Heterogeneity and Specificity of Nanoscale Adhesion Forces

Article pubs.acs.org/Langmuir

Heterogeneity and Specificity of Nanoscale Adhesion Forces Measured between Self-Assembled Monolayers and Lignocellulosic Substrates: A Chemical Force Microscopy Study Baran Arslan,† Xiaohui Ju,‡ Xiao Zhang,‡ and Nehal I. Abu-Lail*,† †

Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington 99164-6515, United States ‡ Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Bioproducts’ Science and Engineering Laboratory, Washington State University, Richland, Washington 99354-1670, United States S Supporting Information *

ABSTRACT: Lack of fundamental understanding of cellulase interactions with different plant cell wall components during cellulose saccharification hinders progress toward achieving an economic production of biofuels from renewable plant biomass. Here, chemical force microscopy (CFM) was utilized to quantify the interactions between two surfaces that model either hydrophilic or hydrophobic functional groups of cellulases and a set of lignocellulosic substrates prepared through Kraft, sulfite, or organosolv pulping with defined chemical composition. The measured forces were then decoupled into specific and nonspecific components using the Poisson statistical approach. Heterogeneities in the distributions of forces as a function of the pretreatment method were mapped. Our results showed that hydrophobic domains and chemical moieties involved in hydrogen bonding and polar interactions were homogeneously distributed on all substrates but with distribution densities that varied with the type of the pretreatment method used to prepare substrates. In addition, we showed that increasing surface lignin coverage increased the heterogeneity of the substrates. When forces were decoupled, our results indicated that xylan reduced the strength of hydrogen bonding between the hydrophilic model surface and substrates. Permanent dipole−dipole interactions dominated the adhesion of the hydrophilic model surface to lignosulfonates, whereas hydrophobic interactions facilitated the adhesion of the hydrophobic model surface to Kraft lignin. We further showed that the structure of lignin determines the type of forces that dominate lignocellulosic interactions with other surfaces. Our findings suggest that nonproductive binding of cellulases to lignocellulosic biomass can be reduced by altering the hydrophobicity and/or chemical moieties involved in the polar interactions and by utilizing organosolv as a pretreatment method.

1. INTRODUCTION

controlled chemical composition and efficient polysaccharide degrading enzymes are needed.5 To investigate the specific effects of individual biomass components on the efficiency of enzymatic hydrolysis of plant cellulose, we have previously developed a set of reference substrates. These reference substrates have a controlled level of physicochemical properties including length, width, cell wall thickness, lumen diameter, cellulose degree of polymerization, etc., with one or few substrate characteristics such as lignin and xylan content selectively altered.6,7 Similar to lack of quality substrates, the lack of highly effective enzymes represents a major bottleneck in the conversion of biomass to biofuels. As such, researchers are aiming at minimizing enzymes’ cost by improving their activity.8 To improve enzymes’ activity,

Lignocellulosic materials with the three major components cellulose, hemicellulose, and ligninare of interest for conversion into fermentable sugars and other value-added chemicals. Deconstruction of lignocellulosic biomass is however limited by the recalcitrance of the plant cell wall.1,2 Biomass recalcitrance can be attributed to various factors including the biomass content, structure, and distribution of hemicellulose and lignin, cellulose crystallinity, and degree of polymerization and substrate accessibility to cellulase.3 In addition, the intricate structure and heterogeneous chemical composition of resulting biomass after pretreatments limits our ability to understand the effects of individual substrate characteristics on enzymatic hydrolysis. Pretreatments aim at reducing the complexity of lignocellulosic biomass and increasing the accessibility of cellulases to the biomass during enzymatic hydrolysis.4 To overcome biomass recalcitrance, homogeneous substrates with © 2015 American Chemical Society

Received: July 16, 2015 Revised: August 31, 2015 Published: September 1, 2015 10233

DOI: 10.1021/acs.langmuir.5b02633 Langmuir 2015, 31, 10233−10245

Article

Langmuir Table 1. Chemical Composition and Surface Lignin Coverage of Reference Substrates6,7 chemical composition (%) reference substratesa KP40 SP40 OPP KP0 SP0 OPP0 a

lignin 5.90 5.90 2.36 N/D N/D N/D

cellulose (as glucan) 77.23 82.87 80.53 82.20 87.41 91.97

xylan

characterization acetone extractives b

15.44 9.21 1.91 16.78 9.65 1.28

N/D N/D 14.54 N/D N/D 3.18

surface lignin coverage (%)

moisture content (%)

45.53 39.23 44.18 N/D N/D N/D

67.47 66.72 69.26 83.22 83.58 84.91

KP and SP substrates were originally characterized in ref 6 while OPP and OPP0 were characterized in ref 7. bN/D = not detected.

nanoscale mapping of the distribution of chemical moieties on surfaces of substrates pretreated differently, the quality of pretreatment technologies and inconsistency in experimental results reported on enzymatic hydrolysis of lignocellulosic biomass in the literature can be evaluated. Third, SAM surfaces that model the hydrophobic and polar components of cellulases were used to elucidate the types of forces that govern possible interactions of cellulases with biomass. Fourth, the Poisson statistical model was applied to the adhesion force data to decouple the contributions of Lifshitz−van der Waals (LW) forces, hydrophobic and polar interactions, and hydrogen bonding on lignocellulosic interactions. Identifying the types and strengths of forces acting between reference substrates and model cellulase surfaces can guide efforts of researchers aiming at designing new enzymes capable of hydrolyzing lignocellulosic biomass effectively. Fifth, the heterogeneity in the chemical properties of the reference substrates was characterized. Obtaining meaningful results and conclusions relies heavily on working with homogeneous substrates. Finally, measurements were carried under liquid media. This is important to avoid the effects of the capillary forces on interactions and to mimic the native environment of the interactions mapped.

fundamental knowledge of the main forces that govern enzymatic interactions with biomass is needed. Interaction forces are largely affected by the surface physiochemical properties of the two interacting surfaces as well as environmental conditions.9 Enzymatic interactions with biomass can be measured using atomic force microscopy (AFM) or its module chemical force microscopy (CFM). The use of AFM to investigate biological interactions is well documented in the literature due to its high lateral, normal, and spatial resolutions and its ability to measure forces in native biological mimicking environments.10,11 When it comes to biomass recalcitrance, AFM and CFM were used to investigate biomass interactions to a variety of surfaces that model enzymes. For example, CFM was used in air to investigate the lignocellulosic interactions with −CH3 and −COOH modified cantilevers to localize cellulose and lignin on pulped fiber surface.12,13 The authors concluded that the −CH3 modified probe was sensitive to lignin, whereas the −COOH modified cantilever was sensitive to cellulose due to high adhesion forces. Although these studies were interesting, they were performed in air where capillary forces mask any possible fundamental interactions between the pulped fiber and the modified cantilevers and as such limit the localization of lignin and cellulose on the surfaces. In another CFM study, an −OH modified cantilever was used to study the interfiber hydrogen bonding in pulped fibers in aqueous media.14 However, due to lack of well-defined homogeneous substrates and detailed analysis on the type of forces governing the interactions, the major mechanism of how lignocelluloses interact with relevant surfaces remains unknown. Other studies in the literature utilized AFM to quantify interactions between a carbohydrate-binding module (CBM) and various plant cell wall surfaces15−17 or adsorbed soluble cellulose and lignin and cellulases.18 These studies are interesting because they are attempting to quantify the direct enzymatic interactions in liquid media. However, mechanistic information about the contributions of different lignocellulosic components to enzymatic interactions is still missing. Here, CFM was utilized to measure the interactions between self-assembled monolayers (SAMs) that model hydrophobicity and polarity of cellulases and a set of reference substrates. In designing our experiments, we have attempted to address issues of biomass recalcitrance at a fundamental level. Our study is unique in six aspects. First, by using reference substrates with well-defined composition, the mechanistic roles of surface lignin coverage, lignin type, and xylan content on enzymatic hydrolysis can be investigated.6 Up to date, the precise mechanism of interactions between individual components of lignocellulosic materials and cellulases remains unknown. Second, three different pretreatment methods (Kraft, organosolv, and sulfite) were used to prepare our substrates. By

2. MATERIALS AND METHODS 2.1. Preparation and Characterization of Reference Lignocellulosic Substrates. All of the reference substrates used in this study were originally prepared and characterized as previously described.6,7 Modified organosolv, sulfite, and Kraft pulping were used to produce reference substrates from poplar. Briefly, organosolv pulping (OPP) substrates were prepared by treating wood chips in 50% (w/w) aqueous ethanol solution with H2SO4 as a catalyst at 170 °C for 60 min. In comparison, sulfite pulping (SP40) substrates were obtained by cooking wood chips in acidic magnesium sulfite solution (5.46% (w/w) SO2, pH 3.8) at 170 °C for 40 min. Finally, Kraft pulping (KP40) substrates were prepared by applying a 16% (w/w) Na2O active alkaline and a 25% (w/w) Na2S solution to poplar wood chips at 170 °C for 37 min. OPP0, SP0, and KP0 are delignified substrates of OPP, SP40, and KP40, respectively. Delignification was performed with an acid chlorite solution at room temperature for 24 h according to a protocol previously described.19 The chemical composition of the reference substrates was previously analyzed via Technical Association of the Pulp and Paper Industry (TAPPI) test methods (T236, T204, T222, T211, and T249)6 (Table 1). Substrate lignin coverage was characterized by X-ray photoelectron spectroscopy (XPS, Physical Equipment Inc., Chanhassen, MN) (Table 1). The reader is referred to ref 6 for the details of XPS analysis of surface lignin coverage. It is important to note that the substrates used in this study were never dried, and their moisture content was kept always above fiber saturation point for all substrates (Table 1). Because of that, our substrates do not swell and their confirmation remains constant during the measurements. 2.2. Sample Preparation for AFM Experiments. Fibers of reference lignocellulosic substrates were attached to silicon wafers coated with poly-L-lysine (PLL) (molecular weight 70 000−150 000, 10234


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Figure 1. Schematic representation of the various methods used in this paper. (a) A simplified schematic representation of a chemically modified AFM cantilever with SAMs of −CH3 or −OH interacting with a substrate surface. Green-dashed line shows a cellulose microfibril, and the whitesolid line shows a globular lignin particle on the substrate surface. (b) A schematic of the AFM cantilever on an AFM 3D micrograph of KP40. (c) Example of an adhesion map generated based on the adhesion events measured between KP40 and −CH3 SAMs. (d) The inset represents retraction curves measured on KP0 and KP40. Each pixel in (c) represents the maximum adhesion force in the retraction curve measured at that pixel. The histograms represent the distribution of absolute values of all adhesion forces measured between −CH3 modified cantilever and KP0 and KP40 in sodium acetate buffer. Gray bars are stacked onto black bars. The red and gray solid lines are the theoretical Poisson distribution fits to adhesion force data of KP0 and KP40, respectively (r2red = 0.99 and r2gray = 0.82). (e) An example of the linear relationship expected between the mean and the variance of the adhesion forces measured between SP0 and −OH modified cantilever. The solid linear regression line was used to calculate the specific and nonspecific force components of adhesion, where the slope is specific forces (Fi) and the minus sign of the intercept is the multiplication of specific and nonspecific forces (−FiF0). Error bars represent the standard error of the mean. r2 for the linear regression is 0.95. Immersion of SAMs in buffer is not expected to affect their structure and group termination based on long-term studies of structure characterization and stability in the literature.20 Contact angle and peak current density measured from cyclic voltammetry of SAMs remained unchanged after immersion of SAMs’ coated strips, particularly SAMs of undecanethiol, into PBS for a day. In addition, immersion of SAM-coated strips into degassed PBS for 35 days did not change the results obtained by XPS and cyclic voltammetry of SAMs’ content.20 A single cantilever was used for each experiment. 2.4. AFM Force Measurements. A PicoForce scanning probe microscope with a Nanoscope IIIa controller (Bruker Inc., Santa Barbara, CA) was utilized in the force−volume (FV) mode for all AFM force measurements. Force measurements were performed under a 50 mM sodium acetate aqueous buffer solution adjusted to a pH of 4.8 under ambient temperature and humidity. The force spring constant of each cantilever was determined prior to force measurements from the power spectral density of the thermal noise fluctuations in buffer.21 On average, the spring constant was found to be 0.123 ± 0.002 N/m (n = 6), very close to the nominal 0.12 N/m spring constant reported by the manufacturer. Deflection sensitivities of the cantilevers were determined on a surface of pure silicon wafer, and then cantilevers were moved to the fiber surface for the collection of force−distance curves (Figure 1b). 32 × 32 array FV and topography images were collected simultaneously over a scan size of 5 × 5 μm2 (Figure 1b). A scan rate of 1.5 Hz with a z-ramp size of 1 μm and a relative trigger of 12 nN were used in all measurements. Three different locations were investigated over two different fibers of each reference substrate. 2.5. Analysis of FV Maps for the Quantification of Adhesion Forces and the Generation of Adhesion Maps. Spatially collected

Sigma-Aldrich, St. Louis, MO). Prior to attachment, silicon wafers were sonicated in deionized (DI) water followed by 95% anhydrous ethanol solution (Ward’s Science, Rochester, NY) for 5 min each. To ensure that the silicon wafers were not contaminated with any organic molecules, the wafers were then immersed into piranha solution (4:1 H2SO4:H2O2) for 45 min and then washed copiously with 95% anhydrous ethanol solution and DI water, successively. Caution: piranha solution should be handled with extreme care due to its high reactivity to organic solvents! Cleaned silicon wafers were then coated with PLL by immersing the wafers into 0.01% PLL solution for 1 h. The wafers were then sonicated in 95% anhydrous ethanol solution to remove excessive PLL and dried in an oven for 30 min at 100 °C. Water suspensions (1 mg/mL) of the fibers were prepared. A 50 μL aliquot of a fiber suspension of interest was added to a PLL-coated silicon substrate and then dried in a vacuumed desiccator overnight. 2.3. Modification of AFM Cantilevers with Self-Assembled Methyl and Hydroxyl Monolayers. Silicon nitride (Si3N4) cantilevers (DNP-10, Bruker Inc., Camarillo, CA) were coated with a 5 nm chromium adhesive layer followed by a 40 nm gold layer using an Auto 306 sputter coating system (base pressure 9 × 10−7 Torr) (Edwards, Sanborn, NY). The gold-coated cantilevers were then cleaned in pure methanol (J.T. Baker) and 95% anhydrous ethanol solution for 15 min each, respectively. Cantilevers were then immersed for 2 h at room temperature into 1 mM ethanolic solution of 1octadecanethiol (CH3(CH2)17SH, Sigma-Aldrich) or 11-mercapto-1undecanol (OH(CH2)11SH, Sigma-Aldrich) for methyl and hydroxyl termination, respectively (Figure 1a). Cantilevers were then rinsed with 95% anhydrous ethanol solution to remove loosely attached functional groups and gently dried in a N2 stream. The hydroxyl- and methyl-terminated cantilevers were used immediately after drying. 10235


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Figure 2. Representative maps of adhesion forces measured between reference substrates and an AFM cantilever decorated with −CH3 SAMs: (a) KP0, (b) SP0, (c) OPP0, (d) KP40, (e) SP40, and (f) OPP. Topography images of corresponding adhesion maps: (g) KP0, (h) SP0, (i) OPP0, (j) KP40, (k) SP40, and (l) OPP. Color bars indicate the z-scale. force−distance retraction curves were processed using the Nanoscope Analysis 1.5 software (Bruker, Camarillo, CA) to determine the number of adhesion events and the magnitudes of adhesion forces measured between the methyl and hydroxyl terminated cantilevers and fibers of a given sample. Each retraction curve was considered individually due to the heterogeneity observed in the measured forces. Absolute values were used for attractive adhesion forces. Adhesion maps were generated using an in-house built MATLAB software by assigning the maximum adhesion force measured in the investigated given force−distance curve to the designated pixel in the FV image (Figure 1c). Histograms of all adhesion forces quantified for a given

substrate are then plotted (Figure 1d). The mean and standard deviation of adhesion forces for each FV experiment were then computed. Statistical tests were used to determine if the median adhesion strength is different among samples investigated. Kruskal− Wallis One Way Analysis of Variance on Ranks with multiple pairwise group comparison procedures (Dunn’s method) available in SigmaPlot 11.0 (Systat Software Inc., San Jose, CA) was applied to the data. 2.6. Heterogeneity Index. Relative chemical heterogeneities of the substrate surfaces were quantified from adhesion force data using a heterogeneity index (HI) that was defined previously by our group.22 A HI was defined as the ratio of the standard deviation (SD) of 10236


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Table 2. Summary of the Adhesion Forces Measured between Reference Substrates and −CH3 Modified and −OH Modified AFM Cantileversa −CH3 KP0 KP40 SP0 SP40 OPP0 OPP

−OH

meanb (nN)

SD (nN)

SEM (nN)

no. of adhesion peaks

meanb (nN)

SD (nN)

SEM (nN)

no. of adhesion peaks

0.295 0.614 0.275 0.419 0.245 0.411

0.241 0.561 0.186 0.350 0.152 0.458

0.003 0.007 0.003 0.004 0.002 0.009

4827 5797 4689 6713 5175 2532

0.238 0.491 0.269 0.650 0.235 0.317

0.162 0.518 0.198 0.503 0.236 0.311

0.002 0.007 0.003 0.007 0.006 0.005

4297 5878 5548 4912 1690 3242

a

SD and SEM refer to standard deviation and standard error of the mean, respectively. bPairwise statistical difference was observed between all of the substrates (p < 0.05). adhesion force data for a given substrate to the maximum SD of adhesion force data reported among all substrates tested. This way, the heterogeneities among substrates were compared relative to each other. It is worth noting that HI was multiplied by 100 in order to represent it as a percentage. 2.7. Types of Interaction Forces Decoupled via the Poisson Statistical Model. Total adhesion forces were decoupled into specific and nonspecific components via the Possion statistical model. Details of the decoupling method and the types of the decoupled specific and nonspecific forces are given in the Supporting Information. Briefly, the specific forces were identified as hydrophobic interactions in the −CH3 modified cantilever system, whereas in the −OH modified cantilever system, specific forces were hydrogen bonding or dipole− dipole interactions depending on the chemical functionalities on the substrate surface. Dipole−dipole interactions refer here to acid−base interactions. Although hydrogen bonding can be considered a form of dipole−dipole interactions, we opted to use hydrogen bonding when they represent the form of interactions in the system. In both hydrophobic and hydrophilic systems, LW forces were the nonspecific forces.

difference in the density of hydrophobic groups between lignincontaining substrates and lignin-free substrates was dependent on the treatment method with KP and OPP substrates experiencing the highest and lowest differences, respectively. Similarly, Figure 3 shows the topography images and the maps of spatial distributions of maximum adhesion forces measured between an −OH functionalized cantilever and various reference substrates in buffer. As can be seen qualitatively from Figure 3, substrates with lignin (bottom row of images), especially SP40, have more bright pixels in their maps compared to lignin-free substrates (top row of images). The bright colored pixels indicate high adhesion forces and chemical groups involved in dipole−dipole interactions while the dark colored pixels indicate low adhesion forces and hydrophobic domains. Our results thus indicate that the strength of dipole−dipole interactions of the lignin-containing substrates as measured with the −OH modified cantilever is higher than the hydrogen bonding interactions of the ligninfree substrates. Similar to the density of hydrophobic domain term defined in the −CH3 system, a density of chemical moieties involved in hydrogen bonding interactions was also defined as the ratio of the number of pixels with greater than 0.250 nN adhesion forces to the total number of pixels. The 0.250 nN cutoff was chosen because the typical strength of a hydrogen bond varies from 0.1 to 0.4 nN,23 and the midpoint of this range is 0.25 nN. Adhesion force values above 0.250 nN for lignin-containing substrates were considered as permanent dipole−dipole interactions since the major contribution to adhesion forces will be due to permanent charge of lignin residues. However, it is important to note that the strength of hydrogen bonds and −O−H (δ+)···(δ−) π interactions depend on the structure and the polarity of interacting molecules.24,25 The density of groups involved in dipole−dipole interactions quantified from adhesion maps of KP40, SP40, and OPP was 63%, 78%, and 22%, respectively. Similarly, the density of groups involved in hydrogen bonding in lignin-free substrates was quantified as 31%, 35%, and 8% for KP0, SP0, and OPP0, respectively. The interactions in lignin-free substrates were defined as hydrogen bonds since there are no aromatic groups on their surface that can contribute to the π dipole−dipole charge interactions. When lignin-containing substrates were compared, the densities of groups involved in dipole−dipole interactions on their surfaces were different although their surface concentration of lignin was almost constant (Table 1). This indicates that the density of the groups involved in dipole−dipole interactions on the lignocellulosic substrates depends on the treatment methodology used to prepare the substrates, which was also true for the density of hydrophobic domains.

3. RESULTS 3.1. Mapping Locations and Density of Hydrophobic Groups and Moieties Involved in Hydrogen Bonds and Dipole−Dipole Interactions on Lignocellulosic Substrates. Figure 2 shows the maps of spatial distributions of maximum adhesion forces measured between a hydrophobic cantilever (−CH3 functionalized) and various reference substrates in buffer. Corresponding topography images are also give in Figure 2. We were not able to observe any correlation with the features of topography images and the magnitudes of adhesion forces. As can be seen qualitatively from Figure 2, lignin-containing substrates (bottom row of images) have more bright pixels in their maps compared to lignin-free substrates (top row of images). The bright colored pixels indicate high adhesion magnitudes or hydrophobic locations on the surfaces while dark colored pixels indicate low adhesion forces or hydrophilic locations on the surfaces. Figure 2 shows that the hydrophobic groups are well dispersed on the surface of substrates investigated. To quantify the distribution of the hydrophobic domains on the substrates, a density term was defined as the ratio of the number of pixels with greater than 0.250 nN adhesion forces to the total number of pixels. The 0.250 nN cutoff was chosen because it is close to the mean of all adhesion forces measured on the lignin-free substrates (0.259 nN, Table 2). The densities of hydrophobic groups quantified from adhesion maps of KP40, SP40, and OPP were 64%, 51%, and 33%, respectively. Similarly, the densities of hydrophobic groups on lignin-free substrates were quantified as 32%, 39%, and 25% for KP0, SP0, and OPP0, respectively. Our results thus indicate that the 10237


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Figure 3. Representative maps of adhesion forces measured between reference substrates and an AFM cantilever decorated with −OH SAMs: (a) KP0, (b) SP0, (c) OPP0, (d) KP40, (e) SP40, and (f) OPP. Topography images of corresponding adhesion maps: (g) KP0, (h) SP0, (i) OPP0, (j) KP40, (k) SP40, and (l) OPP. Color bars indicate the z-scale.

to lignin-free substrates (2.40 ± 0.32-fold). The highest HI was determined for the KP40 substrate (Figure 4). 3.3. Distribution of Adhesion Forces. Figure 5 shows the distributions of the probabilities of adhesion affinities measured between the chemically modified AFM cantilevers and the lignocellulosic substrates in buffer. The Poisson statistical model (Supporting Information eq S1) was able to fit the distributions well for all substrates investigated (r2 = 0.95 ± 0.02). To further describe the adhesion forces reported in these histograms, the means of all adhesion forces were calculated

3.2. Heterogeneities of Reference Substrates. Figures 2 and 3 show that high forces were uniformly distributed on the lignocellulosic substrates; however, pixels varied in the range of forces they represented from 0 to 2 nN. To describe the heterogeneities in the adhesion forces and compare them among various lignocellulosic substrates, heterogeneity indexes of adhesion forces were calculated. As can be seen from Figure 4, the heterogeneity indexes of substrates increased linearly with the increase in adhesion forces. The heterogeneity indexes were also higher for the lignin-containing substrates compared 10238


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defined in the Supporting Information, specific forces which represent the hydrophobic interactions present between the −CH3 modified AFM cantilever and reference substrates were compared. Our results indicate that the specific forces quantified for KP40, SP40, and OPP were 2.46-, 1.32-, and 1.12-fold higher than those quantified for their corresponding lignin-free substrates, KP0, SP0, and OPP, respectively (Table 3). When specific forces were compared among lignincontaining substrates, KP40 had ∼1.5-fold higher specific forces compared to SP40 and OPP, both of which were similar in their specific forces (Table 3). The specific forces quantified for OPP0 were 1.46- and 1.14-fold higher than those quantified for KP0 and SP0, respectively (Table 3). Similarly, nonspecific forces were compared. The effect of lignin on the nonspecific forces can be inferred from comparing the differences in the nonspecific forces quantified for lignincontaining and lignin-free substrates. Our results show that the highest difference in nonspecific forces was measured for Kraft lignin (Table 3) and was 4.30- and 3.34-fold higher than differences observed for substrates with lignosulfonates and organosolv lignin, respectively. Finally, when specific and nonspecific forces were compared for all substrates interacting with the −CH3 modified cantilever,

Figure 4. Heterogeneity index of the reference substrates mapped with −CH3 modified AFM cantilevers. Solid line in the figure refers to the linear fit of the data. The equation for the line is y = 0.178x − 6.101, r2 = 0.86.

and compared. On average, the adhesion forces for lignincontaining substrates were 1.76 ± 0.16- and 1.94 ± 0.31-fold higher than those measured for lignin-free substrates using −CH3 and −OH modified AFM cantilevers, respectively (Table 2). 3.4. Decoupling of Specific and Nonspecific Forces Using Poisson Statistical Model Using the −CH 3 Modified Cantilevers. With the nomenclature of forces

Figure 5. Distribution of adhesion forces between a modified AFM cantilever and reference substrates in sodium acetate buffer. Gray bars are stacked onto black bars. The dashed dark gray and gray solid lines are the theoretical Poisson distribution fits to adhesion force data (a) −CH3 modified and (b) −OH modified AFM cantilevers, respectively. Insets in the figures show representative retraction curves measured between reference substrates and chemically modified cantilevers. On average, r2 for the fitting of Poisson distribution was computed as 0.95 ± 0.02. 10239


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Table 3. Summary of Poisson Statistical Model Decoupled Specific and Nonspecific Forces Estimated for Reference Substrates Using −CH3 Modified and −OH Modified AFM Cantilevers −CH3

−OH differencea

KP0 KP40 SP0 SP40 OPP0 OPP

differencea

SFb (nN)

NFc (nN)

SF (nN)

NF (nN)

SF (nN)

NF (nN)

SF (nN)

NF (nN)

0.202 0.497 0.259 0.342 0.296 0.331

0.178 0.305 0.166 0.196 0.177 0.215

0.295

0.127

0.153

0.030

0.146

0.080

0.035

0.038

0.133 0.286 0.179 0.259 0.126 0.164

0.093

0.083

0.310 0.403 0.379 0.525 0.292 0.406

0.114

0.038

a The difference between the substrates with lignin and lignin-free substrates of a given treatment, for example, specific forces of KP40-specific forces of KP0. bSpecific forces. cNonspecific forces.

specific forces were on average 1.55 ± 0.02-fold greater than nonspecific forces (Table 3). 3.5. Decoupling of Specific and Nonspecific Forces Using Poisson Statistical Model Using the −OH Modified Cantilevers. With the nomenclature defined in the Supporting Information, the types of forces present in this system were investigated. When specific forces were compared among lignin-free substrates, the specific forces calculated for SP0 were 1.30- and 1.22-fold higher than those calculated for KP0 and OPP0, respectively. Similarly, specific forces were compared among lignin-containing substrates. Our results indicate that the specific forces quantified for SP40 were ∼1.30-fold greater than those quantified for KP40 and OPP. The specific forces quantified for lignin-containing substrates were always higher than those quantified for the lignin-free substrates (Table 3). The difference in the specific forces obtained for the SP substrates was 1.57- and 1.28-fold greater than the difference obtained for the KP and OPP substrates, respectively (Table 3). Similarly, the effect of lignin on nonspecific forces was similar to the case when the −CH3 modified cantilever was used. The differences in nonspecific forces between lignin-free and lignincontaining substrates followed the same trend observed for the −CH3 modified cantilevers where KP substrates had the highest difference compared to SP and OPP substrates. When all substrates were considered, the mean of specific forces was 2.11 ± 0.15-fold higher than the mean of nonspecific forces. It was also determined that the difference in the specific forces of KP substrates probed with a −CH3 cantilever was 2.02-fold higher than the difference in the specific forces of SP substrates probed with a −OH cantilever.

substrates.30−32 The above supports the higher number of bright pixels observed for the lignin-free substrates compared to the lignin-containing substrates (Figure 2). Our results agree well with macroscale wettability literature in that increasing lignin content of pretreated wheat straw substrates was shown to increase the contact angle of water and thus indicate a more hydrophobic substrate.33 Previously, it was also shown that adsorption of lignin on a bleached softwood Kraft pulp fiber surface increased the contact angle of water.30 In addition to increasing hydrophobicity of substrates, the presence of lignin increased the adhesion forces due to the presence of strong dipole−dipole interactions as was measured with the −OH modified cantilever. Chemical groups on lignocellulosic substrates that may be involved in forming dipole−dipole and hydrogen bonding interactions are aromatic rings, hydroxyl groups, carboxyl groups, and sulfonate groups. Depending on the substrate content of lignin, xylan, and cellulose as well as lignin chemistry, the distribution of such chemical groups on the substrate can vary significantly.34,35 The introduction of sulfonic groups after sulfite pulping makes lignin highly water-soluble largely due to increased electronegativity resulting from the permanent charged π interactions.36 Sulfonic acid groups contribute to the hydrophilicity of lignosulfonates whereas the aromatic skeleton makes them hydrophobic.27 Such highly water-soluble and negatively charged sulfonate groups present in SP40 are responsible for the high strength of dipole−dipole interactions formed between the −OH modified AFM cantilever and substrate surface in comparison to KP40 and OPP. As can be seen from Figures 2 and 3, hydrophobic domains and groups involved in hydrogen bonding and −O−H (δ+)··· (δ−) π interactions are well spread on all lignocellulosic substrates investigated. This uniformity supports the quality of all pretreatment methods used to prepare reference substrates with homogeneous physicochemical properties including fiber length and coarseness (weight at a given length), cell wall thickness, lumen diameter, crystallinity index (CrI), and cellulose degree of polymerization.6,7 Such homogeneity in the surface properties of lignocellulosic materials allows for AFM experiments to be carried out repeatedly with a high degree of reproducibility. 4.2. Heterogeneity of Nanoscale Adhesion Forces Measured between Reference Substrates and Chemically Modified Cantilevers. a. Effect of Lignin. Heterogeneities in the spatial maps of the adhesion forces measured at different locations on a given substrate simply reflect heterogeneities in the surface composition of the substrate as

4. DISCUSSION 4.1. Hydrophobicity of Lignocellulosic Substrates. Since polar groups attract polar groups and apolar groups attract apolar groups,9,26 the density of hydrophobic domains on the substrates investigated can be quantified and compared among substrates. Independent of the cantilever modification used, the presence of lignin on the substrate increased the strengths of hydrophobic interactions, LW forces, and dipole− dipole interactions. In general, lignin contains both hydrophobic groups (aromatic skeleton and carbon chain) and hydrophilic groups (phenolic and aliphatic hydroxyl groups).27−29 However, contact angle measurements showed that lignin is more hydrophobic than cellulose, and as such, increasing its surface concentration is expected to lead to an increase in surface hydrophobicity compared to lignin-free 10240


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linearly with adhesion largely due to an increase in the surface density of molecules interacting with the cantilever.22 b. Effect of Xylan. KP40 has a similar surface lignin coverage compared to SP40 and OPP (Table 1), yet it was shown to be more heterogeneous both using −OH and −CH3 modified cantilevers. Such high heterogeneity observed for the KP40 is likely due to its high xylan content. Xylan has branched side chains and several functional moieties such as carboxyl, hydroxyl, acetyl, arabinosyl, and glucuronic acid substituents.50 These functional groups can interact differently with the cantilevers, leading to a more heterogeneous population of forces. c. Effect of Cellulose. From a structural point of view, cellulose has large number of linear D-glucose repetitive units linked by β-1,4-glycosidic bonds.51 Those repetitive units have similar functional groups, such as hydroxyl and methylene, throughout the cellulose chains. Therefore, it is expected that cellulose is the most homogeneous molecule in its chemical moieties and structure among molecules constituting the lignocellulosic substrates. Lignin-free substrates mainly consist of cellulose with the highest content measured in OPP0 (Table 1). With OPP0 characterized as our least heterogeneous substrate, it supports the homogeneity of cellulose and its homogeneous distribution on the fiber surface (Figures 2, 3, and 4). 4.3. Distribution and Strength of Adhesion Affinities. Figure 5 shows that histograms of adhesion affinities measured for lignin-containing substrates were wider, skewed to the right, and more heterogeneous compared to the histograms of the lignin-free substrates. This was true for both −OH and −CH3 modified cantilevers. Our results also agree well with a previous study that showed narrower histograms of adhesion forces for delignified pulp fibers compared to nontreated wood fibers.12 This is to be expected, as discussed earlier, due to the heterogeneities in the chemical compositions of lignocellulosic substrates.37 Another reason behind the longer tailed histograms observed for the lignin-containing substrates is likely due to introduction of higher attractive forces to modified cantilevers. Strong forces quantified between the −CH3 cantilever and the lignin-containing substrates agree well with the previous studies. It was shown that sulfite pulped fibers of Acacia mearnsii and Eucalyptus grandis species had 338 and 476 nN adhesion forces to −CH3 modified cantilever measured in air whereas their counter nonpulped fibers (with lignin) had 1434 and 1492 nN, respectively.12 The large adhesion forces measured in the study mentioned above are likely due to capillary forces since measurements were taken in air. Therefore, the magnitudes of adhesion forces cannot be compared with our values since our measurements were taken in a buffer solution and no capillary forces were involved in the adhesion forces. In another study, adhesion forces of fully bleached Kraft pulps were mapped with −CH3 and −OH modified cantilevers in a buffer, and the magnitudes were generally less than ∼1 nN.38 Several pulp fibers were also probed in an aqueous solution with a −OH modified cantilever and the mean adhesion force values varied from 0.29 to 1.08 nN.14 These results also agree fairly well with our mean values of adhesion forces measured with −CH3 and −OH modified cantilevers (Table 2). 4.4. Specific Forces Acting between −CH3 SAMs and Lignocellulosic Substrates. a. Effect of Lignin. Higher specific forces of lignin-containing substrates in comparison to lignin-free substrates can be attributed to the higher hydro-

it interacts with the chemically modified cantilever in a given solvent. The increased heterogeneity with adhesion as lignin surface concentration increased can be attributed to several factors. These include a higher density of side groups of surface molecules available for interactions with cantilevers, a higher contact area with the cantilever, and variations in the size of lignin precipitates on the surface. The high heterogeneity of lignin-containing substrates is likely due to the introduction of various side groups present in the lignin structure to the substrate surface.37 Lignin-containing substrates have ∼45% surface lignin coverage whereas ligninfree substrates have more than 80% cellulose content (Table 1). One can expect that introducing a new molecule (lignin) with several functional groups will increase the heterogeneity of the substrate surface. Our results also agree well with a previous study where the removal of lignin reduced the standard deviation of adhesion forces measured between the lignincontaining surface and −COOH or −CH3 modified cantilevers.12 In another study, FV images of fully bleached Kraft pulped substrates with almost no lignin in them were homogeneous independent of the SAM modification of the cantilevers used to map them.38 This also supports our finding that lignin-free substrates have a uniform surface with similar functional groups whereas lignin-containing substrates have complex surface chemistry. Several AFM phase imaging studies showed that lignin forms globular shaped particles on cellulose microfibrils after pulping or pretreatment steps.39−42 However, due to the heterogeneous nature of chemical pulping/pretreatment technologies and the complex structure of the fiber network, the size distribution of lignin precipitates may vary. Such variations in the size distribution of lignin precipitates can cause variations in the contact area of adhesion between the AFM cantilever and lignin molecules and thus result in a high heterogeneity index. It is also expected that the availability of surface groups contributing to the adhesion forces will be influenced by the size of lignin precipitates. Previously, it was shown that at low kappa number the standard deviation in the precipitate diameter was smaller than that observed for particle size of lignin precipitates at high kappa number.39 In addition, the size distribution of lignin particles was shown to vary significantly for various treatment methods.40 This indicates that the variation in the lignin content and pretreatment methods change the size distribution of lignin precipitates and thus the heterogeneity of the surface composition of the pulp substrates. Therefore, it is likely that the pulping methods used in this study resulted in different sizes of lignin precipitates and increased the heterogeneity of pulp substrates. However, one can expect that due to the high hydrophobicity of Kraft lignin, its monomers tend to aggregate to form larger particles compared to lignosulfonates and organosolv lignin particles. With aggregation between Kraft lignin particles, heterogeneity is expected to increase. This hypothesis is also supported by other studies which quantified the diameter of Kraft lignin precipitates to be between 15 ± 5 and 37 ± 16 nm40 and the mean diameter of lignosulfonate molecules to be ∼8 nm.43 Although heterogeneities in nanoscale adhesion forces on pulp substrates as related to hydrophobicity were not reported previously in the literature, heterogeneities in AFM nanoscale adhesion measurements on biological surfaces are well documented in the literature.22,44−49 We have previously observed that heterogeneities in nanoscale adhesion energies measured between bacterial surface biopolymers and a model surface of silicon nitride increased 10241


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presence of a larger amount of xylan, a branched polysaccharide, may interfere with the formation of hydrogen bonding between cellulose and −OH modified cantilever and thus reduce the specific forces. Recently, it was shown that the addition of xylan to amorphous cellulose films reduced adhesion forces of −OH modified cantilever to surface.55 This also supports the effect of xylan on reducing the specific forces of −OH modified cantilever. A similar analogy can be used to explain the low forces observed for the OPP0 substrate compared to the SP0 substrate. Acetone extractives in OPP0 will likely interact with water molecules in the media, lowering the probability of interactions with the −OH modified AFM cantilever and as such lowering the specific forces. Since the levels of hydrophilicity of acetone extractives and xylan are expected to be different from each other, the strengths of specific forces quantified between the −OH modified cantilever and the substrate surfaces were different. b. Effect of Lignin on Specific Forces. The dipole−dipole interactions formed between the −OH modified cantilevers and lignin-containing substrates are likely due to the formation of −O−H(δ+)···(δ−) π bonds with the available aromatic groups (syringyl, guaiacyl, and p-hydroxylphenylpropane units) of the lignin with the appropriate directionality. Since the substrates are not purely lignin, the hydrogen bonding between cellulose and xylan to −OH cantilever will also be present. Since the partial charge density (δ−) of lignin can vary from substrate to substrate, the strength of dipole−dipole interactions can vary as well. For example, introducing −HSO3 groups to lignin can increase the negative charge density of (δ−) of lignin. Consequently, one can expect that the type of lignin and its structure to affect the strength of dipole−dipole interactions forming between −OH modified AFM cantilever and a substrate surface. The contribution of lignin to dipole−dipole interactions was quantified by taking the difference in specific forces between the lignin-free substrates and the lignin-containing substrates. Since this difference was higher for SP substrates than for the KP and OPP substrates, one can attribute it to the high charge density of lignosulfonates (δ−) that enables them to form stronger dipole−dipole interactions with the −O−H (δ+) groups than either organosolv lignin or Kraft lignin.36 4.6. Nonspecific Forces Acting between SAMs and Lignocellulosic Substrates. Since the substrates are not purely hydrophilic, nonspecific interactions between the −OH modified AFM cantilever or the −CH3 modified AFM cantilever and substrate surface can be described with LW forces. a. Effect of Lignin Type on Nonspecific Forces. Our results suggest that Kraft lignin contributes to the LW forces more significantly in comparison to lignosulfonates and organosolv lignin. This effect is quantified by taking the difference in nonspecific forces between lignin-containing and lignin-free substrates for both cantilever types (Table 3). LW forces between two surfaces depend proportionally on the Hamaker constant estimated based on the surface tensions of the two surfaces, the media of interactions, and the size of the interacting surfaces and inversely on the separation distance between the surfaces.26 Since the media is constant in all our investigations, we will exclude its effects on LW forces. Different lignin types are expected to vary in their Hamaker constants. Few studies determined the Hamaker constant of Kraft lignin in water, vacuum, and hexane.56,57 To our knowledge, there are no studies which quantified Hamaker

phobicity of lignin compared to cellulose and xylan (Table 3). Hydrophobic attractions between lignin moieties on the substrates and −CH3 cantilever can be likely attributed to the attractive forces between the −OCH3 groups in the lignin structure and the −CH3 groups on the cantilever. The higher specific forces shown for the KP40 substrate compared to the SP40 and OPP substrates likely indicate that Kraft lignin is richer in its active −OCH3 groups.52 Lower specific forces estimated for SP40 in comparison to KP40 might be due to the presence of the hydrophilic sulfonate groups. Similarly, the lower specific forces observed for OPP in comparison to KP40 is likely due to the hydrophilicity of acetone extractives in OPP.53 Previous research also showed that lignin in OPP has more hydroxyl and carboxyl groups than organosolv-dissolved lignin; as such, those chemical groups reduce the hydrophobicity of the substrate.35 This explains why OPP substrates in our study were the least hydrophobic as quantified from the difference in specific forces between lignin-containing and lignin-free substrates. Our results suggest that Kraft lignin is more hydrophobic than lignosulfonates and organosolv lignin, and such hydrophobicity leads to higher forces. Our results also agree with previous research showing the high hydrophobicity of Kraft lignin in comparison to lignosulfonates.52 OPP lignin also contains low molecular weight phenolic compounds (acetone extractives) which are also less hydrophobic than that of Kraft lignin (Table 1). In addition to lignin, KP40 is characterized by a higher xylan content compared to SP40 and OPP (1.68- and 8.08-fold, respectively). This is interesting, as one will expect xylan to contribute to strong hydrophilic interactions with the buffer due to its chemical nature. Such interactions are expected to minimize the solvent exclusion effect and as such lower specific forces, which is not the case (Table 3). Our findings strongly suggest that hydrophobic components of Kraft lignin dominate the interactions of the substrates with the −CH3 modified cantilever and are stronger than the contributions made by xylan toward the specific interactions. Since Kraft lignin has 2.62-fold higher xylan content than lignosulfonates and organosolv lignin (Table 1), our results also suggest that KP40 has to be more hydrophobic than SP40 and OPP to counterbalance the extra hydrophilic contributions of xylan. b. Effect of Xylan. Our results showed that the higher the xylan content of substrates, the lower the specific forces of lignin-free substrates. Hydrophilic xylan minimizes the solvent exclusion effect. OPP0, with the lowest content of xylan and thus the lowest density of hydrophilic groups, has the highest specific force among the lignin-free substrates (Table 1). 4.5. Specific Forces Acting between −OH SAMs and Lignocellulosic Substrates. a. Effect of Xylan and Acetone Extractives on Specific Forces. The hydrogen bonding interactions formed between the −OH modified cantilevers and lignin-free substrates are likely due to the formation of O− H(δ+)···(δ−)O−H and/or H(δ+)···(δ−)O−H bonds with available hydroxyl groups in cellulose and xylan with the appropriate directionality.54 Since the number of hydroxyl groups available for interactions can vary from substrate to substrate, the strength of hydrogen bonds can vary as well.24 As mentioned before, the high xylan content in KP0 can be held responsible for the relatively low specific forces observed compared to SP0. Xylan will be expected to interact partially with water molecules in the buffer, thus reducing the probability of −OH groups on AFM cantilever to interact with the substrate surface, resulting in low specific forces. The 10242


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biomass can reduce the heterogeneity of the substrates and thus provide substrates that can be utilized in obtaining reproducible results. We also showed that the hydrophobic domains available on the surfaces of substrates and the chemical moieties involved in hydrogen bonding and dipole−dipole interactions can be located by adhesion maps generated using −CH3 and −OH modified cantilevers. Localization of such domains on the substrate surface will enable researchers to evaluate the effect of hydrophobicity and polar interactions on cellulase adsorption to substrates’ surfaces. If one can design an experiment to locate the hydrophobic domains and the polar sites on the surface as well as locating the sites where the cellulase enzymes adsorb to a substrate surface, specific effects of such interactions on cellulase adsorption can be understood. Understanding the interactions between cellulases and lignocellulosic surfaces can help researchers design efficient enzymes capable of hydrolyzing biomass in a competitive manner. It can also lead to new and efficient pretreatment technologies targeted at altering the hydrophobicity of the substrates or the ability of lignocellulose to form dipole−dipole interactions to cellulases. It was found that the LW forces play a less significant role in the interactions of reference substrates to hydrophobic and hydrophilic surfaces in comparison to hydrophobic interactions and dipole−dipole interactions. Since dipole (δ+)−dipole (δ−) bonds govern the interactions between the −OH modified cantilever and lignosulfonates and hydrophobic forces dominate the interactions between the −CH3 modified cantilever and Kraft lignin, one can propose that the chemical structure of the lignin available on the substrate surface determines the type of interactions that dominate how lignocellulosic surfaces interact with other surfaces of interest. Altering the structure of lignin on the biomass surface by reducing its hydrophobicity or the number of chemical moieties involved in dipole−dipole interactions can minimize the nonproductive binding of cellulases to substrate surface. In addition, enzymes with high partial negative charge (δ−) may lead to repulsive forces to lignin (δ−) and thus provide less nonproductive binding. An alternative approach to reduce the nonproductive binding of cellulases to lignin might be designing new enzymes with less hydrophobicity but high affinity to cellulose. It is important to note that the hydrophobic interactions of lignin were stronger than the dipole−dipole interactions. As such, reducing the hydrophobicity of lignin can be a priori toward the prevention of nonproductive binding of cellulases. This can be also supported with the results that in the presence of hydrophobic lignin the contribution of hydrophobic interactions to overall interactions was more important than the contribution of dipole−dipole interactions in the presence of lignosulfonates and organosolv lignin. Our results also support our previous observation that Kraft lignin contributes to nonproductive binding of cellulases more than lignosulfonates due to the high hydrophobic nature of Kraft lignin.6 In general, the differences of specific and nonspecific forces for OPP substrates were less than both the KP and SP substrates. Therefore, one can conclude that organosolv lignin has less ability to form dipole− dipole interactions, low strength of LW, and hydrophobic forces, and thus it is likely that organosolv lignin has less nonproductive binding to cellulases. We also showed that the presence of xylan hinders the substrates to form hydrogen bonding to the −OH modified cantilever in aqueous media due to its high affinity to water molecules in the media and its partial ability to hinder hydrogen

constants of isolated lignin from Kraft, sulfite and organosolv pulping in the literature. However, it is likely that Kraft lignin has a larger Hamaker constant in comparison to lignosulfonates and organosolv lignin, and this increases the LW forces between the hydrophobic cantilever and the substrates’ surface. In addition to Hamaker constant, contact area between the two surfaces plays an important role in LW forces. It is important to note that the Hamaker constant is a function of dispersion energy between molecules,58,59 and therefore the density of molecules on the fiber surface. As discussed before, it is likely that Kraft lignin has larger precipitate size40 in comparison to lignosulfonates43 and organosolv lignin. Such large precipitates result in high density of lignin polymers on the surface and thus likely strong LW forces. Our results also showed that the lignincontaining substrates have higher nonspecific forces in comparison to lignin-free substrates. This can also be explained by the stronger LW forces which is likely due to larger Hamaker constant values of lignin in comparison to cellulose in aqueous media57 as well as due to larger size of lignin precipitates on the substrates’ surfaces.40,43 b. Effect of Xylan on Nonspecific Forces. The nonspecific forces were relatively similar among the three lignin-free substrates for both cantilevers’ modifications. This indicates that the conformational properties of xylan and cellulose on these surfaces are similar and independent of the treatment technology. Our observation is supported by the similar values of the LW components of interfacial tension of the substrates. For example, the LW component of the surface energy of Kraft pulp and neutral sulfite semimechanical pulp fibers obtained from Eucalyptus regnans were 43.6 and 45.7 mJ/m 2 , respectively.60 4.7. Types of Forces That Dominate Interactions between SAMs and Lignocellulosic Substrates. Since the specific forces estimated between the hydrophobic (−CH3) modified cantilever and lignocellulosic substrates were stronger than the nonspecific forces estimated for the same couple, one can suggest that the interactions are dominated by hydrophobic forces and solvent exclusion effects and not by LW forces. Similarly, since the specific forces estimated between the hydrophilic (−OH) modified cantilever and lignocellulosic substrates were always stronger than the nonspecific forces between the same couple, one can propose that the interactions between the −OH modified cantilever and lignocellulosic substrates are dominated by dipole−dipole interactions compared to LW forces. Finally, since the difference of specific forces quantified for KP substrates with a −CH3 modified cantilever is significantly higher than the differences of specific forces quantified for SP substrates with a −OH modified cantilever, one can suggest that the hydrophobic interactions play a dominant role on the adhesion forces to lignin in comparison to dipole−dipole interactions.

5. IMPLICATIONS WITH REGARD TO LIGNOCELLULOSIC TREATMENT AND ENZYMATIC HYDROLYSIS We successfully showed that CFM can be used to map the heterogeneity of lignocellulosic biomass at the nanoscale. By comparing the heterogeneity of substrates, one can evaluate the quality of pretreatment methods for further utilization of biomass. It was shown that lignin and xylan contribute to the chemical heterogeneity of the reference substrates in a positive manner. However, the effect of lignin was more significant than that of xylan. It can be concluded that removing lignin from 10243


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Recalcitrance Factors Using Reference Substrates. Appl. Microbiol. Biotechnol. 2014, 98 (10), 4409−4420. (8) Himmel, M. E. Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy; Blackwell Pub.: Oxford, 2008. (9) van Oss, C. J. Interfacial Forces in Aqueous Media, 2nd ed.; CRC Press: Boca Raton, FL, 2006. (10) Dufrêne, Y. F. Recent Progress in the Application of Atomic Force Microscopy Imaging and Force Spectroscopy to Microbiology. Curr. Opin. Microbiol. 2003, 6 (3), 317−323. (11) Dufrêne, Y. F.; Martinez-Martin, D.; Medalsy, I.; Alsteens, D.; Müller, D. J. Multiparametric Imaging of Biological Systems by ForceDistance Curve-Based AFM. Nat. Methods 2013, 10 (9), 847−854. (12) Klash, A.; Ncube, E.; Meincken, M. Localization and Attempted Quantification of Various Functional Groups on Pulpwood Fibres. Appl. Surf. Sci. 2009, 255 (12), 6318−6324. (13) Klash, A.; Ncube, E.; du Toit, B.; Meincken, M. Determination of the Cellulose and Lignin Content on Wood Fibre Surfaces of Eucalypts as a Function of Genotype and Site. Eur. J. For. Res. 2010, 129 (4), 741−748. (14) Yan, D.; Li, K. Evaluation of Inter-Fiber Bonding in Wood Pulp Fibers by Chemical Force Microscopy. J. Mater. Sci. Res. 2012, 2 (1), 15−22. (15) Zhang, M.; Chen, G.; Kumar, R.; Xu, B. Mapping out the Structural Changes of Natural and Pretreated Plant Cell Wall Surfaces by Atomic Force Microscopy Single Molecular Recognition Imaging. Biotechnol. Biofuels 2013, 6 (1), 147. (16) Zhang, M.; Wang, B.; Xu, B. Measurements of Single Molecular Affinity Interactions between Carbohydrate-Binding Modules and Crystalline Cellulose Fibrils. Phys. Chem. Chem. Phys. 2013, 15 (17), 6508−6515. (17) Zhang, M. M.; Wu, S. C.; Zhou, W.; Xu, B. Q. Imaging and Measuring Single-Molecule Interaction between a CarbohydrateBinding Module and Natural Plant Cell Wall Cellulose. J. Phys. Chem. B 2012, 116 (33), 9949−9956. (18) Qin, C.; Clarke, K.; Li, K. Interactive Forces between Lignin and Cellulase as Determined by Atomic Force Microscopy. Biotechnol. Biofuels 2014, 7 (1), 65. (19) Browning, B. L. Methods of Wood Chemistry; John Wiley & Sons Inc.: New York, 1967; Vol. II. (20) Flynn, N. T.; Tran, T. N. T.; Cima, M. J.; Langer, R. Long-Term Stability of Self-Assembled Monolayers in Biological Media. Langmuir 2003, 19 (26), 10909−10915. (21) Hutter, J. L.; Bechhoefer, J. Calibration of Atomic-Force Microscope Tips. Rev. Sci. Instrum. 1993, 64 (7), 1868−1873. (22) Park, B.-J.; Abu-Lail, N. I. Atomic Force Microscopy Investigations of Heterogeneities in the Adhesion Energies Measured between Pathogenic and Non-Pathogenic Listeria Species and Silicon Nitride as They Correlate to Virulence and Adherence. Biofouling 2011, 27 (5), 543−559. (23) Pauling, L. The Nature of the Chemical Bond and the Structure of Molecules and Crystals: An Introduction to Modern Structural Chemistry, 3rd ed.; Cornell University Press: Ithaca, NY, 1960. (24) Kamya, P. R. N.; Muchall, H. M. Revisiting the Effects of Sequence and Structure on the Hydrogen Bonding and π-Stacking Interactions in Nucleic Acids. J. Phys. Chem. A 2011, 115 (45), 12800− 12808. (25) Saggu, M.; Levinson, N. M.; Boxer, S. G. Experimental Quantification of Electrostatics in X−H···π Hydrogen Bonds. J. Am. Chem. Soc. 2012, 134 (46), 18986−18997. (26) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1992. (27) Qiu, X.; Kong, Q.; Zhou, M.; Yang, D. Aggregation Behavior of Sodium Lignosulfonate in Water Solution. J. Phys. Chem. B 2010, 114 (48), 15857−15861. (28) Ouyang, X.; Qiu, X.; Chen, P. Physicochemical Characterization of Calcium Lignosulfonatea Potentially Useful Water Reducer. Colloids Surf., A 2006, 282−283, 489−497.

bonding to cellulose. Because of its high attractive potential to water, xylan likely increases the accessible area to cellulases by swelling the cellulose fibrils. It was previously shown that xylan increased the hydrolysability of reference substrates due to its ability to swell cellulose fibrils in the presence of water.6 Future studies targeted at performing real-time AFM imaging of substrates with various xylan contents in the presence of cellulases could help quantify the swelling effects of xylan in aqueous media. Finally, we are currently working on measuring the interaction forces between CBM and reference substrates in buffer. By combining the knowledge gained here and our ongoing specific enzymatic measurements, the precise mechanism of cellulase binding to variable components of lignocellulosic biomass can be elucidated.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02633. Description of the decoupling of the total adhesion forces into specific and nonspecific components using Poisson statistical analysis; description of the type of forces involved in lignocellulosic interactions (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel 509-335-4961 (N.I.A.-L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by National Science Foundation (NSF), Grant 1067012. The authors acknowledge Muhammedin Deliorman for in-house MATLAB software and Jing Li for his assitance in silicon wafer preparation.

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REFERENCES

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