Enzymatic hydrolysis
It has previously been found that cellulose is not completely enzymatically hydrolyzed at low cellulase concentration [1].
In this study, the saccharification rate reached a maximum at 142 h (Fig. 2), as was found previously [1]. The saccharification rate was lower at 196 h than at 168 h, but the difference was not significant.
Weight-averaged DP
The hydrolysis of cellulose by endoglucanase causes the weight-averaged DP (DPw) to decrease rapidly [13]. This is because endoglucanase cuts cellulose chains at random points in amorphous regions. In this study, the DPw decreased rapidly in the first hour of incubation but the saccharification rate remained below 20% at that time (Fig. 2). This indicated that the cellulose chains had been cut into shorter molecules but that not much glucose was produced at the beginning of the hydrolysis process. The DPw then slowly decreased but the saccharification rate increased rapidly, mainly through the actions of two cellobiohydrolases.
TEM
The cellulose microfibrils before saccharification had been performed were more than several micrometers long. The ends of the microfibrils could not be seen, so it was not possible to determine the actual lengths of the microfibrils (Fig. 3a). The microfibrils had become drastically shorter after 1 h of saccharification (Fig. 3b), and the microfibrils were slightly shorter still after 196 h of saccharification (Fig. 3c). The lengths and widths of the microfibrils were determined using Image J software. The microfibrils became shorter during the first phase of the saccharification process, which lasted ~ 24 h. The saccharification process caused the cellulose microfibrils to become both shorter and narrower (Fig. 4). This agreed with the decrease in DPw. The saccharification residues became aggregated (Fig. 3b, c). Large cellulose aggregates were also visible by eye when the saccharification process had been occurring for 24 h.
SAXS analysis
Structural changes in the cellulose caused by cellulase were identified by performing SAXS analysis of an aqueous sample. The cellulose residue after 142 h of saccharification was subjected to SAXS analysis. The saccharification rate had reached a maximum at 142 h, as shown in Fig. 2, so it was reasonable to use the residue after 142 h of saccharification as a substitute for the residue after 196 h of saccharification. The q-region of the SAXS signal was limited, so we qualitatively interpreted the q–I curve shown in Fig. 5. The SAXS data were prepared by subtracting the SAXS profile of pure water from the SAXS profile of the aqueous residue sample.
A shoulder feature was found at around q = 1 nm−1 for cellulose both before and after treatment with cellulase. This could be explained by the presence of highly dispersed cellulose microfibrils with sharp interfaces with the solvent, as was found in previous studies [16, 17]. The higher q-region of this shoulder feature (1.5–2.5 nm−1) was successfully fitted using the power law in the form I (q) ∝ q−D, where D = 4 for both samples, as shown in Fig. 5. This could be interpreted as indicating that scattering occurred at the cellulose interface with a surface fractal dimension of 2 (i.e., 6 − D). The cellulose interface (i.e., the cellulose microfibril surface) was assumed to be, on average, smooth before and after hydrolysis by cellulase. The lack of change in this parameter indicated that the surfaces of the cellulose microfibrils that remained after 142 h of cellulase hydrolysis were as smooth as the surfaces of the microfibrils before they were hydrolyzed. A similar study with more frequent sampling times will need to be performed to allow a more detailed interpretation to be made.
Another important feature of the SAXS profile was monotonic scattering decay in the range q = 0.2–0.7 nm−1. This q-region was fitted to the power law I (q) ∝ q−D, and the values of D for the 0- and 142-h hydrolysis time samples were 2.2 and 2.6, respectively. Perfectly dispersed plant cellulose microfibrils would be expected to exhibit scattering decay along a q−1 asymptote in this q-region, as found in a previous study [17]. The D values used in this study were > 2, clearly indicating that the cellulose microfibrils were aggregated rather than dispersed. Hydrolysis by cellulase clearly increased the D value in this q-region. The increase in the D value indicated that the structures at the size corresponding to the q values became denser. This change in the SAXS profile probably indicated that cellulose microfibrils were more densely packed after hydrolysis by cellulase than before. This agreed with the conclusion drawn from the TEM images that cellulase hydrolysis removed dispersed long microfibrils and caused cellulose microfibrils with similar lengths (~ 200 nm) to form aggregates (Fig. 3a, c).
IR spectra of deuterated bagasse cellulose
Cellulose has numerous hydroxyl groups that form intracellular and intercellular hydrogen bonds with each other. However, free hydroxyl groups are exposed on cellulose microfibril surfaces, and the hydrogen atoms in these groups can easily be replaced with deuterium when the cellulose is stored in deuterium oxide. It is difficult to acquire an IR spectrum of deuterated cellulose because the OD groups in deuterated cellulose easily return to being OH groups when the system is exposed to H2O in the air. IR spectra of deuterated cellulose in deuterium oxide vapor have been acquired [11, 18]. In this study, a cellulose suspension in deuterium oxide was applied to an ATR prism and a spectrum was acquired while the cellulose was enveloped in a dry nitrogen atmosphere. No absorbance was found at ~ 2500 cm−1 (absorbance by OD) before the cellulose was deuterated (Fig. 6). However, the spectrum of the deuterated cellulose both before and after 196 h of saccharification contained an OD absorption peak (Fig. 6). As more saccharification occurred, the OD ratio decreased from > 40 to < 20% (Fig. 2). It has previously been found that cellulase binds to hydrophobic planes of cellulose and then starts to hydrolyze the cellulose [19, 20]. The exposed hydroxyl group ratio decreased and more hydrophobic planes became exposed as cellulose I was hydrolyzed from the hydrophobic planes (Fig. 7). Cellulose I has two crystal forms, cellulose Iα and cellulose Iβ [21]. Cellulose Iβ is dominant in sugarcane, a higher plant. The cellulose microfibrils are synthesized by the cellulose synthase enzyme complex visualized in the plasma membrane as rosettes. It has been proposed that the microfibril consists of 36 molecular chains [22]. In recent years, various models (e.g., 18 molecular chains, 24 molecular chains) have been proposed by some researchers [23,24,25]. In this study, theoretical OD ratios were calculated for the conventional 36 molecular chains’ model. A cellulose microfibril consisting of 36 molecules will have the cross-section before saccharification shown in Fig. 7a. The saccharification rate was 15%, and the microfibrils became shorter after an hour of saccharification. The cellulose molecules have been hydrolyzed from two hydrophobic planes, and the cross-sections would have the structures shown in Fig. 7b. The microfibrils were then saccharified further and the hydrophobic plane areas increased. Once the saccharification reached the limit, the short microfibrils aggregated through hydrophobic effects (Fig. 7c). This was consistent with the cellulose residue aggregation observed by TEM. The ratio of hydroxyl groups exposed on the outsides of the microfibrils was calculated (Fig. 8). The ratio before saccharification was 31%, although the ratio of deuterated hydroxyl groups determined from the IR spectrum was 47%. The discrepancy was caused by microfibrils having some disordered parts with hydroxyl groups that could be deuterated. As saccharification progressed, the ratio of exposed hydroxyl groups gradually decreased to ~ 20%, as did the ratio of deuterated hydroxyl groups (Fig. 8). The accessibility of the cellulose to cellulase decreased as the hydrophilicity decreased and aggregation increased, and this explained the cellulose residues becoming more difficult to hydrolyze. Cellulose IIII can be more easily hydrolyzed than cellulose I, and does not have a saccharification limit [1]. This is because cellulose IIII has wider moderately hydrophobic planes that enzymes can become attached to and hydrolyze [26]. Moderately hydrophobic planes also have free hydroxyl groups on their surfaces (Fig. 9). The ratio of free hydroxyl groups on moderately hydrophobic plane surfaces will probably not decrease even though enzymes will hydrolyze cellulose IIII along its planes. It is likely that the remaining hydroxyl groups on cellulose IIII crystals prevent them from spontaneous aggregation, which leads to the complete degradation. Recently, it was reported that the saccharification residue of cellulose II at a low cellulase concentration was cellulose I [1]. This is a case of the aggregation of cellulose I by increase of hydrophobic planes.