Characterization of chemically and enzymatically treated hemp fibres using atomic force microscopy and spectroscopy

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Contents lists available at ScienceDirect

Applied Surface Science

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haracterization of chemically and enzymatically treated hemp fibressing atomic force microscopy and spectroscopy

ichael Georgea, Paolo G. Mussonea, Zeinab Abbouda,b, David C. Bresslera,∗

Biorefining Conversions and Fermentations Laboratory, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB,anada T6E 2P5Department of Physics, University of Guelph, Guelph, ON, Canada N1G 2W1

r t i c l e i n f o

rticle history:eceived 28 April 2014eceived in revised form 9 June 2014ccepted 12 June 2014vailable online xxx

eywords:oughnessdhesion forcesemp fibres modificationFMulfonic acidsnzymes

a b s t r a c t

The mechanical and moisture resistance properties of natural fibre reinforced composites are depend-ent on the adhesion between the matrix of choice and the fibre. The main goal of this study was toinvestigate the effect of NaOH swelling of hemp fibres prior to enzymatic treatment and a novel chem-ical sulfonic acid method on the physical properties of hemp fibres. The colloidal properties of treatedhemp fibres were studied exclusively using an atomic force microscope. AFM imaging in tapping moderevealed that each treatment rendered the surface topography of the hemp fibres clean and exposed theindividual fibre bundles. Hemp fibres treated with laccase had no effect on the surface adhesion forcesmeasured. Interestingly, mercerization prior to xylanase + cellulase and laccase treatments resulted ingreater enzyme access evident in the increased adhesion force measurements. Hemp fibres treated withsulfonic acid showed an increase in surface de-fibrillation and smoothness. A decrease in adhesion forcesfor 4-aminotoulene-3-sulfonic acid (AT3S) treated fibres suggested a reduction in surface polarity. Thiswork demonstrated that AFM can be used as a tool to estimate the surface forces and roughness for

modified fibres and that enzymatic coupled with chemical methods can be used to improve the surfaceproperties of natural fibres for composite applications. Further, this work is one of the first that offerssome insight into the effect of mercerization prior to enzymes and the effect on the surface topography.AFM will be used to selectively screen treated fibres for composite applications based on the adhesionforces associated with the colloidal interface between the AFM tip and the fibre surfaces.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Hemp fibres are characterized by low density and manufactur-ng costs, low abrasion on processing equipment and comparablepecific mechanical properties relative to glass fibre composites1]. Recent demonstrated applications of hemp fibres in industrialroducts include applications such as reinforcement for matri-es for the automotive and housing industry [1]. Despite thesedvances, the widespread, large scale utilization of hemp and otheratural fibres is limited due to their limited compatibility with

ndustrial petroleum based polymeric matrices [2,3]. The underly-

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ng cause for these challenges is commonly attributed to the surfacehemical characteristics of the major components of natural fibresncluding, cellulose, hemicellulose and lignin. For each molecule of

∗ Corresponding author. Tel.: +1 780 492 4986; fax: +1 780 492 8914.E-mail addresses: [email protected], [email protected]

D.C. Bressler).

ttp://dx.doi.org/10.1016/j.apsusc.2014.06.080169-4332/© 2014 Elsevier B.V. All rights reserved.

�-glucose in the complex linear cellulose structure, there are threefree hydroxyl groups within and on the surface which contributesto a high surface polarity. This structural arrangement rendersnatural fibres incompatible with non-polar matrices, resulting inpoor stress transfer from the matrix to the fibres and consequentlychallenges meeting mechanical and impact standard requirements[1]. Additionally, these hydroxyl groups lead to moisture absorp-tion accelerating the composite property degradation over time[1]. These limitations can be overcome by protocols such as sur-face modification including alkylation, benzoylation and silanation[4–6] and the use of compatabilizers [7]. While these methods havedemonstrated in the laboratory and at the industrial scale, limiteddepth information is available above the effect of these methodson the surface physical properties and topology. These are criticalparameters as the uppermost layer of the fibres is in direct contact

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with the matrices in composite materials.The microstructure of hemp fibres consists of the primary

cell wall which surrounds the secondary cell wall (S1, S2 andS3) and the middle lamella. This fibrilliar arrangement lends to

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Table 1Characteristics of each enzyme system with corresponding activity at optimumconditions.

Enzyme Optimum conditions Activity*

pH Temperature (◦C)

Xylanase 7 70 1000 AXU/gXylanase + cellulase 6 50 2500 FXU-S/gLaccase 7 50 1000 LAMU/g

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he high strength and compact nature of the fibres. The primaryell wall consists of cellulose and hemicellulose and constitutespproximately 10% of the cell’s cross-sectional area [8]. A majorontribution of the mechanical properties associated with naturalbres results from the high cellulosic content of the secondary cellall. This model structure sub-divides into three sections namely

1, S2 and S3, with S2 being the major section. An understanding ofow this fibrilliar assembly is affected after fibre modification is ofreat importance to further improve upon the previous research inhis area [8].

Atomic force microscopy (AFM) has emerged as a very usefulool for probing interfaces to determine properties such as rough-ess, thickness and morphology. Balnois et al. [8] investigated theffect of different chemical treatments on the microscopic anddhesion properties of flax samples. Their results indicated thathe treated fibre surfaces were smoother and less heterogeneousn topology. Interestingly, none of the chemical treatments (NaOH,aOH with acetic anhydride and formic acid) yielded any changes

n roughness. In their opinion the adhesion force between the tipnd sample was due mainly to capillary forces. Han and Choi [9]tudied the effect of electron beam at different intensities on theorphology of henequen fibres and AFM was one of the techniques

sed to study the changes. It was found that surface impuritiesnd the pectin layer were removed resulting in striations in theurface of the natural fibres. Wang et al. [10] were interested inhe morphological changes from the micro to nanoscale for chem-cally treated hemp fibres. They investigated how high pressureefibrillation and chemical treatments affected the fibre networkt different size scales.

Findings included from the microscopy study lead to a uniqueorphology of interconnected web-like structure of hemp fibres

or each treatment. This paper adds a different dimension, in thatifferent methods, chemical, enzymatic and a combination of bothere used to treat hemp fibres and the effect on the surface chargesere measured.

The goal of this work was to study the effect of mercerization,nzymatic and chemical modifications, individually and in combi-ation on the adhesion force and roughness of hemp fibres. Thetudy was outlined based on two hypotheses. The first hypothe-is involved using a green and specific method of modification vianzymes to remove the hygroscopic hemicellulosic content. Thishould indirectly result in a more uniform and less hygroscopicbre system. Second, the use of a novel chemistry involving twoulfonic acids was studied.

The main aim was to graft the aromatic based acids onto the sur-ace of the hemp fibres via an esterification reaction so as to reducehe surface polarity. The measurements presented here identifiedhe fundamental physical and chemical surface microstructuralhanges in the natural fibres that play a key-role in determiningolid–solid interactions between fibres and polymeric matrices.n addition, the data collected in this paper constitutes a usefulramework to establishing suitable reinforcement candidates forpplications based on the availability of surface physical and chem-cal characterization.

. Experimental

.1. Materials

Mechanically processed hemp fibres were provided by thelberta Biomaterials Development Centre located in Vegreville,

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lberta. The samples were placed in air tight bags and stored at◦C. Enzymes used were provided by Novozymes (Bagsvaerd,enmark) and stored at 4 ◦C. Sodium acetate (99%, mol wt.2.03 g/mol), glacial acetic acid (99.7%, mol wt. 60.05 g/mol),

* AXU is the acid xylanase number, FXU is the farbe xylanase unit, LAMU is thelaccase myceliophthora units

sodium phosphate dibasic (99%, mol wt. 141.96 g/mol) and sodiumhydroxide (99%, mol wt. 40.00 g/mol) were obtained from FisherScientific. Sodium citrate monohydrate (99%, mol wt. 214.11 g/mol)was purchased from Sigma-Aldrich. Sodium phosphate monobasic(99%, mol wt. 119.98 g/mol) was obtained from Acros Organics.Citric acid (99%, mol wt. 192.13 g/mol) was sourced from EMDChemicals. Aniline-2-sulfonic acid (95.5%, mol wt. 173.19 g/mol)and 4-aminotoulene-3-sulfonic acid (95.5%, mol wt. 187.22 g/mol)were sourced from Sigma. Distilled water was used for all analyses.

2.2. Fibre treatment

2.2.1. Enzymatic treatmentApproximately 1.0 g of fibre was weighed into a 125 ml Erlen-

meyer flask. All experiments were carried out in triplicate.Optimal conditions for each enzymatic reaction were provided byNovozymes (Table 1). The procedure outlined by George et al. [11]was used for each enzymatic treatment and is summarized in thelines to follow. For each system, 6% (% w/v) of enzyme was added toeach flask. For all experiments, the liquid (ml) to fibre (g) ratio wasmaintained at 50:1 so as to facilitate complete wetting of fibres.Fibres were exposed to each enzyme for 90 min and agitated at80 rpm at the enzymes’ optimum temperature in a standard waterbath. Enzymes were deactivated by heating at 90 ◦C for 10 min.Fibres were washed with excess warm water to remove traces ofenzyme and buffer reagents [11]. All samples were dried at 80 ◦C for5 h and stored in polyethylene bags for subsequent analysis [12].

2.2.2. Chemical treatments2.2.2.1. Mercerization. The hemp fibres were treated with 10%(w/v) of NaOH as outlined by Kalia et al. [13]. Fibres were treatedat 50 ◦C for 90 min. The fibres were washed with distilled waterand then neutralized using 0.5% (w/v) glacial acetic acid. Sampleswere dried at 80 ◦C in an oven for 5 h and subsequently stored foranalysis.

2.2.2.2. Sulfonic acid treatment. Two sulfonic acids, aniline-2-sulfonic acid and 4-aminotoulene-3-sulfonic acid were investi-gated. These acids were studied because of the presence of thearomatic functionality and the acid groups which technically allowgrafting onto the surface of the hemp fibres. The main goal was toreduce the surface polarity of the hemp fibres by reacting the fibressurface hydroxyl groups with the acid groups via an esterificationreaction. These acids are characterized with limited solubility inwater, hence all treatments were conducted at a concentration levelof 0.01 M (the maximum in water). No prior work was reported onthese acids; hence the parameters adopted were from the litera-ture with similar chemical treatments such as acetylation [4,14].The parameters included exposing fibres to the acids (fibre to solu-tion was 1:50 w/w) at a 50 ◦C for 90 min in a 250 ml round bottom

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flask fitted to a condenser [14]. A water bath was used to maintainthe temperature during the course of the reaction. After comple-tion of the reaction, fibres were washed with acetone and dried inat 80 ◦C for 5 h.

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.2.3. Hybrid treatmentHemp fibres were treated with 10% NaOH as outlined above and

reated using enzymes. The procedure for enzyme treatment washe same as outlined in Section 2.2.1. The objective here was toetermine whether initial swelling of the macrostructure would

ead to better enzyme access.

.3. AFM characterization

.3.1. Sample preparationAt least four (4) fibre samples were randomly selected from each

reatment for analysis. Double sided tape was used to attach thesebres onto 5 mm AFM specimen disc. The samples were prepared

n a room maintained (AFM equipment housed there) at ambientonditions. Samples were then mounted on the sample stage forubsequent analysis.

.3.2. AFM analysisAtomic force microscopy and spectroscopy were performed

sing a Bruker Dimension Edge TM AFM and a JPK NanoWizard®I AFM, respectively. All AFM measurements were made in ambi-nt conditions in a room with climate control. Specifically theemperature and humidity of the room were maintained withinhe temperature of 22 ± 1 ◦C and relative humidity of 70 ± 5%. Themages and force measurements were collected using tapping andontact modes, respectively. For tapping (contact) mode, a siliconitride cantilever with force constant 42 (0.2) N/m and resonance

requency of 320 (13 kHz) purchased from NanoWorld Point ProbeCH was used. As for the samples treated with the sulfonic acidsnd both base and enzyme, a Bruker non-conductive silicon nitrideantilever coated with reflective gold with a spring constant of.58 N/m and a resonance frequency between 40 and 75 kHz wassed. A tip with a higher spring constant was used for these samplesecause in some cases during scanning, the higher adhesion forcesesulted in the tip getting stuck to the sample. The force appliedo the tip was maintained around 20 ± 5 nN for the enzyme andaOH + enzyme treated sample. The force was varied within ‘±’ecause some of the sample surfaces (specifically the sulfonic acidreated fibres) were overly ‘sticky’. Adhesion measurements were

ade after the contacting the surface and scanning the topogra-hy. Force curves on different areas were randomly collected for aotal of 10 force curve per sample. The entire force–distance curveas collected for each sample. Given that initially the cantilevereflections and sample deformations were not known, the onlyistance that can be controlled was the Z distance (the displace-ent of the piezo). The cantilever deflection was converted into a

orce measurement by employing the retraction deflection curvef the contact region. As reported by Balnois et al., Hooke’s law

= −k �Z where k is the spring constant and was used to estimatehe force. It should be noted, for each measurement, the zero forceas when the tip was retracted from the surface. To ensure good

eproducibility in the fibre preparation, for each treated fibre type,nalysis was performed on four replicas and for each replica threeeasurements were taken [15–18].The root mean square roughness (RMS) was determined from

he tapping mode images using NanoScope Analysis (Version 1.40).he raw height images were subjected to the flatten function, whichemoved unwanted features such as bow, tilt and noise, and cleanmage function that removed spikes and streaks. The roughness washen measured for areas from 1 to 100 �m2 [8,15]. The calculationone was based on the equation below, where Zi represents theeight of the ith pixel, Zm was the mean image height and n was

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he total pixel in the Image8.

MS =√∑n

t=1(Zi − Zm)2

n

Fig. 1. Surface roughness for NaOH and xylanase treated hemp fibres(Xyl = xylanase).

Force curves collected from the JPK system were analyzed usingJPK DP, data processing software (version 4.0).

2.4. Statistical analysis

All experiments were replicated at least three times and resultswere expressed as mean value ± standard deviation. The statisticalanalyses of the data were conducted using the statistical softwarepackage SAS Version 9.4. It should be noted for the enzyme sys-tems, each system was compared to the control and not among eachother given the difference in activity for the enzymes and the dif-ferent mode of substrate attack. To identify significant differencesbetween mean values for control (raw) and a given system, theKruskal Wallis Test was applied to the data populations involved,with a 95% confidence level (p < 0.05).

3. Results and discussion

3.1. Enzyme and mercerized treated hemp fibres

The enzymes investigated were xylanase, xylanase + cellulaseand laccase. One of the main aims of this research study was toinvestigate individually and in combination, the effect of mer-cerization and enzymatic treatments on the surface topographyfeatures and adhesive properties. Mercerization is known to alterthe fibre macrostructure via hydrogen bonding disruption and net-work swelling [19]. Hence, mercerization was used to swell themacrostructure of the hemp fibres to allow greater penetration ofthe enzymes into the bulk material. The enzymes used in this workare known to degrade the hemicellulosic (xylanase) [20] and lignin(laccase) [21] fractions of the hemp fibres. However, to the bestof our knowledge this is the first study aiming to measure surfaceroughness and adhesion forces as a function of these treatmentsindividually and in combination. Fig. 1 compares the surface rough-ness for different spot areas for hemp fibres treated with Xylanase.

Hemp fibres treated with NaOH when compared to the con-trol/raw fibres, showed a significant (p < 0.05) reduction in surfaceroughness plausibly because of bundle disruption and networkswelling [10,19]. This pattern can be observed from the heightimages in Fig. 2, where the hemp fibres appeared to be de-fibrillated and bundles more exposed when exposed to NaOH.Xylanase is known to degrade the xylan network found predomi-nantly in the primary cell wall leaving exposed uniform crystallinecellulose [20,22]. For the hemp fibres studied here, xylanase treat-ment resulted in a reduction of surface roughness at 1, 25 and

2

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100 �m spot areas. Both of these observations were supportedby the increase in adhesion forces observed in Table 2, lending toan increase in surface hydrophilicity. The removal of the hemi-cellulosic content possibly exposed the more uniformly packed

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ig. 2. AFM height images (5 × 5 �m) of hemp fibres after different chemical and enreated hemp fibres.

ellulosic hydroxyl groups [20] which interacted more with theolar tip. The increase adhesion forces for the NaOH treated hempbres plausibly was due to the swelling of the structure of the hempbres which could have exposed a greater surface area of predom-

nantly cellulose while the xylanase treatment plausibly removedhe hemicellulosic content exposing the cellulose backbone. Whenxposed to base, the hydroxyl groups of hemp fibres were con-erted into the alkoxide form and the structure of the cellulosicetwork was changed, thereby allowing for better interaction withhe polar silicon nitride tip. Fibres treated with base has shownhanges in the form of cellulose and the bonding within the net-ork [4,13]. As a result, to balance this charge and to account for theisrupted bonding, the sodium ions would be solvated by the waterolecules present. Removal of hemicellulose exposes the cellulosic

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ackbone, which is more hydrophilic. This results in more moistureetention on the surfaces of the fibres. As a result, it is plausible toonclude that the increase in adhesion forces for xylanase treated

able 2ffect of different treatments on the adhesion force for hemp fibres.

Sample Force (nN) mean ± SD

Raw/control 9.34 ± 0.94a

NaOH 32.67 ± 6.48b

Xylanase 29.69 ± 6.90b

NaOH + xylanase 7.84 ± 0.76a

Xylanase + cellulase 13.45 ± 2.50c

NaOH + (xylanase + cellulase) 21.24 ± 4.24b

Laccase 10.85 ± 1.32c

NaOH + laccase 14.11 ± 2.00a

,b,c Treatments with each enzyme type were compared to the control/raw samplend among each other in the group. Treatments with the same denotation are notignificantly different at p < 0.05 from each other.

ic treatments. Images for (a) raw, (b) 10% NaOH, (c) xylanase and (d) NaOH xylanase

hemp fibres could be due to increased capillary and van der Waalsforces [17]. It is noted that adhesion force measurements can becomplimented with surface properties especially surface tensionor contact angle. As reported in an earlier publication from ourgroup, the use of the enzymes (xylanase, xylanase + cellulase andlaccase) removed the specific components targeted and resultedin significant changes in contact angle. In fact, the removal of thehemicellulosic content and lignin resulted in a significant reductionin the contact angle. This supports the claims made here, where it’sreported that the systems treated with enzymes exhibited moreinteraction with polar tip when compared to the control [11].

Force measurements did not detect any significant differencesbetween the sequential treatment (NaOH + enzyme) and the con-trol experiment. There was a reduction in surface roughness whencompared to the xylanase treated fibres but an increase when com-pared to the mercerized sample. A plausible reason for this couldbe the initial swelling of the structure, resulted in better enzymeaccess (larger surface area) hence aiding removal of a larger portionof the primary cell wall exposing the cellulose and lignin sec-ondary structure. Lignin is aromatic and much more heterogeneousin structure when compared to cellulose and would exhibit lessinteraction with the polar tip.

Hemp fibres treated with xylanase + cellulase and NaOH showedthe same trend as the hemp fibres treated with xylanase. The onlyexception was when the hemp fibres were treated with enzyme(xylanase + cellulase) the roughness (Fig. 3) increased relative tothe previous enzyme system (xylanase) and was characterized bylower adhesion forces (Table 2). de Vries and Visser [22] outlined

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the simultaneous degradation of the xylan backbone and celluloseusing an enzyme system of cellulases and xylanases. They con-cluded that the xylanases degraded the xylan network, exposingthe cellulosic bundles for the cellulases. In this study, the decrease

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lignocellulosic material to acids results in degradation of the pri-

ig. 3. Surface roughness for NaOH and Xyl Cel treated hemp fibres (Xylel = xylanase cellulase).

n surface roughness for the enzyme treatment relative to the rawample may plausibly be due to removal of random amorphousramework exposing the cellulose bundles [23].

This observation could explain the lower force detected forhe xylanase + cellulase treated hemp fibres when compared tohe xylanase treated hemp system. The removal of hemicellu-ose by the xylanase system exposed the accessible cellulose. Onhe other hand, the removal of hemicellulose + accessible cellu-ose (xylanase + cellulase) as reported by George et al. [11] exposedhe least polar crystalline cellulose. This explains the reductionf the adhesion force for the xylanase + cellulase treated hempbres because of the reduction in interaction with the polar sil-

con nitride tip [24]. Hemp fibres treated with NaOH and hybridNaOH + xylanase cellulase) systems were characterized by theame trend as observed for the previous system. Similar mode ofction (xylanase vs xylanase + cellulase) and enzyme dosage foroth systems may account for this. On the other hand, hemp fibres

n the presence of NaOH and the enzyme system, was character-zed by a significant (p < 0.05) increase in adhesion force relativeo the raw fibres. As mentioned before, NaOH possibly improvedhe effectiveness of the enzyme treatments because of disruptiono the fibre network and swelling which increases the surface area10]. As a result, more of the xylan network bonded to the cellu-osic bundles were significantly degraded (p < 0.05) increasing theurface forces [24].

Finally, hemp fibres treated with NaOH + xylanase when com-ared to NaOH + (xylanase + cellulase) showed a significantly lowerdhesion force. One plausible reason for this may be the greaterenetrating action of the NaOH + (xylanase + cellulase) systemhich removed the hemicellulosic content and exposed the acces-

ible cellulose. According to Mohanty et al. [1], accessible celluloses much more hydrophilic than the other components. Hence thisxplains the increased adhesion forces. Also, the arrangement of thebre component may have influenced the reaction as well. Specif-

cally, removal of hemicellulose could have exposed the randomlyrranged lignin fragment which will in turn have less of an interac-ion with the polar probe for the NaOH + xylanase treated fibres. Its worth noting that the specific attack of these enzymes are influ-nced by the geometrical arraignment of the components withinach fibril [11] and the access of these enzymes to the targetedomponent [1].

The roughness data and force measurements for the hemp fibresreated with Laccase are shown in Fig. 4 and Table 2, respectively.

adhavi and Lele [21] and Kunamneni et al. [25] studied the effectf Laccase during the degradation of recalcitrant lignin and con-

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luded the removal of lignin exposes the cellulose backbone ofhe lignocellulosic material. The main aim in this study was to usehe Laccase system to remove the lignin fraction from the hemp

Fig. 4. Surface roughness for NaOH and laccase treated hemp fibres (Lac = laccase).

fibres and investigate how this impacted the surface roughness andbonding at the uppermost 5 nm.

Interestingly, hemp fibres treated with laccase were charac-terized with no significant increase in surface forces (p < 0.05) asshown in Table 2 and when compared to the raw fibres, exhib-ited similar surface roughness. One plausible reason for this maybe the hierarchical nature of the hemp fibres, with the lignin pri-marily confined to the inner regions of the primary cell wall andlumen [8]. In a similar study, Balnois et al. [8] demonstrated thatthe lignin fraction of flax fibres were intact because of its geomet-rical arrangement in the lumen of the fibres and resistance to thechemical methods studied. On the other hand, the hybrid treatment(NaOH + laccase) resulted an increase in the adhesion force, indi-cating an increase in surface polarity. The use of the base disruptedthe fibre network allowing the laccase to access the lignin network.The degradation of lignin, which acts as an adhesive medium for theentire macrostructure of the fibres, exposed the hemicellulosic andcellulosic content, hence increasing the surface polarity which cor-relates to a higher interaction with the tip. This observation was alsosupported by the significant (p < 0.05) reduction in surface rough-ness for the hybrid treatment because the removal of the ligninfound in the intercellular regions resulted in a smoother surface.

3.2. Hemp fibres treated with derivative of sulfonic acid

The hypothetical reactions for the two sulfonic acids with amolecule of �-d-glucose are highlighted in Fig. 5. The reactionmechanism involves the production of the acid chloride deriva-tive of each acid, given that in this form the reaction is kineticallyfavoured because Cl atoms are better leaving groups that thehydroxyl groups [25]. In the second step, the acid chloride reactswith one of three hydroxyl groups (C-2, C-3 and C-6) found on eachmolecule of �-d-glucose of the cellulose exposed on the surface.The reaction may proceed either with the presence of cellulosic orhemicellulosic hydroxyl groups. Natural fibres possess 4% of thetotal hydroxyl groups on the surface [25]. These are the majorcontributors to poor interfacial adhesion to hydrophobic basedmatrices [1]; hence, these acids were grafted onto the surfaceto improve the hydrophobicity. Pyridine was used as a catalystbecause Bledzki et al. [26] showed when flax fibres were reactedwith acetic anhydride, that in the absence of a catalyst, esterifica-tion does not result in uniform product.

The influence of the two acids on the surface roughness for thetreated hemp fibres is given in Fig. 6. As expected with any acidsystem as demonstrated in the bioethanol area of study, exposing

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mary cell wall [26–28]. This degradation corresponds to a cleanersurface with exposed cellulosic OH groups for grafting of the sul-fonic derivatives. A reduction in the forces on the surface signalled a

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F cid (AT3S) with a �-d-glucose molecule hypothetically found on the surface of the hempfi s it is produced.

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Table 3Effect of different treatments on the adhesion force for hemp fibres treated with thesulfonic acids.

Sample Force (nN) mean ± SD

Raw 9.34 ± 0.94a

A2S 8.06 ± 0.77a

AT3S 7.38 ± 0.78b

ig. 5. Reactions for aniline-2-sulfonic acid (A2S) and 4-aminotoulene-3-sulfonic abres. Pyridine was used to drive the reaction forward by chelating with the H-Cl a

eduction in the free OH groups on the surface. As shown in Table 3,T3S significantly (p < 0.05) reduced the surface forces plausiblyue to the reaction outlined in Fig. 5.

Based on the main aim of this part of the study, it has beenemonstrated that a water soluble and organic solvent free sul-onic acid system can be used to enhance hemp fibres for compositepplications. In fact, it was demonstrated that the reaction of theseoieties results in significant reduction in adhesion force between

he polar tip and the sample. This signals that the overall surface

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olarity has decreased. This study will compliment and allow for aetter understanding of the influence of any given chemical methodn the microstructure and physical properties of hemp fibres.

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ig. 6. Surface roughness for hemp fibres treated with derivatives of sulfonic acidsA2S = aniline-2-sulfonic acid and AT3S = 4-aminotoulene-3-sulfonic acid).

a,b Treatments with the same denotation are not significantly different at p < 0.05.

4. Conclusion

This study demonstrated, through the use of a colloidal probethat hemicellulases can be used to reduce the surface roughnesswhile increasing the adhesion forces on the surface. Mercerizationtreatment prior to enzymatic treatment plausibly resulted inbetter enzyme access to the macrostructure of the hemp fibres.This was especially noticeable for hemp fibres treated with laccasebecause NaOH allowed access to the inner structure that is ligninfilled. Grafting of sulfonic acids onto the surfaces of the hempfibres resulted in lower adhesion forces when compared to theraw fibres. This may be due to a reaction involving the hydroxylgroups on the surface of the fibres, exposing the hydrophobicaromatic rings of the acids to the polar silicon nitride tip. Thisstudy highlights that in order for natural fibres to be a competitiveoption for polymer reinforcement, the use of enzymes, a green

f chemically and enzymatically treated hemp fibres using atomicoi.org/10.1016/j.apsusc.2014.06.080

and specific method of modification can be used to selectivelyremove hygroscopic hemicellulose. Similarly, the use of novelchemistry involving cheap and reactive sulfonic acids can be used

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o significantly reduce the surface polarity via grafting of theromatic artefacts onto the surfaces of the fibres.

cknowledgements

The authors acknowledge the financial support provided byhe Natural Sciences and Engineering Research Council of CanadaNSERC), Alberta Innovates-Bio solutions Corporation (AI- BIo)grant no. AF 770) and the Alberta Livestock and Meat AgencyALMA) (grant no. AF 607). The authors are grateful to Albertaiomaterials Development Centre and Novozymes for kindly sup-lying the hemp fibres and enzymes, respectively, needed for thistudy. The experimental assistance provided by Shiau-Yin Wu athe Integrated Nanosystems Research Facility at the University oflberta is much appreciated.

eferences

[1] A.K. Mohanty, M. Misra, G. Hinrichsen, Biofibres, biodegradable polymers andbiocomposites: an overview, Macromol. Mater. Eng. 276 (2000) 1–24.

[2] N.M. Barkoula, S.K. Garkhail, T. Peijs, Biodegradable composites based onflax/polyhydroxybutyrate and its copolymer with hydroxyvalerate, Ind. CropsProd. 31 (2010) 34–42.

[3] S.K. Chattopadhyay, S. Singh, N. Pramanik, U.K. Niyogi, R.K. Khandal, R. Uppaluri,A.K. Ghoshal, Biodegradability studies on natural fibers reinforced polypropyl-ene composites, J. Appl. Polym. Sci. 121 (2011) 2226–2232.

[4] L.Y. Mwaikambo, M.P. Ansell, The effect of chemical treatment on the propertiesof hemp, sisal, jute and kapok for composite reinforcement, Angew. Makromol.Chem. 272 (1999) 108–116.

[5] A. Bledzki, A. Mamum, M. Gabor, V. Gutowski, The effects of acetylation onproperties of flax fibre and its polypropylene composites, Polym. Lett. 2 (2008)413–422.

[6] A. Anyakora, O. Abukare, Effect of silane treatment on the impact strength prop-erties of oil pal empty fruit bunch fiber reinforced polyester composites, WorldJ. Eng. Pure Appl. Sci. 1 (2) (2001) 40–46.

[7] Mariano Pracella, Md. Minhaz-Ul Haque, Vera Alvarez, Functionalization, com-patibilization and properties of polyolefin composites with natural fibers,Polymers 2 (2010) 554–574.

Please cite this article in press as: M. George, et al., Characterization oforce microscopy and spectroscopy, Appl. Surf. Sci. (2014), http://dx.d

[8] E. Balnois, F. Busnel, C. Baley, Y. Grohens, An AFM study of the effect of chemicaltreatments on the surface microstructure and adhesion properties of flax fibres,Compos. Interfaces 14 (2007) 715–731.

[9] S.O. Han, H.Y. Choi, Morphology and surface properties of natural fibre treatedwith electron beam, Microsc.: Sci., Technol., Appl. Educ. 3 (2010) 1880–1887.

[

PRESSScience xxx (2014) xxx–xxx 7

10] B. Wang, M. Sain, K. Oksman, Study of structural morphology of hemp fiberfrom the micro to the nanoscale, Appl. Compos. Mater. 14 (2007) 89–103.

11] M. George, G.P. Mussone, C.D. Bressler, Surface and thermal characterization ofnatural fibres treated with enzymes, Ind. Crops Prod. 53 (2014) 365–373.

12] Y. Karaduman, D. Gokcan, L. Onal, Effect of enzymatic pretreatment on themechanical properties of jute fiber-reinforced polyester composites, J. Compos.Mater. 47 (2013) 1293–1302.

13] S. Kalia, B.S. Kaith, I. Kaur, Pretreatments of natural fibers and their applicationas reinforcing material in polymer composites—a review, Polym. Eng. Sci. 49(2009) 1253–1272.

14] V. Tserki, C. Panayiotou, N.E. Zafeiropoulos, A study of the effect of acetylationand propionylation on the interface of natural fibre biodegradable composites,Adv. Compos. Lett. 14 (2005) 65–71.

15] Q. Zhong, D. Inniss, K. Kjoller, V.B. Elings, Fractured polymer silica fiber sur-face studied by tapping mode atomic-force microscopy, Surf. Sci. 290 (1993)L688–L692.

16] Y. Seo, W. Jhe, Atomic force microscopy and spectroscopy, Rep. Prog. Phys. 41(2008) 1–23.

17] A. Noy, D. Vezenov, C. Lieber, Chemical force microscopy, Annu. Rev. Mater. Sci.27 (1997) 381–421.

18] B. Cappella, G. Dietler, Force–Distance Curves by Atomic Force Microscopy, vol.34, Elsevier, 1999, pp. 1–104.

19] J. Liu, F. Wang, Influence of mercerization on micro-structure and propertiesof kapok blended yarns with different blending ratios, J. Eng. Fibers Fabr. 6 (3)(2011) 63–68.

20] D.F. Bateman, H.G. Basham, Degradation of Plant Cell Walls and Membranes byMicrobial Enzymes, 1976, pp. p. 355.

21] V. Madhavi, S.S. Lele, Laccase, Properties and applications, Bioresources 4(2009) 1694–1717.

22] R.P. de Vries, J. Visser, Aspergillus enzymes involved in degradation of plantcell wall polysaccharides, Microbiol. Mol. Biol. Rev. 65 (2001), 497±.

23] R.P. de Vries, H.C.M. Kester, C.H. Poulsen, J.A.E. Benen, J. Visser, Synergybetween enzymes from Aspergillus involved in the degradation of plant cellwall polysaccharides, Carbohydr. Res. 327 (2000) 401–410.

24] A. Pietak, S. Korte, E. Tan, A. Downard, M.P. Staiger, Atomic force microscopycharacterization of the surface wettability of natural fibres, Appl. Surf. Sci. 253(2007) 3627–3635.

25] A. Kunamneni, F.J. Plou, A. Ballesteros, M. Alcalde, Laccases and their applica-tions: a patent review Recent Patents on Biotechnology, 2, 2008, pp. 10–24.

26] A.K. Bledzki, A.A. Mamun, M. Lucka-Gabor, V.S. Gutowski, The effects of acety-lation on properties of flax fibres and its polypropylene composites, Exp. Polym.Lett. 2 (6) (2008) 413–422.

27] C.S.R. Freire, A. Silvestre, A. Gandini, C. Neto, New materials from cellulose

f chemically and enzymatically treated hemp fibres using atomicoi.org/10.1016/j.apsusc.2014.06.080

fibres. A contribution to the implementation of the integrated biorefinery con-cept, O Papel 72 (2011) 91–99.

28] J. Iranmahboob, F. Nadim, S. Monemi, Optimizing acid-hydrolysis: a criticalstep for production of ethanol from mixed wood chips, Biomass Bioenergy 22(2002) 401–404.