0.1 m sodium hydroxide solution

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.

The synthesis of BNC was carried out on an up-scaled pilot plant as described by Beekmann et al. [39 (link)]. Briefly, BNC fleeces were obtained by static cultivation of Komagataeibacter xylinus (DSM 14666, German Collection of Microorganisms and Cell Cultures DSMZ, Germany) in Hestrin-Schramm medium in a 1 m² pilot plant at 28 °C for seven days. After synthesis, fleeces were boiled in 0.1 M sodium hydroxide solution (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) and washed with deionized water until neutral pH for purification. Circular BNC fleeces with a 15 mm diameter were punched out of the BNC layer and finally sterilized by autoclaving (121 °C, 20 min, 2 bar). The mass and dimensions of BNC fleeces were characterized to calculate surface area and volume according to Müller et al. [40 (link)].