Article pubs.acs.org/Biomac
Xyloglucan−Cellulose Nanocrystal Multilayered Films: Effect of Film Architecture on Enzymatic Hydrolysis Carole V. Cerclier,† Aurélie Guyomard-Lack,† Fabrice Cousin,‡ Bruno Jean,§ Estelle Bonnin,† Bernard Cathala,*,† and Céline Moreau*,† †
INRA, UR1268 Biopolymères Interactions Assemblages, 44316 Nantes, France Laboratoire Léon Brillouin, CEA-CNRS Saclay, 91191 Gif sur Yvette, France § Centre de Recherche sur les Macromolécules Végétales (CERMAV-CNRS), 38041 Grenoble, France ‡
S Supporting Information *
ABSTRACT: Understanding the hydrolysis process of lignocellulosic substrates remains a challenge in the biotechnology field. We aimed here at investigating the effect of substrate architecture on the enzymatic degradation process using two different multilayered model films composed of cellulose nanocrystals (CNCs) and xyloglucan (XG) chains. They were built by a spin-assisted layer-by-layer (LbL) approach and consisted either of (i) an alternation of CNC and XG layers or of (ii) layers of mixed (CNC/XG) complexes alternated with polycation layers. Neutron reflectivity (NR) was used to determine the architecture and composition of these films and to characterize their swelling in aqueous solution. The films displayed different [XG]/[CNC] ratios and swelling behavior. Enzymatic degradation of films was then performed and investigated by quartz crystal microbalance with dissipation monitoring (QCM-D). We demonstrated that some architectural features of the substrate, such as polysaccharide accessibility, porosity, and cross-links, influenced the enzymatic degradation.
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use of thin films as model substrates.11−16 Thin films offer highly dense and heterogeneous media that can mimic some conditions encountered by enzymes when they hydrolyze solid lignocellulosic substrates. These approaches are also encouraged by the emergence of efficient techniques to monitor degradation at the nanoscale such as quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance.11−16 Recent studies have not only demonstrated that the evaluation of cellulose thin film hydrolysis can be quantitatively achieved, but that QCM-D can provide relevant information about the degradation process as well, especially phenomena related to film surface saturation with enzymes that have an impact on hydrolysis efficiency, also known as crowding effects.17 QCM-D was also used in combination with other techniques to give a detailed description of film structure and enzyme action.18,19 Nevertheless, a major limitation of these pioneering works is that they are restricted to single-component films, which do not reflect either the chemical or the structural complexity of the biomass. For this reason, recent studies have proposed more complex model films that incorporate various lignin/cellulose ratios that
INTRODUCTION Plant cell walls are a major part of the lignocellulosic biomass, which is used in a wide variety of industrial processes in the textile and paper industries, for example, and for bioenergy applications. The first step in the industrial processing of biomass may involve partial or total degradation of the polymeric fraction with a combination of chemical and enzymatic treatments. In the particular case of biorefineries, oligosaccharides and monosaccharides are produced and can be further hydrolyzed or fermented.1,2 However, despite the amount of research undertaken, the understanding of the hydrolysis processes of lignocellulosic substrates is far from being complete. Many parameters have been identified as limiting factors of enzyme action.3−5 For example, the presence of other polymers such as hemicelluloses and lignin has been reported to limit cellulose degradation, but the physical state of the polymers in the cell wall can also be a relevant parameter to be evaluated.6,7 Due to the high architectural and compositional complexity of the cell wall, simplified systems such as reconstructed model systems can be helpful in identifying the role of individual parameters or the effect of the architectural organization. In addition, interlinked phenomena can be disconnected by the specific addition or the removal of one component in model experiments.8−10 In the case of the study of cell wall enzymatic hydrolysis, a recent trend involved the © 2013 American Chemical Society
Received: July 3, 2013 Revised: September 6, 2013 Published: September 9, 2013 3599
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Figure 1. Schematic illustration of film method preparations on solid supports previously coated with an anchoring layer of cationic PAH polymer. Both methods are based on alternate LbL deposition assisted by a spin-coating procedure consisting of 5 min of adsorption time before spinning at 3500 rotations per minute (rpm) for 40 s for each layer deposition from (a) individual CNC dispersion followed by the XG solution, to construct multilayer (CNC-XG)n films, and (b) deposition of mixed (CNC/XG) dispersion followed by the PAH solution, to construct multilayer (PAH− CNC/XG)n films, with n the number of bilayer.
corresponding swelling ratio. The susceptibility of these films to cellulolytic hydrolysis was then probed in situ using the QCM-D technique. Hydrolysis patterns are discussed in light of the structural data extracted from neutron reflectivity data.
induce the modulation of the surface morphology that influences the efficiency of the enzyme.20,21 With a similar goal in mind, we describe here the preparation of two films composed of cellulose nanocrystals (CNCs) and xyloglucan (XG) chains that differ by their architecture. Among its numerous components, the plant cell wall contains a network of load-bearing cellulose microfibrils cross-linked with hemicelluloses. Cellulose microfibrils are composed of parallel and aligned β-1,4-glucan chains and exist in cell walls as highly ordered crystalline zones separated by amorphous zones. In our model system, cellulose nanocrystals resulting from the sulfuric acid hydrolysis of native cellulose and corresponding to the crystalline parts of microfibrils were used. Xyloglucans are hemicelluloses contained in primary cell walls of dicotyledonous plants. The XG backbone is a β-1,4-D-glucan on which 75% of the glucose residues (Glc) are substituted by mono-, di-, or trisaccharide side chains. The first sugar of these side chains is always a α-D-xylopyranose (Xyl) that can carry β-Dgalactopyranose, or α-L-fucopyranose-(1,2)-β-D-galactopyranose disaccharide. XG chains display a high affinity for cellulose, and this strong and irreversible adsorption is thought to occur through concomitant hydrogen bonding and van der Waals forces,22−24 even though the role of hydrophobic interaction should be considered.25 XG, as well as cellulose, can be degraded by the synergistic action of different cellulolytic enzymes that hydrolyze β-1,4-glucan chains to obtain glucose that can be further used in bioconversion technologies. The global purpose of this work is to tentatively establish the relationship between the architecture of two model films and their degradation patterns. Films were built according to two different preparation methods, as shown in Figure 1: the first one consisted of alternating layers of CNC and XG, and the second one was composed of layers of a CNC and XG mixture (CNC/XG) alternated with layers of a cationic polymer, poly(allylamine hydrochloride) (PAH). The organization of the polymeric constituents at the nanoscale was studied using mechanical profilometry combined with neutron reflectivity (NR) measurements. In particular, NR experiments were carried out on films containing either hydrogenated or deuterated CNCs to extract the internal composition of the films whenever possible. Further studies on films in contact with an aqueous solution give the
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EXPERIMENTAL SECTION
Materials. Poly(allylamine hydrochloride) (PAH; average Mw = 70000 g mol−1) was provided by Sigma-Aldrich and was used without further purification to prepare a 0.5 g L−1 aqueous solution. XGs from Tamarindus indica were provided by Dainippon Pharmaceutical Corporation (Osaka, Japan) and purified according to Gidley’s method.26 The weight-average and number-average molar masses of XGs, determined by size exclusion chromatography coupled with laser light scattering, were Mw = 670000 g mol−1 and Mn = 394000 g mol−1, respectively. The gyration radius was 51 nm. All solutions were prepared with Milli-Q water and filtered prior to use. Deuteration of Cellulose. First, cotton cellulose from Whatman paper (20 CHR) was deuterated using the hydrothermal intracrystalline deuteration method described by Nishiyama et al.27 Whatman paper (6 g) were immersed in a 0.1 M NaOD/D2O solution and placed in an autoclave for 2 h at 210 °C, resulting in the replacement of intracrystalline OHs by OD groups. The occurrence of this deuteration was checked by Fourier transform infrared spectroscopy because OH and OD stretching bands are separated by approximately 1000 cm−1. Fourier transform infrared (FTIR) spectra from CNC samples before and after deuteration of cellulose are shown in Figure S1 in the Supporting Information. Preparation of Cellulose Nanocrystals. Deuterated and protonated celluloses were then hydrolyzed following a protocol described in the literature28 and inspired by Revol et al.29 Briefly, hydrolysis of cotton cellulose was carried out with 50% (w/w) sulfuric acid at 70 °C for 40 min with constant stirring. The hydrolysis reaction was then stopped by diluting the suspension 10-fold with water before performing washing steps by centrifugation, dialysis against water, and finally, placing the suspension on a mixed-bed resin (Sigma, TMD-8). The concentration of the resulting suspensions was approximately 1% w/w. Sodium azide (0.01%) was added to the suspension to prevent microbial spoilage. The length of the nanocrystals was determined by the analysis of transmission electron microscopy micrographs using Image J software. The number average length (Ln), length average length (Ll), and length polydispersity index (PL) resulting from the analysis of approximately 400 nanoparticles were Ln = 215 nm, Ll = 243 nm, and PL = 1.13, respectively. The CNC surface charge was determined by conductometric titration with 0.01 mM NaOH, and was equal to 0.04 e nm−2. Enzymatic Solutions. A commercial powder of cellulase mixture, Cellulyve, was provided by Lyven (Colombelles, France) and used to 3600
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prepare the enzymatic solution at 5 mg mL−1 in 10 mM acetate buffer, pH 5. This commercial enzyme mixture obtained from Trichoderma reesei fungus consists of 1,4-β-endo-D-glucanase, cellobiohydrolase and β-glucosidase. The enzyme mixture had a maximum activity in mild acidic conditions (pH ∼ 5). The indications given by the manufacturer are 49 mg protein per g of powder and an activity of 4880 nkat g−1 on Whatman CC31 cellulose at 50 °C and pH 4.8, 1 nkat being defined as the amount of enzyme required to release 1 nmol of reducing groups per second under standard assay conditions. The enzymatic activity was assayed toward the substrates used in this study, namely, XG and CNCs, prepared at 1 and 5 mg mL−1 in the same buffer, respectively. The assay was performed by incubating the enzyme solution with the substrate solution at 20 °C for 10 min. The release of reducing sugars was measured by the method of Nelson30 using glucose as the standard. To be used as a negative control, the enzyme solution was inactivated by heating in a water bath at 100 °C for 5 min. The activity toward XG was 1.2 nkat mL−1, whereas no activity was detected in these conditions on CNC dispersion. Experimental conditions (temperature, time of hydrolysis and concentrations) of enzymatic assay were guided by QCM-D results (see Results). Film Preparation. Multilayered films were assembled on polished circular disks of silicon ⟨100⟩ wafers (diameter of 5 cm) obtained from ACM (Villiers Saint Frederic, France). The wafers were first cleaned in a piranha solution for 30 min and then repeatedly rinsed with deionized water. The samples were prepared immediately after support cleaning. Two types of films (Figure 1) were built with a prototype spin-coater: the first one consisted of alternating layers of CNCs and XG, and the second one was composed of alternating layers of a CNC and XG mixture (CNC/XG) and PAH. For the first type, 2 mL of a CNC suspension (5 g L−1) was poured onto a stationary wafer previously coated with PAH, which was then accelerated after 5 min of adsorption and spun at 3600 rotations/min (rpm) for 40 s. The film was then rinsed with 2 mL of deionized water and spun. A quantity of 2 mL of XG solution (1 g L−1, semidilute regime28) was poured on the cellulose layer, spun, and rinsed, following the same procedure. A bilayer is defined as a single CNC deposition step followed by a XG deposition step and includes the rinsing steps. Films composed of alternating layers of CNC/XG and PAH (0.5 g L−1) were built in the same manner using a mixed CNC/XG aqueous suspension. To obtain the CNC/XG mixture, CNC dispersion and XG solution were mixed at final concentrations of 5 g L−1 for CNCs and 1 g L−1 for XG, and sonicated for 3 min before use. The films are referred to as (CNC-XG)n and (PAH-CNC/XG)n, respectively, where n is the number of bilayers. Both films were built with either protonated (CNCh) or deuterated (CNCd) cellulose nanocrystals for neutron reflectivity measurements. Mechanical Profilometry. Film thickness was obtained by measuring the groove depth on a surface scratched with a razor blade using a stylus-based mechanical profilometer (Dektak 8, Veeco). The scratched surfaces were scanned along multiple straight lines using a 5 μm radius hemispherical tip carbide stylus and a contact force of 1 mg. Thickness values are averages of eight measurements. Neutron Reflectivity. Specular neutron reflectivity experiments were carried out at the Orphée reactor on the EROS time-of-flight reflectometer at the Laboratoire Léon Brillouin (CEA Saclay, France).31 A broad momentum transfer (Q) range from 0.008 to 0.1 Å−1 was obtained by collecting data at two fixed angles with a neutron white beam covering wavelengths from 3 to 25 Å. For the air/solid geometry, the angles were 0.93 and 2.2°. For measurements in solution, silicon wafers coated with multilayered films were placed in a liquid−solid cell. For this geometry, the angles chosen were 1.34 and 2.5° for the D2O/solid interface. In air/solid geometry, the incident neutron beam goes through the film side to the interface, whereas in liquid/solid geometry, it goes through the silicon side. Details on characteristics and performance of both of these geometries can be found in Cousin et al.31 A standard treatment was applied to the raw data to obtain reflectivity curves on an absolute scale. The data were then analyzed using a “box” model, which consisted in dividing the thin film into a series of layers. Each layer was characterized by a finite thickness (d), scattering length density (SLD), and interfacial
roughness (σ) with the neighboring layer. Details about the fitting procedure, the SLD profile determination, and calculations of the volume fractions of polysaccharides and air or D2O content, as well as surface concentrations, can be found in the Supporting Information. QCM-D Experiments. Quartz crystal microbalance with dissipation monitoring (E4 instrument; Q-Sense AB, Sweden) was used to study the swelling of thin films deposited on sensors (AT-cut goldcoated quartz crystals) and their degradation by a mixture of cellulases. The crystal was excited to oscillate at its fundamental resonance frequency (f 0 = 5 MHz) through a voltage applied across the electrodes. Any material adsorbed on the crystal surface induces a decrease of the resonance frequency (Δf). If the adsorbed mass is evenly distributed, rigidly attached, and small compared to the mass of the crystal, Δf is directly proportional to the adsorbed mass (Δm) using the Sauerbrey equation:32 Δm = − C
Δf n
(1)
where C (= 0.177 mg m−2 Hz1− at f 0 = 5 MHz) is the constant for the mass sensitivity of the quartz crystal and n is the overtone number. Strictly speaking, the Sauerbrey relation is not valid in liquids, especially for adsorbed polysaccharides for which the energy dissipated to the environment is greatly increased due to coupled water. Thus, the dissipation factor, D, simultaneously recorded with the resonant frequency of the crystal, is a measure of the frictional losses due to the viscoelastic properties of the adsorbed layer. In this way, the relative stiffness or conformation of the adsorbed layer can be evaluated. High dissipation values reflect a thick and loose adsorbed layer, whereas a thin and rigid layer that vibrates with the crystal is indicated by a low dissipation factor. Before use, gold-coated quartz crystals ( f 0 = 5 MHz) were cleaned in a piranha solution (∼30−60 s), followed by Plasma Cleaner treatment for 20 min. (PAH−CNC/XG)2 and (CNC-XG)4 films were spin-coated onto these quartz crystals, as described in Film Preparation and subsequently mounted in the QCM-D modules. The QCM liquid chamber was temperature-controlled to 20 ± 0.1 °C during the measurements. Experiments were conducted in flow mode, that is, the acetate buffer and enzymatic solutions continuously circulated into the QCM cell modules at a flow rate of 0.1 μL min−1. Before each measurement, the films were swollen in a buffer solution (10 mM acetate, pH 5) until equilibrium was reached, that is, when the QCM frequency baseline was stabilized (changes in Δf n/n < 0.03 Hz min−1, and in ΔDn/n < 0.01 min−1) after 2 h of swelling. Enzymatic solutions were then injected and changes in the frequency, Δf, and dissipation, ΔD, signals were simultaneously recorded at five frequencies (5, 15, 25, 35, and 45 MHz, corresponding to n = 3rd, 5th, 7th, and 9th harmonic) as a function of time (min) for approximately 25 min, after which the enzyme solution was replaced by acetate buffer for about 1 h. The third overtone, Δf 3 (15 MHz), was chosen for data processing.
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RESULTS Architecture of Multilayered Films. Multilayered films based on CNCs and XG were designed according to two preparation methods (Figure 1). Concentrations were kept constant for (CNC-XG) and (PAH-CNC/XG) films (5 g L−1 CNC dispersion, 1 g L−1 XG, and 0.5 g L−1 PAH solutions). The thickness values of both films measured by mechanical profilometry as a function of the number of bilayers n deposited, with 1−4 deposition cycles, are shown in Table S1 (Supporting Information). On the basis of these values, film growths are linear for both types of films but proceed with unambiguously different construction processes, as shown by the different estimated thickness increment per bilayer (i.e., 17 nm for (CNC-XG) vs 31 nm for (PAH-CNC/XG) films). The first method takes advantage of hydrogen bonds and van der Waals forces that exist between XG and cellulose nanocrystals.23,24 Alternate deposition of CNC and XG layers 3601
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(CNC-XG) was done using the well-known LbL technique33 that had already been used in recent works.28,34−38 As indicated in Table S1, spin-coated film showed a linear growth with an increment of approximately 16 nm per bilayer, as previously reported.28 With respect to the average thickness of cotton nanocrystals (i.e., ∼6−7 nm),39,40 it can be concluded that each deposition cycle results in the adsorption of two monolayers of CNCs, separated by a thin layer of XG. When dipping was used as a preparation method, single CNC layers (7−8 nm thick) were adsorbed.37 This finding is similar to that of our recently published results where such variations in slope growth in the case of CNC/polycation systems can be tuned by the polyelectrolyte conformation.41 In the case of XG, a neutral polymer, the basic physicochemical process responsible for these changes is probably linked to the modification of interaction parameters between cellulose nanocrystals and to the deposition method (i.e., spin assisted layer-by-layer). This point will be detailed in a future paper. The second method to build nanostructured films is less common. (PAH-CNC/XG) multilayered films were built by the alternate spin-coating assisted deposition of mixed CNC/ XG complexes and polycation chains (PAH). In the mixed CNC/XG suspension, CNC and XG concentrations were the same as the ones used for the first LbL method (i.e., CCNC = 5 g L−1 and CXG = 1 g L−1). After the formation of a first CNC/XG layer by spin-coating, the subsequent deposition of CNC/XG mixture was only possible if a new layer of PAH was added on top of the previously spin-coated CNC/XG layer. Without deposition of a PAH layer, the thickness of the film did not evolve linearly and reached a plateau value after two or three depositions. This feature suggests that (i) some negative charges arising from sulfate ester groups on the CNC surfaces are still available for the cationic polymer to be adsorbed via electrostatic interactions, despite the probable decoration of CNCs with XG chains, and (ii) a positively charged intermediate layer is required to build the second layer of CNC/XG. After adsorption of the PAH layer, CNC/XG deposition was again possible, as indicated by the thickness values shown in Table S1. Thus, this atypical design seems to involve both the cellulose−xyloglucan specific interactions that maintain the cohesion between CNCs and XG, as well as electrostatic interactions between PAH and CNCs. The average increment of thickness per deposited bilayer was approximately 31 nm (Table S1), which is nearly 2-fold higher than the one obtained with the first system (17 nm). On the basis of these results, (CNC-XG)n with n = 4 bilayers and [PAH-(CNC/XG)n] with n = 2 bilayers of (CN/XG) and one layer of PAH between, were selected to be studied by neutron reflectivity and QCM-D. They in fact present similar thickness values, that is, 65.5 and 57 nm for (CNC-XG)4 and (PAH-CNC/XG)2, respectively, and are thick enough to eliminate possible substrate effects and fall in the appropriate thickness range for neutron reflectivity investigations. Internal Structure and Composition of Multilayered Films Probed by Neutron Reflectivity. Neutron reflectivity (NR) is able to provide information about the internal structure and polymer composition of thin films adsorbed onto solid supports. It makes it possible to extract the variation of the neutron refractive index of the film along the z-direction normal to the reflecting surface. Fitting the experimental spectra with those that have already been calculated34 makes it possible to evaluate the average thicknesses (d), scattering length densities (SLD), and roughness (σ) of the films. The
whole NR procedure is detailed in the Supporting Information. In this work, NR data from both films will be used to (i) determine, whenever possible, the mean composition of (CNCXG)4 and (PAH-CNC/XG)2 films, by variation of the isotopic contrast using hydrogenated (CNCh) or deuterated (CNCd) cellulose nanocrystals; and (ii) evaluate the swelling behavior of both types of films by measuring their thicknesses in air and in deuterated water. Neutrons are scattered differently depending on the isotopic composition of the sample. Native CNCs and XG display quite similar SLD values (ρ) that make it difficult to distinguish between them (Table S2, Supporting Information). To eliminate this limitation, two types of films were constructed using either native or deuterated CNCs. NR data from these two types of films that share similar structural features but different SLDs make it possible to extract the volume fraction (ϕ) of each component in the film that will be used to analyze the degradation patterns of both architectures by enzymes. Neutron reflectivity spectra and scattering length density profiles for both film architectures, incorporating either CNCd or CNCh, were recorded at the film/air interface (Figure 2). Reflectivity curves display well-pronounced fringes due to constructive and destructive interferences (Figure 2). These fringes, also referred to as Kiessig fringes, are typical of a sharp interface characterized by low roughness between two media with different SLDs, that is, the support/film and the film/air interfaces. Experimental spectra were fitted with calculated curves, assuming that a film consisted of multiple layers and given the calculated scattering length densities of each pure component: 1.9 × 10−6, 3.3 × 10−6, and 1.6 × 10−6 Å−2 for CNCh, CNCd, and XG, respectively. The fitting procedure of neutron reflectivity data is detailed in the Supporting Information with fitting results given in Tables S3 and S4. On the basis of this investigation, the volume fraction of each component in the film and, consequently, individual concentration surfaces of CNC and XG were determined for both film architectures. Figure 2 insets reveal the SLD profiles obtained from the best fits of the measured reflectivity curves (expanded graphs are presented in Figure S2, Supporting Information). Table S5 in the Supporting Information presents the average NR parameters extracted from each SLD profile, that is, average thickness of dry films (ddry) with their respective SLD (ρfilm) and interfacial roughness (σ) values. Thicknesses measured by NR on both hydrogenated (ddry H) and deuterated (ddry D) dry films are in good agreement with those measured by mechanical profilometry (Table S1), confirming the reliability of the results and suggesting that the replacement of CNCh by CNCd has very little effect on the film structure. Assuming that deuterated and protonated films display the same structure, NR parameters extracted from reflectivity curves make it possible to determine the chemical composition of each film (Table S5). Volume fractions (ϕ) of CNC, XG, and air were calculated from the average measured SLD of the films (ρfilm) consisting of XG and either CNCh or CNCd. By combining the volume fraction (ϕ), the thickness (d) of the film and assuming a density of 1400 kg m−3 for XG and 1550 kg m−3 for CNCs, it is thus possible to determine the surface concentration of each polymer. It should be mentioned that the contribution of PAH layer between the two (CNC/XG) layers (see Figure 1b) to the calculation of the volume fractions of polysaccharides has no significant impact (calculation details are given in the Supporting Information, Table S5). However, to make the 3602
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(CNC-XG)4 and (PAH-CNC/XG)2 samples, the relative volume fraction of polymers in both films varied, emphasizing the role of the preparation method (i.e., alternated deposition of CNC and XG layers or deposition of CNC/XG complexes). XG and CNC mass concentrations are roughly identical in the (CNC-XG)4 film ([XG]/[CNC] = 0.89), whereas in the (PAH−CNC/XG)2 film, the XG concentration is approximately twice as high as that of the CNCs ([XG]/[CNC] = 2.15). However, XG concentrations in both films are within the same range: 3.75 μg cm−2 for (CNC-XG)4 versus 4.69 μg cm−2 for (PAH-CNC/XG)2. If we assume that the coverage of the cellulose surface is maximum (i.e., the cellulose surface is fully covered by XG), then a higher proportion of the XG chains would be adsorbed onto the CNC surface in the (CNC-XG)4 system compared to the (PAH-CNC/XG)2 system. It is noteworthy that the methods used to produce the films involve strikingly different organizations. In one case, the sequential deposition of XG and CNCs is related to conventional surface adsorption processes that induce a low [XG]/[CNC] ratio. On the contrary, for the (PAH-CNC/XG)2 film, XG adsorption occurs in the bulk prior to the spin-coating step and leads to a higher [XG]/[CNC] ratio after deposition. It is likely that either the length of the XG moieties adsorbed on the CNCs are longer in the case of (CNC-XG)4 than in the case of (PAHCNC/XG)2, or the trains adsorbed on the cellulose surface are of similar length for both preparations but are more numerous for the (CNC-XG)4 film. Accordingly, in both cases, the distance between the adsorbed segments of the XG is longer in the (PAH-CNC/XG)2 film than in the (CNC-XG)4 film. This point will be further discussed in a future article dealing with the tethered model described in the literature.42,43 Nevertheless, in the context of enzymatic degradation, this point has to be emphasized. XG is much more sensitive to enzyme hydrolysis than crystalline CNCs, at least at the time scale of the QCM-D experiment and in our experimental conditions. It can therefore be assumed that the nonadsorbed tails or loops of XG chains are more easily accessible for enzyme hydrolysis than the polymer parts adsorbed on the cellulose surface. Consequently, the amount of unbound XG can be a critical criterion for the understanding of enzyme degradation. To summarize, bound XG can be estimated from the cellulose content, allowing the calculation of the cellulose surface chains that interact in a 1/1 ratio with XG. This estimation will provide the amount of bound and unbound XG fraction since the total amount of XG is known from NR data. The minimum amount of nonadsorbed chains, that is, the minimum accessible XG fraction, can be established by assuming that XG chains cover the entire surface of the cellulose nanocrystals. Knowing the geometrical dimensions of the CNCs (length, 189 nm; width, 13 nm; and thickness, 6 nm40), the ratio of cellulose surface chains to the total number of chains can be estimated at 0.25. The minimum amount of XG nonadsorbed to the CNC surface can thus be estimated by the following formula: [XG]nonadsorbed = [XG]total − ([CNC] × 0.25). As a result, the nonadsorbed fraction of XG can be estimated at approximately 2.70 μg cm−2 ([XG]adsorbed = 1.05) for the (CNC-XG)4 system, and at 4.14 μg cm−2 ([XG]adsorbed = 0.54) for the (PAH-CNC/ XG)2 system. These results will be useful for the comparison of enzyme accessibility in the film and during the degradation process. Swelling Behavior of the Films. Swelling of films that incorporate deuterated CNCs was studied by NR after immersion in heavy water. Figure 3 presents the resulting
Figure 2. Reflectivity curves (symbols) and best fits (lines) for (CNCXG)4 and (PAH-CNC/XG)2 films prepared from hydrogenated (CNCh; a) and from deuterated (CNCd; b) CNCs in the dry state. The insets show the respective SLD profiles normal to the film surface (expanded graphs of NR profiles are shown in Figure S2A,B in the Supporting Information).
comparison between both films, we chose to present these without taking PAH into account. As suggested from the average SLD of the films, the total polymer content (ϕCNC + ϕXG) is nearly identical in both films since they roughly present the same air volume fraction (ϕ = 0.10−0.14). However, the most important feature is that the ratios between XG and CNC contents are quite different. Although the starting concentration volumes of CNC suspension and XG solution were the same to prepare 3603
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(CNC-XG)4. This can be explained either by a higher amount of nonadsorbed XG that forms longer tethers between CNC or by a less cross-linked CNC/XG network in (PAH-CNC/XG)2. This latter parameter has already been reported to be crucial for hydrogels of carboxymethylcellulose that present lower water uptake when their cross-linking levels increase.47 Results from neutron reflectivity provided a great deal of information about the composition of both thin films. It reveals that they have similar thicknesses and XG contents, whereas the CNC contents differ considerably, leading to distinct architectures that display variations in water uptake and probably cross-linking levels. QCM-D Study of Enzymatic Degradation of Films. Enzymatic degradation of both films (CNC-XG)4 and (PAHCNC/XG)2, was investigated in real-time using the QCM-D technique. Films were spin-coated onto gold-coated quartz crystal surfaces and then allowed to swell in buffer solution in the flow cell for approximately 2 h before injection of the enzyme solution. To facilitate the task, frequency and dissipation signals were offset to zero just before injection of the enzyme solution. Typical changes in frequency and dissipation for the third overtones as a function of time for a quartz crystal covered by (CNC-XG)4 and (PAH-CNC/XG)2 films in the presence of enzyme solution (50, 125, and 208 μg mL−1 at 20 °C) under a continuous flow rate of 100 μL min−1 are shown in Figure 4A−C. As already reported from the hydrolysis of simple cellulosic systems with cellulase cocktails45,48−50 or individual enzymes,17,18,51 three main stages of the degradation process can be distinguished from QCM-D profiles of (PAH-CNC/XG)2 film: (i) A few minutes after injection of the enzyme solution (Figure 4; stage a), the adsorption of cellulases onto the film surface can be experimentally visualized via the occurrence of a frequency decrease for the (PAH-CNC/ XG)2 film. The minimum values of this frequency decrease, Δfads, are about 2 Hz at approximately ∼2−3 min after enzyme injection. (ii) Immediately after enzyme adsorption, the enzymatic degradation process begins (Figure 4; stage b), as reflected by the gradual increase of Δf 3/3 that reveals a mass decrease due to the effective hydrolytic action of the enzymes. Concomitantly, a sharp and rapid increase of ΔD3 up to a maximum, ΔDmax, from ∼3.0−8.9 × 10−6 is observed. This dissipation increase, which occurs at about 3−5 min after enzyme adsorption, is indicative of a more “viscous” structure, at least transiently, induced by the penetration of enzymes into the film and accommodating more and more water/electrolyte molecules, consequently swelling the film structure. (iii) Finally, the degradation of XG by cellulases is the major process (region between b and c in Figure 4), with the resulting products and water/buffer molecules being eliminated through continuous flow as reflected by the gradual mass loss (increase of Δf 3/3) and by the film destructuration (ΔD3 decrease). The film is thus gradually hydrolyzed until plateau values are reached, corresponding either to depletion of XG chains (since CNCs were shown to be resistant to enzyme action in these experimental conditions, especially at ambient temperature and at the time scale of QCM experiments) or to the lack of accessible substrate.
Figure 3. Reflectivity curves (symbols) and best fits (lines) for (CNCXG)4 and (PAH-CNC/XG)2 films exposed to D2O solution. The inset shows the respective SLD profiles normal to the film surface (expanded graphs of NR profiles are shown in Figure S2C in the Supporting Information).
curves and fits, as well as SLD profiles. As expected, upon exposure to D2O, the amplitude of the fringes becomes smeared when compared to the dry state. This observation suggests a D2O uptake and an increase in the roughness of the interface between the film and heavy water. Moreover, it can be qualitatively seen from the closer distance between the minima of the fringes that the thicknesses of both types of films considerably increased. As expected, NR analysis confirms D2O uptake in both films. The thickness of the (CNC-XG)4 film increased from 65 nm in air to 126 nm in D2O, whereas an increasing thickness of approximately 131 nm was found for the (PAH-CNC/XG)2 film (Table S5). A rapid evaluation of the swelling ratio (Sw) of each film from film thicknesses in air and D2O media, as defined by Sw = dswollen/ddry, gives a swelling ratio of 1.9 and 3.4 for (CNC-XG)4 and (PAH-CNC/XG)2 samples, respectively. While the swelling ratio of the CNC-XG sample is comparable to those of amorphous cellulose (approximately 1.6019 or 1.9744), those of the PAH-CNC/XG film is surprisingly much higher. It is assumed that only surface groups (hydroxyl and sulfate) of CNCs interact with water and, as a result of the crystalline nature of the crystal core, water molecules probably do not penetrate inside the structure.45 Kittle et al.46 studied the swelling behavior of regenerated cellulose (RC) and CNC thin films. They concluded that the RC samples were analogous to a gel, whereas the water content of the stiff CNC networks is part of the porosity of the film. It is likely that our films present intermediate behavior because the stiffness of the CNCs creates a porous network that can swell as a result of amorphous and flexible XG chains. The XG/CNC network should behave as an amorphous phase, capable of swelling up to some extent, which is limited by polymer entanglement or cross-linking between CNCs and XG and likely by the presence of the PAH layer. Thus, the difference observed between both architectures can be related to a looser structure in the case of (PAH-CNC/XG)2 than in the case of 3604
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Figure 4. Changes in Δf 3/3 (left axis) and ΔD3 × 10−6 (right axis) signals from (CN-XG)4 (solid lines) and (PAH-CN/XG)2 (dashed lines) films under the continuous flow of enzyme solution at (A) 50, (B) 125, and (C) 208 μg mL−1, corresponding to 0.012, 0.03, and 0.05 nkat mL−1 at 20 °C, respectively, as a function of time. Stages indicated by dashed lines correspond to (a) injection of enzyme solution; (b) adsorption of enzymes onto the film surface (when visible, the small increase in Δf 3/3 is designated as Δfads, and the increase in ΔD3 is designated as ΔDmax) with subsequent hydrolytic processes; and (c) plateau values of frequency (Δf plateau) and dissipation (ΔDplateau) corresponding to the end of the enzymatic degradation process before replacing the continuous flow of enzyme solution with acetate buffer solution.
The hydrolysis process, that is, the gradual increase of Δf 3/3, takes place in less than 8 min after enzyme injection for the three enzyme concentrations used. While comparison with QCM-D data from the literature is difficult due to the various experimental conditions used, lower degradation times (from 5 to 60 min) found in previous works concern amorphous cellulose with an accessible surface area more available to
enzymes than CNCs.13,17,19,45,48,50 In their work, Ahola et al.48 found a higher degradation time of about 300 min for CNC film (at 40 °C), supporting that cellulases mainly hydrolyzed XG chains in our films. The plateau values of Δf 3/3 and ΔD3 remain constant for approximately 10 min under the continuous flow of enzyme solution before replacing it with acetate buffer. During this period, no significant changes in 3605
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Table 1. Slope of Δf 3/3 Increase and Δf 3/3 Values after Degradation and Rinsing Steps (Δf plateau) for (CNC-XG)4 and (PAHCNC/XG)2 Films at Different Enzyme Concentrations slope of Δf 3/3 increase (s−2) −1
a
Δf plateaua (Hz)
cellulase concentration (μg mL )
(CNC-XG)4
(PAH-CNC/XG)2
(CNC-XG)4
(PAH-CNC/XG)2
50 125 208
0.47 ± 0.00 0.72 ± 0.02 0.72 ± 0.04
0.52 ± 0.01 1.35 ± 0.04 1.48 ± 0.01
195.3 ± 4.7 181.3 ± 2.3 194.2 ± 7.8
129.0 ± 2.4 214.9 ± 3.7 164.0 ± 1.2
After buffer rinsing step.
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either Δf plateau and ΔDplateau were observed, indicating that the degradation process is terminated. If enzyme molecules were entrapped in the film and still active, it could be assumed that the degradation process had continued, even after the enzyme flow was stopped. Furthermore, no change in the film viscoelastic properties was induced by the rinsing step. Compared to the (PAH-CNC/XG)2, the QCM-D profile of (CNC-XG)4 displays a distinct feature. In fact, no cellulose adsorption phenomenon is visible from the frequency response before the increase of Δf 3/3 due to the enzymatic degradation process. Nevertheless, a short increase of the frequency (Δf 3/3 = 1.1−4.7 Hz) and dissipation (ΔDmax = 1.0−2.9 × 10−6) signals occurred just before the Δf 3/3 increase, probably due to the adsorption of cellulases onto the film surface, inducing a small and rapid change of the dissipative nature of the film surface. The slopes of frequency increase corresponding to the enzymatic degradation rate (linear part of the Δf 3/3 increase, that is, just after the adsorption step (stage b) and before the plateau value for each profile) were measured for three different cellulase concentrations (50, 125, and 208 μg mL−1), and the results are summarized in Table 1. As can be seen, the slope of the enzymatic degradation is similar for both films at an enzyme concentration of 50 μg mL−1 (approximately 0.5 s−2). However, when increasing the enzyme concentration, the degradation slope strongly increases up to approximately 0.75 s−2 slope for (CNC-XG)4, whereas a 1.4 s−2 for (PAH-CNC/XG)2 film is found. This result indicates that a greater amount of material is degraded (or removed) from the surface for (PAH-CNC/XG)2 at high enzyme concentrations compared to (CNC-XG)4. Increasing enzyme concentration from 125 to 208 μg L−1 does not significantly enhance the hydrolytic rate. The Δf 3/3 value after degradation and rinsing steps was also measured for the three enzyme concentrations (Table 1). Values are rather stable in the case of (CNC-XG)4, suggesting that the amount of degraded materials is almost identical for all three enzyme concentrations tested. In the case of (PAHCNC/XG)2, the Δf 3/3 values are not reproducible. This suggests that either the degradation is not complete or that nonspecific adsorption occurs, probably due to impurities in the enzyme preparation. This might be explained by the presence of the PAH layer between the two CNC/XG layers. PAH is not degraded by cellulase and can be a physical barrier for the degradation, as previously demonstrated in the case of the degradation of amylopectin52 or as an anchor layer for adsorption of impurities in the enzyme preparation. Thus, the presence of remaining materials and the amount of degraded product are not easy to investigate and will be the focus of a future study. As a consequence, the discussion will only focus on the comparison of the degradation slopes that represent the speed of degradation of the accessible substrate.
DISCUSSION In nature, it is assumed that cellulose microfibrils and XG assemble in muro to form a load-bearing network responsible for the mechanical properties of the growing plant cell walls of nongraminaceous land plants. The model usually reported in the literature proposes that XG chains act as tethers between cellulose microfibrils.42,53 The different parts of the XG chains are not all equivalent with respect to their accessibility to enzymes. In fact, only the loops, tails, or cross-links between cellulose microfibrils are accessible, whereas the part of the chains that are either adsorbed on the cellulose surface42,54 or entrapped in the cellulose crystals43 are inaccessible. In the case of the model films reported in this study, only XG adsorption on the CNC surface occurs, and entrapment of XG in the crystalline structure should probably be ruled out. Thus, in order to discuss the differences observed during enzymatic hydrolysis, the adsorbed and nonadsorbed part of the XG chains will be considered to be inaccessible and accessible to enzymes, respectively. Moreover, the XG will be considered as the unique substrate of the enzyme because the CNCs were found to be insensitive to enzyme action at the time scale of the enzymatic hydrolysis run in this study. In our conditions, no enzyme activity was detected at 20 °C on CNC dispersion (at 5 g L−1) during the 10 min period that roughly corresponds to the hydrolysis time of the QCM-D experiments. The results obtained from neutron reflectivity experiments showed contrasted architectures linked to the processing method used for film preparation. In the case of (PAHCNC/XG)2, XG and CNCs were mixed prior to the spincoating step, which yields layers with a [XG]/[CNC] ratio of 2.15. The minimum amount of XG nonadsorbed on the cellulose surface is very high since it represents approximately 88% of the total XG, and XG domains inaccessible to enzymes are very small (less than 12%). In the case of successive deposition of XG and CNCs used to build the (CNC-XG)4 film, the enzyme-accessible fraction is much lower since its minimum amount represents 71% of the total XG, consequently revealing that the adsorbed part is large and may reach up to 29%. The small fraction of adsorbed chains for (PAH-CN/XG)2 compared to (CN-XG)4 may explain the larger swelling because it is likely that the XG tethers that crosslink CNCs are likely longer in the first structure than in the latter. It is thus possible to suppose that the pore size and, as a result, the accessibility of the enzyme to the film is greater in the more swollen structure. The films display different behaviors when they are submitted to enzyme hydrolysis. Given that CNC is insensitive to enzymatic hydrolysis in our experimental conditions, the mass loss observed by QCM-D can be interpreted within the framework of the hydrolysis of the XG cross-links between CNCs alone. Thus, a single hydrolysis event could remove a nanocrystal from the film if its connection was achieved by only one XG chain so that the total 3606
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Figure 5. Simplified pictures of the arrangement of CNCs and XG within the two film systems. The light gray cylinders represent CNCs, whereas XG chains are represented by a black segment; the layers of PAH onto CNC surfaces is described by light gray segments adsorbed on the CNC surface (a) (CNC-XG) bilayer and (b) (CNC/XG; PAH-CNC/XG) bilayer, as inferred from both film architectures, (CNC-XG)4 and (PAH-CNC/ XG)2, respectively. In the first case, a greater amount of XG is adsorbed on the CNC surface, and the tethers are therefore shorter. The second system is looser due to lower adsorption of XG on the CNC surface. The XG chains are more abundant.
(CNC-XG)4 film. This argument can be proposed as an explanation of the differences observed in the degradation rates. Nevertheless, it should be observed that both structures are highly hydrated and that their nanometric dimensions suggest that the diffusion times of the enzyme in the films are fast enough in both cases with respect to the enzyme size. Accordingly, the limitation of the hydrolysis by a porosity effect would occur only in heterogeneous structures if some XG were located in crowded zones due, for example, to the high density of cross-links. However, the homogeneous water content within the structures after swelling (see Figure S2C, Supporting Information) instead suggests a homogeneous structure. Finally, the last parameter that may be invoked to explain the differences in the enzymatic hydrolysis process is the number of XG cross-links between CNCs. As mentioned above, the disruption of the film could result from the cleavage of the XG and not inevitably from the total hydrolysis of the XG chains. Thus, with respect to the low amount of XG adsorbed in the case of (PAH-CNC/XG)2 (12% maximum), it is likely that a single chain of XG cannot cross-link several nanocrystals and cannot be linked to the same CNCs with several trains. On the contrary, in the case of (CNC-XG)4, it is more probable that the same chain can be adsorbed on a single nanocrystal with several trains or connected to several nanocrystals. Thus, disruption of the film would require more hydrolysis events and, accordingly, the degradation rate would be slowed down. A simplified scheme depicting the organization of XG/CNC networks in both films is reported in Figure 5. The picture displays the (CNC-XG)4 (Figure 5a) architecture, where the XG chains are more frequently adsorbed on the CNC surface, resulting in a higher cross-linking density. In the case of the (PAH-CNC/XG)2, the lowest adsorption of XG on CNCs results in a looser and more porous structure that is more rapidly degraded by enzymes (Figure 5b).
hydrolysis of the XG chain is not necessarily required to break the films. The evaluation of the degradation rate on the basis of the measurement of the frequency increase slope could therefore reflects the disruption of the film but not necessarily the total hydrolysis of XG chains. The frequency increase slope was found to be higher in the case of (PAH-CNC/XG)2 than of (CNC-XG)4, especially at the highest enzyme concentrations (Table 1). At least three main parameters may be involved to explain this behavior: (i) the amount of substrate available (i.e., accessible parts of the XG chains); (ii) the porosity of the film (i.e., the accessibility of the substrate); and (iii) the number of cross-links between the CNCs and XG in the films responsible for film cohesion. They are described in more detail below. In the case of (CNC-XG)4, the amount of XG accessible is 2.6 μg cm−2 and represents 71% of the total XG. In the case of (PAH-CNC/XG)2, the accessible XG is 4.1 μg cm−2 (88% of total XG) if the entire layer is considered, or 2.05 μg cm−2 if only the upper layer is taken into account due to the presence of the PAH layer that can act as a barrier. On the basis of this estimation, the amount of substrate to be hydrolyzed for (CNC-XG)4 film is either (i) roughly half of the XG accessible in the (PAH-CNC/XG)2 structure when all the (PAH-CNC/ XG)2 layers are considered, or (ii) in the same range if only the upper layer is degraded. By keeping in mind that the enzyme concentrations used for each hydrolysis experiment are identical for both structures and enzymes are in large excess due to the continuous flow through the cell, two potential events can be hypothesized. First, if the accessibility and crosslink numbers were comparable for both types of films, the disruption of the films in the case of (CNC-XG)4 would be expected to be more rapid since less XG is present (case i) or similar if identical accessible XG is considered in both systems (case ii). However, the reverse effect is observed (Table 1). It can therefore be concluded that the amount of XG available is not the only parameter to be taken into account in the degradation process of the film. The second parameter that can be proposed to account for the description of the enzymatic hydrolysis is the porosity of the hydrated films and, as a result, the associated diffusion of the enzyme. To support this hypothesis, it has been reported that this structural parameter can in some cases be a major factor of cell wall recalcitrance to enzyme action. In the case of the films studied here, an indirect means to approach film porosity is the swelling behavior since dry films have similar thicknesses. Determination of the swelling ratio indicates that (PAH-CNC/XG)2 (Sw = 3.55) swells more than (CNC-XG)4 (Sw = 2.0). This certainly results in a lower porosity of the
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CONCLUSIONS In summary, we have developed two thin films made of xyloglucan and cellulose and investigated their architecture and their enzymatic degradation. Results from neutron reflectivity provided a great deal of information about the composition of both thin films. It reveals that they have similar thicknesses and XG contents, but CNC content differs considerably, leading to two distinct architectures that display variations in water uptake and probably cross-linking levels as well. Our results demonstrated that the architecture of the substrate, in addition to its chemical composition, has an impact on the degradation 3607
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process. We have also shown that the enzymatic degradation process can be used as a tool to investigate polymer organization in the plant cell wall. Comparison with model substrates can provide prospective schemes to improve the use of enzymes in biorefineries.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
S Supporting Information *
Sample data for cellulose nanocrystal films, sample data from neutron reflectivity experiments, including details about the procedure for curve fitting and profile determination, as well as for the determination of polysaccharide volume fractions, and QCM-D profiles of the enzymatic degradation of films. This material is available free of charge via the Internet at http:// pubs.acs.org. Corresponding Author
*E-mail:
[email protected]; celine.moreau@ nantes.inra.fr. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the French National Research Agency (ANR) through the Reflex Program (ANR P2N 2011) for its financial support.
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