Adsorption of Arabinoxylan on Cellulosic Surfaces: Influence of

ARTICLE pubs.acs.org/Biomac

Adsorption of Arabinoxylan on Cellulosic Surfaces: Influence of Degree of Substitution and Substitution Pattern on Adsorption Characteristics ‡ € Tobias K€ohnke,*,† Åsa Ostlund, and Harald Brelid† †

Forest Products and Chemical Engineering and ‡Applied Surface Chemistry, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden ABSTRACT: This study presents results that show that the fine structure of arabinoxylan affects its interaction with cellulosic surfaces, an important understanding when designing and evaluating properties of xylan
cellulose-based materials. Arabinoxylan samples, with well-defined structures, were prepared from a wheat flour arabinoxylan with targeted enzymatic hydrolysis. Turbidity measurements and analyses using NMR diffusometry showed that the solubility and the hydrodynamic properties of arabinoxylan are determined not only by the degree of substitution but also by the substitution pattern. On the basis of results obtained from adsorption experiments on microcrystalline cellulose particles and on cellulosic model surfaces investigated with quartz crystal microbalance with dissipation monitoring, it was also found that arabinoxylan adsorbs irreversibly on cellulosic surfaces and that the adsorption characteristics, as well as the properties of the adsorbed layer, are controlled by the fine structure of the xylan molecule.

’ INTRODUCTION Hemicelluloses such as xylans, mannans, and xyloglucans are, together with cellulose, the primary polysaccharides that build up the cell wall of vascular plants. Xylans are the most common hemicelluloses and thus are one of the most abundant biopolymers on earth. Despite their great abundance, xylans have mainly been proposed for niche applications such as food additives, adhesives, and gums.1 Nevertheless, in the current trend for a more effective utilization of biomass that includes issues of sustainability, greater attention has been paid to the exploitation of xylans as biopolymer resources. This interest is aided by the fact that xylans are readily available in residues and side-streams in the forestry/pulp and agricultural industry.1,2 Relatively new discoveries, such as the film-forming properties and oxygen barrier properties of xylans,3 and rediscoveries, such as the use of xylans as a strength enhancing additive to pulp and paper,4 have also increased the interest in the enhanced utilization of xylans. One reason why xylans have attracted little interest in the past as potential raw material in the development of new materials and products is molecular structure heterogeneity. The variations in xylan structure from different parts of a plant, from plants grown under various conditions or during different seasons may be as significant as interspecies variations.5 This diversity raises the requirements of fundamental knowledge of xylan structure
property relationships. However, the influence of xylan structure on different properties, such as solubility, conformation in solution, and interaction with other polysaccharides has not been studied systematically enough to be fully understood. r 2011 American Chemical Society

The conformation and flexibility of arabinoxylan in an aqueous solution have been debated for quite some time, and no consensus has been reached. Results from the most recently conducted research point to the fact that arabinoxylans can be considered as fairly flexible polysaccharides that behave as random coils,6
8 but the influence of arabinofuranosyl (Araf) side groups on the conformation is still under discussion.8,9 Nevertheless, the fact that the number of Araf side groups affects arabinoxylan solubility in water is undebatable; several studies have clearly shown that the removal of Araf residues gives rise to aggregation and precipitation.10 The influence of the pattern of substitution on solubility, however, is not as clear. The xylan backbone has an affinity to cellulose and will adsorb irreversibly on cellulosic surfaces, a feature that plays an important role in biosynthetic processes in plants11 as well as in industrial processes.12 This propensity also provides great opportunities for the use of xylans in cellulose-based composites13 and in the modification of cellulosic surfaces.14
16 Paananen et al.17 and Tammelin et al.18 suggest that the driving force for the adsorption of xylan on cellulose is a combination of the inherent entropy increase associated with the release of solvent molecules when polymers are adsorbed and weak van der Waals attraction rather than the formation of hydrogen bonds, as has been repeatedly cited. Adsorption is known to depend on the molecular structure of xylan, particularly when it comes to the content Received: March 30, 2011 Revised: May 18, 2011 Published: May 20, 2011 2633

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Figure 1. Principal structure of the wheat flour arabinoxylan (WAX) used in this study.

of uronic acid groups.19,20 The influence of Araf substituents has not been as frequently studied, but there are indications in the literature that Araf substitution has an impact on the adsorption of xylan on cellulose surfaces,21 where low substituted xylans show preferential adsorption.16,22 It has clearly been demonstrated that Araf substitution affects the film-forming properties as well as the material properties of arabinoxylan films.23 Consequently, knowledge of the substitution pattern of xylan and an understanding of its relation to xylan
cellulose and xylan
xylan interactions, are thus important when designing new xylan
cellulose-based materials. The current study was undertaken to gain a better understanding of the relationship between the fine structure of arabinoxylan, in terms of the degree of substitution and the substitution pattern, and its interaction with cellulosic surfaces. This puts heavy demands on the preparation of arabinoxylan samples that possess well-defined molecular structures. A water-soluble arabinoxylan isolated from wheat flour (WAX) was chosen as the starting material because of its relatively high degree of substitution and its even distribution of substituents. This WAX has a degree of substitution of ∼0.6, where roughly one-third of the substituents are linked to C3 of monosubstituted xylopyranose (Xylp) residues and the remaining two-thirds to C2 and C3 of disubstituted Xylp (Figure 1). The original WAX structure was redesigned by targeted enzymatic modification performed by selective hydrolysis using two different kinds of arabinofuranosidases to obtain arabinoxylans with the same chain length (i.e., degree of polymerization (DP)) but different Araf/Xylp ratios as well as arabinoxylans with the same DP and Araf/Xylp ratio but different substitution patterns. The main focus of the study is to give a systematic illustration of how the fine structure of arabinoxylan affects its solution properties and how these structure
property changes are related to the adsorption characteristics of xylan on cellulosic surfaces and the properties of the adsorbed layers.

’ EXPERIMENTAL SECTION Materials. Arabinoxylan (WAX), isolated from wheat flour by alkaline extraction, was purchased from Megazyme (P-WAXYM, Lot 40302a). According to the product specification, the WAX has a carbohydrate composition of 62% xylose, 38% arabinose, and negligible amounts of other sugars. Furthermore, an Mw of 300 kDa is reported. R-L-Arabinofuranosidases (EC 3.2.1.55) from Aspergillus niger (CAZy GH51) and Bifidobacterium adolescentis (CAZy GH43) were purchased from Megazyme (E-AFASE Lot 70303 and E-AFAM2 Lot 31001). According to the supplier, the two enzymes have a specific activity on wheat flour arabinoxylan of 0.7 and 28.3 U/mg. Microcrystalline cellulose (MCC) with an average particle diameter of 20 μm was obtained from FMC BioPolymer (Avicel PH-105). According to the product specification, the material contains 0.25%

water-soluble substances, which were determined to be mainly composed of xylose-containing compounds. Because this was known to interfere with the adsorption measurements, the MCC was purified by alkaline (1.25 M NaOH) extraction according to a procedure described by van de Steeg et al.24 The purification step removed >99% of the watersoluble xylose-containing substances. The carbohydrate composition of the alkaline-extracted MCC was determined to be: 97.8% glucan, 1.2% xylan, and 1.0% mannan. Cellulose-coated QCM-D crystals were purchased from Q-Sense (QSX 334). According to the manufacturer, the cellulose crystals were prepared by spin-coating of microfibrillated cellulose25 on SiO2 sensors using poly(ethylene imine) as an adhesive layer.26 The surface contains both crystalline cellulose I and amorphous regions. The manufacturer also reports a cellulose thickness of 6 nm and a surface roughness of 3 to 4 nm. For NMR characterization, the following deuterated solvents and salt were used: dimethyl sulfoxide (DMSO-d6, 99.8 atom % D, ARMAR Chemicals), deuterium oxide (D2O, 99.8 atom % D, ARMAR Chemicals), and LiBr (g99%, Sigma-Aldrich). Enzymatic Hydrolysis. The WAX was dissolved in deionized H2O (1% w/v) according to instructions from the supplier. Equal volumes of WAX solution and a sodium phosphate buffer (100 mM, pH 6) containing a specified amount of arabinofuranosidase from Bifidobacterium adolescentis, or a sodium acetate buffer (100 mM, pH 4) containing a specified amount of arabinofuranosidase from Aspergillus niger, were mixed and incubated at 40 °C. Aliquots were removed at various time intervals, and the enzyme activity was terminated by keeping the samples in a boiling water bath for 10 min. Preparation of WAX Samples. On the basis of the results of the enzymatic hydrolysis, the following treatments were performed to design four WAX samples with specific degrees of substitution and substitution patterns: A, No enzymatic treatment B, An equal volume of a 1% (w/v) WAX solution and a sodium phosphate buffer (100 mM, pH 6) containing 1000 nkat/g WAX of arabinofuranosidase from Bifidobacterium adolescentis were mixed and incubated at 40 °C for 24 h. C, An equal volume of a 1% (w/v) WAX solution and a sodium acetate buffer (100 mM, pH 4) containing 50 nkat/g WAX of arabinofuranosidase from Aspergillus niger were mixed and incubated at 40 °C for 9 h. D, An equal volume of a 1% (w/v) WAX solution and a sodium acetate buffer (100 mM, pH 4) containing 250 nkat/g WAX of arabinofuranosidase from Aspergillus niger were mixed and incubated at 40 °C for 24 h. All samples (including sample A) were kept in a boiling water bath for 10 min, dialyzed (Spectra Por 4, MCO: 12
15 kDa) for 72 h against deionized H2O, and finally freeze-dried. Turbidity. The turbidity (in this case reported as the transmittance of light with a wavelength of 700 nm, referenced to deionized water) of enzymatically treated WAX solutions (500 mg/dm3) was measured using an Analytic Jena SPECORD 205 UV
vis spectrophotometer. 2634

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Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectra for structure determination were collected from each xylan sample dissolved in a mixture of DMSO-d6 and D2O (90/10 v/v) containing 50 mM LiBr. For each spectrum, 32 numbers of transients were accumulated on a Bruker 500 MHz Avance III spectrometer at 28 ((0.1) °C. Self-diffusion measurements of the xylan samples were performed in deuterated water using NMR diffusometry (NMRd).27,28 This technique was recently applied to determine the hydrodynamic radius of glucuronoxylan in solution.29 By using the stimulated echo-decay pulse sequence,30 the apparent self-diffusion coefficient, Da, may be extracted from a stretched exponential version of the Stejskal
Tanner relation,31 eq 1 I ¼ expð
kDa Þβ I0

ð1Þ

where I and I0 are the signal intensities with and without magnetic field gradients, g, and k = γ2g2δ2(Δ
δ)/3, where γ is the gyromagnetic ratio for the nucleus, δ is the gradient pulse length, and Δ is the observation time. The detected self-diffusion of a molecule at infinite dilution, D0, at the temperature, T, can be used to calculate the hydrodynamic radius, RH, of an assumed random coil conformation following the Stokes
Einstein relation, eq 2, where the value for viscosity, η, for deuterated water is 1.132 mPa s at 25 °C. RH ¼ kB T=6πηD0 1

ð2Þ

For H NMRd studies, the different xylan samples were dissolved at 0.12% (w/w) in D2O. The measurements were performed at 25 ((0.1) °C on a Bruker 500 MHz Avance III spectrometer equipped with a Diff30 diffusion probe. The parameters were set to δ 1.5 ms and Δ = 100 ms, and g was increased linearly from 0.8 to 8.0 T/m in 24 steps, where 32 numbers of transients were accumulated at each gradient step. The mean values of the self-diffusion coefficients, Ds, of the WAX samples in deuterated water were calculated from the apparent diffusion coefficient, Da, in eq 1, with β-correction according to Walderhaug et al.31 Both Da and β were extracted by CORE.32,33 MCC Characterization. To estimate the amount of water present inside the MCC particles under the conditions used during the adsorption experiments, the fiber saturation point (FSP) was measured with the solute exclusion technique according to Stone and Scallan,34 using Dextran T2000 purchased from Pharmacosmos, Denmark. The specific surface area of the MCC used in this study was determined by N2 adsorption (the BET technique) using a Micromeritics TriStar 3000 apparatus. When applying the N2 adsorption technique to estimate the surface area of cellulosic materials in the water-swollen state, sample preparation is crucial because the method requires samples in the dry state. Drying cellulosic materials from polar liquids, such as water, causes the collapse of pores and the loss of internal surface, resulting in a determination of the external surface area. The use of different solvent exchange systems has been shown to be an effective way to remove water in a manner that prevents the collapse of pores during drying35 and enables the determination of the total surface area in the water-swollen state. In the present work, a solvent exchange procedure was applied in which water was first replaced with acetone, a water-miscible organic solvent, and then acetone with cyclohexane, a nonpolar solvent, followed by drying in a nitrogen stream overnight.4,36 Adsorption of WAX on MCC. We suspended 400 mg (dry weight) MCC in 4 mL of 2 mM NaHCO3 and let it swell overnight. We added 4 mL of a freshly prepared aqueous solution containing a specified amount of arabinoxylan. The suspension was shaken for 24 h at a temperature of 23 °C. Part of the liquid was then separated from the suspension after the sedimentation of the MCC particles. The amount of adsorbed arabinoxylan was calculated from the difference in the xylose and arabinose concentrations of the separated solution and a

corresponding sample not containing MCC. Because MCC particles swell in contact with water and contain pores with radii smaller than the arabinoxylan molecules, the concentration of arabinoxylan will not only be affected by adsorption but also by the so-called solute exclusion effect. To make a correction for this effect, we estimated the excluded volume to the FSP. Carbohydrate Analysis. The neutral carbohydrate composition was analyzed, after acid hydrolysis, using ion chromatography with pulsed amperometric detection (HPAEC-PAD).16 The separation was performed isocratically in deionized water on a CarboPac PA1 column (Dionex). The detection was enhanced by the post column addition of a NaOH solution. QCM-D Measurements. The adsorption of WAX on cellulosic surfaces was also studied using a quartz crystal microbalance with dissipation (QCM-D model E4) from Q-Sense. QCM-D enables realtime measurements of molecular adsorption on surfaces where the adsorbed mass (including the solvent coupled to the adsorbed material) is measured as the change in oscillating frequency (Δf). The technique also allows the simultaneous measurement of the structure and viscoelastic properties of the adsorbed layer by monitoring the changes in dissipation (ΔD). The basic principles of the instrument have been ok et al.38 The viscoelastic properties described by Rodahl et al.37 and H€o€ of the adsorbed film were estimated by fitting Δf and ΔD from several overtones to a Voigt-based model included in the program Q-Tools from Q-Sense.39 It was assumed that the density and viscosity of the bulk liquid were those of water. In the modeling of the xylan film properties, the density of the film was assumed to be constant (1200 g/dm3), and the thickness (h), the shear elastic modulus (μ), and the viscosity (η) were adjusted to give the best possible fit using the least-squares method.18 The cellulose-coated sensors were rinsed according to recommendation from the supplier. Stable zero frequency and dissipation were ensured by allowing the cellulose films to swell and stabilize overnight at 23 °C in the electrolyte solution to be used in the measurements (1 mM NaHCO3). The constant f and D signals were offset to zero frequency and dissipation, which were then monitored for an additional 15 min to obtain the experimental baseline. After the replacement of the electrolyte solution with a freshly prepared corresponding solution containing arabinoxylan (0.1 g/dm3), the changes in f and D were measured as a function of time for 180 min. The solution was then replaced again to the background electrolyte solution to study possible desorption. In the reported measurements, the solution was injected at a constant flow of 100 μL/min at a temperature of 23 °C. All measurements were repeated at least twice. The repetition curves followed the same trend.

’ RESULTS AND DISCUSSION Preparation of Enzymatically Engineered WAX Samples. When studying the influence of arabinoxylan substitution and the substitution pattern on the adsorption on cellulosic surfaces, the preparation of xylan samples with well-defined molecular structures is central. In this study, the aim was to obtain WAXs with the same chain length (i.e., the same degree of polymerization (DP)) but with different Araf/Xylp ratios as well as WAXs with the same DP and Araf/Xylp ratio but with different substitution patterns. Enzymes are valuable tools for the modification of biopolymers, especially for targeted modification such as selective hydrolysis. The arabinofuranosidase from Bifidobacterium adolescentis releases only C3-linked Araf residues from disubstituted Xylp residues40,41 and can consequently be used to design WAX only composed of monosubstituted Xylp. The arabinfuranosidase from Aspergillus niger is known to hydrolyze the Araf of monosubstituted Xylp and to hydrolyze slowly the Araf of disubstituted Xylp and can consequently be used to design 2635

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Figure 2. Release of arabinofuranosyl (Araf) groups from wheat arabinoxylan (WAX) by enzymatic hydrolysis using arabinofuranosidase from Aspergillus niger (in 50 mM sodium acetate buffer, pH 4) or Bifidobacterium adolescentis (in 50 mM sodium phosphate buffer, pH 6) at 40 °C.

WAX with low Araf/Xylp ratios as well as WAX mainly composed of disubstituted Xylp. The reaction properties of the two different enzymes were investigated to determine the parameters suitable for the modification of WAX into desired structures. Figure 2 shows the release of Araf as a function of time under the conditions chosen for this purpose. The treatment with arabinofuranosidase from Bi. adolescentis (1000 nkat/g WAX) seems to remove all of the R-L-Araf (1f3)linked substituents from the β-D-Xylp units substituted with two Araf units relatively rapidly. Treatment with arabinofuranosidase from A. niger at a dosage of 50 nkat/g WAX was shown to be suitable when the termination of hydrolysis with a high precision at a specific level of released Araf is desired. A greater addition of enzyme was needed to reach enhanced levels of degradation. On the basis of these findings, four different WAX fractions were prepared (cf. the Experimental Section). Chemical Structure of the Prepared WAX Samples. The R-anomeric region of the 1H NMR spectra of the four prepared WAX samples is presented in Figure 3. The spectrum of the original WAX fraction (A), that is, the WAX sample not treated with arabinofuranosidase, shows three major peaks. The peak at 5.30 ppm (A3m) indicates the presence of R-L-Araf (1f3) substituents linked to monosubstituted β-DXylp residues. The signal at 4.94 ppm (A3d) also originates from (1f3)-linked R-L-Araf substituents but is linked to disubstituted β-D-Xylp units in the main chain. The peak at 5.09 ppm indicates the presence of R-L-Araf (1f2) substituents linked to disubstituted β-D-Xylp residues. Because this peak is overlapping the signal of R-L-Araf (1f2) substituents linked to monosubstituted β-D-Xylp residues (5.11 ppm), it is simply denoted A2. Other small overlapping R-anomeric signals, which broaden the signals, are typical for complex structures of polymeric arabinoxylans.42 From the spectrum of fraction B, that is, the WAX fraction treated with arabinofuranosidase from Bi. adolescentis, it is obvious that the enzyme released the (1f3)-linked R-L-Araf substituents of disubstituted β-D-Xylp units more or less completely. The

Figure 3. R-Anomeric region of 1H NMR spectra of (A) the original WAX fraction and (B
D) the prepared samples in DMSO-d6/D2O/ LiBr. Peak assignments: A3m = R-L-Araf (1f3) mono, A2 = R-L-Araf (1f2) di and mono, and A3d = R-L-Araf (1f3) di. Relative peak intensities are presented in Table 1.

spectrum of fraction C, corresponding to WAX treated with arabinofuranosidase from A. niger, shows a considerable reduction of R-L-Araf (1f3) substituents linked to monosubstituted β-D-Xylp residues, and no pronounced effect on the relation between the signals from R-L-Araf substituents of disubstituted β-D-Xylp units. Finally, fraction D, that is, the WAX fraction treated with a greater addition of arabinofuranosidase from A. niger than C, shows a total release of R-L-Araf (1f3) substituents linked to monosubstituted β-D-Xylp residues, and, as in the C case, there is no pronounced effect on the relation between the signals from R-L-Araf substituents of disubstituted β-D-Xylp units. The relative peak intensities of the different Araf R-anomeric protons were measured and are shown in Table 1, together with Araf/Xylp ratios, determined by carbohydrate analysis. On the basis of these data, hypothetical structures of the prepared WAX fractions can be drawn, as shown in Figure 4. Note the following: (i) A and B have about the same number of unsubstituted Xylp but different numbers of Araf; (ii) B and C have different numbers of unsubstituted Xylp but about the same number of Araf; and (iii) D has the highest number of unsubstituted Xylp and the lowest number of Araf. Fine Structure of Xylan Affects Its Solution Properties. The solution properties of the adsorbing polymer are of great 2636

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Table 1. Arabinose-to-Xylose Ratio and Relative Intensities of the Different Araf Units, As Well As the Fraction of Unsubstituted Xylose Residues (uXylp), of the Original WAX Fraction (A) and the Prepared Samples (B
D) sample

Araf/Xylpa

A3m (%)b

A2 (%)b

A3d (%)b

uXylp (%)a,b

A

0.55

33

33

34

63

B

0.41

50

46

4

61

C

0.40

12

42

46

78

D

0.30

4

46

50

84

a

Determined by carbohydrate analysis using HPAEC-PAD. b Determined from 1H NMR peak integrals for R-anomeric protons (cf. Figure 3).

Figure 5. Turbidity, reported as transmittance at 700 nm, as a function of Araf/Xylp ratio. The degree of substitution is varied by enzymatic hydrolysis (cf. Figure 2). WAX concentration is 500 mg/dm3. A
D represent the different WAX samples used in this study (cf. Figure 4).

Figure 4. Schematic tentative presentations of structures of (A) the original WAX fraction and (B
D) the prepared samples.

importance when studying polymer adsorption on surfaces. The driving force of polymer adsorption is always enhanced if solubility is decreased.43 Solubility also affects polymer conformation in the solution as well as on the surface.44 Furthermore, if solubility is poor, the polymer might phase separate, aggregate, and accumulate on surfaces. These features are especially important when investigating xylans because they possess a relatively moderate solubility in water18 and show a tendency to aggregate in aqueous solutions.20 To investigate the influence of the degree of substitution and the substitution pattern of arabinoxylans on solubility in an aqueous solution, the turbidity (reported as the transmittance of light with a wavelength of 700 nm) was measured as a function of Araf/Xylp ratio (Figure 5). The original WAX fraction (A) is fully water-soluble under the applied conditions. The enzymatic release of R-L-Araf (1f3)linked substituents from disubstituted Xylp residues to an Araf/ Xylp ratio of 0.41 (B) using the arabinofuranosidase from Bi. adolescentis did not affect the turbidity of the solution at all. WAX treated with arabinfuranosidase from A. niger, which preferentially hydrolyzes Araf of monosubstituted Xylp and slowly hydrolyzes Araf of disubstituted Xylp, shows completely different behavior. At an Araf/Xylp ratio of ∼0.47, the transmittance suddenly decreases rather steeply until an Araf/Xylp ratio of ∼0.40 is reached (C), after which the rate of decrease is lower. Considering the structure analyses performed by 1H NMR spectroscopy (cf. Table 1), this trend may be explained by the rate at which unsubstituted Xylp is formed. The initial hydrolysis preferentially releases Araf of monosubstituted Xylp, resulting in the formation of one unsubstituted Xylp per every hydrolyzed

Table 2. Mean Values of Self-Diffusion Coefficients (DS) and Hydrodynamic Radius (RH) of the Original WAX Fraction (A) and the Prepared Samples (B
D) in Deuterated Water at 25°C sample

DS (m2/s)

RH (nm)

A

(8.03 ( 0.02)  10
12

24.02 ((0.06)

B

(9.05 ( 0.02)  10
12

21.30 ((0.04)

C D

(9.49 ( 0.04)  10
12 (20.86 ( 0.17)  10
12

20.33 ((0.08) 9.24 ((0.07)

Araf. Subsequent hydrolysis releases Araf of disubstituted Xylp, resulting in one unsubstituted Xylp per every second hydrolyzed Araf. Consequently, it is not the number of Araf substituents that determines the solubility of arabinoxylan but rather the number of unsubstituted Xylp units. In other words, to predict the solubility of arabinoxylan in water, knowledge of the Araf/Xylp ratio is not sufficient, as the substitution pattern is essential. The influence of Araf side groups on the conformation and flexibility of xylan has been under discussion for quite some time.8,9 Solution property analyses of the enzymatically modified WAX samples prepared in this study (A
D) that possess the same degree of polymerization but different degrees of substitution and substitution patterns provide the opportunity to obtain further information regarding this matter. To study the influence of Araf substituents on the conformational behavior of arabinoxylan in an aqueous solution, self-diffusion coefficient measurements were performed using NMRd. Diffusion coefficients, from eq 1, of each WAX sample dissolved in water are presented in Table 2, where it can be seen that Araf substitution does indeed have an impact on the estimated hydrodynamic properties of arabinoxylan, calculated from eq 2. The selfdiffusion coefficient increases as the Araf/Xylp ratio decreases. 2637

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Biomacromolecules Furthermore, a significant difference can be seen between sample B and C, indicating that the substitution pattern may also affect conformational properties. This could be explained by an increase in the flexibility of the xylan chain in accordance with Pitk€anen et al.,8 but the reduced solvency of arabinoxylan at a relatively low degree of Araf substitution must also be taken into consideration because this is expected to lead to a denser polymer coil. The finding that the hydrodynamic size of arabinoxylan in water is affected by the substitution pattern implies that such effects must be considered when size exclusion chromatography data from arabinoxylan analyses are evaluated. Fine Structure of Xylan Affects Its Adsorption to Cellulosic Surfaces. The well characterized WAX preparations (A
D), with different degrees of substitution and substitution patterns, were adsorbed on MCC in order to determine if the structure of arabinoxylan influences interaction with cellulose surfaces. As can be seen in Figure 6a, the structure of xylan definitely has an effect on adsorption characteristics. It is evident that a decrease in the Araf/Xylp ratio increases the amount of adsorbed xylan on the MCC particles. Furthermore, considering the difference in adsorption isotherms between sample B and C, it is also evident that the substitution pattern has an impact on the amount adsorbed. Figure 6b shows the corresponding Langmuir plots. A clear difference in the apparent equilibrium constant between the original WAX fraction (A) and the modified samples (B
D) can be seen. This does not only mean that there is an actual difference in the dynamic equilibrium between WAX adsorption and desorption, but could also be an effect of molecular structure heterogeneity.45 In any case, Figures 6a and b indicate higher adsorption, and an increase in the affinity of WAX on cellulosic surfaces, with a decrease in the Araf/Xylp ratio. The conformation of polymers at the solid/solution interface is generally controlled by their equilibrium solution concentration, molecular weight, and their interactions with the surface and the solvent quality.43 It is the net effect of these parameters that determines the amount of adsorbed polymer and the properties of the adsorbed layer. Considering the applied system, together with results from the turbidity measurements and NMRd, it is evident that the solution properties of the adsorbing WAX must have a significant impact on adsorption characteristics. The effects observed can thus partially be referred to as a consequence of the decrease in WAX solvency upon a decrease in the Araf/ Xylp ratio, making the driving force for adsorption stronger and making it easier for approaching xylan molecules to accumulate on the cellulose surface due to a weaker effective repulsion between polymer segments. Decreasing the Araf/Xylp ratio even further would probably impair the solvency of xylan to such an extent that extensive aggregation and finally phase separation would be induced. Such precipitation would most likely result in a multilayer xylan deposition and assembly on cellulose surfaces, as observed by Linder et al.20 and Tammelin et al.,18 who studied glucuronoxylan sorption on cellulosic surfaces under conditions where xylan solubility is poor. There is, however, yet another phenomenon to consider in the evaluation of the adsorption data, taking into account the fact that MCC cannot be considered as solid particles under the conditions used but rather as partially amorphous,46 water-swollen, porous,47 cellulosic structures. A thorough characterization of the alkaline-extracted MCC used in this study showed an FSP of 0.47 ( 0.02 g/g, a total surface area in the fully water-swollen state of 47 ( 1 m2/g, and an external area of 2.1 ( 0.1 m2/g. Consequently, the MCC should definitely be considered as a

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Figure 6. (a) Adsorption isotherms of WAX on MCC at 23 °C and (b) the corresponding Langmuir plots. MCC concentration is 50 g/dm3 in 1 mM aqueous NaHCO3 solution. A
D represent the different WAX samples used in this study (cf. Figure 4).

water-swollen cellulosic particle with a porous structure. Because the hydrodynamic size of arabinoxylan seems to be dependent on Araf substitution (Table 2), xylan substitution may have an influence on the amount of accessible surface area for adsorption, which naturally would affect the adsorbed amount. Considering the magnitude of Γmax, it is plausible to assume that the adsorption is restricted to monolayer coverage of the external surface of the MCC particle, at least for samples A
C. Nevertheless, the remarkable decrease in the hydrodynamic size of sample D might result in a larger accessible surface area due to possible partial diffusion into the MCC particles. To eliminate this effect, we adsorbed the different WAX samples on cellulosic model surfaces using the QCM-D technique. Fine Structure of Xylan Affects the Properties of the Adsorbed Layer. The QCM-D technique offers the potential for in situ investigation of adsorption kinetics and adsorbed mass. Simultaneous dissipation measurements also enable analysis of 2638

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Figure 7. Change in QCM (a) normalized frequency (third overtone) and (b) dissipation as a function of time for arabinoxylan (0.1 g/dm3) adsorbing from 1 mM NaHCO3 aqueous solution on cellulosic model surfaces. A
D represent the different WAX samples used in this study (cf. Figure 4).

the structural properties of the adsorbed molecular layer. Figure 7a shows QCM-D frequency curves for the adsorption of the different WAXs on water-swollen microfibril model surfaces. Some general reflections can be made. First of all, the adsorption can be considered to be rather slow and does not reach a plateau value within the time studied (3 h). However, the initial adsorption rate (as determined from df/dt) is higher at a lower Araf/Xylp ratio. Second, the adsorption can be considered to be irreversible because no apparent desorption (i.e., change in frequency) occurs upon rinsing. The frequency profiles obtained are dependent on the molecular structure of the injected xylan. In agreement with the adsorption study on MCC, results show that a decrease in the Araf/Xylp ratio of the starting material increases the amount of adsorbed xylan. Furthermore, considering the difference in adsorption between samples B and C, it is also

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evident that the substitution pattern has an impact on adsorption characteristics. Surprisingly, sample D shows a lower response in frequency change than sample C, which differs from the results of the adsorption isotherms on MCC. However, this comparison is only valid if there is an identical relation between frequency changes and mass for all of the samples, which will be discussed below. The corresponding QCM-D energy dissipation curves are presented in Figure 7b. Because the dissipation value reaches >1  10
6 per 10 Hz, the adsorbed layer can be considered to be rather soft and water-swollen, which indicates the formation of a viscoelastic xylan layer. These results are in accordance with observations by Paananen et al.17 and Tammelin et al.,18 who studied the adsorption of glucuronoxylan on Langmuir
Blodgett/Langmuir
Schaefer cellulose films. The change in dissipation increases in the studied range of time from A to C, most probably due to a larger amount of adsorbed material, which increases the thickness of the adsorbed layer. Further information about the adsorbed layer can be obtained from the frequency and dissipation data by plotting ΔD as a function of Δf (so-called D
f plots), which qualitatively shows changes in polymer conformation as the layer builds up. This facilitates a more direct comparison of the properties of adsorbed layers of different xylans than separate plots of the time dependencies of ΔD and Δf. The steeper the slope of the D
f curve, the more the adsorbed layer dissipates energy per frequency unit and the softer the adsorbed layer. If the relation between ΔD and Δf has a constant slope, then the adsorbed polymer does not change conformation with an increase in the adsorbed amount. Conversely, if the relationship is nonlinear or has a discontinuity, then there is a change in the conformation of the adsorbed layer during the adsorption process. Although D
f plots do not explicitly show the time dependency of ΔD or Δf, they do contain information about adsorption kinetics. The adsorption process can be considered to be “fast” if all data points recorded under the experiment form a cluster or “slow” if the data points form a continuous curve. Figure 8 contains the D
f plots for the adsorption of the different WAXs. All samples show relatively continuous curves, demonstrating rather slow adsorption. The curves of samples A
C have more or less the same slope, indicating that the adsorbed layers virtually possess the same viscoelastic properties. The lower gradient of the D
f plot for adsorption of sample D indicates that the adsorbed xylan has a more compact conformation, forming a more rigid, less waterswollen layer. This tendency is consistent with the results obtained from NMRd. It can also partially explain why sample D shows a relatively lower degree of adsorption when studied with QCM-D than the measured adsorption on MCC (Figure 7a vs Figure 6a). Changes in frequency acquired with QCM-D measure coupled water as an inherent mass via direct hydration, viscous drag, or entrapment in cavities in the adsorbed film. It should also be mentioned that the D
f curves of samples A
C show a small kink in the slope, indicating a change in conformation, a decrease in the hydration of the adsorbed layer, or both as the surfaces approach their saturation point. The QCM-D technology allows a quantitative analysis of the thickness (h), the shear elastic modulus (μ), and the viscosity (η) of the adsorbed films. This is achieved by combining frequency and dissipation measurements from multiple harmonics (overtones) and applying simulations using a Voigt-based viscoelastic model. The simplicity of the model implies that whereas the results can be compared relative to each other the absolute values of 2639

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’ CONCLUSIONS The degree and the pattern of arabinoxylan substitution affect the water solubility and the hydrodynamic properties of arabinoxylan. On the basis of the results obtained from adsorption experiments on MCC and cellulosic model surfaces using the QCM-D technique, it can be concluded that: (i) arabinoxylan adsorbs on cellulosic surfaces; (ii) the adsorption is apparently irreversible; (iii) the adsorption is dependent on the degree of substitution; (iv) the adsorption is dependent on the substitution pattern; and (v) the properties of the adsorbed layer are dependent on the degree of substitution. Consequently, this investigation gives information about how the fine structure of arabinoxylan influences its interaction with cellulose, vital knowledge when designing and evaluating the properties of xylan
cellulose-based materials. ’ AUTHOR INFORMATION Corresponding Author

*Phone: þ46 (0)31 7725097. E-mail: [email protected]. Figure 8. Change in dissipation as a function of change in normalized frequency (third overtone) for arabinoxylan (0.1 g/dm3) adsorbing from 1 mM NaHCO3 aqueous solution on cellulosic model surfaces. t = 0
195 min. A
D represents the different WAX samples used in this study (cf. Figure 4).

Table 3. Simulated Mean Shear Elastic Modulus (μ), Viscosity (η), and Thickness (h) of the Adsorbed Layers during the Rinsing Period (t = 210
270 min)a sample

μ (Pa)

η (kg/ms)

h (nm)
3

2.0 ( 0.1

A

(1.2 ( 0.2)  10

B

(1.3 ( 0.2)  105

(1.5 ( 0.04)  10
3

2.4 ( 0.1

C

(1.1 ( 0.1)  105

(1.4 ( 0.01)  10
3

4.4 ( 0.03

D

(1.7 ( 0.3)  10

5

5

(1.4 ( 0.05)  10

(1.8 ( 0.07)  10

’ ACKNOWLEDGMENT This work has been carried out within the framework of Avancell
Centre for Fibre Engineering. The authors gratefully acknowledge financial support from S€odra. The Industrial NMR Centre at KTH, Stockholm, Sweden, is acknowledged for granting NMR spectrometer time and facilities. Dr. Romain Bordes at Chalmers University of Technology, Gothenburg, Sweden, is acknowledged for assistance during the QCM-D measurements.


3

2.4 ( 0.1

a

A
D represent the different WAX samples used in this study (cf. Figure 4).

the viscosities and elasticities obtained should not be given too much significance.48 Table 3 reports the obtained viscoelastic properties and the hydrodynamic thickness of the layers during the rinsing process (t = 210
270 min) as calculated by the application of the Voigt model. The results are in accordance with the qualitative interpretations made based on the D
f plot; that is, the layers of samples A
C possess more or less the same viscoelastic properties, whereas the adsorbed layer of sample D has a more rigid, less water-swollen structure. Consequently, the degree of arabinoxylan substitution has an effect not only on the adsorption kinetics and the adsorbed amount but also on the structure and thereby the properties of the adsorbed layer. The choice of biomass for xylan extraction as well as isolation method and pre/post treatments will affect the structure of the xylan polymers. These factors will accordingly influence the xylan retention on cellulosic surfaces, a parameter of great importance in the large scale cellulosic fiber modifications performed in the pulp and paper industry.16 Furthermore, these factors will also determine if the adsorbed xylan layer is extended and water swollen or compact and rigid, features that will affect the properties of a xylan
cellulose-based materials.

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