Novel Approach to Prepare Ultrathin Lignocellulosic Film for

Research Article pubs.acs.org/journal/ascecg

Novel Approach to Prepare Ultrathin Lignocellulosic Film for Monitoring Enzymatic Hydrolysis Process by Quartz Crystal Microbalance Chong Jiang, Tingyue Cao, Wenjuan Wu, Junlong Song, and Yongcan Jin* Laboratory of Wood Chemistry, Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China ABSTRACT: The applications of quartz crystal microbalance (QCM) rely heavily on the preparation of ultrathin films. So far, techniques on direct lignocellulosic film making without components isolation have been hardly investigated. In this work, a novel approach was developed to prepare spin-coated ultrathin films based on the complete dissolution of ball-milled wood in LiCl/DMSO solvent system. The surface analysis and elemental composition of the films respectively using atomic force microscopy and X-ray photoelectron spectroscopy proved that an even-textured lignocellulosic film could be formed on QCM gold sensors. The prepared ultrathin films were successfully applied on monitoring the enzymatic hydrolysis process in situ and in real time by QCM. The changes of QCM frequency showed clearly that the enzymatic hydrolysis of lignocellulosic materials could be divided into three stages, including cellulase adsorption, fast substrate hydrolysis, and slow substrate hydrolysis. The adsorption and hydrolysis processes were fitted with Lagergren and Boltzmann-sigmoidal kinetic models, respectively, indicating that cellulase adsorption on lignin and cellulose is competitive and that lignin inhibits the enzymatic hydrolysis of cellulose. KEYWORDS: Ultrathin film, Lignocellulosic biomass, LiCl/DMSO solvent system, Quartz crystal microbalance (QCM), Cellulase adsorption, Enzymatic hydrolysis

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INTRODUCTION As the most abundant source of renewable carbon on the planet, the utilization of lignocellulosic biomass for producing sustainable green materials and fuels has gained increasing interest worldwide. The bioconversion of lignocellulose is an attractive and environmentally friendly alternative to sugar and bioethanol platforms.1 The efficient bioconversion of lignocellulose to bioethanol via the sugar platform involves three key steps: pretreatment, enzymatic saccharification, and fermentation or catalytic conversion of sugars.2,3 The purpose of pretreatment is to remove lignin and/or hemicellulose, reduce cellulose crystallinity, and increase the porosity of the materials. The key of the biochemical processing for lignocellulose is the enzymatic depolymerization of cellulose and hemicelluloses into monomeric and oligomeric sugars that are subsequently fermented and processed into fuels or other chemicals.4 However, the structure of lignocellulose and its chemical composition impact the enzymatic hydrolysis, including the accessibility, surface area, the crystallinity, and degree of polymerization of cellulose, as well as the presence and distribution of lignin.5,6 Due to the complexity of lignocellulose, most studies in this field have focused on bulk materials and measured its overall sugar yield after enzymatic hydrolysis. However, these methods fail to directly monitor the dynamics of enzymatic hydrolysis © 2017 American Chemical Society

and surface activities, especially in the initial stages of the process.7 To overcome these limitations, sensing techniques such as quartz crystal microbalance (QCM) have been proposed, which is very useful to monitor in situ and in real time the binding and/or catalytic activities of cellulases on lignocellulosic substrates.8,9 Recently, lignin and cellulose bicomponent ultrathin films prepared from acetylated lignin and trimethylsilyl cellulose have been proposed to study hydrolysis events on surfaces containing both of these biopolymers.10 However, these films were not likely to contain lignin−carbohydrate complexes and had no chemical interactions.11 Therefore, it is of great interest to develop a novel and green method for the direct preparation of ultrathin films by using lignocellulosic biomass without assembling the chemical components. Some attempts have been done to prepare ultrathin film using high-pressure homogenized or fluidized lignocellulosic nanofibrils (LCNFs). For example, Martin-Sampedro et al.8 investigated the rapid enzymatic saccharification of LCNF prepared from refined fibers processed in a high-pressure fluidizer by monitoring nanoscale changes in mass via QCM. Kumagai et al.12,13 prepared thin Received: November 29, 2016 Revised: March 5, 2017 Published: March 14, 2017 3837

DOI: 10.1021/acssuschemeng.6b02884 ACS Sustainable Chem. Eng. 2017, 5, 3837−3844

Research Article

ACS Sustainable Chemistry & Engineering Table 1. Main Chemical Composition of Poplar, GL-Poplar, and Regenerated Poplar and GL-Poplara lignin (%) materials

regeneration yield (%)

poplar GL-poplar regenerated poplar regenerated GL-poplar a

78 84

KL 23.3 21.4 28.8 23.9

± ± ± ±

polysaccharides (%) ASL

0.1 0.2 0.2 0.2

2.6 1.5 1.2 0.9

± ± ± ±

0.0 0.0 0.0 0.0

gluan 44.1 57.3 55.0 64.6

± ± ± ±

0.2 0.2 0.4 0.1

xylan

lig/cel

± ± ± ±

0.44 0.32 0.48 0.34

15.4 14.5 7.9 9.4

0.2 0.2 0.2 0.1

Data are the mean of two measurements. (Fritsch Pulverisette 7, Idar-Oberstein, Germany) at a fixed frequency of 600 rpm. Two zirconium dioxide bowls (80 mL for each) with 25 zirconium dioxide balls (1 cm diameter) in each bowl were used in the milling. The milling was conducted at room temperature, and 15 min intervals were provided between every 5 min of milling to prevent overheating. The milling times for poplar and GL-poplar were 4 and 1 h, respectively. The ball-milled wood samples were vacuum-dried at 40 °C for further treatment. Ball-milled samples were suspended into the solvent system of 8 wt % LiCl/DMSO at a mass concentration of 0.5% and magnetically stirred overnight at room temperature. The scheme of LiCl/DMSO dissolution preparation is involved in Figure 1.

films using LCNFs fibrillated by a high-pressure homogenizer to evaluate the effect of chemical composition on the enzymatic adsorption and degradation behavior using a QCM. For preparation LCNFs, disk mill or PFI treatment is usually necessary before high-pressure homogenization or fluidization to enhance fiber accessibility and fibrillation efficiency. Meanwhile, high-pressure homogenization or fluidization needs to be repeated several times at a very high pressure. Complete dissolution of wood using an appropriate solvent offers a simple alternative to prepare ultrathin lignocellulosic films. Lithium chloride and dimethyl sulfoxide (LiCl/DMSO) was reported to be an effective solvent system for dissolving both ball-milled wood and straw materials at room temperature.14−16 Reports also indicate that little changes of lignin structure occurred during the processes of ball-milling, LiCl/ DMSO dissolution, and water regeneration.14,15 Poplar is a typical and resource-rich hardwood species. The effects of green liquor (GL) pretreatment on the chemical composition and enzymatic hydrolysis of poplar have been investigated in our previous works.17,18 In this work, a novel method for the preparation of even and smooth ultrathin lignocellulosic films on QCM gold sensors was investigated using solutions of ball-milled poplar and GL-pretreated poplar in LiCl/DMSO solvent system. The obtained films were used to monitor the processes of cellulase adsorption and enzymatic hydrolysis in situ and in real time by a QCM instrument.

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Figure 1. Schematic illustration of ultrathin lignocellulosic film preparation.

Preparation of Ultrathin Lignocellulosic Films. Ultrathin lignocellulosic films were prepared on gold-coated QCM sensors (Västra Frölunda, Sweden). QCM gold sensors were first cleaned via treatment with 25% ammonia solution/30% hydrogen peroxide/water (1:1:5, v/v/v) at 75 °C for 5 min, rinsed with Milli-Q water, and dried with nitrogen. The prepared poplar or GL-poplar LiCl/DMSO solution was coated on QCM gold sensors with a spin coater (KW-4A, Shanghai Daojing Instrument Plant, China) operated at 3000 rpm for 1 min. The spin coating process was repeated twice. Most DMSO could be evaporated from the obtained films at room temperature in 48 h. The films were then immersed into Milli-Q water to dissolve LiCl and residual DMSO. Milli-Q water was carefully replaced every 2 h until no Cl− was detected by 0.1 M AgNO3 aqueous solution. Ultrathin lignocellulosic films were obtained after vacuum drying at 40 °C (Figure 1). The poplar or GL-poplar film attached to the QCM gold sensors was used as a sensing element to monitor enzyme activities in QCM. Characterization of Ultrathin Films. The morphology, roughness, and material distribution of poplar and GL-poplar film were characterized by an atomic force microscopy (AFM) (Dimesion Edge, Bruker, Saarbrücken, Germany). The images were scanned in tapping mode using a J-scanner and silicon cantilevers. At least two different films obtained for each condition were prepared, and at least two different areas were analyzed on each of them. AFM images flattened following first-order conversion. Image analysis was performed using NanoScope analysis software (version 1.40, Bruker, Saarbrücken, Germany) from which root-mean-square (RMS) roughness was determined.

MATERIALS AND METHODS

Materials. Poplar trees (Populus deltoides) were harvested from the north of Jiangsu Province, China. Air-dried poplar was cut into chips with the size of 3−5 cm in length, 2−3 cm in width, and 1−2 mm in thickness. The wood chips were sealed in plastic bags and stored in a refrigerator at 4 °C before use. GL-pretreated poplar (GL-poplar) was prepared according to the method described by Meng et al.18 The total titratable alkali charge (TTA, the sum of Na2S and Na2CO3 as Na2O) on the basis of bone dry material was 24%. The sulfidity of the pretreatment liquor, defined as the percentage of Na2S as Na2O to TTA, was 25%. The ratio of liquor to biomass was 4 mL g−1. Poplar and GL-poplar were grounded with a Wiley mill, and the fractions between 40 and 80 mesh were collected. The poplar meals were extracted with a mixture of benzene−ethanol (2:1, v/v) for 8 h to remove solvent extractives. The extractive-free poplar and GL-poplar meals were dried under air and subsequently under vacuum for further treatment. The main chemical composition of these two samples is given in Table 1. Cellic CTec2, a kind of cellulase complex for degradation of cellulose to fermentable sugars, was provided by Novozymes A/S (Bagsværd, Denmark). It is a blend of aggressive cellulases, a high level of β-glucosidases, and hemicellulose.19 Cellulase activity in terms of “filter paper unit” (FPU) and protein content of CTec2 was 200 FPU mL−1 and 155.6 mg mL−1, respectively. All the reagents used were of analytical grade and purchased from Nanjing Chemical Reagent Co., Ltd. of China. Dissolution of Poplar and GL-Poplar in LiCl/DMSO. Vacuumdried wood meal (4 g per bowl) was milled in a planetary ball mill 3838


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ACS Sustainable Chemistry & Engineering The surface chemical composition of poplar and GL-poplar films was quantified using an X-ray photoelectron spectroscopy (XPS) spectrometer (AXIS Ultra DLD, Kratos Analytical, Manchester, U.K.) with monochromated Al Kα X-ray source. To obtain stable ultrahigh vacuum conditions, all samples were pre-evacuated overnight. At least three different points of two parallel samples were analyzed. The atomic concentrations were calculated from the photoelectron peak areas by Gaussian deconvolution. The C 1s spectra were divided into four different contributions of bonded carbon, i.e., C−C and C−H, with a binding energy at 285 eV, C−O at 286.7 eV, O−C−O at 288 eV, and O−CO at 289.5 eV.20,21 Both the O/C atomic ratios and the C 1s high-resolution curve fits were used in chemical analysis. Enzymatic Treatment in QCM. Cellulolytic activity on these films was monitored in situ by a quartz crystal microbalance (QCM-D E4 model, Biolin Corp., Gothenburg, Sweden). The poplar or GL-poplar deposited on the sensors was applied on QCM. Right before starting the measurement, acetate buffer (pH 4.8) was introduced into the measuring chambers by a peristaltic pump at a flow rate of 0.1 mL min−1. After the unit was filled with the buffer solution and the frequency of vibration was monitored continuously until a stable signal was reached. Thereafter, cellulase solution (protein concentration 0.1 mg mL−1) was injected at a same flow rate of 0.1 mL min−1, and then the flow was stopped until reaching stable conditions (indicating a complete adsorption and/or hydrolysis). Buffer solution was introduced again to rinse the system after plateau signal. Frequency (Δf) and dissipation changes (ΔD) for the fundamental frequency (5.0 MHz) and its overtones (n = 3, 5, 7, 9, 11, and 13) were monitored simultaneously. However, only the third overtone (n = 3) was used in the data evaluation. The temperature was maintained at 40 °C in all experiments, and each condition was tested at least three times. The film treated by cellulase in QCM was performed post-treatment by CTec2 under the same condition in QCM. Conventional Enzymatic Hydrolysis. Poplar and GL-poplar dissolved in LiCl/DMSO were regenerated in deionized water. The regenerated materials were washed with deionized water several times by centrifugation until no Cl− was detected in the centrifugate. The main chemical components of these two samples are given in Table 1. Enzymatic hydrolysis and the determination of monomeric sugars were performed following the method described by Wang et al.15 Cellic CTec2 was used for enzymatic hydrolysis, and the enzyme loading was 10 FPU per gram of cellulose in the substrate based on cellulase activity. Analytical Methods. Cellulase activity of cellulase CTec2 was determined by filter paper method using Whatman No. 1 filter paper as a standard substrate.22 The enzyme protein content was determined using the Bradford method.23 Lignin and sugar contents of the samples were analyzed using the NREL protocol.24 Acid-soluble lignin (ASL) was measured by absorbance at 205 nm in a UV−vis spectrometer (TU-1810, Beijing Puxi, China) and 110 L g−1 cm−1 as absorptivity value was used. The monomeric sugars were quantitatively measured with a high-performance liquid chromatography (HPLC, Agilent 1200 Series, Santa Clara, CA) equipped with the refractive index detector (RID). The HPLC analysis was carried out using a Bio Rad Aminex HPX-87H 20n exclusion column (300 × 7.8 mm, Bio-Rad Laboratories, Hercules, CA) with a Cation-H Refill Cartridge guard column (30 × 4.6 mm, Bio-Rad Laboratories, Hercules, CA). Data of glucose and xylose contents were corrected to anhydro units, i.e., glucan and xylan. Each data point was the average of duplicate experiments.

example, Hoeger et al.11 dissolved trimethylsilyl cellulose (TMSC) and lignin acetate (LAc) in chloroform to form a precursor bicomponent film with intermixed phase domains, then converting TMSC to cellulose and LAc to lignin. Compared to actual interactions between cellulose and lignin in the cell wall, these bicomponent films are lacking lignin− carbohydrate complexes have but weak physical interactions. Therefore, it is of great importance to develop a new method to prepare ultrathin films using “whole” lignocellulosic materials without component isolation and derivatization instead of using “assembled” ones. Wang et al.14 first reported that ball-milled lignocellulosic materials, including hardwood (beech) and softwood (spruce), can be completely dissolved in 6 wt % LiCl/DMSO solvent. Similar results with wheat straw and rice straw were obtained using 8 wt % LiCl/DMSO as solvent.15,16 In this work, 8 wt % LiCl/DMSO was used as solvent to dissolve ball-milled poplar and GL-pretreated poplar. The obtained solutions were used to prepare ultrathin films on the surface of QCM gold sensors by spin coating, as described earlier. The most important innovation of this approach, compared with the reported methods, is that these novel films consist of all cell wall components including lignin, cellulose, and hemicellulose. The chemical structure of the cell wall components, as well as the covalent bonds and physical interactions between them at the molecular level, could be fairly well kept in the prepared ultrathin films. In addition, isolation and derivatization of cell wall components is omitted during the preparation of lignocellulosic ultrathin film, avoiding the use of chemicals which may be toxic or harmful to health and environment. Such a novel system can also be used in combination with other surface sensitive platforms including AFM, XPS, ellipsometry, and so on. Surface Characterization of Poplar and GL-Poplar Thin Film. Spin-coating a film of a single polymer generally produces a smooth, unstructured thin film, but if a mixture of polymers is used then the two polymers will usually phase separate. Phase separation is a complex nonequilibrium process, and the outcome is very sensitive to the solvent used and the spinning conditions.26 The topographic images of poplar and GL-poplar films observed by AFM are presented in Figure 2. It is apparent from the AFM images that there was no phase separation for the preparation of lignocellulosic film using LiCl/ DMSO as solvent. Poplar and GL-poplar were dissolved in LiCl/DMSO in the form of very fine complexes, which contained lignin and wood polysaccharides. Therefore, unlike the mixed polymers, the natural interactions between lignin, hemicellulose, and cellulose were well-kept. Since these complexes were not a mixture of isolated wood components, they were much more miscible than the individual polymers. As a result, phase separation was avoided in the process of spincoating. The RMS’s of poplar and GL-poplar films were 3−5 nm and 6−8 nm, respectively. These were slightly higher than those of pure cellulose film (2−3 nm),27 pure lignin film (≤1 nm), and lignin/cellulose bicomponent film (1−2 nm).10,11 This should be mainly caused by their different concentrations and chemical compositions, as well as different spin-coating conditions (speed and time). However, it should be highlighted that these obtained films consist of all cell wall components as no component isolation and derivatization were carried out during the process. The surface of poplar and GL-poplar ultrathin films were analyzed by XPS. The atom oxygen/carbon (O/C) ratio and

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RESULTS AND DISCUSSION Preparation of Ultrathin Films Using LiCl/DMSODissolved Lignocellulosic Biomass. QCM has been used to constantly monitor the adsorption and desorption of enzyme on the surface of lignin and cellulose in situ and in real time.9,25 However, due to the different dissolvability of lignin and polysaccharides, most studies have focused on monocomponent films prepared by isolated lignin or cellulose and bicomponent films prepared with their derivatives. For 3839


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than the true ones. Therefore, the O/C ratios of poplar and GL-poplar films were smaller than the true ones.34 Cellulolytic Reactions of Poplar and GL-Poplar Ultrathin Films Monitored by QCM. The enzymatic binding and hydrolysis of these films were monitored by following QCM frequency shifts (Δf). The relationship between Δf and Δm (the change of mass) was illustrated by the viscoelastic model.35 Lignin and cellulose films were applied to QCM analysis successfully.36 Using lignocellulosic films as a sensing element, the knowledge of enzymatic hydrolysis can be expanded. The prepared ultrathin lignocellulosic film was applied to QCM analysis for monitoring its enzymatic hydrolysis process. Figure 4 shows the binding and catalytic activities of cellulases on poplar and GL-poplar films. The negative shift of QCM frequency was related to the amount of adsorbed enzyme. After enzymatic hydrolysis, the system was rinsed with buffer solution, and the effective contribution of enzyme treatment was discerned with the final values of frequency. As shown in Figure 4, the whole process of the enzymatic hydrolysis can be clearly divided into three stages: cellulase adsorption on substrate (SI), fast substrate hydrolysis (SII) and slow substrate hydrolysis (SIII).37 SI is the first decrease in Δf 3 and the increase in ΔD, caused by cellulase adsorption on the films. Subsequent SII is the stage that the hydrolysis of the film becomes prominent, with a rapid increase of Δf 3 and ΔD. When ΔDmax occurs, the structure of the film is the most exposed, and the accessibility of cellulase to the film is the largest; thus, enzymatic hydrolysis rate reaches the maximum. After the ΔDmax, ΔD begins to decrease and the increase rate Δf 3 slows down gradually until reaching a plateau,37 which is the final stage, slow substrate hydrolysis (SIII). At this stage, the substrate is hydrolyzed by cellulase at a very slow rate. The QCM frequency changes during enzymatic binding and hydrolysis was fitted with an empirical model.25 The frequency values corresponding to film swelling and signal stabilization were taken before the baseline (starting at 0 min), and the modeling was applied to the time range after enzyme binding was detected (upon enzyme injection). Enzymatic binding and hydrolysis take place concurrently as soon as enzymatic activity is initiated, so the model presented here involves two different equations, the Lagergren kinetic equation (eq 1) for binding and Boltzmann-sigmoidal equation (eq 2) for cellulose hydrolysis. The fitted parameters include the binding rate (k) and maximum binding capacity (Mmax) as well as the parameters related to the hydrolysis of the substrate, namely, the frequency at which hydrolysis ceases (B), the time required to reach (A + B)/2(V50), and the hydrolysis rate (1/c).37

Figure 2. AFM 5 × 5 μm2 height images of poplar thin film (left) and GL-poplar thin film (right) after spin-coating from solution on gold sensors.

the concentration of C−C groups were calculated from the respective XPS intensities.21 The O/C ratio of cellulose and lignin is different. The theoretical O/C value is 0.83 for cellulose,28 0.34 for milled wood lignin,29 and 0.26 for organosolv lignin.30 With respect to the concentration of C− C groups, pure cellulose does not have a C−C signal theoretically. However, there is always a small contribution because of sample contamination.31 The C−C contributions of milled wood and organosolv lignin are 49 and 55%, respectively.30,32 Correlating the O/C value and the concentration of C−C groups in lignocellulose samples can get more reliable data.33 The O/C ratios of poplar and GL-poplar films was 0.64 and 0.68, while the atom carbon contents were 60.9 and 59.6%, respectively (Table 2). As calculated from Figure 3, Table 2. Atom Content and the O/C Ratio of Lignocellulosic Films

poplar film GL-poplar film

element

atom content (%)

peak area

C 1s O 1s C 1s O 1s

60.9 39.0 59.6 40.3

127894 216303 148984 266194

O/C 0.64 0.68

Δf = M max (1 − e−kt )

the percentages of C−C bonds in poplar and GL-poplar films were 29.0 and 23.8%. The lignin-to-cellulose (lig/cel) ratio of poplar was 0.52 (28.8/55.0), and it became 0.37 (23.9/64.6) after GL pretreatment (Table 1). Therefore, the relatively lower C−C % and higher O/C ratio of GL-poplar film compared with those of untreated poplar film is mainly due to the differences in chemical components. However, it is important to consider that the effect of extractives on the XPS spectra. Although the relative amount of extractives is extremely low, they may cause a significant contribution to the XPS spectra. Due to the reprecipitation of extractives during dissolving or washing, they are enriched at the surface. The amount of alkyl (C−C) carbon decreases in the order of extractives > lignin > carbohydrates; the C−C amount of poplar and GL-poplar films became larger

Δf = A +

(1)

B−A 1 + e(V50− t )/ c

(2)

After treatment by ball-milling and dissolution/regeneration, the differences in enzymatic hydrolysis processes of poplar and GL-poplar are mainly due to the difference of main chemical components. The adsorption of cellulase on substrates was the prerequisite step for enzymatic hydrolysis.38 In the adsorption stage, the negative shift of frequency (Δf) of GL-poplar film (−21.5 Hz) was similar to that of poplar film (−20.3 Hz); however, this could not represent the real adsorption amount due to the enzymatic hydrolysis of substrate. In fact, the adsorption rate of GL-poplar film was faster than poplar film (Table 3). This is mainly because cellulase adsorption on 3840


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Figure 3. XPS spectra for poplar film (A) and GL-poplar film (B). Shown in this figure are the carbon (C 1s) high resolution spectra.

Figure 4. QCM frequency and dissipation changes in the process of enzymatic hydrolysis of poplar (A) and GL-poplar (B) films. Three clear stages are observed in the whole process: cellulase adsorption on substrate (SI), fast substrate hydrolysis (SII), and slow substrate hydrolysis (SIII).

parameters in Table 4: The hydrolysis extent and rate of poplar film were lower than those of GL-poplar film. Cellulase

Table 3. Parameters Describing Enzyme Binding on Lignocellulosic Films Used in QCM GL-poplar film poplar film

−Mmax (Hz)

k (min−1)

R2

21.5 20.3

6.02 3.66

0.97 0.91

Table 4. Parameters Describing Cellulolytic Activity on Lignocellulosic Films Used in QCM GL-poplar film poplar film

cellulose and lignin most likely takes place concurrently, but lignin hinders the binding of cellulase to cellulose.39 The lignin content (30.0%) and the lignin/cellulose ratio of poplar film (0.48) were higher than those of GL-poplar film (24.8%, 0.34); therefore, poplar film exhibited more hindrance to the binding of cellulase to cellulose than GL-poplar film and thus decreased the adsorption rate.39,40 Many factors can affect the enzymatic hydrolysis of lignocellulosic substrates, such as overcrowding of enzymes,41 substrate porosity, and accessibility.42 In this work, a series of steps were taken to prepare ultrathin lignocellulosic films, including ball-milling, dissolving, spin-coating, solvent displacement, and vacuum-drying. Therefore, it is reasonable to assume that the porous structure of poplar and GL-poplar films are similar. The lignin/cellulose values of regenerated poplar and GL-poplar were 0.48 and 0.34, respectively (Table 1). Our previous research indicated that lignins in poplar and GLpoplar are structurally different.43 Lignin is not the only inhibitor of cellulase activity on substrate; however, it becomes a very important factor in the comparison of cellulase behavior on substrates with different lignin content and structure. A faster enzymatic hydrolysis was observed on GL-poplar films. These observations were further corroborated by the quantitative assessment performed and reported by the key

B (Hz)

V50 (min)

1/C (min−1)

R2

175.3 115.2

2.26 5.24

2.50 0.58

0.91 0.97

adsorption on lignin and cellulose is competitive, and the adsorption of lignin is irreversible, which decreases the accessibility of cellulase to cellulose.44,45 The inhibition effect of lignin was more serious for poplar film, mainly due to its higher content and altered structure of lignin. The energy dissipation profiles are also shown in Figure 4. No changes occurred after the introduction of enzyme-free buffer solution. This reveals that the film of cellulose is thin and relatively rigid. When enzyme solution injected, energy dissipation increased rapidly due to the adsorbed enzyme and its coupled water.25 ΔDmax is the most distinguished difference among enzymatic hydrolysis behaviors of poplar and GL-poplar films. ΔDmax values of poplar and GL-poplar films were 10.47 × 106 and 2.34 × 106, respectively. After ΔDmax, ΔD began to decrease. The reduction of ΔD was related to a severe change in the film structure. Cellulase had a little impact on the changes of GL-poplar film structure; however, it showed a distinct influence on the changes of poplar film structure during enzymatic hydrolysis.37 The results illustrate that the enzymatic hydrolysis behavior of poplar film was not the same as that of GL-poplar film. An important reason is that lignin in poplar 3841


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ACS Sustainable Chemistry & Engineering changed in both structure and content after GL pretreatment43 causing the difference in irreversible adsorption of cellulase on lignin. After QCM analysis, the films were dried under vacuum. XPS analysis of the dried films shows that the relative concentrations of N in hydrolyzed poplar and GL-poplar films were 7.3 and 5.8%, respectively. As no N was detected in the films before QCM analysis (Table 2), it verifies that cellulase adsorbed irreversibly on the substrate. Poplar film adsorbed more enzyme than GL-poplar one. As most carbohydrates were hydrolyzed, the difference in the structure of residual lignin should be the most important factor that affects the irreversible adsorption of enzyme. Post-Treatment of Cellulase on Ultrathin Films. From XPS analysis of hydrolyzed poplar and GL-poplar films, the O/ C ratio of hydrolyzed poplar and GL-poplar films were 0.57 and 0.42, and the percentages of C−C bonds were 43.1 and 49.7%, respectively. As the O/C ratios of pure cellulose and lignin were reported as 0.83 and 0.34,28,29 respectively, it can be deduced that some cellulose remained in the hydrolyzed film. To prove the existence of carbohydrates in the hydrolyzed residues, sugar release of regenerated poplar and GL-poplar in conventional enzymatic hydrolysis was also investigated (Table 5). Though

Table 6. Parameters Describing Enzyme Binding on Lignocellulosic Films Used in QCM GL-poplar film poplar film

glucan (%)

xylan (%)

total sugar (%)

77.0 84.1

96.6 98.1

81.0 86.3

k (min−1)

R2

29.1 23.0

0.40 0.19

0.93 0.90

the residual cellulose mainly covered by lignin; therefore, cellulases were very easily adsorbed by lignin in post-treatment with a relatively low rate and high amount.40 This result can be used to explain why enzymatic hydrolysis is slow or even stagnant when a certain amount of cellulose in substrate is hydrolyzed. In post-treatment, the adsorption amount and rate of GL-poplar film were larger and faster than those of poplar film, which may be due to not only different content (more carbohydrates depolymerized in GL-poplar) but also different structure between poplar and GL-poplar lignin.43 However, the mechanism of lignin inhibiting the substrate enzymatic hydrolysis is not completely elucidated so far and needs further investigation. Though the prepared lignocellulosic films were successfully applied on QCM for monitoring the process of the binding and catalytic activities of cellulases on substrates, there still exist some differences in structure between the regenerated film and plant cell walls. For example, the distribution of lignin and the crystal structure of cellulose are altered due to lignocellulose being treated by ball-milling and LiCl/DMSO dissolution/ regeneration. Our attempt focuses on the chemical composition of a “pseudo-wall” surface. However, one needs to be aware that so far there are no better models that are able to mimic the structural complexity of the plant cell wall accurately by its chemistry.

Table 5. Sugar Conversion of Regenerated Poplar and GLPoplar in Conventional Enzymatic Hydrolysis regenerated poplar regenerated GL-poplar

−Mmax (Hz)

most xylan in regenerated solids was hydrolyzed into monomers, the enzymatic conversion of glucan was 77.0 and 84.1%, respectively for regenerated poplar and GL-poplar. The result confirms that cellulose in regenerated samples could not be completely hydrolyzed. To understand the effect of lignin on the enzymatic hydrolysis of substrates in the final stage, posttreatment of the enzymatic treated ultrathin films was carried out, and the process was monitored by QCM. In Figure 5, a clear process of cellulase adsorption on substrates is observed, but little substrate hydrolysis feature is found. The frequency change of post-treatment was also closely fitted Lagergren kinetic equation. The adsorption rate of both films in posttreatment reduces obviously compared with those in enzymatic hydrolysis (Table 6). A reasonable explanation is that most cellulose was depolymerized in enzymatic hydrolysis and that

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CONCLUSIONS Ultrathin lignocellulosic films were prepared using milled lignocellulose dissolved in LiCl/DMSO solvent by spin-coating. Surface characterization of the films proved that a complete and uniform surface coverage was formed. The developed lignocellulosic film was applied on QCM analyses. Real-time measurements of enzymatic binding and hydrolysis were fitted to Lagergren and Boltzmann-sigmoidal kinetic models. Cellulase adsorption on lignin and cellulose is competitive, and lignin hinders the binding of cellulase to cellulose. Meanwhile, lignin inhibits the enzymatic hydrolysis of cellulose through the irreversible adsorption of cellulase to lignin, and

Figure 5. QCM frequency and dissipation changes in the process of post-treatment of enzymatic treated poplar (A) and GL-poplar (B) films. No hydrolysis feature, but only enzyme binding was observed. 3842


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ACS Sustainable Chemistry & Engineering hydrolytic reactions may be completely inhibited in the final stage.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86(25)8542 8163. Fax: +86(25)8542 8689. ORCID

Yongcan Jin: 0000-0002-0748-3629 Funding

This work was supported by National Key Technology Research and Development Program of China (Grant 2015BAD15B09), National Natural Science Foundation of China (Grant 31370571), Specialized Research Fund for the Doctoral Program of Higher Education, China (Grant 20133204110006), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China. Notes

The authors declare no competing financial interest.

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Novel Approach to Prepare Ultrathin Lignocellulosic Film for

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