Nh4 2so4

Chemicals and media components were purchased from manufacturers as follows: yeast extract, proteose peptone, tryptone, casitone (pancreatic digest of casein), and soy peptone, (Becton–Dickinson, USA); peptone (Difco, USA); glucose, K₂HPO₄, MgSO₄, (NH₄)₂SO₄, KCl, NaCl, Fe₂(SO₄)₃, MnSO₄, CuSO₄, and sodium citrate (POCH, Poland); sodium succinate (Avantor, USA); LB, MOPS, l-leucine (Leu), l-valine (Val), l-glutamic acid (Glu), glycine (Gly), l-serine (Ser), and l-threonine (Thr) (Bioshop, Canada); glycerol (VWR International, USA); hexadecane (Sigma-Aldrich, Germany); and rapeseed oil (ZT Kruszwica S.A., Poland).

A loop full of material was used to inoculate 20 mL of YPD medium in 100 mL Erlenmeyer flask. All cultures were grown in triplicate at 15 °C, 20 °C, 22 °C or 25 °C, and agitated at 180 rpm in Lab Companion SI-600R bench top shaker.
Overnight cultures were diluted in 100-mL Erlenmeyer flasks containing 20 mL of production medium [10 (link)]: 20 g/L glucose (in selected cases 30 g/L or 40 g/L), 2 g/L (NH₄)₂SO₄ (Avantor, Gliwice, Poland), 1 g/L KH₂PO₄, 0.5g/L MgSO₄ (Avantor), 0.1 g/L CaCl₂ (Avantor), 2 g/L yeast extract, pH 4.9, to a final concentration of 10⁶ cells/mL, and were cultivated for 4 days at 15 °C, 20 °C, 22 °C or 25 °C and agitated at 180 rpm. All cultures were grown in the Lab Companion SI-600R bench top shaker. All experiments were performed in four parallel cultivations.

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).