N propanol

Methacrylic acid (99%) (MAA), titanium (IV) isopropoxide (97%) and zirconium (IV) propoxide in n-propanol (70 wt.%) were purchased from Merck and n-propanol from Alfa Aesar and were used as received. Acrylamide (Aam), 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Lysil-lysine (Lys-Lys), Methacrylic acid (MAA), N-tert-butylAcrylamide (TBAm), N,N′-methylene bisAcrylamide (BIS), N,N,N′,N′-tetramethyl ethylenediamine (TEMED), ammonium persulfate (APS), sodium dodecyl sulfate (SDS), Tris (hydroxymethyl)-aminomethane (TRIS), sodium dihydrogen phosphate, disodium monohydrogen phosphate, hydrochloric acid, sodium hydroxide, sodium chloride, Tween-20, 3-Aminopropyl)Triethoxysilane, acetonitrile, acetic acid, ethanol, were from Sigma–Aldrich (Darmstadt, Germany). The proteins human serum transferrin (HTR), horseradish peroxidase (HRP), trypsin were from Sigma–Aldrich (Darmstadt, Germany). A 40% w/w APS stock solution was freshly prepared prior to polymerization.

Consumption of GOSP was determined by measuring the GOS fractions remaining in the post-fermentation supernatants of lactobacilli isolated from the IL and AC as previously described [39 (link)]. Briefly, 2 μL of a 1% (v/v) carbohydrate standard or bacterial post-fermentation supernatant was spotted onto TLC silica gel plates (Sigma-Aldrich, St. Louis, MO, USA). Glucose, galactose (Sigma-Aldrich), lactose (Fisher Scientific) and GOSP were included as carbohydrate standards. The TLC developing solvent was composed of a 2:1:1 mixture of n-propanol (Fisher Scientific), acetic acid (Fisher Scientific) and water. TLC plates were further prepared by spraying an ethanol based solution containing 0.5% (w/v) α-naphthol (Alfa Aesar) and 5% (v/v) H₂SO₄ (Sigma-Aldrich). TLC plates were visualized by heating at 100°C for 5 min.

Indanthrene Blue RS and other anthraquinone dyes (Vat Green XBN, Vat Brown R, and Vat Yellow 5G) were obtained from Cuttack, Odisha, India. Tartaric acid, n-propanol, ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)], NADH, methyl red, catechol were purchased from Fisher Scientific, India. Agricultural residues like rice straw and husk, sugarcane bagasse, and wood scrapings were collected from local industries in Odisha, India. These residues were washed and dried and then were milled into a uniform size and stored at room temperature before use. All the chemicals and media components used have been supplied from Hi-media (India) and of analytical grade. The alkaline pH of the media was maintained by using sterilized NaOH (1 M). The textile wastewater sample was collected from local textile processing units, Odisha, India.

Thin-layer chromatography (TLC) was performed as described [49 (link)]. Briefly, overnight cultures of B. subtile, B. bifidum, B. adolescentis, or Bacteroides thetaiotaomicron were harvested by centrifugation and resuspended in Ringer’s solution. Reaction mixtures were prepared by dissolving indicated concentrations of bile acids in BHI medium. Bacterial suspensions were added in a 1:1 ratio to the reaction mixture; control solutions were incubated without bacterial suspension. After anaerobic incubation overnight at 37°C, samples centrifuged and the supernatant was filtered, lyophilized and residues were redissolved in 1 mL methanol. After centrifugation, 3 μl of supernatant was spotted onto a TLC sheet (Polygram™ SilG precoated with 0.25 mm silica gel; Machery-Nagel, Germany) (stationary phase) and dipped into the mobile phase. The mobile phase consisted of isoamyl acetate, propionic acid, n-propanol, and water (40:30:20:10; Fisher Scientific, Ottawa ON). After the run was completed, TLC sheets were dried for 3 min at 110°C, and bile acids were located by spraying sheets with 10% phosphomolybdic acid in ethanol (Fisher Scientific, Ottawa ON). R_f-values were determined by dividing the distance traveled by the bile acid by the distance traveled by the solvent front.

Commercially available methanol, ethanol, isopropanol, n-propanol, and acetone were obtained from Fisher Scientific (Hampton, NH, USA) and acetaldehyde was obtained from Thermo Scientific (Waltham, MA, USA). Heavy isotopes were obtained from the following sources: methanol-d4, ethanol-d6, acetone-d6 (Cambridge Isotopes, Tewksbury, MA, USA), isopropanol-d8 (Thermo Chemicals, Waltham, MA, USA), and acetaldehyde-d4 (Fisher Scientific, Hampton, NH, USA).