Carboxylation of Bacterial Cellulose (CBC)

In general, the carboxyl functional groups are created on the nanofibrils of the BC by the TEMPO-oxidation procedure. As previously described³¹, to obtain the carboxylated BC (CBC), 250 mg of dried BC was dispersed in 150 ml of Milli-Q water, which contained dissolved TEMPO (7.8 mg) and NaBr (78 mg). To start the oxidation, 11 wt% NaOCl with the ratio of 8:1 (mmol/g BC) was added to the solution, and pH was adjusted to 10.5 by adding 0.1 M HCl. Since the pH of the solution decreased during the oxidation reaction, it was kept constant at a pH of 10.5 by adding 0.1 M NaOH. After an hour of gentle mixing at room temperature, the reaction was stopped by adding ethanol to reach pH 7. The CBC was separated from the reaction solution and washed sufficiently with ethanol, and then thrice washed with plenty of Milli-Q water. The obtained CBC was finally preserved in Milli-Q water at 4 °C.

To determine the content of created carboxyl groups on the BC fibers, conductometric titration was performed on the as-oxidized BC³². Accordingly, 50 mg of dried and oxidized BC was dispersed in 50 ml of Milli-Q water. Afterward, all carboxyl groups were protonated by adding 0.1 M HCl to reach a pH of around 2.5. Subsequently, the suspension was titrated with 0.01 M NaOH, and the changes in the conductivity of the solution were recorded using the Oakton PC2700 Benchtop Meter. It was finally terminated once pH attained 11. Correspondingly, the carboxyl group content was estimated by drawing the titration curve.