Article pubs.acs.org/Biomac
Thin Film of Lignocellulosic Nanofibrils with Different Chemical Composition for QCM‑D Study Akio Kumagai,† Seung-Hwan Lee,*,†,‡ and Takashi Endo*,† †
Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Hiroshima 739-0046, Japan ‡ Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, South Korea ABSTRACT: Thin films of lignocellulosic nanofibrils (LCNFs) with different chemical compositions were prepared for real-time observation of their enzymatic adsorption and degradation behavior by using a quartz crystal microbalance with dissipation monitoring (QCM-D). LCNFs were obtained by disk milling followed by high-pressure homogenization of Hinoki cypress. The lignin contents were adjusted by the sodium chlorite treatment. The film thickness was adjusted by controlling the concentration of the LCNF suspension, which was determined from its proportional relationship to the UV absorbance of lignin. The enzymatic degradation behavior was investigated with a commercial enzyme mixture. The results of the QCM-D showed that changes in frequency and dissipation in the initial reaction stage were different from the typical changes reported for pure cellulose. To the best of our knowledge, this is the first report of the preparation of thin films of LCNFs with high lignin and hemicellulose contents and their application in a QCM-D study.
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INTRODUCTION Bioethanol from lignocelluloses has attracted much attention as a potential source of sustainable energy. Enzymatic saccharification is known to be an environmentally benign method;1 however, direct hydrolysis of lignocellulose with enzymes is difficult because of its robust structure.2,3 Thus, chemical or physical pretreatments are generally performed before enzymatic saccharification. Various methods have been developed for efficient hydrolysis; our research group has reported a method employing mechanical fibrillation after loosening the cell-wall structure of lignocellulose with different pretreatments.4−6 The resultant lignocellulosic nanofibrils (LCNFs) showed very high yields and rates of enzymatic saccharification at lower enzyme loadings, which is mainly attributable to the high specific surface area (SSA). Complete enzymatic hydrolysis of cellulose to glucose can be achieved by the complementary activity of several different cellulases. Cellulases are generally classified into three types; endo-β-1,4-glucanase (EG), cellobiohydrolase (CBH), and βglucosidase (BGL).7 EG randomly hydrolyzes internal β-1,4glycosidic bonds in the cellulose chain. CBH releases cellobiose from the ends of cellulose chains. It is also known that EG mainly acts on amorphous or disordered regions of cellulose, while CBH acts on crystalline regions. BGL hydrolyzes cellobiose and other soluble cello-oligosaccharides to glucose. During the enzymatic hydrolysis process, these different types of cellulases play individual roles, and efficient hydrolysis of crystalline cellulose is accomplished by several enzyme © 2013 American Chemical Society
components working synergistically. Synergism is defined as the increase in activity exhibited by the mixtures of enzyme components compared with the sum of the activities of each component evaluated individually.8 Therefore, a synergistic enzyme system is essential for the efficient hydrolysis of lignocelluloses,9,10 and various previous experiments have attempted to elucidate the complex hydrolysis mechanism of synergism.11−13 Recently, interactions between enzymes and cellulose have been observed in real time using a quartz crystal microbalance with dissipation monitoring (QCM-D).14−18 The techniques used to prepare thin films consisted of cellulose on QCM-D sensors able to analyze cellulose hydration,19−21 and enzymatic kinetics of cellulose hydrolysis.15,17,18 Cellulose model films prepared from regenerated cellulose have been frequently used in investigations of swelling behaviors and enzymatic hydrolysis, including synergism.14−16,18−20 Films of amorphous cellulose made from chemically modified trimethylsilyl cellulose (TMSC) were also studied.17,21−24 Additionally, bicomponent amorphous cellulose/lignin films were prepared, and enzymatic hydrolysis by cellulase mixture and purified monocomponent cellulases were investigated.25,26 Although these films can be used to investigate cellulose hydration and hydrolysis behaviors in the presence of lignin but not other properties such as Received: April 19, 2013 Revised: May 22, 2013 Published: May 30, 2013 2420
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hemicellulose contents and fibril dimensions, there is still a certain difference between them and native lignocellulose. Ahola et al. reported a method to prepare native cellulose model films with both amorphous and crystalline cellulose regions by spin coating cellulose nanofibril dispersions.22 The native cellulose model films had a fibrillar structure unlike the regenerated and amorphous cellulose model films, and their enzymatic hydrolysis by cellulase mixture indicated fast ́ reaction.16 More recently, Martin-Sampedro et al. reported a QCM-D study on thin films of LCNF containing high hemicellulose and low lignin contents.27 However, to the best of our knowledge, films prepared from LCNFs containing high lignin and hemicellulose contents have not yet been reported. In this study, we modified the preparation method of cellulose nanofibril model films reported by Ahola et al.22 by using our mechanical fibrillation technology to produce model LCNF thin films with different chemical compositions. The LNCF films were then analyzed using QCM-D to evaluate the enzymatic hydrolysis of LCNFs.
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rinsed with Milli-Q water at each step. A 1 wt % PEI/water solution was used as an anchoring substance. PEI is considered to improve the coverage of LCNFs on the sensor by its cationic nature. The cleaned sensor was immersed in the PEI/water solution for 15 min and rinsed with Milli-Q water before spin coating. The LCNF suspension was dropped on the PEI-absorbed sensor, incubated for 1 min, and then spin coated at 3000 rpm for 1 min using a spin coater (Opticoat MSA100, Mikasa, Tokyo, Japan). Finally, the spin-coated sensor was heated to 80 °C for 10 min in an oven. Chemical Composition Analysis. Structural carbohydrates and Klason lignin contents were determined using the Laboratory Analytical Procedure (LAP) of the National Renewable Energy Laboratory (NREL) with some modifications.29 A dried sample (50 mg) was swollen in 72 wt % sulfuric acid (600 μL) and hydrolyzed by shaking at 30 °C for 90 min before diluting to 4 wt % with additional Milli-Q water (16.8 mL). The diluted sample was heated to 120 °C for 1 h in an autoclave. After autoclaving, the supernatant was used to determine the structural carbohydrates, and the residue was used to determine the Klason lignin contents. The supernatant was neutralized with saturated barium hydroxide, and the monomeric sugars in the supernatant were analyzed using an HPLC system consisting of an LC-2000 Plus (Jasco) equipped with an Aminex HPX-87P column (Bio-Rad Laboratories, Hercules, CA) at 60 °C. Milli-Q water was used as the eluent at a flow rate of 0.25 mL/min. The vacuum-filtered residue was washed with hot Milli-Q water until the pH of the filtrate was neutral; then, the Klason lignin content of the residue was quantified after complete drying using a vacuumdrying apparatus. Characterization of LCNFs. For SSA measurement, dried LCNFs were prepared by freeze-drying after solvent exchange. The Milli-Q water of the LCNF suspension was exchanged to tert-butyl alcohol by repeated centrifugations because the aggregation of LCNFs was prevented by the freeze-drying process. The SSA of the LCNFs were measured using a BEL-SORP-Max (BEL Japan, Osaka, Japan), and the SSA values were determined from a Brunauer−Emmett−Teller plot of the nitrogen adsorption−desorption isotherm.30 The zeta potentials of the LCNFs were measured using a nanoparticle analyzer (nanoPartica SZ-100 nanoparticle analyzer, HORIBA, Kyoto, Japan), and the pH values of the LCNF dispersions were determined using a pH meter (twin pH compact pH meter, HORIBA). AFM Imaging. AFM imaging (JSPM-5200, JEOL, Tokyo, Japan) was performed to evaluate the morphology, roughness, and thickness of the prepared films by using the AFM tapping mode in air at 25 °C and relative humidity of ∼30%. AFM images were also obtained to compare the changes in the morphology of films before and after enzymatic hydrolysis. Images of sizes 5 μm × 5 μm and 25 μm × 25 μm were taken in at least three different areas of each sensor. The roughness was calculated with the WinSPM software (JEOL). The thicknesses of the layer of anchoring PEI substrate and the prepared film were determined by the scratching method with a needle, as described by Ahola et al.16 A 5 μm × 5 μm portion of the scratched area was scanned again, and the film thickness was determined using at least 20 height profiles. QCM-D Experiment. The QCM-D experiment (Q-Sense E1, Qsence AB, Götenborg, Sweden) was performed to investigate the enzymatic adsorption and degradation behavior of the LCNF films. The sensor coated with the LCNF films was placed in 50 mM sodium acetate buffer (pH 5.0) overnight to swell completely. The swollen sensor was mounted in the QCM-D flow cell, which was then filled with the buffer solution, and the sensor was swelled again until no appreciable frequency shifts were observed. Afterward, the enzyme solution, 0.05 mg/mL Acremonium cellulase in 50 mM sodium acetate buffer (pH 5.0), was introduced into the QCM-D flow cell with a peristaltic pump at a flow rate of 50 μL/min. The pump was stopped after the enzyme solution was completely loaded for 10 min. Here all QCM-D experiments were conducted under batch conditions. The enzyme solution was introduced in the cell until the initial buffer solution was fully replaced by the enzyme solution, which was twice the volume of the flow, and the measurement was performed in the
EXPERIMENTAL SECTION
Materials. Hinoki cypress (Chamaecyparis obtusa) softwood powder less than 0.2 mm in size was used as the starting material for the LCNFs. Commercial microfibrillated cellulose (CMFC) Celish KY-100G (Daicel Chemical Industries, Tokyo, Japan) was used as a reference for the pure cellulose nanofibril. Sodium chlorite and acetic acid were used for delignification. Sulfuric acid and barium hydroxide were used for the chemical composition analysis. Thirty-percent polyethyleneimine (PEI) solution was used as an anchoring substance for spin coating of the LCNFs on the QCM-D gold sensor. Ammonia solution (25%) and hydrogen peroxide (30%) were used for cleaning the sensors. The commercial enzyme mixture used was Acremonium cellulase (derived from Acremonium cellulolyticus; Meiji Seika, Tokyo, Japan). All chemicals were purchased from Nacalai Tesque (Kyoto, Japan) or Wako Pure Chemical (Osaka, Japan). Preparation of LCNFs. Wood powder of 1 wt % was soaked overnight in water. The soaked wood powder was fibrillated by repeating disk milling (CRENDIPITOR MKCA6-2, Masuko Sangyo, Saitama, Japan) 10 times with a clearance of 150 μm and rotation speed of 1800 rpm. Delignification of the disk-milled product was performed using a sodium chlorite treatment (SCT) in a water bath at 75 °C according to a modified Wise method.28 Sodium chlorite (0.666 g/g wood powder) and acetic acid (0.133 mL/g wood powder) were added to the disk-milled product at 1 wt %. A series of LCNFs with different lignin contents was prepared by changing the reaction time (10 min, 4 h, 7 h, and 8 h) and the amounts of reagents used. After the treatment, partially delignified samples were vacuum-filtered with filter paper (no. 5A, ADVANTEC Toyo, Tokyo, Japan) and washed with water until the washings were colorless. The filtrated product was suspended in water again without drying. Further fibrillation was conducted using a high-pressure homogenizer (MASSCOMIZER MMX-L100, Masuko Sangyo) at a pressure of approximately 200 MPa. The resulting product was diluted to 0.5 wt %, ultrasonicated for 1 min to prevent their aggregation and to improve their dispersibility, and centrifuged for 30 min at 10 000 rpm. The supernatant was used for spin coating to prepare the thin film. Preparation of the Thin Films. The UV absorbance of the supernatants at 202 nm attributable to the stretching vibration of double-bonded carbon in lignin was measured using a UV spectrophotometer (UV−visible-NIR spectrophotometer, V-670, Jasco, Tokyo, Japan) to obtain uniform concentrations. Before spin coating, the QCM-D gold sensor (QSX 301, Q-sence AB, Götenborg, Sweden) was cleaned with UV/ozone Pro Cleaner Bioforce Nanoscience, Ames, IA) for 10 min, followed by boiling in a mixture of 25% ammonia solution and 30% hydrogen peroxide (1:1:5 with Milli-Q water by volume) at 75 °C for 5 min. Then, the sensor was cleaned with UV/ozone Pro Cleaner for a further 10 min. The sensor was 2421
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absence of flow. The frequency and dissipation changes at the fundamental resonance frequency (5 MHz) and its overtones (15, 25, 35, 55, and 75 MHz) were monitored simultaneously; the third overtone (15 MHz) was used in the data evaluation. No significant changes in frequency and dissipation were detected in the pump operation at the third overtone. The measurement was continued for more than 6 h, even if no appreciable changes in frequency and dissipation were observed. The enzymatic saccharification with the enzyme solution derived from A. cellulolyticus has often been investigated at 45−50 °C as optimal temperature to ensure thermostability.31 However, the maximum allowable temperature of QCM-D equipment is 40 °C. Therefore, the temperature of the QCMD flow cell was maintained at 40 °C during the measurement of the enzymatic reaction. All experiments were repeated at least twice.
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RESULTS AND DISCUSSION Adjustment of Chemical Composition and Concentration of LCNFs. LCNFs with different chemical compositions were prepared from Hinoki cypress, a typical softwood in Japan. The chemical composition was adjusted by SCT using disk-milled samples, and the obtained results are summarized in Table 1. SCT was conducted mainly to remove lignin. The
Figure 1. UV absorption spectra of LCNF suspensions [lignin contents of 26.4% (●), 16.7% (▲), 10.8% (■), and 6.5% (◆)] and soluble sodium lignosulfate.
cycles of homogenization was observed by AFM to confirm the uniformity of the film. After a number of investigations, more than 20 homogenization cycles (passes) were found to be required to prepare a film with uniform morphology, showing even oscillations across the entire overtone frequency range in QCM-D. The detailed morphological characteristics of the thin films will be discussed later. Table 2 shows the pH, zeta potential, and SSA of the LCNF supernatants along with CMFC, which was also prepared by the
Table 1. Chemical Composition of the LCNFs Used in This Study SCT timea lignin (%) glucose (%) xylose (%) galactose (%) arabinose (%) mannose (%) a
untreated
10 min
4h
7h
8h
27.2 48.0 2.5 1.4 1.7 11.4
26.4 48.3 2.5 1.4 1.8 11.7
16.7 55.5 2.3 1.2 1.5 10.8
10.8 60.7 2.8 1.2 1.7 11.3
6.5 67.1 2.3 1.2 1.5 11.4
Table 2. Zeta Potential at Different pH and SSA of CMFC and LCNFs SCT timea
SCT: sodium chlorite treatment.
pH zeta potential (mV) SSA (m2/g)c
lignin contents decreased with the length of the SCT, whereas the composition of the constituent sugars of hemicellulose was not significantly changed. After adjusting the chemical composition, disk-milled samples were further fibrillated with a high-pressure homogenizer, and after centrifugation, the supernatants were used to obtain fine fibrils for spin coating of the QCM-D gold sensor. However, their LNCF concentrations were different because of the differences in the degree of fibrillation and chemical composition. Therefore, the concentration needed to be adjusted to obtain the same thickness of the resulting films. Suchy et al. have reported that the thickness of spin-coated films can be controlled by changing the concentration of a TMSC suspension in toluene, and the change in film thickness induced different QCM-D results.24 Thus, to remove the effect of any other factors, it is better to have a uniform film thickness in all samples to investigate the effect of chemical composition on the enzymatic adsorption and degradation. The concentration of the supernatant was adjusted using the UV absorption of lignin. Figure 1 shows the UV absorption spectra in the 190−300 nm wavelength range. The relationship between the absorbance at 202 nm and the concentration of soluble sodium lignosulfate for the calibration was measured, and thus the lignin content of the supernatant was determined. Because each LCNF has different lignin content, the concentrations of every sample had to be adjusted on the basis of the lignin content measured by wet chemistry (Table 1). The morphology of the fibrillated samples after every five
CMFCb
10 min
4h
7h
8h
6.0 −47.3 101.1
5.6 −45.5 86.3
5.7 −44.6 189.1
5.9 −46.7 161.5
6.0 −43.6 201.0
a SCT: sodium chlorite treatment. bCMFC: commercial microfibrillated celluose. cSSA: specific surface area.
same fibrillation procedure as the LCNFs. The preparation method of LCNF films was based on the method for pure cellulose nanofibril model film by Ahola et al.22 In the present study, PEI with a branched structure and high cationic nature as an anchoring substance was adsorbed on the sensor before spin coating to improve the coverage of LCNFs, which are generally anionic. There was a concern about the effect of lignin in LCNFs on the zeta potential because of its hydrophobicity. The zeta potential is the electrochemical border between the immobile layer of the adsorbed counterions and the diffuse layer on the surfaces and is directly related to the surface charge density.32 As shown in Table 2, the zeta potentials of all LCNFs were found to be in the range −43.6 to −46.7 mV in the pH range of 5.6 to 6.0, which is similar to that of CMFC (zeta potentials of −47.3 mV at pH 6.0). These small differences in the zeta potential would be helpful in the preparation of uniform films on the cationic PEI-adsorbed QCM-D gold sensors. The SSA of the LCNFs was found to be in the range of 161.5−201.0 m2/g, except for 86.3 m2/g for the LCNF with a high lignin content of 26.4% (SCT time, 10 min). These SSAs are considered to be similar to those of partially delignified or fibrillated LCNFs reported in other studies.33,34 The SSA values 2422
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film showed an average thickness of 14.3 ± 0.7 nm and an average roughness of 6.23 ± 0.78 nm, but the cellulose nanofibril model films reported by Ahola et al. had values ranging from 4 to 6 nm and 2 to 4 nm, respectively.16,22 Thus, the thickness of the LCNF films was greater than that reported by Ahola et al., but the roughness was comparable. QCM-D Study of Enzymatic Hydrolysis. Using a series of the prepared LCNF sensors, enzymatic hydrolysis was performed using the commercial enzyme mixture. Figure 3
of LCNFs obtained by nitrogen adsorption method are probably larger than those that the enzymes can actually access because the enzymes are much larger than a nitrogen molecule. However, these SSA values can be used as the criteria to estimate the enzymatic accessibility. Figure 2a−e shows AFM height images of the film surfaces from the LCNFs and CMFC. The morphology of all LCNFs
Figure 2. AFM height images (5 μm × 5 μm) of nanofibrils on the QCM-D gold sensors before (a−e) and after (f−j) enzymatic hydrolysis. Lignin content: 26.4% (a,f), 16.7% (b,g), 10.8% (c,h), and 6.5% (d,i). CMFC (e,j).
Figure 3. Frequency (a) and dissipation (b) changes for the enzymatic hydrolysis of CMFC and LCNFs [lignin content of 26.4% (●), 16.7% (▲), 10.8% (■), and 6.5% (◆)] by 0.05 mg/mL enzyme mixture in 50 mM sodium acetate buffer (pH 5.0) at 40 °C.
was nanoscopic, and they were well-dispersed on the PEIadsorbed surface of the QCM-D gold sensor. The size distribution of the LCNF diameters was found to range from 3 to 20 nm, corresponding to elementary cellulose fibrils to cellulose microfibril bundles.35 All film surfaces from the LCNFs (Figures 2a−d) were similar to that of CMFC (Figures 2e). Table 3 summarizes the average values of the thickness and roughness of the thin films determined by AFM. The average thickness and roughness of the LCNF films were found to be 10.6 ± 0.8 to 13.5 ± 0.8 nm and 3.34 ± 0.13 to 4.60 ± 0.16 nm, respectively. Only small differences were observed between LCNF films with different chemical compositions. The CMFC
shows the change in the frequency and dissipation monitored over 6 h at 40 °C with a 50 mM sodium acetate buffer (pH 5.0) after complete swelling of the film. The optimum concentration of the enzyme mixture was found to be 0.05 mg/mL to obtain a clear change in frequency and dissipation in the initial stage of the reaction. The enzyme mixture derived from A. cellulolyticus used in this study contains EG, CBH, and BGL, along with hemicellulases such as xylanase and mannase.36 Fujii et al. reported that this enzyme mixture showed higher mannanhydrolyzing activity than Accellerase 1000 derived from
Table 3. Thickness and Roughness of CMFC and LCNF Films Determined by AFM SCT timea thickness (nm) roughness (nm) a
CMFCb
10 min
4h
7h
8h
14.3 ± 0.7 6.23 ± 0.78
13.5 ± 0.8 4.60 ± 0.16
12.9 ± 0.7 4.20 ± 0.18
12.6 ± 0.6 3.64 ± 0.18
10.6 ± 0.8 3.34 ± 0.13
SCT: sodium chlorite treatment. bCMFC: commercial microfibrillated cellulose. 2423
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Trichoderma reesei.36 Therefore, Acremonium cellulase was suitable for degrading softwood Hinoki cypress, in which the main components of hemicellulose are mannans, including galactoglucomannan and glucomannan (Table 1).37 CMFC showed a typical QCM-D response for enzymatic reactions. The decrease in the frequency corresponds to enzyme adsorption on the substrate, and this was followed by a rapid increase, indicating the release of hydrolysis products and desorption of enzymes from the substrate. This increase in frequency continued until the hydrolysis did not proceed further; then, the frequency finally reached a plateau. Meanwhile, the dissipation rapidly increased in response to the addition of the enzyme solution. The change in dissipation reveals information about changes in the viscoelasticity and morphology of the substrate on the QCM-D gold sensor and serves as a measure of the thickness of the substrate. The first rapid increase in dissipation indicates an increase in the thickness of the substrate caused by adsorption of the enzyme. After reaching its maximum, the dissipation started to decrease, which results from the decrease in thickness of the substrate. Finally, the dissipation reached a plateau along with the frequency.14−18 Comparing the plateau values of frequency and dissipation in LCNFs, the values were only slightly different, except for the LCNF with a lignin content of 26.4%. This indicates that the differences in film thickness in this study can be considered to be small. As shown in Table 2, the LCNF films showed similar thicknesses. Thus, the plateau frequency values seem to be correlated with the film thickness. The low values for the LCNF with a lignin content of 26.4% probably indicate low enzymatic degradability because of the high lignin content and low SSA, as mentioned above. Figure 2f−i shows the AFM height images of LCNF films after enzymatic hydrolysis. None of the LCNF films was completely degraded by the enzyme, indicating that cellulase could not completely degrade LCNFs containing lignin and hemicellulose. In particular, the LCNFs with the high lignin content of 26.4% were much more persistent and remained evenly distributed across the entire surface of the sensor after enzymatic hydrolysis, in contrast with those with lignin contents of less than 16.7%. This may also be because of the lower SSA (86.3 m2/g). This result is in agreement with the results of the QCM-D analysis previously mentioned. Thus, it can be concluded that the presence of lignin and hemicellulose inhibits the access of cellulases to cellulose. However, CMFC without lignin and hemicellulose was completely removed from the sensor under the same reaction conditions. Figure 4 shows the changes in frequency and dissipation in the initial stage of the enzymatic reaction. In the case of CMFC, the initial decrease in frequency is caused by adsorption of enzymes; then, the frequency increases because of the enzymatic degradation of the substrates in the earlier stage (Figure 4a). Meanwhile, the change in dissipation of CMFC showed only an initial increase because of the adsorption of the enzyme and its associated water, followed by a decrease caused by degradation of the substrates and dissociation of the enzymes. These changes in frequency and dissipation are ubiquitous in QCM-D studies of cellulose film hydrolysis by cellulase.15−17 The enzymatic interaction is very complex because several phenomena such as enzyme adsorption and degradation on substrates and dissociation of hydrolysates and enzymes from substrates occur simultaneously during enzymatic reactions. In a general enzyme reaction, we can
Figure 4. Frequency (a,b) and dissipation (a,c) changes in the early stage of the enzymatic hydrolysis of CMFC (a) and LCNFs (b,c) [lignin content of 26.4% (●), 16.7% (▲), 10.8% (■), and 6.5% (◆) by 0.05 mg/mL enzyme mixture in 50 mM sodium acetate buffer (pH 5.0) at 40 °C].
hypothesize that the following phenomena occurred in this initial stage. The adsorption of enzymes, more so than the degradation of substrates, caused the decrease in frequency; then, the frequency started to increase as degradation of the substrates and dissociation of the enzymes exceeded adsorption of the enzymes. Compared with CMFC, the changes in the frequency and dissipation of LCNFs in the initial stage showed different trends. The frequency of all LCNFs first increased slightly and then decreased before increasing again (Figure 4b). The surface of LCNFs consisted of a smaller amount of cellulose than CMFCs because the LCNFs contained large amounts of lignin and hemicellulose adsorbed on the cellulose.38 Therefore, the adsorption of cellulase may not occur noticeably in the initial stage, when only a very small amount of cellulase can be 2424
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than those in frequency. The initial decrease in dissipation after the enzyme addition probably indicates the primary degradation of partial hemicelluloses and the release of lignin from the surface of cellulose microfibrils. The subsequent increase in dissipation after the initial decrease suggests that the amount of cellulases adsorbed on the newly exposed cellulose surface and water uptake (swelling) is higher than the mass loss because of the initial degradation. To understand the complex hydrolysis mechanism in more detail, other information through QCM-D studies using purified monocomponent enzymes such as CBH, EG, and hemicellulases for LCNFs is needed. Additionally, non-QCM-D experiments using the same LCNFs and enzymes and detailed kinetic analyses may be also beneficial to compliment the QCM-D results. These investigations should help to provide a better understanding of the enzymatic adsorption and degradation behavior of LCNFs.
adsorbed on the small surface area of the exposed cellulose. It was reported that lignin inhibited the enzymatic hydrolysis of lignocellulose owing to the adsorption of cellulases onto lignin fragments.39,40 However, remarkable changes in the cellulase adsorption on lignin were not detected by the QCM-D method. One reason might be that the lignin contained in LCNFs in this study was likely to have little chemical structure attributable to the adsorption of cellulases. It was also reported that chemically altered lignin structures with some sort of pretreatment such as hydrothermal treatment, organosolv pretreatment, acid treatment, and alkali treatment contributed to the nonproductive binding of cellulases to lignin.40−43 Recently, the nonproductive adsorption of cellulases onto the structurally changed lignin caused by steam explosion pretreatment was monitored using QCM-D by Rahikainena et al.44 In this study, the increase in the initial frequency was clearly observed in the LCNFs containing the highest lignin content (26.4%). The delignification treatment time with sodium chlorite for the sample preparation was only 10 min, indicating that lignin was likely to have few chemical structural changes. Therefore, we concluded that the cellulases hardly adsorbed on the LCNF surfaces. A possible explanation for this result could be related to the important role of hemicellulases. The presence of lignin− carbohydrate complexes formed by covalent linkages between lignin and hemicellulose residues has been previously reported.45,46 It was reported that the hydrolysis of hemicellulose by hemicellulase enhanced delignification by chemical reactions.47 Moreover, hemicellulases were found to facilitate the total hydrolysis of lignocellulose by enhancing the accessibility of cellulases to cellulose.48,49 Considering these previous reports, we thought that the accessible cellulose surface for the enzyme adsorption was exposed by the hemicellulases during the initial stage, resulting in the increase in enzyme adsorption beyond the degradation amount. Moreover, the initial stage in the hydrolysis of cellulose microfibrils by cellulases has been reported to improve the reactivity of the highly ordered and tightly packed regions of cellulose microfibrils by increasing the cellulose surface, making it more accessible to the cellulases.50 Therefore, following the frequency decrease after small frequency increase may be caused by cellulase adsorption on the newly exposed cellulose surface. These frequency changes after the initial frequency increase were more prominent at lower lignin contents. This means that enzymes can more easily approach hemicellulose and newly exposed cellulose microfibrils in the case of LCNFs with low lignin content. Finally, the frequency increased again because of the main degradation of the accessibility-improved cellulose microfibrils by cellulases. On the other hand, similar initial increase in frequencies has been also observed for the hydrolysis of films of amorphous cellulose by EG.14,26 Although the composition of films and used enzymes differed from each other, amorphous cellulose was one component of LCNF, and EG was contained in the enzyme mixture used in this study. Therefore, we also considered the possibility that the initial increase in frequency was because of the dominant hydrolysis of amorphous cellulose by EG reported in previous studies.14,26 Dissipation of all LCNFs decreased just after the addition of the enzyme (Figure 4c) and then increased again in the early stage before finally decreasing to reach a plateau. The initial changes in the dissipation of LCNFs more clearly indicate the characteristic behavior of the enzyme mixture on lignocellulose
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CONCLUSIONS LCNFs with different chemical compositions were prepared for QCM-D sensors by combining SCT and mechanical fibrillation. LCNFs coated onto QCM-D sensors were well-dispersed with a nanoscopic morphology, and they exhibited smooth surfaces comparable to those of CMFC. The effect of the chemical composition of LCNF thin films on the enzymatic saccharification using an enzyme mixture was investigated with QCMD. In the initial reaction stage, characteristic frequency and dissipation changes observed for LCNFs were different from the typical changes seen for pure cellulose. These differences seem to be caused by the presence of hemicellulose and lignin on the surface of the cellulose microfibrils. The increase in frequency and decrease in dissipation in the earliest stage may be because of the degradation of hemicelluloses and the release of lignin from the surface of the cellulose microfibrils by the initially adsorbed enzymes, and the amounts of hemicellulose degraded and lignin released are considered to be greater than the mass increase caused by enzyme adsorption. The subsequent decrease in frequency and increase in dissipation may be caused by the adsorption of cellulase on the newly exposed cellulose surface. These different phenomena in the early enzymatic reaction stage between LCNFs and pure cellulose microfibrils may provide important information about the enzymatic adsorption and degradation behavior of the enzymatic hydrolysis of LCNFs.
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +82-33-250-8323; E-mail:
[email protected] (S.-H.L.). Tel: +81-82-493-6890; Fax: +81-82-420-8278. Email:
[email protected] (T.E.). Funding
Japan-U.S. cooperation project for research and standardization of Clean Energy Technologies. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Japan-U.S. cooperation project for research and standardization of Clean Energy Technologies. 2425
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ABBREVIATIONS QCM-D, quartz crystal microbalance with dissipation monitoring; LCNF, lignocellulosic nanofibril; CMFC, commercial microfibrillated cellulose; EG, endo-β-1,4-glucanase; CBH, cellobiohydrolase; BGL, β-glucosidase; TMSC, trimethylsilyl cellulose; PEI, polyethyleneimine; SCT, sodium chlorite treatment; SSA, specific surface area
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