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Surface Modification of Sisal FibersUsing Cellulase and Microwave-AssistedGrafting: A Study of Morphology,Crystallinity, and Thermal StabilitySusheel Kalia a & Shalu Vashistha ba Department of Chemistry , Shoolini University, Bajhol, Dist. ,Solan , (H.P.) , Indiab Department of Chemistry , Singhania University , Pacheri Bari ,IndiaAccepted author version posted online: 07 May 2012.Publishedonline: 24 Sep 2012.

To cite this article: Susheel Kalia & Shalu Vashistha (2012) Surface Modification of Sisal Fibers UsingCellulase and Microwave-Assisted Grafting: A Study of Morphology, Crystallinity, and Thermal Stability,International Journal of Polymeric Materials and Polymeric Biomaterials, 61:14, 1130-1141, DOI:10.1080/00914037.2011.617342

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Surface Modificationof Sisal Fibers UsingCellulase andMicrowave-AssistedGrafting: A Study ofMorphology, Crystallinity,and Thermal Stability

Susheel Kalia1 and Shalu Vashistha2

1Department of Chemistry, Shoolini University, Bajhol, Dist., Solan, (H.P.), India2Department of Chemistry, Singhania University, Pacheri Bari, India

Surface modification of sisal fibers (Agave sisalana) using microwave-radiation-inducedgrafting and cellulase has been carried out in this paper. The modified fibers werecharacterized by various techniques to determine their morphology, crystallinity, andthermal stability. It has also been found that microwave-radiation-assisted graftcopolymerization results in enhanced thermal stability and has a diminutive effect onthe crystalline structure of sisal fibers. Cellulase enzyme treatment also results inenhanced thermal stability with negligible change in percentage crystallinity.

Keywords cellulase enzyme, crystallinity, microwave radiations, morphology, sisal,thermal stability

Received 23 May 2011; accepted 21 August 2011.The authors are highly thankful to Sisal Research Station (ICAR), Bamra, Dist.Sambalpur, Orrisa (India), for providing sisal fibers.Address correspondence to Susheel Kalia, Department of Chemistry, ShooliniUniversity, Bajhol-173229, Dist., Solan, (H.P.), India. E-mail: susheel_kalia@yahoo.com

International Journal of Polymeric Materials, 61:1130–1141, 2012

Copyright # Taylor & Francis Group, LLC

ISSN: 0091-4037 print=1563-535X online

DOI: 10.1080/00914037.2011.617342

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INTRODUCTION

The surface and pore structure of cellulose fibers have a significant impact on

the properties and performance in applications. Cellulase enzymatic hydroly-

sis of cellulose fibers can result in changes to the surface and pore structure,

thus providing a useful tool for fiber modification [1]. The enzymatic modifi-

cation of cellulose has been an important research topic over the last several

decades [2–5]. In order to improve the interaction between natural fibers

and the matrix, it is necessary to modify the natural fibers to compatibilize

them, which is required for the design of truly green composites. Grafting is

one of the best methods to modify the properties of natural fibers [6–13]. Graft

copolymerization of acrylonitrile on sisal fibers was studied by Mishra et al.

[14], using a combination of NaIO4 and CuSO4 as an initiator in an aqueous

medium at temperatures between 50�C and 70�C. Kalia and Kaith [15] graft

copolymerize methyl methacrylate onto flax fibers and synthesized flax-g-

copolymers were used as filler in composite materials. It has been reported

that modified flax fibers results in enhanced mechanical properties of

phenol-formaldehyde composites.

These chemical treatments are important and effective methods to modify

the fiber; however, the process can alter the crystalline structure of the cellu-

lose, resulting in a degradation of mechanical properties. An alternative

method to modify the fiber is retting by micro-organisms. Some of the bacteria

produce both cellulose and cellulase. Both bacterial cellulose and cellulase

were used for the modification of natural fibers. Enzymatic technology pro-

vides a natural solution for various problems encountered in fiber processing.

Cellulase treatment results in decreased hydrophilicity of natural fibers,

which is very essential for fiber-reinforced composites. Research activities

on cellulase enzymes have been going on since the 1950s. It has been shown

that although enzymatic treatment of fibers decreased the amount of amorph-

ous and gel-like polysaccharide layer on the surface, it did not affect the

amount of fines [16]. On the other hand, the enzyme may behave in a

manner similar to retention aids and polymer facilitating the flocculation of

the small fiber particles [17].

Cellulase enzymes have broad applications in the bioprocessing of natu-

ral fibers such as biopolishing of cotton fabrics to improve softness, appear-

ance, and paper and fabric properties. In cotton fabrics, the protruding

fibers are removed by biopolishing the fabric surface using cellulase [18].

The more successful use of commercial cellulase has been in the textiles

area [19].

Pretreatments of natural fibers aimed at improving the interfacial

adhesion and decreased hydrophilicity of natural fibers. Modified fibers might

lead to composites with enhanced properties and much better durability. So in

this paper, attempts have been made to modify the sisal fibers through graft

Surface Modification of Sisal Fibers 1131

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copolymerization and cellulase enzyme and a comparative study of the modi-

fied fibers has been reported.

EXPERIMENTAL

MaterialsSisal cellulose fibers (SCF) were obtained from Sisal Research Station,

Bamra (Sambalpur), Orissa (India). Bacteria strain Streptomyces albaduncus

(MTCC No. 1764) was purchased from the Institute of Microbial Technology,

Chandigarh, India. Glucose, methyl methacrylate (MMA), vinyl acetate (VA),

ethyl acrylate (EA), acrylic acid (AA), acrylonitrile (AN) (Merck, India), and

NaCl (Qualikems Fine Chemicals, India) were used as received.

MethodsPurification of Sisal Fibers

Sisal cellulose fibers were washed with detergent in order to remove impu-

rities and then Soxhlet extracted with acetone for 72 hours in order to remove

waxes, lignin, and other impurities.

Graft Copolymerization of Binary Vinyl Monomer Mixtures onto Sisal

Fibers

Sisal fibers (500mg) were activated by immersing them in 100ml distilled

water for 24 hours. A definite ratio of FAS-H2O2 mixture was added to the reac-

tion medium, which was followed by addition of the binary monomer mixture

keeping MMA as the principal monomer. The reaction mixture was stirred

and transferred to the microwave equipment operating at 210W microwave

power for a specific time interval. Different binary vinyl monomer mixtures

used were: MMAþEA, MMAþAN, MMAþAA andMMAþVA. However, opti-

mum reaction conditions for maximum graft yield were obtained with grafting

of the principal monomer (MMA) onto the sisal fiber prior to the grafting of

binary vinylmonomermixtures onto sisal fiber. The graft copolymerwas soxhlet

extracted with acetone for about 5-6 hours so as to remove the homopolymer of

poly(MMA). Other homopolymers such as poly(EA), were removed by extraction

with alcohol, whereas poly(AN) was removed with DMF. Poly(VA) was removed

with CHCl3 extraction, while poly(AA) was removed with hot water extraction.

Graft copolymer obtained was dried in an oven at 50�C until a constant weight

was obtained. The percent graft yield (% G) was calculated as follows:

%G ¼ W2 �W1

W1� 100

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where W1 and W2 are the weights of original and grafted sisal fibers,

respectively.

Modification of Sisal Fibers Using Bacteria Streptomyces Albaduncus

For the bacterium growth, the standard growth medium for Streptomyces

albaduncus (MTCC No. 1764) was prepared and pH was adjusted to 7.4 with

sodium hydroxide. The starter culture was first autoclaved at 121�C for 45

minutes and then inoculated with the bacterium strain in static conditions

at 29� 1�C in an incubator. Glucose (2 g) was added into the culture medium

to produce culture media. After five days of Streptomyces albaduncus incu-

bation in static cultures, some of the suspension material was used to start

agitated cultures. Under these conditions, strings of materials started appear-

ing on the five days of Streptomyces albaduncus and were harvested by filter-

ing with gauze on the third day. All products were kept in vacuumed

desiccators with anhydrous calcium sulfate until characterization.

Sisal fibers (0.5 g, 5 cm long) were put in 250mL Erlenmeyer flasks con-

taining 90ml of culture medium composed of 4 g=L glucose, 4 g=L yeast

extract, 10 g=L beef extract, and 20 g=L agar for Streptomyces albaduncus.

This formulated media was found to promote the bacterial medication of sisal

fibers with stable pH. After autoclaving at 121�C for 20 minutes, the flasks

were inoculated with 10ml of a five-day-old broth of a previous culture of

Streptomyces albaduncus. The fermentation was conducted under agitated

conditions on a shaking plate (150 rpm) in an environmental chamber at

30�C for one week.

After the fermentation, the modified sisal fibers were purified in 0.1M

NaOH at 80�C for 20 minutes to remove all microorganisms, medium compo-

nents, and soluble polysaccharides. After filtration, they were then thoroughly

washed in distilled water until neutral pH [5].

Characterization of Modified Sisal Fibers

FTIR spectra of the sisal and modified sisal fiber were taken with KBr

pellets on a Perkin Elmer RXI spectrophotometer. Morphological studies of

original and modified sisal fibers were carried out on an electron microscopy

machine (LEO 435 VP). Thermal analysis was carried out in nitrogen atmos-

phere at a heating rate of 10�C=minute using Perkin Elmer (Pyris Diamond)

thermal analyzer.

X-ray diffraction studies were performed under ambient condition on an

X-ray diffractometer (Brucker D8 Advance). Crystallinity was determined by

using the wide angle X-ray diffraction counts at 2h angle close to 22� and

18�. The counter reading at peak intensity at 22� is said to represent the

crystalline material and the peak intensity at 18� corresponds to the

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amorphous material in cellulose. Percentage crystallinity (%Cr) was calcu-

lated as follows:

%Cr ¼ I22I22 þ I18

� 100

where I22 and I18 are the crystalline and amorphous intensities at 2h scale

close to 22� and 18�, respectively.

RESULTS AND DISCUSSION

Sisal fibers were immersed in water for 24 hours so as to open the active sites

for graft copolymerization. Grafting has been carried out using

solvent-monomer mixtures rather than pure monomer, which enhances the

deep penetration of the monomer inside the polymer matrix. The optimized

reaction conditions for grafting of MMA onto sisal fibers were monomer con-

centration¼ 1. 96� 10�3mol=lt, initiator ratio (FAS:H2O2)¼ 1:2 and time¼ 30

30 minutes and pH¼ 7.0.

Cellulase enzyme results in degrading cellulose in fiber wall structure,

initiates wall stripping, and fines generation by the mechanism of a peeling

effect, leaving the fibers less hydrophilic. Refining then delaminates cell walls,

causes the wall to collapse, and starts fibrillation [20].

Effect of Concentrations of Binary Vinyl MonomerMixtures on Percent GraftingIt is evident from Figures 1–4 that graft copolymerization of binary

vinyl monomer mixtures such as MMAþEA, MMAþAN, MMAþAA, and

MMAþVA onto sisal fibers under the influence of microwave radiations

Figure 1: Effect of MMAþ EA concentration on percent grafting under optimum reactionconditions.

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Figure 2: Effect of MMAþAN concentration on percent grafting under optimumreaction conditions.

Figure 3: Effect of MMAþAA concentration on percent grafting under optimumreaction conditions.

Figure 4: Effect of MMAþVA concentration on percent grafting under optimum reactionconditions.

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using MMA (1.96� 10�3mol L�1) as the principal monomer showed maximum

grafting of 118.8% (EA¼ 2.30� 10�3mol L�1) (Figure 1), 110.4% (AN¼ 3.79�10�3mol L�1) (Figure 2), 108.6% (AA¼ 2.91� 10�3mol L�1) (Figure 3), and

70.4% (VA¼ 1.62� 10�3mol L�1) (Figure 4). The higher percentage grafting

in the case of these binary monomer mixtures can be explained by the

fact that addition of electron acceptor monomers (EA, AN) to MMA increases

the reactivity of MMA towards grafting [21]. High percentage grafting

has been observed in the case of the MMAþEA and MMAþAN binary

mixture, which is due to the presence of a strong acceptor monomer in the

binary mixtures MMAþEA and MMAþAN. However, with the low graft

yield in the case of MMAþVA binary mixtures, two monomers with

electron accepting and electron donating ability enter into a charge transfer

complex formation, thereby reducing the activity of monomers towards

grafting [22,23].

Modification of Sisal Fiber Utilizing CellulaseAction of cellulase on sisal fibers resulted in stripping of fiber surface

through hydrolysis of the b 1, 4- glycosidic bond, which removes subsequent

layers or fibrils of the fiber by the mechanism of peeling effect, leaving the

fiber less hydrophilic and easier to drain [24,25]. Increase in drainage has also

been attributed to decrease in the amount of amorphous and gel-like polysac-

charide layer on the surface, yet it did not affect the amount of fines, and

removal of fuzz formation increases the commercial value of sisal fiber

[16,18]. Slow kinetics of enzymatic degradation of crystalline cellulose allow

fabric and fiber properties to be improved without excessive damage [26].

Treatment of sisal fibers with bacteria Streptomyces albadancus was

observed for five days, at the pH 7.4 and glucose concentration 2 g, which

results in enhanced softness and brightness due to the removal of gum mate-

rials and small fibrils protruding from the fiber surface [27]. The effect of pH

on the bacterial modification was tested in the range pH 6.7 to 7.7. Amount

of extracellular protuberant structures on the fiber due to cellulase pro-

duction on the fiber increased linearly up to pH 7.4 and then decreased. This

is due to the fact that high pH value deactivated bacteria, thereby inhibiting

the bacterial cellulase. In general, glucose has been used as a carbon source

for bacteria Streptomyces albaduncus. The optimum glucose concentration

used for bioprocessing by bacteria Streptomyces albaduncus was 2.0 g. At

higher glucose concentrations, the amount of gluconic acid increased during

the cultivation period. The total amount of gluconic acid produced corre-

sponds to the amount of glucose consumed in the period during which glu-

conic acid was increasing. This suggested that glucose not consumed by

bacteria was metabolized to gluconic acid and other substances. With

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increase in glucose concentration, the accumulation of gluconic acid also

increased and no more glucose was available for the bacteria to grow

[5,28]. Moreover, the accumulated gluconic acid also lowered the pH of

the culture media and inhibited cellulase production by deactivating the

bacteria.

The amount of extracellular protuberant structures on the fiber due to cel-

lulase production by bacteria Streptomyces albaduncus increased linearly

with culture time up to the fifth day and then decreased. This is due to the fact

that, after the fifth day, most of the glucose was metabolized via gluconic acid

to other substances and hence no more glucose was available for the bacteria

to grow. Moreover, the accumulated gluconic acid also lowered the pH of the

culture media which deactivated the bacteria, resulting in inhibition of cellu-

lase enzyme [5,28].

Characterization of Biologically and ChemicallyModified Sisal Fibers

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectrum of sisal and modified fibers has been observed. IR spec-

trum of sisal fiber showed peaks at 3441.22 cm�1 due to the bonded –OH

group and at 2918.70 cm�1, 1645.20 cm�1, and 1245.86 cm�1 arising from

–CH2, C-C, and C-O stretching, respectively. However, in the case of sisal-g-

poly(MMAþEA), sisal-g-poly(MMAþAN), sisal-g-poly(MMAþAA), and

sisal-g-poly(MMAþVA), additional peaks at 1782.45 cm�1, 2258.29 cm�1,

1681.79 cm�1 and 1749.16 cm�1 due to >C¼O of EA, -C�N of AN, >C¼O

of AA and VA, respectively, have been observed. An additional peak due to

–OH of AA has been observed at 2695.07 cm�1. This suggests that binary

vinyl monomers have been grafted onto the sisal through covalent linkages.

However, in the case of bacterial strain Streptomyces albaduncus, peaks at

3463.79 cm�1, 2917.14, 2851.76 cm�1, 1611.98 cm�1 and 1111.49 cm�1 due to

–OH, -CH2, C-C and C-O stretching have been observed. In addition to this,

IR spectrum of bacteria Streptomyces albadancus showed an extra peak at

3513.45 cm�1 due to –OH group stretching.

Scanning Electron Microscopy (SEM)

Figures 5a–5f shows the morphology of sisal fibers, sisal-g-copolymers,

and bacterial-treated sisal fibers. It is quite evident from Figs. 5b–5e that

there has been a sufficient deposition of copolymers onto sisal fibers. Compari-

son of the scanning electron micrographs of sisal-g-copolymers reveals a

clear-cut distinction between the original and grafted fibers. Morphology of

sisal fibers was changed through both bacterial and chemical treatments.

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Surface of grafted sisal fibers (Figures 5b–5e) is rough and amorphous [29],

whereas biologically modified sisal fibers showed the enhanced softness and

smooth appearance due to the presence of extracellular protuberant struc-

tures of cellulase, which results in the removal of gum materials and small

fibrils protruding from the fiber surface (Figure 5f) [30,31].

Figure 5: SEM of (a) original sisal fibers; (b) sisal-g-poly(MMA=EA); (c) sisal-g-poly(MMA=AN); (d) sisal-g-poly(MMA=AA); (e) sisal-g-poly(MMA=VA); and (f) cellulaseenzyme-treated sisal fibers.

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Thermal Analysis (TGA=DTA)

TGA of original and modified sisal fibers was carried out at a heating

rate of 10�C=minute in air as a function of percentage weight loss versus tem-

perature. It is evident that initial and final decomposition temperatures of

the sisal fiber are 250�C and 363�C, respectively, whereas, in the case of sisal

graft copolymers sisal-g-poly(MMAþEA), sisal-g-poly(MMAþAN), sisal-g-

poly(MMAþAA), sisal-g-poly(MMAþVA), and cellulase enzyme-treated sisal

fibers, the initial decomposition temperatures (IDTs) are near to 250�C and

the final decomposition temperatures (FDTs) are 419�C, 424�C, 425�C,

430�C, and 425�C, respectively. In the case of sisal fiber, sisal-g-poly

(MMAþEA), sisal-g-poly(MMAþAN), sisal-g-poly(MMAþAA), sisal-g-poly

(MMAþVA), and bacterial cellulase-treated sisal fibers, two-stage decompo-

sition has been observed. Maximum thermal stability has been found in the

case of modified sisal fibers followed by original sisal fibers. Thus chemical

treatments enhanced the thermal stability of sisal fibers [32]. In the case of

graft copolymers, additional vinyl monomer incorporated on the surface of

the backbone further strengthens and increases the thermal resistance due

to an increase in the number of covalent bonds. In the case of cellulase enzyme

treatment, thermal stability is enhanced due to the removal of gum materials

and fibrillation, which provides strength to fibers with more bonding sites.

Crystallinity of Original and Modified Fibers

The percentage crystallinity of sisal fiber, sisal-g-poly(MMAþEA), sisal-g-

poly(MMAþAN), sisal-g-poly(MMAþAA), sisal-g-poly(MMAþVA), and cellu-

lase enzyme-treated sisal fibers was 82, 72.3, 73.71, 73.87, 72.6, and 81.18%,

respectively. It has been observed from Table 1 that percentage crystallinity

of sisal was decreased a little with grafting of the binary monomer mixture.

Decrease in crystallinity is due to the increase in amorphous content by the

addition of vinyl monomers. There is negligible change in % Cr of sisal fibers

on cellulase enzyme treatment. A small decrease on % Cr is due to the

Table 1: Crystallinity of sisal, sisal-g-copolymers, and bacterial cellulase-treatedsisal fibers

At 2h Scale

Sr. No Sample I22 I18 % Cr C.I.

1 Sisal fiber 1025 225 82.0 0.782 Sisal-g-poly(MMAþ EA) 1283 493 72.3 0.613 Sisal-g-poly(MMAþAN) 1237 348 73.7 0.714 Sisal-g-poly(MMAþAA) 984 348 73.8 0.645 Sisal-g-poly(MMAþVA) 1020 360 72.6 0.646 Bacterial cellulase-treated sisal fibers 1079 225 81.1 0.77

Surface Modification of Sisal Fibers 1139

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production of extracellular cellulase, which hydrolyses cellulose by

exo-cleavage of b-1, 4-glycosidic linkage [33]. Exo-acting cellulases are more

active on the crystalline regions of cellulose, thereby disturbing the crystalline

structure. In chemical modification, it is due to the grafting of copolymers,

which disturbed the crystalline structure of sisal fibers because of impreg-

nation of monomer chains in the matrix, and the fiber becomes more amorph-

ous [34,35] and hence results in decreased %Cr.

Crystallinity index of sisal fiber, sisal-g-poly(MMAþEA), sisal-g-poly

(MMAþAN), sisal-g-poly(MMAþAA), sisal-g-poly(MMAþVA), and biologi-

cally modified sisal fiber was 0.7804, 0.6157, 0.7186, 0.6463, 0.6470, and

0.77, respectively. A lower crystallinity index in the case of biologically and

chemically modified sisal fiber means poor order of cellulose crystals in the

fiber. This is due to misorientation of the cellulose crystals to the fiber axis

during treatment with bacteria and MMA, as indicated by their lower crystal-

linity index [21]. This clearly indicates that the cellulose crystals are better

oriented in sisal fibers, followed by biologically and chemically modified fibers.

CONCLUSIONS

We describe simple methods to reduce the hydrophilicity of natural fibers with

enhanced thermal stability. It has been found that graft copolymerization has

a diminutive effect on the crystalline structure of sisal fibers. Microwave-

radiation-induced grafting and cellulase enzyme treatment results in

enhanced thermal stability of sisal fibers. SEM micrographs confirm that

the morphology of modified fibers was totally different from original fibers.

The surface of sisal fibers becomes rough on grafting with a binary vinyl mono-

mer, whereas bacterial treatment resulted in a smooth and shiny surface due

to bioprocessing.

Since both treatments result in enhanced thermal stability and negligible

disturbances in crystallinity with different morphological structure and

reduced hydrophilicity, so modified fibers can be used as fillers in composites

for enhancement in mechanical properties.

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