Total carbohydrate determination

The determination of carbohydrate release was conducted using the Anthrone sulfuric acid assay [45]. Freeze-dried biomass was resuspended in deionized water in concentrations ~ 0.1–0.2 g·L⁻¹. On parallel, fresh starch aqueous solutions with concentrations ranging from 0.02 g·L⁻¹ to 0.4 g·L^− 1 were prepared from starch powder (Merck 1.01257) to be used as standards and processed in a similar manner with the samples. The anthrone reagent was prepared on the day of the experiment by dissolving anthrone (Merk 1.01468) in 95% sulfuric acid (AnalaR NORMAPUR: VWR Chemicals 20,700) at a final concentration of 0.1% w/v. 400 μL of diluted sample or standard along 800 μL of anthrone reagent were mixed in 1.5 mL Eppendorf Safe Lock tube. After 5 min incubation in ice, the sample was placed into a thermo-incubator pre-heated at 95 °C and shaken at 300 rpm for 16 min followed by cooling down on ice for again 5 min. Optical density of the cooled samples was measured at 625 nm in a spectrophotometer (Genesys 10S UV–Vis, Thermo Scientific) and carbohydrate concentration was calculated using the standard curve and considering the dilution factors. All measurements were performed in duplicate.