Journal of Environmental Chemical Engineering 4 (2016) 1743–1752

The development of antibacterial and hydrophobic functionalities innatural fibers for fiber-reinforced composite materials

Kamini Thakura, Susheel Kaliab,*, B.S. Kaithc, Deepak Pathaniaa, Amit Kumara,Pankaj Thakura, Chelsea E. Knitteld, Caroline L. Schauerd, Grazia Totaroe

aDepartment of Chemistry, Shoolini University, Solan, H.P., IndiabDepartment of Chemistry, Army Cadet College Wing, Indian Military Academy, Dehradun, 248007 UK, IndiacDepartment of Chemistry, Dr. B.R. Ambedkar National Institute of Technology, Jalandhar, 144011 Pb., IndiadDepartment of Materials Science and Engineering, Drexel University, Philadelphia, PA 19047, USAeDipartimento di Ingegneria Civile, Chimica, Ambientale e dei Materiali, Università di Bologna, Via Terracini 28, Bologna 40131, Italy

A R T I C L E I N F O

Article history:Received 29 November 2015Received in revised form 21 January 2016Accepted 26 February 2016Available online 3 March 2016

Keywords:BiograftingFibersLaccaseAntibacterial propertyBiocomposites

A B S T R A C T

Green surface modification of coconut fibers was performed using laccase biografting of eugenol for thedevelopment of antibacterial functionalities and fiber-reinforced polymer composites. Fourier transforminfrared analysis, X-ray diffraction and surface morphology of grafted fibers were utilized to confirm thebiografting of eugenol. Antibacterial, hydrophobicity and thermal properties were evaluated by colonyforming unit (CFU) method, moisture absorption and thermogravimetric analysis, respectively. Thegrafted surfaces were found to be antibacterial, hydrophobic and thermally more stable. Grafted fiberswere reinforced in a poly(butylene succinate) matrix to improve the mechanical properties of thebiocomposites. The mechanical properties were improved even with a low content of biografted coconutfibers.

ã 2016 Elsevier Ltd. All rights reserved.

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Journal of Environmental Chemical Engineering

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1. Introduction

Natural fiber reinforced polymer composites are one of themost rapidly growing fields of research in composite science andtechnology [1]. Natural fibers usually display poor microbial andmoisture resistance therefore biografting is utilized as a novelgreen method to impart antibacterial and hydrophobic properties.Enzyme-assisted surface modification of lignocellulosic fibers canbe used to improve fibers for composite materials and otherindustrial applications [2–6]. In natural fibers, cellulose is themajor constituent with a percentage of lignin varying from 0–40%of the total material [7]. Biografting of organic molecules is mainlytargeted at the lignin leaving most of the surface of fibersunmodified, if the percentage of lignin is very low. Biografting aphenolic group makes natural fibers more hydrophobic andtherefore more compatible with synthetic polymer matrices forthe development of composite materials [8].

* Corresponding author.E-mail address: susheel.kalia@gmail.com (S. Kalia).

http://dx.doi.org/10.1016/j.jece.2016.02.0322213-3437/ã 2016 Elsevier Ltd. All rights reserved.

Attempts have been made to develop antibacterial andhydrophobic lignocellulosics through biografting methods[8–14]. Laccase-assisted coating of flax fibers with ferulic acidand hydroquinone resulted in enhanced antimicrobial activity [13].Laccase assisted biografting of ferulic acid was also used for surfacemodification of sisal pulp. Effect of the laccase–ferulic acid systemon the refined and unrefined pulp fibers was investigated.Increased grafting and handsheets with improved strengthproperties were obtained with refining before the enzymetreatment [15]. Surface functionalization of sisal and flax pulpfibers using the laccase induced grafting has been performed usingsimple phenols like syringaldehyde, acetosyringone, p-coumaricacid, coniferaldehyde, sinapaldehyde, ferulic acid and sinapic acid[16]. Dong et al. [17] used laccase catalyzed grafting of dodecylgallate onto the jute fibers, increasing their hydrophobicity, andprepared polypropylene composites with modified jute fiber.Mechanical property enhancement was observed using thebiografted jute fibers.

Coconut fibers are lignin-rich fibers and can easily be modifiedthrough laccase biografting. The main objective of this study is toinvestigate laccase biografting of phenolic compounds on coconutfibers and their use as a novel reinforcing material.

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2. Experimental

2.1. Materials

Coconut fibers were extracted from the skin of coconut palm(Cocos nucifera), which was purchased from a local market of Solan(Himachal Pradesh, India). Extracted fibers were washed with milddetergent followed by distilled water to remove the water-solubleimpurities. Clean fibers were then extracted with acetone in aSoxhlet extractor for 24 h to remove other impurities. Most of thecoconut fibers used in present study were 6–15 cm length with anaverage diameter of 0.25 mm. Laccase (from Trametes versicolor)(98% pure) and eugenol (EG) (99% pure) were purchased fromSigma–Aldrich. Citric acid (Himedia, extra pure), sodium citrate(Himedia, extra pure), nutrient broth (Himedia, M002), nutrientagar (Himedia, M001), and poly(butylene succinate) (PBS)(Sigma–Aldrich) were used as received.

2.2. Laccase biografting of eugenol

Biografting of eugenol was carried out in an Erlenmeyer flaskcontaining 40 mM citrate buffer (pH 4), 3.5% (w/w) eugenol, 40 Ulaccase and coconut fibers (200 mg). This reaction mixture wasincubated at 50 �C for 12 h with constant rotation at a rate of30 rpm. A control reaction was also used under identical conditionsin the absence of enzyme. Modified fibers were washed thoroughlywith distilled water until a neutral pH and then Soxhlet extractedwith acetone for 12 h to remove the fraction of unreacted EG. Fibersamples were then dried in a vacuum oven at 40 �C to a constantweight. Optimization of reaction conditions were determined byvarying three reaction parameters such as enzyme concentration,phenol concentration and incubation period [18].

Fig 1. Schematic representation of preparation of b

2.3. Quantitative analysis of biografting

Quantitative analysis of biografted coconut fibers was deter-mined by the weighing method. The percentage of biografting wascalculated by using the following equation:

Biografting %ð Þ ¼ w2 � w1

w1� 100

where w2is the weight of biografted fibers and w1is the finalweight of control sample [17,18].

2.4. Characterization techniques

FTIR technique was used to identify the chemical groups ofunknown composition and intensity of absorption spectraassociated with molecular composition of the chemical group.FTIR spectra of coconut fibers were taken with KBr pellets ona PerkinElmer RXI Spectrophotometer over a range of 400–4000 cm�1. Surface morphology of coconut fibers was examined byusing a Jeol JSM-6610LV electron microscopy machine. Thermalbehavior of fibers was studied using a PerkinElmer TGA in an inertatmosphere from 50 to 800 �C at a heating rate of 10 �C/min. XRDstudies were done on a Brucker D8 Advance X-ray diffractometerunder ambient conditions. Crystalline and amorphous material innatural fibers is represented by peak intensities at 22� and 18�

respectively. Percentage crystallinity (% Cr) was calculated asfollows:

% Cr ¼ I22I22 þ I18

� 100

where I22 and I18 are the crystalline and amorphous intensities at2u scale close to 22� and 18� respectively [19,20].

iocomposites reinforced with biografted fibers.

K. Thakur et al. / Journal of Environmental Chemical Engineering 4 (2016) 1743–1752 1745

2.5. Antibacterial property

The antibacterial property of coconut fibers was studied bycolony forming unit method. Coconut fibers (0.05 g) were added to10 mL of nutrient broth followed by the addition of 1 mL inoculatedbacterial culture and incubated at 37 �C. The colonies were countedafter 24–48 h periods by seeding the aliquot of incubated sampleon nutrient agar plates and compared with the control. TheCFU mL�1 was calculated as follows [18,20]:

CFUmL

¼ Colony count on plate � Dilution factorAmount of sample poured on plate

Fig. 2. Mechanism of laccase-catalyzed bio

2.6. Moisture absorption study

The moisture absorption study of coconut fibers was carried outin a humidity chamber. Coconut fibers were dried at 80 �C for 4 hand then exposed to 55% and 75% relative humidity at 23 �C in ahumidity chamber. The weight of fibers was recorded every houruntil a constant weight was obtained. The increase in weight offibers was calculated as follows [18,20].

Weight gain% ¼ wf � wi

wi� 100

where wi and wf are the initial and final weight of coconut fibers,respectively.

grafting of eugenol on coconut lignin.

Fig. 3. Optimization of reaction parameters: (a) phenol concentration, (b) laccase concentration and (c) incubation period.

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2.7. Synthesis of biocomposites reinforced with coconut fibers

The biocomposites were synthesized by mixing the dried poly(butylene succinate) and 3 cm long coconut fiber samples (0.5 wt%and 1 wt%) in the mold cavity of a hot press. This mold cavity wasthen heated in three cycles; room temperature to 70 �C for 15 min,

Fig. 4. FTIR spectra of raw and

71–100 �C for 30 min and 101–125 �C for 20 min. It was thenallowed to cool naturally to room temperature. The risk ofstructural disorder was avoided by maintaining the constantpressure (100 MPa) during thermal and cooling treatments. Theresulting composite material (a rectangular strip of 100 � 10 � 3mm3 dimensions) was used for tensile and flexural testing in a

biografted coconut fibers.

Fig. 5. XRD of raw and biografted coconut fibers.

Table 1Crystallinity of raw and biografted coconut fibers.

Sr. no. Fibers At 2u scalea % Cr

I22 I18

1 Coconut fibers (CF) 1177 1953 62.42 CF-g-EG(1) 1384 854 61.83 CF-g-EG(2) 1516 915 62.44 CF-g-EG(3) 1550 974 61.4

a I22 and I18 represent the crystalline and amorphous intensities at 2u scale closeto 22� and 18�, respectively.

K. Thakur et al. / Journal of Environmental Chemical Engineering 4 (2016) 1743–1752 1747

universal testing machine (5 kN, H5KT Tinius Olsen) at roomtemperature with a crosshead speed of 5 mm/min [18,20]. Fig. 1shows the schematic representation of fabrication of biograftedfiber reinforced biocomposites.

3. Results and discussion

3.1. Mechanism

The proposed mechanism for laccase catalyzed biografting ofeugenol onto lignin is shown in Fig. 2. The mechanism of laccasecatalyzed biografting of phenolic compounds on lignocellulosicsdepends upon two aspects; stability of phenoxy radical andgeneration of mediator species [21]. The laccase assisted mecha-nism starts with the formation of a phenoxy radical cation. Theenergy of the highest occupied molecular orbital gives the mostinformation about the ionization potential of the molecule. Thisionization potential supports the presence of one methoxy groupat ortho-position. Due to the electron-donating effect of themethoxy group, the release of an electron becomes easy andformation of phenoxy radical cation takes place by hydrogenabstraction. Electron donating groups at ortho and para positionswill decrease the bond dissociation energy and electron with-drawing groups at the same positions will increase the bonddissociation energy. Methoxy group is at ortho-position in eugenol,which comforts the formation of phenoxy radical through therelease of a proton, a rate determining step [22–24]. The phenoxyradical covalently bonded to the lignin radical and results in astabilized structure.

3.2. Optimization of reaction parameters for biografting

Biografting is a way of tailoring the surface of lignocellulosics toproduce required functionalities under optimum conditions. Threeparameters were optimized to study the extent of laccase-catalyzed biografting onto coconut fibers, i.e., phenol concentra-tion, laccase concentration and incubation period. In this study,

phenol concentration was varied as 2.5%, 3.5%, 4.5% and 5.5% (w/w). Maximum biografting (21.4%) was observed at 4.5% (w/w) ofphenol concentration [CF-g-EG(1)] (Fig. 3a). The percentage ofbiografting was increased with the increase in the concentration ofphenol. The enzymatic reaction mechanism demonstrated anincrease in reaction rate with an increase of reactant or substrate toform the enzyme substrate complex until it approaches itsmaximum velocity. The formation of phenoxy radicals in thereaction media was increased with an increase in the concentra-tion of phenol, which speeds up the covalent coupling of phenoxyradicals with lignin radicals until maximum biografting occurs andthereafter self-polymerization reaction occurs. This causes thedeposition of self-polymerized units onto fiber surface to make itrough and coated [25–27]. The effect of enzyme concentration onbiografting was studied at different enzyme concentrations, i.e.,20 U, 40 U, 60 U and 80 U. Maximum biografting (17.8%) wasattained at 40 U of enzyme concentration [CF-g-EG(2)] (Fig. 3b). Alow concentration of enzyme was able to catalyze the reactioneffectively. More and more phenoxy radicals were formed with theinitial increase in laccase concentration. The lignin degradationand generation of different products in reaction media occur at ahigher laccase concentration, which results in decreased percent-age biografting [28]. The effect of incubation period on the activityof laccase was studied at 12 h, 24 h and 36 h. Maximum percentagebiografting (21.5%) was observed at 24 h of incubation period[CF-g-EG(3)] (Fig. 3c). Maximum biografting was due to the growthof biografting at a longer incubation period of 24 h. After optimizedincubation period, the laccase-mediated depolymerization reac-tions turned to be predominant. Inactivation of enzyme occurred ata higher incubation time period and results in decreasedpercentage biografting [28–31].

3.3. FTIR spectra

Laccase catalyzed functionalization of eugenol onto coconutfiber surface has been characterized by FTIR spectra (Fig. 4). IRspectra of raw and biografted coconut fibers show the broad bandat 3400 cm�1 and 3370 cm�1, respectively, which is characteristicof O��H stretching corresponding to its structure and position insample [32]. The bands at 2923 cm�1 and 2924 cm�1 were due toC-H stretching in the aliphatic region [33]. The bands due to thecarbonyl group were observed at 1737 cm�1 and 1735 cm�1 in thecase of raw and biografted coconut fibers, respectively. Bands at1638 cm�1 and 1598 cm�1 were the characteristic of eugenol,which represent the presence of C¼C (alkene) and C��C¼Csymmetric stretching. These bands were completely absent inthe IR spectra of raw coconut fibers [33–35]. Bands at 1510 cm�1,1463 cm�1 were due to the C¼C aromatic vibrations. The bands at1353 cm�1 and 1231 cm�1 correspond to ��CH3 and C��O

Fig. 6. SEM of raw and biografted coconut fibers: (a) coconut fibers, (b) CF-g-EG(1), (c) CF-g-EG(2), and (d) CF-g-EG(3).

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stretching, respectively [33]. The band at 1142 cm�1 was due to thepresence of ��OCH3 (ether linkage). The bands observed in therange of 913 cm�1 and 597 cm�1 were in agreement with the IRspectra of eugenol [34].

3.4. X-ray diffraction

The X-ray diffraction pattern of coconut fiber and biograftedcoconut fibers is depicted in Fig. 5. It has been observed thatcrystallinity of coconut fibers is not disturbed due to thebiografting process. The relative intensities and % crystallinity ofraw and modified fibers are given in Table 1. Biografting of EG ismainly targeted at lignin and cannot be applied to other majorconstituents of the lignocellulose material such as cellulose,thereby leaving most of the lignocellulosic surface unaffected. Anegligible change in the crystallinity of coconut fibers was noticedbecause coconut fiber is a lignocellulosic complex, whosecrystallinity is affected by the hydroxyl groups [36]. A little change

Fig. 7. TGA of raw and

in crystallinity was due to change in intensity and position ofhydroxyl group due to laccase-catalyzed biografting.

3.5. Surface morphology

Surface morphology of coconut fiber and biografted coconutfibers are depicted in Fig. 6. Fig. 6a shows the regular smoothsurface of the coconut fiber. In Fig. 6b, coating of biografted EG onthe surface of coconut fibers can be seen. The availability ofPh��OH group increases with the increase in phenolic concentra-tion and results in the generation of phenoxy radicals by oxidation[26]. This was followed by biografting of EG on fiber surface, whichgives the surface a rough and coated appearance. Fig. 6c and dindicates that the surface of biografted coconut fiber was coated byirregularly arranged flakes or particulates, which may result inbetter compatibility with the polymer matrix. This attachment ofphenolic units onto the surface of fiber was due to the covalentbonding during the process of biografting [31]. A complete change

biografted fibers.

Fig. 8. Antibacterial behavior against E. coli: (a) control, (b) coconut fibers, (c) biografted coconut fibers and S. aureus: (d) control, (e) coconut fibers, (f) biografted coconutfibers.

K. Thakur et al. / Journal of Environmental Chemical Engineering 4 (2016) 1743–1752 1749

in the morphology of coconut fibers was observed due tobiografting of EG on surface of fibers.

3.6. Thermogravimetric behavior

Fig. 7 shows thermogravimetric graphs of raw and biograftedcoconut fibers. It has been observed that biografting of EG oncoconut fibers causes a considerable change in thermal behavior.Raw and biografted coconut fibers were observed with twodecomposition stages. In case of raw coconut fiber, CF-g-EG(1),CF-g-EG(2) and CF-g-EG(3), the first decomposition stage wasobserved up to 200 �C (3.8% weight loss), 231 �C (3.3% weight loss),218 �C (5.8% weight loss) and 282 �C (7.9% weight loss), respective-ly. The first stage decomposition was attributed to the dehydrationprocess, i.e., loss of water by evaporation. The maximum weightloss was observed in second stage decomposition of raw andbiografted fibers. The second decomposition stage of raw coconutfibers, CF-g-EG(1), CF-g-EG(2) and CF-g-EG(3) occurred in therange of 220–338 �C (61.3% weight loss), 231–454 �C (65.1% weightloss), 218–474 �C (65.4% weight loss) and 282–428 �C (57.3% weightloss), respectively. The second stage decomposition represents thedegradation of lignin and breaking of covalent bonding between

Fig. 9. Moisture absorption study of raw and biograf

lignin and EG. Biografting of EG onto coconut fibers throughcovalent bonding contributed towards the thermal stabilityof fibers. The increased final decomposition temperature inbiografted fibers confirms the enhancement in thermal stabilityof the fibers due to biografting.

3.7. Antibacterial property

The antibacterial property of biografted coconut fibers wasexamined against the gram negative E. coli and gram positive S.aureus bacteria. The effectiveness in inhibiting the bacterial growthby biografted coconut fibers in comparison to unmodified coconutfibers is depicted in Fig. 8. Biografting of coconut fibers results in aconsiderable decrease in viable microbial count against the E. coliin comparison to S. aureus because of presence of thickpeptidoglycan layer in S. aureus. The CFU/ml was 44 �109 for E.coli and 224 �109 for S. aureus. Antibacterial activity of phenoliccompounds depends on the hydroxyl group and delocalizedelectron system. The phenolic compounds permit the exchange ofprotons across the cell membrane and decrease the gradient,which is followed by the loss of energy and nutrition flow andresults in the death of cell [37]. In case of EG, hydrophobicity also

ted coconut fibers: (a) 55% RH, and (b) 75% RH.

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plays a vital role in the effectiveness of antibacterial activity.Hydrophobicity changes the cell permeability by increasing theproton passive flux across the cell membrane. This is followed byexcessive loss of cell contents, which leads to cell death [38,39].Cytotoxicity of EG is due to its phenoxy radicals, which alter thelipid membrane behavior to kill the cell [40]. EG biografted coconutfibers have effective antibacterial characteristics because of thelipophilic nature of EG towards bacterial cell membrane, whichalters the membrane permeability.

3.8. Moisture absorption study

Presence of hydroxyl groups on the surface of natural fibersmakes them hydrophilic by nature. Covalent bonding of ahydrophobic group with a phenoxy radical creates hydrophobicfibers. Successful binding of EG onto coconut fiber has beenconfirmed by FTIR. The hydrophobicity has been developed incoconut fibers through the biografting process (Fig. 9a and b). Inmoisture absorption, there is diffusion of moisture from the outeratmosphere to the surface of fibers followed by diffusion from thesurface to the individual fiber surface. Finally, diffusion to theinterior of fiber took place [41]. Due to the coating of a hydrophobiccompound onto the fiber, surface diffusion of moisture isprohibited. Biografting of EG has greatly affected the fiber surfacefunctionalization and hence moisture absorption capacity. Phe-noxy radicals were generated during the biografting process,which undergo coupling and polymerization reactions in parallel

Fig. 10. Tensile load–strain curves of neat PBS, PBS + coconut fibers and PBS + CF-g-EG:

strength.

on the surface of fibers and block the various moisture vulnerablesites [17].

3.9. Mechanical properties of synthesized biocomposites

The effect of biografting of EG was studied based on themechanical properties of biocomposites depicted in Fig. 10. Thebiocomposites reinforced with biografted coconut fibers requiredmore tensile load to break in comparison to neat polymer and rawcoconut fiber reinforced biocomposites (Fig. 10a and b). The effectof fiber content on the mechanical properties of biocomposites isdepicted in Fig. 10c. The biocomposite reinforced with 1%biografted fibers showed maximum tensile strength (34.96 MPa)in comparison to biocomposites reinforced with 0.5% biograftedfibers (23.18 MPa), 1% raw fibers (13.64 MPa) and 0.5% raw fibers(10.48 MPa). Similar results were observed in case of flexuralstrength of biocomposites (Fig. 11). The biocomposites reinforcedwith 1% biografted fibers showed maximum flexural strength(22.08 MPa) followed by biocomposites reinforced with 0.5%biografted fibers (14.18 MPa), 1% raw fibers (8.9 MPa) and 0.5%raw fibers (7.68 MPa). Tensile and flexural strengths were found toimprove in case of reinforcements with biografted fibers. In case offiber contents, there was a huge improvement in mechanicalproperties even with the slight enhancement in biografted fibercontents.

The biografting of eugenol incorporates hydrophobicity incoconut fibers, which in turn improved the mechanical properties

(a) 0.5% fiber content, (b) 1% fiber content and (c) effect of fiber content on tensile

Fig. 11. Flexural load–strain curves of neat PBS, PBS + coconut fibers and PBS + CF-g-EG: (a) 0.5% fiber content, (b) 1% fiber content and (c) effect of fiber content on flexuralstrength.

Fig. 12. Fractured surface morphology of biocomposites: (a) coconut fiber/PBS biocomposites, and (b) CF-g-EG/PBS biocomposites.

K. Thakur et al. / Journal of Environmental Chemical Engineering 4 (2016) 1743–1752 1751

of the biocomposites due to the superior interphase compatibility,interlocking and even stress distribution between fibers andpolymer matrix (Fig. 12) [42]. Three different heating cycles incomposite manufacturing also contributed towards the desirablesoaking of fibers with polymer resin for a superior interphase andmechanical properties.

4. Conclusions

Surface modification of coconut fibers was achieved success-fully through laccase biografting of eugenol. Through laccasebiografting, antibacterial and hydrophobic properties are

developed in the coconut fibers. Biografted fibers can successfullybe used as reinforcing materials for the development of polymercomposites. The mechanical properties of poly(butylene succinate)composites were improved with the small reinforcement percent-age of biografted fibers.

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