Phosphoric acid

Olive stones were harvested in Granada, Spain, and were used as the raw material for the activated carbon preparation. The olive stones were dried at 80 °C until constant weight and sieved to 1.6–2.4 mm size. Potassium hydroxide (reagent grade ≥90%, Sigma-Aldrich, St. Louis, MO, USA) and phosphoric acid (mass fraction of 85% in H₂O, Sigma-Aldrich, St. Louis, MO, USA) were used for the chemical activation of the olive stones before carbonization, while melamine (99%, Sigma Aldrich, St. Louis, MO, USA) and phosphoric acid (mass fraction ≥85% in H₂O, Sigma-Aldrich, St. Louis, MO, USA) were used for the modification of surface chemistry. Chlorihydric acid was used for the washing protocol (mass fraction of 37% in H₂O, Sigma-Aldrich, St. Louis, MO, USA). Acetonitrile (mass fraction of 99.8% in water, Sigma-Aldrich, St. Louis, MO, USA) was employed for rhamnolipid quantification. Tryptone glucose extract agar (TGEA) and bushnell haas broth (BH, Sigma Aldrich, St. Louis, MO, USA) were used for the bacterial culture. A Colombian light crude oil (33° API), deionized water and NaCl (99%, Merck KGaA, Darmstadt, DE, Germany) were used for preparing oil in brine emulsions. Benzene, toluene, and phenol (≥98%, CARLO ERBA Reactive-SDS, Barcelona, ES, Spain) were used for the assays of the removal of aromatics hydrocarbons.

Nitrite/nitrate production was measured in cell culture supernatants as described previously [95 (link),96 (link)]. Following cell incubation, 50 µL of cell culture supernatant was added to 25 µL 1% (w/v) sulfanilamide in 2.5% (v/v) phosphoric acid and 25 µL of 0.1% (w/v) N-naphthyl-ethylenediamine in 2.5% (v/v) phosphoric acid (all from Sigma–Aldrich, St. Louis, MO, USA). The OD was read at 540 nm. The nitrite concentration was calculated with a sodium nitrite standard curve (0–10 µM).

Nitrite/nitrate production, an indicator of nitric oxide synthesis, was measured in cell culture supernatants as described previously [60 (link)]. Following incubation 100 μL of cell culture supernatant was added to 100 μL 1% (w/v) sulfanilamide in 2.5% (v/v) phosphoric acid and 100 μL of 0.1% (w/v) N-naphthyl-ethylenediamine in 2.5% (v/v) phosphoric acid (all from Sigma–Aldrich, St. Louis, MO, USA). The O.D. was read at 540 nm.

Nitrite/nitrate production, an indicator of nitric oxide synthesis, was measured in cell culture supernatants as described previously (Chadzinska et al. 2009 (link)). Following 24 h incubation, 100 μL cell culture supernatant was added to 50 μL 1% (w/v) sulfanilamide in 2.5% (v/v) phosphoric acid and 50 μL of 0.1% (w/v) N-naphthyl-ethylene-diamine in 2.5% (v/v) phosphoric acid (all from Sigma-Aldrich, St. Louis, MO, USA). The OD reading at 540 nm (with 690 nm as a reference) was taken using the cRPMI++ medium as blank. Nitrite concentration was calculated by comparison with a sodium nitrite standard curve (0–10 μM).

Phosphoric acid (85 wt.% in H2O), obtained from Sigma-Aldrich (Milwaukee, WI,
USA), was diluted in a 50/50% water-propylene glycol solution to obtain a 35%
experimental Phosphoric acid. A thickening agent was added to increase the
solution viscosity and make it more resistant to flow, avoiding the etching of
undesirable spots. In order to produce an experimental Phosphoric acid
containing PA, grape seed extract (GSE, Vitis vinifera, PA≥95%, Farmácia de
Manipulação Luva Ervas, Caieiras, SP, Brazil) was dissolved in the 35%
experimental acid solution prior to the thickening step, to a final
concentration of 10% w/v of PA. The resulting mixture underwent magnetic
stirring for 24 h under room temperature until complete dissolution of the GSE,
followed by addition of the thickening agent. The final solutions were stored at
4ºC.

The solvents used: acetone, ethanol, methanol, n-hexane, chloroform, 2-propanol, and ethyl ether were purchased from Carlo Erba Reagenti (Milan, Italy). Chloroform was purified by standard procedures. Resveratrol or 3,5,4’-trihydroxy-trans-stilbene (MM = 22,825 g/mol), D-Mannose (MM = 180,156 g/mol), ciprofloxacin (MM = 331,346), methacrylic acid (MAA), 85% formic acid (FA), 85% phosphoric acid (AP), sodium nitrite (SN), dicyclohexylcarbodimide (DCC), dimethylaminopyridine (DMAP), dimethyl sulfoxide (DMSO), potassium chloride (KCl), hydrochloric acid (HCl), sodium phosphate (Na₃PO₄), citric acid, disodium hydrogen phosphate (Na₂HPO₄), sorbitan monooleate (Span 80) and polyoxyethylene sorbitan monooleate (Tween 80), N,N′-ethylene bis-acrylamide (EBA) were purchased from Sigma-Aldrich (Sigma Chemical CO, St. Louis, MO, USA).