Disodium hydrogen phosphate

All reagents and solvents were either HPLC or analytical grade. Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid (98%), quercetin (98%), artemisinin (98%), Folin–Ciocalteu reagent, ascorbic acid, β-carotene, and linoleic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anhydrous sodium sulfate, potassium ferricyanide, trichloroacetic acid (TCA), ferric chloride, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, and hydrochloric acid were bought from VWR Chemicals (Radnor, PA, USA). Hexane, dichloromethane (CHCl₃), ethyl acetate (EtOAc) and methanol (technical and HPLC) were purchased from VWR International (Fontenay-sous-Bois, Val-de-Marne, France).

Bacterial nanocellulose (BNC) was produced by cultivation of Komagataeibacter xylinus (K. xylinus) strain DSM 14666, deposited at the German Collection of Microorganism and Cell Cultures (DSMZ, Braunschweig, Germany). For cultivation, a preculture of K. xylinus in the Hestrin–Schramm culture medium (HSM) was used [34 (link)]. HSM contains 2% glucose (Carl Roth, Karlsruhe, Germany), 0.5% peptone (Carl Roth, Karlsruhe, Germany), 0.5% yeast extract (Carl Roth, Karlsruhe, Germany), 0.34% disodium hydrogen phosphate (VWR, Radnor, PA, USA) and 0.115% citric acid (Carl Roth, Karlsruhe, Germany). One liter of preculture was added to 4 L HSM and maintained for 7 days at 28 °C in a pilot-scale plant (JeNaCell, Jena, Germany). Static cultivation was used, characterized by forming a homogenous three-dimensional nanostructured cellulose network at the boundary layer between medium and air over time [23 (link),35 (link)]. The BNC fleece thus obtained was subsequently harvested and deposited in 0.1 M sodium hydroxide solution (Carl Roth, Karlsruhe, Germany) overnight and washed several times afterward with water for injection until pH neutrality. Finally, the purified BNC was mechanically cut into 1.9 cm² round pellicles and sterilized by autoclaving (121 °C, 20 min, 2 bar) for further experiments.

A 0.6 molar sodium phosphate buffer at 8 different pH values (5.8, 6.0, 6.3, 6.5, 6.8, 7.2, 7.4, and 7.8) was employed. The buffer components were sodium di-hydrogen phosphate (monohydrate; Merck KGaA, Darmstadt, Germany) and di-sodium hydrogen phosphate (anhydrous; VWR, Radnor, PA, USA). After preparation, and after two weeks of storage, the buffer solutions were filtered over a 0.2 µm Supor®-200 filter (Pall Corporations, Port Washington, NY, USA). This buffer system was chosen to use literature data provided by Taratuta et al. [13 (link)] as a validation set. The pH value was set with a five-point calibrated pH meter (HI-3220, Hanna® Instruments, Woonsocket, RI, USA), which was equipped with a SenTix® 62 pH electrode (Xylem Inc., White Plains, NY, USA). The pH value of all buffers was checked throughout the study and was maintained within ± 0.05 pH unit of the desired pH values.

Norfloxacin, 95%, was purchased from abcr GmbH (Karlsruhe, Germany). Commercially available, cellulose-based paper (Soft & Sicher, dm-drogerie markt GmbH + Co. KG, Karlsruhe, Germany) was utilised as the paper matrix. Sucrose, peptone, beef extract, sodium chloride and potassium chloride were acquired from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Agar was purchased from Sigma-Aldrich Pty Ltd. (Darmstadt, Germany). Magnesium sulfate heptahydrate, magnesium chloride hexahydrate, calcium chloride dihydrate, sodium hydrogen carbonate, di-potassium hydrogen phosphate and di-sodium hydrogen phosphate were acquired from VWR International GmbH (Darmstadt, Germany). Aliivibrio fischeri (A. fischeri) bacterial strain (ATCC 7744/ NCMB 1281) was obtained from Dr. G. Schuchardt^® (Göttingen, Germany). Purified water was freshly obtained from a PURELAB Flex 2 (ELGA LabWater, Veolia Water Technologies GmbH, Celle, Germany).

Lyophilized (with no additives) and purified type II ASNase from E. coli (P1321-10000; 10,000 IU) were supplied by Deltaclon S.L., Spain. Carbon xerogels with different pore sizes, namely 4, 13, and 30 nm (CX-4, CX-13, and CX-30, respectively), were prepared by polycondensation of resorcinol and formaldehyde, using different pH values (6.0, 5.2, and 5.0, respectively), according to a procedure already described in detail [27 (link)]. L-asparagine (≥99.0%), tris(hydroxymethyl)aminomethane (TRIS) (≥99.0%) and disodium hydrogen phosphate (≥99.0%) were purchased from VWR International, LLC. Trichloroacetic acid (TCA) (≥99.0%) was obtained from J.T. Baker. Citric acid (≥99.5%) and Nessler’s reagent (potassium tetraiodomercurate (II)) were supplied by Merck Chemical Company (Darmstadt, Germany). Nessler’s reagent is an acutely toxic substance that can be fatal by ingestion, skin absorption, or inhalation. Only trained personnel with appropriate training and proper personal protective equipment (PPE) should handle this material. Sodium hydroxide (≥98.0%) and hydrochloric acid (37%) were acquired from Sigma-Aldrich.

For our experiments, CAPE (Sigma-Aldrich, Buchs, Switzerland); sodium dodecyl sulfate; crystal violet; peptone; yeast extract (Merck, Germany); agar-agar (Fluka, Buchs, Switzerland); a modified version of RPMI 1640 medium (containing dextrose 1.8% (w/v), MOPS 3.4% (w/v), and adenine 0.002% (w/v)) (Sigma-Aldrich, St. Louis, MI, USA); potassium dihydrogen phosphate; disodium hydrogen phosphate (Reanal, Budapest, Hungary); dimethyl sulfoxide; ethanol (VWR Chemicals, Paris, France); sodium chloride (VWR Chemicals, Debrecen, Hungary); glucose (VWR Chemicals, Leuven, Belgium); adenine; calcium chloride; magnesium chloride; potassium chloride (Scharlau Chemie S.A, Sentmenat, Spain); Z-VAD-FMK (Biovision, Milpitas, CA, USA); glutardialdehyde solution; osmium tetroxide; propylene oxide; Durcupan (R) ACM components A/M, B, C, and D (Sigma-Aldrich, Darmstadt, Germany); 0.22 µm vacuum filters (Merck Millipore, Guyancourt, France); sterile 96-well microtiter plates for susceptibility testing (Costar^®, Phoenix, AZ, USA) and for biofilm assays (Sarstedt AG & Co. KG, Nümbrecht, Germany, Catalog number: 83.3924.500); CF^®488A Annexin V and PI apoptosis Kit (Biotium, Fremont, CA, USA); and methanol (Chemolab Ltd., Budapest, Hungary) were used. All of the chemicals used in the experiments were of analytical or spectroscopic grade.