*2.7. Fermentability of Pretreatment Liquids*

Fermentability tests with freeze-dried *S. cerevisiae* yeast (Ethanol Red, Fermentis, Marcq en Baroeul, France) were performed using microtiter plates (maximum well volume 330 µL; Nunc, Roskilde, Denmark). Freeze-dried yeast was rehydrated by suspending it in 5 times its weight of sterile tap water for 30 min at 35 ◦C, and it was then inoculated at an initial loading of 0.2 g (DW)/L. Prior to fermentation, the pH of the pretreatment liquids

was adjusted to 5.5 using a 1-M solution of NaOH. The final volume in each well was 300 µL. The mixture contained either 120 µL pretreatment liquid (diluted with deionized water to 40% of the initial concentration) or 234 µL pretreatment liquid (100% of the initial concentration), 6 µL of a nutrient solution (consisting of 150 g/L yeast extract, 75 g/L (NH4)2HPO4, 3.75 g/L MgSO4·7 H2O, and 238.2 g/L NaH2PO4·H2O), and deionized water. Glucose was added to all the media until a final concentration of 20 g/L. Reference fermentations without any pretreatment liquid were performed, while control samples with only culture medium (no yeast) were also processed in order to confirm that there was no cross-contamination. The microtiter plates were incubated in an orbital shaker at 150 rpm and 30 ◦C.

Yeast growth was monitored by measurements of optical density (OD) at 600 nm using the SpectraMax i3x multimode microplate reader after 0, 12, 24, 36, and 48 h. Growth medium without yeast was used as blank. All experiments were performed in triplicates, and mean values were used in the evaluation. The growth rate in each medium was calculated with the following equation:

$$Groupth\,rate = \frac{\text{Cell density (OD}\_{600})}{Inculation\,time\,(\text{h})} \tag{9}$$

The relative growth rate (RGR) of cultures with a medium of pretreatment liquid was calculated by comparing the growth rate of those cultures to the average growth rate of the reference cultures with sugar-based medium (Equation (10)).

$$RGR = \frac{Growth\ rate\ of\ culture\ with\ PL}{Growth\ rate\ of\ reference\ culture} \tag{10}$$
