Materials
Commercial CNFs of BiNFi-s AMa, BMa, and FMa (Sugino Machine Co., Ltd., Japan) and Rheocrysta (DKS Co. Ltd., Japan) were purchased and used as mechanical nanofibrillated cellulose fibers and isolated single-cellulose nanofibers. Commercially available cellulases, hemicellulases and their mixtures Accellerase 1500 (DuPont Co., USA), Optimash XL (DuPont Co., USA), Sherazyme (Novozyme), Optimash BG (DuPont)) were provided by Genencore Corp. (2011; DuPont Co., USA) and Prof. Watanabe (Ehime University in Japan).
Enzymatic treatment of CNF
Cellulases and hemicellulases were added to the substrate diluted in 50 mM sodium acetate buffer at 48ºC for 1, 6, 12, and 24 hours to achieve an enzyme content of 40 mg-protein/g-substrate, respectively, based on the previous reports. Next, the reaction mixtures were centrifuged at 20,000 × g for 10 min. The supernatant was removed, 1 ml of ultrapure water was added, the mixture was stirred, and centrifugation was performed under the same conditions. After repeating the above procedure three times, the supernatant was removed in the same manner. After sequential solvent replacement with ethanol and t-butyl alcohol, the supernatant was freeze-dried. In the enzymatic reaction without buffer, cellulase and hemicellulase were diluted with ultrapure water to 40 mg-protein/g-substrate, and then reacted at 48ºC for an arbitrary time. The same procedure was followed, as described above.
Preparation of the sheets from enzyme-treated CNFs
Sheets of the enzyme-treated CNF was prepared as previously described (Hideno et al. 2016). Each CNF sample was collected to obtain a dry basis weight of 30 g/m2, diluted with ultrapure water, agitated in a homogenizer, and filtered under reduced pressure through a PTFE membrane filter (pore size of 1 µm, ADVANTEC Co., Japan) to obtain a wet sheet. The wet sheet was transferred onto a stainless wire mesh (#300), sandwiched between a water-absorbing papers and a stainless plates, and hot-pressed at 110ºC for 25 minutes under any pressure (1.1 ~ 1.6 MPa) to obtain a CNF sheet.
Thermal analysis of enzyme-treated CNFs
In accordance with the findings of previous studies (ex. Hideno 2023), freeze-dried powder samples of CNF subjected to enzymes were solidified into tablets of 4.5 mm diameter by a hand press machine and subjected to thermal analysis. For thermal analysis, the pyrolysis weight loss (thermogravimetric: TG) and pyrolysis weight velocity (differential thermogravimetric: DTG) were measured under a nitrogen gas atmosphere (100 ml min− 1) in a thermal analyzer under the following temperature increase conditions: The temperature was increased from 30°C to 110°C at 40°C min− 1, held for 10 minutes, and then increased to 550°C at 10°C min− 1, and held for 5 minutes. The DTG and relative DTG curves were calculated using Equations (1) and (2).
TG (%) = (weight loss due to thermal decomposition / original weight) × 100 (1)
DTG (%/min) = TG (%) / time for increase in temperature (min) (2)
Curve-fitting of the DTG peak separation was carried out using TA7000 version 10.41 (Hitachi High-Tech Science Co., Tokyo, Japan) and Fityk version 0.9.4 (Fityk, Warsaw, Poland). A split Gaussian method and a Levenberg-Marquardt algorithm were used for peak separation and DTG curve fitting, respectively.
Determination of constituent sugars in CNFs
In accordance with previous studies, the LAP-NREL method was used as a reference for constitutive sugar analysis (Sluiter et al. 2008). Briefly described, approximately 0.03–0.04 g of dried sample was weighed into a 2 ml tube, 300 µL of 72 wt% sulfuric acid was added and stirred, and primary hydrolysis was performed at 30°C for 1 h. The hydrolyzed solution was transferred to a sealed glass vessel while diluted with 8.4 mL pure water, and subjected to autoclave at 120°C for 60 min in secondary hydrolysis reaction. After solid-liquid separation with glass-fiber filter paper, the filtrate was meshed up to 10 mL, neutralized with aqueous barium hydroxide, passed through a 0.45 µm filter, and analyzed for monosaccharides by high-performance liquid chromatography. The residue was washed with at least 100 mL of deionized water and dried at 105°C to obtain a weight of acid insoluble matters.
X-ray diffraction (XRD) analysis
The XRD analyses were carried out based on a previous report (Abe and Yano 2009) using a Rigaku RINT2000 or Ultima IV X-ray diffractometer (Rigaku Co., Tokyo, Japan) with Cu Ka radiation at 40 kV and 300 mA. The scan range (2θ) was set to 5–40° at a rate of 1°min-1. The crystallinity index was calculated using Eq. (1) based on the method described by Segal et al. (1959). The peak heights of the 200 peak (I200, 2θ = 22.5°) and the amorphous region (Iam, 2θ = 18.7°) were used.
$$Crystallinity Index \left(\%\right)=\frac{{I}_{200}-{I}_{am}}{{I}_{200}} \times 100$$
1
Measurement of basic physical properties of enzyme-treated CNF sheets
Enzyme-treated CNF sheets were conditioned under constant temperature and humidity (23 ± 1ºC, 50 ± 2% humidity) for at least 4 hours, cut into 10 × 50 mm specimens, and subjected to tensile strength and contact angle measurements. The tensile strength was measured using a tensile and compression testing machine (A&D Corporation) with a load cell of 1 kN and a crosshead speed of 10 mm min− 1 by placing sandpaper between the gripper and specimen. The contact angle was measured 10 s after a drop of pure water was dropped onto the specimen using a solid-liquid interface analysis system (FAMAS, Kyowa Surfaces Science Co.).
Observation of enzyme-treated CNFs by Atomic Force Microscope
The enzyme-treated CNFs were observed using an atomic force microscope (SPM9700, Shimadzu Corporation) equipped with a cantilever (4 µm thick, 125 µm long, 30 µm wide, NCHR-10, NANOWORLD) with a resonance frequency of 320 kHz (SPM9700, Shimadzu Corporation) after dropping and drying on a mica substrate. The observation conditions were a scanning range of 5 × 5 mm and a scanning speed of 1 kHz, and the measurements were performed in the phase mode.