Yeast extract

All the experiments were carried out using B. coagulans MA-13 [24 (link)], a thermophilic and cellulolytic strain previously isolated and cultivated using a medium containing a glycine-buffered Brock’s basal salt solution, which is suitable for the cultivation of thermoacidophilic microorganisms [34 (link)–36 (link)].
LB medium was used as inoculation medium and contained 1% (w/v) tryptone (AppliChem), 1% (w/v) NaCl (AppliChem), 0.5% (w/v) yeast extract (VWR).
The seed medium contained final concentrations of 5% (v/v) molasses, 1% (w/v) yeast extract (VWR), 1% (w/v) peptone (VWR), 0.75% (w/v) (NH₄)₂SO₄ (VWR), 0.35% (w/v) KH₂PO₄ (VWR), 0.07% (w/v) MgSO₄·7H₂O (VWR), and 1× trace metals and 1× vitamins, prepared according to [37 (link)]. To test the pre-adaptation of seed cultures, hydrolysate was added at final concentrations of 30%, 40%, 50%, 70% and 95% (v/v) to the seed medium.
The SSF medium was composed of 10% weight/weight (w/w) water insoluble solids (WIS) supplemented with 1% (w/v) yeast extract (VWR), 1% (w/v) peptone (VWR) and 0.05% (w/v) (NH₄)₂HPO₄ (VWR). With this setup, the approximate concentrations of the microbial growth inhibitors acetic acid, furfural and 5-hydroxymethyl furfural were 0.58 g/L, 0.61 g/L, and 0.21 g/L, respectively. All media were adjusted to pH 5.5 by titration with 3 M NaOH (Merck).

BMGY medium contained 1% yeast extract (VWR, PA), 2% peptone (VWR, PA), 100 mM potassium phosphate buffer (pH = 6.0) (VWR, PA), 4 × 10⁻⁵ % biotin (ThermoFisher, MA), 1.34% Yeast Nitrogen Base (Sunrise Science, PA), and 2% glycerol (VWR, PA). BMMY contained 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer (pH = 6.0), 4 × 10⁻⁵ % biotin, and 1% methanol (VWR, PA). YPD contained 1% yeast extract, 2% peptone, and 2% glucose (VWR, PA).
Binding buffer for Protein A, Protein G, and Blue Sepharose columns contained 20 mM sodium phosphate (pH = 7.0) (Teknova, CA). Elution buffer for Protein A and Protein G columns contained 0.1 M citric acid (pH = 3.0) (VWR, PA). Elution buffer for Blue Sepharose column contained 20 mM sodium phosphate and 100 mM sodium chloride or 2000 mM sodium chloride (VWR, PA).

Two different culture media were used: (1) Supplemented Brain Heart Infusion (BHI) medium: BHI medium (VWR, Avantor, PA, USA), 2% (w/v) gelatin (Sigma-Aldrich, Hamburg, Germany), 1% (w/v) yeast extract (VWR, Avantor, PA, USA), and 0.1% (w/v) soluble starch (Merck, Gernsheim, Germany); (2) New York City III (NYCIII) medium: 0.4% (w/v) HEPES buffer (Merck, Gernsheim, Germany), 1.5% (w/v) proteose peptone (VWR, Avantor, PA, USA), 0.5% (w/v) sodium chloride (VWR, Avantor, PA, USA), 0.5% (w/v) glucose (VWR, Avantor, PA, USA), 2.5% (w/v) yeast extract (VWR, Avantor, PA, USA), and 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, Hamburg, Germany). The incubation conditions used were as follows: 24 h, 10% CO₂, and 37 °C (Binder GmbH, Tuttlingen, Germany).