SO₄, as eluent, was used for the analyzes of glucose, ethanol, glycerol and acetic acid. A UV absorbance detector (Waters 2487), operating at 210 nm wavelength, was used in series with a refractive index (RI) detector (Waters 2414). The morphology of the fungal biomass was determined by visual examination of the 72 h submerged culture (mycelium or pellets). Fungal biomass concentration (dry weight) was determined at the end of the cultivation by washing the pellet or mycelial biomass with deionized water followed by drying at 105 °C for 24 h before weighing. A digital Vernier caliper (Limit, Alingsås, Sweden) with a resolution of 0.01 mm was used to measure the pellet diameter. An average of 50–100 pellets (from shake flask and bench-scale reactor culture, respectively) was measured for the pellet size determination.

Cell dry weight was determined in triplicate by filtering 5 mL of the culture on a pre-weighed 0.45 μm pore size Supor^® membrane disc filter (Pall Corporation, Port Washington, NY, USA). Filters were washed with distilled water and dried for 8 min at 350 W in a microwave oven. To analyze the metabolites, cells were separated by centrifugation at 13,200 rpm for 2 min; the supernatant was filtered through 0.20 μm membrane filters (Toyo Roshi Kaish, Tokyo, Japan) and stored at −20 °C until analysis. Concentrations of glucose, glycerol, acetate, ethanol, HMF, furfural and vanillin were determined by high performance liquid chromatography (Waters, Milford, MA, USA) using a HPX-87H resin-based column (Bio-Rad, Hercules, CA, USA) preceded by a Micro-Guard Cation-H guard column (Bio-Rad). Separation was performed at 45 °C with 5 mM H₂SO₄ at a flow rate of 0.6 mL min⁻¹. All compounds were quantified by refractive index detection (Shimadzu, Kyoto, Japan). For each HPLC run, a seven-point calibration curve was made for each compound.