Furfural

The hydrotrope used for the pretreatment was sodium cumene sulfonate (NaCS, C₉H₁₁NaO₃S) in the form of 40% w/v of Stepanate SCS 40 by Stepan Company (Northfield, Illinois, United States). Other reagents used for pretreatment, i.e. sulfuric acid, sodium hydroxide and ethanol were marked by their analytical grade purity and were manufactured by Merck (Darmstadt, Germany). Chromatographic analyses were conducted using a solution of sulfuric acid, methanol and acetic acid marked by HPLC grade purity and manufactured by Merck (Darmstadt, Germany). The standards of sugars (glucose, galactose and xylose), ethanol, glycerol and pretreatment by-products (5-HMF, furfural, 1,2-dihydrobenzene, 4-hydroxybenzoic acid, vanillic acid, sinapyl alcohol, guaiacol, syringaldehyde, furoic acid) used for chromatographic analyses were marked by their HPLC grade purity and originated from Merck (Darmstadt, Germany).

All materials were provided from mercantile reagent sources and utilized without extra refinement. Nickel(II) chloride hexahydrate (99%), ethylenediamine (99%), ammonium oxalate (99.5%), and sodium perchlorate were purchased from Sigma Aldrich and utilized for the synthesis of [Ni₂(en)₄(ox)](ClO₄)₂. Phenylacetylene (98%), propargyl alcohol (99%), morpholine (99%), piperidine (99%), benzaldehyde (99%), 4-methylbenzaldehyde (97%), furfural (98%), 1-methylpyrrole-2-carboxaldehyde (98%) were purchased from Merck and used for the A³-coupling reaction. All solvents used in this work, consist of N,N-dimethylformamide (DMF), ethanol, methanol, n-hexane, ethyl acetate, toluene, tetrahydrofuran (THF), acetonitrile dichloromethane and dimethyl sulfoxide (DMSO), were of analytical grade. An ATR-FTIR PerkinElmer (model: Spectromrx) apparatus was employed for the determination of IR absorption peaks of complex 1 after and before use it as an A³-coupling reaction catalyst. UV–vis absorption spectrum of the fresh and reused catalyst was recorded by Shimadzu UV-160A spectrophotometer. Energy dispersive X-ray spectrometry (EDS) elemental analysis was performed by a TESCAN (model: MIRA II, detector: SAMX) FESEM apparatus which has an EDS facility attachment.

Orthophosphoric acid (H₃PO₄) (85%), sulfuric acid (95–97%), D-(+)-cellobiose (≥99%), D-(+)-glucose, (≥99.5%), D-(+)-xylose (≥99%), L-(+)-arabinose (≥99%), D-(+)-galactose (≥99%), D-(+)-mannose (≥99%), furfural (≥98.5%), acetic acid (≥99.5%), 5-hydroxymethylfurfural (5-HMF) (≥98.5%), levulinic acid (≥98%), and formic acid (≥99%) were purchased from Merck (Eschenstr. 582024 Taufkirchen, Germany) and used without further purification.

Alginic acid, HPLC grade solvents (methanol, ethanol, dichloromethane, heptane), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodi-n-propylamine (NDPA), N-nitrosopiperidine (NPIP), N-nitrosodi-n-butylamine (NDBA), carvone, linalool, linalyl acetate, syringol, resorcinol, guaiacol, vanillin, propanil, simazine, imazalil, acetochlor, chloropyrifos, thiobendazole, naphthalene, 1-naphthol, 1-napthylamine, aniline, imidazole, caffeine, phenethyl alcohol, furfural, methyl-2-furoate, and decanoic acid were purchased from Merck (Gillingham, Dorset, UK). These standards were dissolved in methanol at a concentration of 5 mg/mL of each nitrosamine. Working solutions were then prepared by an appropriate dilution of this stock solution with methanol. Stock solutions were protected from light and stored at 4 °C. Sodium hydroxide and hydrochloric acid were obtained from Thermo Fisher Scientific (UK).
C18 SPE cartridges (100 mg; 1 mL tube) were purchased from Merck (Gillingham, Dorset, UK). PGC SPE cartridges (100 mg; 1 mL) were obtained from Thermo Fisher Scientific (UK).

The standards used in this study were acetaldehyde (2,3-butanodione), ethyl acetate, 1-propanol, 2-butanol, 2-methylbutanol, 3-methylbutanol, ethyl hexanoate, ethyl octanoate, ethyl decanoate, furfural, isobutanol (2-methylpropan-1-ol), isopentyl acetate (isoamyl acetate) and phenylethyl alcohol (purity of the standards was equal to or higher than 99%, suitable for GC analyses) purchased from Merck (Merck KGaA, Darmstadt, Germany). Reagents used in this study were calcium carbonate (99%), sodium hypochlorite (15%), aqueous iodine (2% w/v of iodine), diatomaceous earth (Chempur, Piekary Śląskie, Poland) and α-amylase from Bacillus sp. solution (amylolytic activity equal to 28,345 units per cm³ of solution, where 1 unit will liberate 1 mg of maltose from starch in 3 min. at pH 6.9 at 20 °C) (Merck KGaA, Darmstadt, Germany).

To analyze saccharides and inhibitory compounds, 1 mL of culture was taken at the indicated time points, centrifuged (3000 rpm for 5 min) and the supernatant was diluted (1:10 in dH₂O) an used for quantification by HPLC, using a Jasco HPLC 2000 plus series (Biolab, Vienna, Austria) with an Aminex HPX 87H column at 65 °C. H₂SO₄ (c = 5 mM) was used as eluent at an isocratic flow rate of 0.8 mL min⁻¹. Sugars were detected with a refractive index and organic acids and inhibitors by a UV detector. Data were analyzed with ChromPass (Version 1.8.6.1, Jasco Europe, Italy). WSHL was analyzed in the same way, but was diluted 1:50. For the preparation of standards, chemicals were obtained from the following suppliers: xylitol from Sigma Aldrich (Steinheim, Germany), glucose, xylose, arabinose, acetic acid and formic acid from Carl Roth (Karlsruhe, Germany), cellulose form Machery-Nagel (Düren, Germany), furfural from Merck (Hohenbrunn, Germany) and HMF from Alfa Aesar (Kandel, Germany). Resulting saccharide and inhibitor yields (g per 100 g WS) were calculated as follows: $\mathrm{g}\mathrm{per} 100 \mathrm{g}\mathrm{WS}=\frac{\left[\mathrm{g}\right]\mathrm{saccharide}\mathrm{per}\mathrm{L}\mathrm{as}\mathrm{measured}\mathrm{by}\mathrm{HPLC}*\mathrm{dilution}\mathrm{factor}}{[\%]\mathrm{dry}\mathrm{mass}\mathrm{of}\mathrm{WS}*\left[\mathrm{g}\right]\mathrm{WS}\mathrm{loading}\mathrm{per}\mathrm{L}}$
Hydrolysates were prepared at 10% solids loading, which means that 100 g WS are contained in 1 L hydrolysate. Therefore, resulting yields in g per 100 g raw material equal g L⁻¹ hydrolysate.

All reagents such as butyraldehyde, dodecanal, octanal, hydro cinnamaldehyde, benzaldehyde, furfural, nitromethane, 1,1,3,3-tetramethylguanidine, nichel chloride hexahydrate (NiCl₂^.6H₂O), sodium borohydride (NaBH₄), ethanolamine, sodium acetate (NaOAc), cerium trichloride heptahydrate (CeCl₃^.7H₂O), sodium iodide (NaI), isophorone diisocyanate (IPDI), PEG 400, 2-hydroxyethyl methacrylate (HEMA), benzophenone and N-methyl diethanolamine were all purchased from Merck. Polyethylene terephthalate (PET) are 2–5 mm particle size pellets obtained by crushing PET bottle scraps. As the first treatment, diols and diisocyanate were dried at 50°C under vacuum conditions for 24 h to remove residual water, and all the aldehydes were previously distilled. In the case of air and moisture-sensitive reactions, the glassware was oven dried at 100°C for more than 2 hours before use, when the reactions were performed under an inert atmosphere (nitrogen gas). For thin-layer chromatography (TLC) analysis, Merck pre-coated TLC plates (silica gel 60 GF254 0.25 mm) were used. Products were observed under UV light or stained in 2,4-dinitrophenyl hydrazine or potassium permanganate solutions.