Measurement of cellulose crystallinity by XRD.

To determine the crystallinity of Avicel PH101 (Sigma-Aldrich) incubated with C. hutchinsonii cells, strains were cultured in Stanier medium to mid-exponential phase. Cells were harvested, washed twice with fresh Stanier medium, and then transferred into Stanier medium supplemented with 0.2% (vol/vol) Avicel PH101. Cells were cultured at 30°C with shaking at 160 rpm. Samples were taken at set intervals (0 h, 24 h, and 48 h) and centrifuged at 5,000 × g for 10 min to harvest the cell pellets. The cell pellets were suspended with 0.2 M NaOH, boiled for 10 min, and centrifuged at 12,000 × g for 10 min to remove the supernatant. The pellets were then washed twice with distilled water and dried at 60°C overnight. The X-ray diffraction (XRD) of samples was observed with a D8 Advance system diffractometer (Bruker, Germany). The XRD crystallinity index (CI_XRD) was calculated as previously described (18): CI_XRD (%) = (I₀₀₂ − I_am)/I₀₀₂ × 100. I₀₀₂ is the height of the crystalline peak at 22°, and I_am is the intensity of the peak at 18°.