ORIGINAL PAPER

Utilizing cellulase as a hydrogen peroxide stabilizerto combine the biopolishing and bleaching proceduresof cotton cellulose in one bath

Longyun Hao • Rui Wang • Li Zhang •

Kuanjun Fang • Yajing Men • Zongzhong Qi •

Peng Jiao • Jianwei Tian • Jingquan Liu

Received: 10 October 2013 / Accepted: 27 November 2013 / Published online: 10 December 2013

� Springer Science+Business Media Dordrecht 2013

Abstract In this research, the stabilization effect of

cellulase on the decomposition of hydrogen peroxide

was investigated for the first time. It was concluded

that, regardless of the decomposition mechanism, the

cellulase protein could contribute significantly to

peroxide stability. This effect stems from the formation

of molecular hydrogen bonding between peroxide and

cellulase protein or direct sequestering of free metal

ions by amino acids in cellulase. Furthermore, based on

this stability, a combined biopolishing and peroxide

bleaching protocol was developed to improve cotton

quality more efficiently. Afterwards, physicochemical

properties such as the weight and strength loss, water

absorbency, and carbonyl and carboxyl group content

of treated cotton cellulose were measured to show the

feasibility of the new method. Fourier-transform

infrared (FT-IR) and X-ray diffraction (XRD) analyses

indicated that the crystallinity index of cotton was

increased due to the preferential hydrolysis of amor-

phous cellulose by cellulase.

Keywords Cellulase � Hydrogen peroxide �Stabilizer � Biopolishing � Bleaching � Cotton

cellulose

Introduction

Chemically, cotton cellulose is a polymer of D-glucose

in which individual glucose units are joined by b-1,4-

glycosidic linkages, forming a long unbranched poly-

mer with a high degree of polymerization (Dourado

et al. 1999). Cotton cellulose has excellent properties

including good moisture absorbency, biodegradabil-

ity, and biocompatibility as well as being comfortable

to wear and easy to color, so the market share of cotton

cellulose in the fiber and apparel industry is fairly large

(Hashem et al. 2010; Ibrahim et al. 2010; Xie et al.

2012). Natural cotton contains approximate 4–12 %

noncellulosic components such as hemicellulose,

protein, pectic matter, ash, colorant, wax, and organic

acids, depending on the type, origin, maturity, weath-

ering, and agricultural conditions of raw cotton

(Hebeish et al. 2009a; Stathakos et al. 2006). Most

of these impurities must be removed by wet prepara-

tion procedures involving elevated temperature and

long treatment duration before subsequent coloring

and finishing operations. A majority of the impurities

can be removed in an alkaline or enzymatic scouring

stage, while natural colorants (some protoplasmic

residues of protein and flavone pigments) have to be

removed by a process called bleaching using certain

oxidants (Abdel-Halim 2012; Abidi et al. 2010; Xu

et al. 2013). Hydrogen peroxide is widely employed as

an environmentally benign oxidant to bleach wood

pulp and raw cotton. Considering the similar treatment

requirements, the alkaline scouring and peroxide

L. Hao (&) � R. Wang � L. Zhang � K. Fang � J. Liu

Chemistry and Environment College, Qingdao University,

Qingdao 266071, China

e-mail: hlyqdu@hotmail.com

L. Hao � Y. Men � Z. Qi � P. Jiao � J. Tian

Sunvim Group Co., Ltd., Gaomi 261500, China

123

Cellulose (2014) 21:777–789

DOI 10.1007/s10570-013-0130-1

bleaching procedures can be incorporated into one

step (usually the bleaching step) for higher efficiency

and lower energy consumption and cost, especially in

the case of high-quality cotton cellulose containing

lower amounts of natural impurities (Li and Hinks

2012; Presa and Tavcer 2009; Priya and Arputharaj

2006). In such one-step preparation, impurities are

effectively removed by the synergetic action of caustic

soda, hydrogen peroxide, and various surfactants. It is

expected that the water absorbency and whiteness of

cotton cellulose will thereby be improved (Brooks and

Moore 2000; Zeronian and Inglesby 1995).

As is well known, pure hydrogen peroxide solution

is stable at low pH, but there is an incremental trend for

the peroxide to decompose with increasing alkalinity.

To achieve the optimum bleaching effect of hydrogen

peroxide, treatment baths are normally activated by

adjusting the pH within the range 10–12. The decom-

position of peroxide to release the active bleaching

species must be carefully controlled because the

presence of even traces of catalysts such as Fe2?,

Cu2?, Mn2?, and Co2? homolytically catalyzes the

decomposition of hydrogen peroxide, making the

critical control of the bleaching system very difficult

(Nicoll and Smith 1955; Zeronian and Inglesby 1995).

Therefore, addition of appropriate stabilizers is vital

for controllable decomposition of hydrogen peroxide

to acquire predictable cotton bleaching quality.

A variety of different chemicals have been selected

in the past as stabilizers of hydrogen peroxide under

various application conditions, among which sodium

silicate has been the most preferred option for many

years. Silicate, when used for stabilization, can form a

stable complex with peroxide that opposes the effects

of catalytic ions, or produce a colloidal complex

(usually together with calcium and magnesium ions)

that is able to adsorb catalytic ions and prevent them

from initiating peroxide decomposition (Nicoll and

Smith 1955). Furthermore, it was also suggested by

some researchers that various organic acids and

phosphates tend to stabilize peroxide bleaching baths

under alkaline- or metal-catalyzed conditions. These

substances, including phthalic, citric, L-ascorbic, eth-

ylenediamine tetraacetic, and aminotrimethylene

phosphonic acid, can strongly sequester/chelate tran-

sition-metal ions to form stable complexes and thus

retard the decomposition rate of peroxide (Jung et al.

2013; Vicente et al. 2011; Watts et al. 2007; Xu and

Thomson 2007).

Besides wet preparation, cotton fabrics are fre-

quently subjected to various postfinishing procedures

to improve their quality, of which biopolishing by

cellulase is the most important. Cotton cellulose used

in fabric manufacturing always has a tendency for

‘‘fuzz’’ formation as well as ‘‘pilling,’’ which are

identified as negative features of cellulosic fabrics. For

this reason, prevention or permanent removal of fuzz

and pilling are desired to enhance their commercial

value. Industrially, this can be achieved by using

cellulases in a process called ‘‘biopolishing’’ (Bhat

2000). Cellulases, mainly based on Trichoderma

reesei fungus in commercial preparations, are natural

catalysts with two-domain architectures (catalytic and

cellulose-binding domains) for modification of cotton

cellulose (Henrissat 1994; Pere et al. 2001). Cellulase

enzymes consist of three major types of enzymes:

endoglucanase (EC 3.2.1.4), exoglucanase (EC

3.2.1.91), and b-glucosidases (EC 3.2.1.21), which

synergistically result in enzymatic hydrolysis of the

cellulose substrate (Al-Zuhair 2008; Cavaco-Paulo

et al. 1996; Hao et al. 2012). With the assistance of

mechanical forces, cellulases can efficiently scission

small fiber ends protruding from the fabric surface to

improve its appearance.

In practice, the biopolishing process can be

scheduled before or after bleaching, depending on

the specific process flow. Notably, the former case is

more promising because it enables combined biopo-

lishing and bleaching of cotton cellulose, decreasing

water and energy consumption. Another advantage

highlighted is that the cellulase protein containing

20 different amino acids in its structure can also

sequester free metal ions from the bleaching bath

just like other organic acids and thus may result in a

cellulase-stabilized hydrogen peroxide bleaching

system. With this in mind, the performance of a

commercial cellulase from Trichoderma reesei for

stabilization of hydrogen peroxide decomposition

was carefully investigated in this research. Then, a

combined biopolishing and bleaching protocol was

developed for treating raw cotton cellulose to

examine the effectiveness of cellulase as a stabilizer

in a real bleaching process. Finally, the physico-

chemical properties of cotton cellulose were

checked to evaluate the impact of this new process-

ing method on the cotton quality. To the best of our

knowledge, such research has not been reported

previously.

778 Cellulose (2014) 21:777–789

123

Experimental

Materials

Pure cotton knit fabric (120 g/m2, 20 tex single yarn)

was obtained from Sunvim Textile Company of China

and rinsed in boiling water for 15 min prior to further

utilization. A commercial cellulase (10,000 ECU/mL

activity) was kindly supplied by Haiyi Chemicals Co.,

Ltd. of China. A nonionic surfactant, based on

polyoxyethylenated linear alcohol, was purchased

from Panya Chemicals Company, Taiwan. Hydrogen

peroxide (30 %) was purchased from the Chemicals

Company of Laiyang, China. Other chemical agents

were all of analytical reagent (AR) grade.

Methods

Effect of cellulase on the decomposition of hydrogen

peroxide

The potential of cellulase to stabilize hydrogen

peroxide was investigated in alkaline baths using

50-mL glass-stoppered test tubes as containers in a

constant-temperature shaking oscillator (50 rpm con-

tinuous agitation). Water (45 mL) and hydrogen

peroxide (30 %, 0.3 mL) were injected into every test

tube, then 0.01–0.1 mL cellulase was added. The pH

of each solution was adjusted to 11 using caustic soda.

Then, some amount of water was supplemented to

constant volume of 50 mL to set the ultimate concen-

trations of hydrogen peroxide and cellulase protein to

6 and 0.2–2 mL/L, respectively. The resulting mix-

tures were heated and kept at 95 �C for 60 min to

decompose the hydrogen peroxide. The residual

hydrogen peroxide in solution was measured by the

redox titration method using a standard solution of

potassium permanganate.

The combined biopolishing and bleaching procedure

The combined procedure was conducted in 250-mL

glass containers in a thermostatic reactor with

mechanical stirring at 120 rpm. First of all, the

biopolishing step was carried out, in which pure

cotton knits (5 g for each piece) were separately

immersed in solutions containing 1 mL/L cellulase

and 0.5 g/L nonionic surfactant at liquor ratio of 30:1.

The pH was set at 5.5 using 0.1 M acetate buffer, and

the temperature kept at 55 �C for 60 min. During this

process, the amounts of enzyme protein adsorbed were

measured using the Bradford (1976) method after

predetermined time intervals. The released reducing

sugars were determined by the 3,5-dinitrosalicylic

acid (DNS) method using glucose for calibration

(Miller 1959).

After biopolishing, some cotton fabrics were

immediately bleached in the same baths by further

adding 4–12 mL/L hydrogen peroxide and regulating

the pH to 11 using caustic soda. The bleaching

temperature was kept at 95 �C for a duration of

60 min. On the other hand, some biopolished fabrics

were immediately taken out of the baths and thor-

oughly washed using hot water for complete removal

of bound cellulase. Subsequently, these fabrics were

bleached in parallel as control using the identical

bleaching recipe in new water. Finally, all the treated

fabrics were thoroughly rinsed using hot water and

then dried in air.

Measurements

The decomposition percentage of hydrogen peroxide

The concentration of hydrogen peroxide remaining in

the solutions was determined under acidic conditions

by the redox titration method using a standard solution

of potassium permanganate as oxidation agent.

Hydrogen peroxide can react stoichiometrically with

permanganate according to the following equation:

2MnO�4 þ 5H2O2 þ 6Hþ ! 2Mnþ2 þ 5O2 þ 8H2O

ð1Þ

The decomposition percentage (DP) of H2O2 was

calculated by Eq. (2).

DP ð%Þ ¼ V0 � V1

V0

� 100; ð2Þ

where V0 is the volume of standard potassium

permanganate solution consumed by 5 mL of initial

bleaching bath and V1 is the volume consumed by

5 mL of reacted bleaching bath (Hou et al. 2010).

Degree of whiteness

The degree of whiteness of the cotton samples was

measured using an X-Rite 8400 spectrophotometer

Cellulose (2014) 21:777–789 779

123

with the D65 illuminant and Commission Internatio-

nale de l’Eclairage (CIE) 1964 standard observer. The

CIE whiteness index (WI) was calculated by Eq. (3).

WI ¼ Y þ 800 0:3138� xð Þ þ 1700 0:3310� yð Þ;ð3Þ

where Y, x, and y are the chromaticity coordinates of

the cotton fabric. Each sample was measured four

times with 90� measurements to give an average value

(Xu et al. 2013).

Fabric wettability

The wettability of the cotton samples was measured by

means of the drop absorbency test according to American

Association of Textile Chemists and Colorists (AATCC)

test method 79-2000. A drop of water was allowed to fall

from a fixed height onto the taut surface of a test

specimen. The time required for the specular reflection of

the water drop to disappear was measured and recorded

as the wetting time (Abou-Okeil et al. 2010).

The weight and strength loss of fabrics

Loss in weight (WL %) was calculated as the

difference in the weight of samples before and after

treatment according to the following equation:

WL ð%Þ ¼ Wbefore treatment �Wafter treatment

Wbefore treatment

� 100:

ð4Þ

The tensile strength of the cotton fabrics was tested

according to ISO 13934-1-1999 using a YG-2 testing

machine (Laizhou, China). Samples were tested five

times, and the average value was used. The loss in

tensile strength (SL %) was calculated according to the

following equation:

SL ð%Þ ¼ Sbefore treatment � Safter treatment

Sbefore treatment

� 100: ð5Þ

All samples were conditioned at 20 �C in 65 %

relative humidity (RH) for 24 h before weight and

tensile strength measurements.

Carboxyl and carbonyl contents

The carboxyl content of the cotton fabrics was

determined according to the previously reported

conductometric method (Zemljic et al. 2008). The

carbonyl group content of the cotton samples was

determined using the potentiometric titration method

(Lewin and Epstein 1962; Wojciak et al. 2007).

FT-IR analysis

FT-IR spectra were recorded on a Fourier-transform

infrared instrument (Magna 560, Nicolet; Thermo

Electron Corp.) in the range of 400–4,000 cm-1 with

resolution of 4 cm-1. The sample was ground into

powder using a fiber microtome, then blended with

KBr before pressing the mixture into ultrathin

pellets.

XRD measurements

XRD patterns of the fabric samples were measured

using an X-ray diffractometer (D/max 2200; Rigaku)

with Ni-filtered Cu Ka radiation at 40 kV and 30 mA.

Dried cellulose samples were mounted onto a quartz

substrate using several drops of diluted glue. This glue

is amorphous when dry and adds almost no background

signal. Scattered radiation was detected in the range of

2h = 5–40� at a scan rate of 2� min-1. Individual

crystalline peaks were extracted by a curve-fitting

process using PeakFit software (Sea-Solve Software

Inc., Richmond, CA), assuming Gaussian functions for

each peak. Iterations were repeated until the maximum

F number was obtained. In all cases, the F number was

[10,000, corresponding to an R2 value of 0.997 (Park

et al. 2010). The crystallinity index (CrI) was obtained

as the ratio of the area arising from the crystalline phase

to the total area (Lu et al. 2012). The Scherrer equation

was used to calculate the crystallite size, D (nm),

perpendicular to the (200) plane as

D ¼ Kkb cos h

; ð6Þ

where K is the correction factor (taken to be 0.9), k is

the X-ray wavelength, h is the diffraction angle, and bis the peak width at half-maximum intensity in radians

(Elazzouzi-Hafraoui et al. 2008).

Microscope analysis

A light microscope (Nikon E600) fitted with a digital

camera was used to observe the fuzz fibers and seed

780 Cellulose (2014) 21:777–789

123

coat on the cotton surface. Scanning electron micros-

copy (SEM) examination was carried out by mounting

the fabric samples on a stub using double-sided

adhesive tape and coating with gold using a sputter

coater unit. The samples were then viewed using a

JSM-6390LV scanning electron microscope (JEOL,

Japan) at accelerating voltage of 10 kV and magnifi-

cation between 10 and 2,000.

Results and discussion

The stabilization effect of cellulase on hydrogen

peroxide in alkaline solution

As mentioned above, the basis of the combined

biopolishing and bleaching procedure lies in the

stabilization effect of cellulase on hydrogen peroxide

in alkaline solution, so it is necessary to investigate the

impact of cellulase on peroxide decomposition. In

these experiments, for clearer observation, the fabrics

were not included in the solutions because the

undetermined ash content in the fabrics would make

the results unreliable. The decomposition percentage

(DP) of hydrogen peroxide after adding different

amounts of cellulase is shown in Fig. 1. It can be seen

that, regardless of the use of deionized or tap water,

addition of cellulase apparently reduced the DP of

hydrogen peroxide in alkaline solution, indicating the

effectiveness of cellulase as a stabilizer.

As suggested previously, hydrogen peroxide

decomposition may proceed in a chain or nonchain

fashion (Brooks and Moore 2000). Regardless of the

mechanism, the decomposition of peroxide to release

the active bleaching species must be carefully con-

trolled to achieve optimum bleaching. In the absence

of metal ions (e.g., in deionized water), a nonchain

process occurs under alkaline conditions and can be

represented by the following reactions (Brooks and

Moore 2000):

OH� þ H2O2 ! HO�2 þ H2O ð7ÞH2O2 þ HO�2 ! HO� þ H2Oþ O2 ð8Þ

H2O2 ! Hþ þ HO�2 ð9ÞIn this mechanism, the perhydroxyl anion is

proposed as the primary species responsible for

bleaching because it may act as a nucleophile to

attack double bonds present in chromophores, thus the

degree of whiteness obtained after textile bleaching is

directly related to the perhydroxyl anion concentration

(Brooks and Moore 2000; Zeronian and Inglesby

1995). The stabilization effect of cellulase on peroxide

decomposition in this condition stems from the

formation of molecular hydrogen bonding between

peroxide and amino acids in the cellulase protein,

which would reduce its reactivity and thus the

decomposition of peroxide in the equations above.

On the contrary, in the presence of metal ions (e.g.,

in tap water), chain propagation of peroxide

0

10

20

30

40

50

60

70

80

90

100

0 0.2 0.5 1 1.5 2

Cellulase conc.(ml/l)

Dec

om

po

siti

on

per

cen

t(%

)

Deionized water

Tap water

Fig. 1 Effect of cellulase on the decomposition percentage of hydrogen peroxide in deionized and tap water (tap water contains

0.3 ppm iron ion, 40 ppm manganese ion, and 1 ppm copper ion)

Cellulose (2014) 21:777–789 781

123

decomposition is generated by metal ions or oxyhy-

droxides formed in alkaline bleaching conditions

(Nicoll and Smith 1955; Petlicki et al. 2005); For

instance, when iron ions exist in the solution, the

following equations may be involved (Watts et al.

2007):

Fe2þ þ H2O2 ! OH � þOH� þ Fe3þ ð10Þ

H2O2 þ Fe3þ ! HO2 � þHþ þ Fe2þ ð11ÞH2O2 þ OH� ! HO2 � þH2O ð12Þ

HO2 � þFe2þ ! HO�2 þ Fe3þ ð13ÞIn this mechanism, the free radicals are responsible

for bleaching (Taher and Cates 1975). With the

addition of cellulase, the free metal ions can be

directly sequestered by the amino acids to form a

stable complex, losing the capability to initiate the

decomposition of peroxide. Moreover, the capture of

free metal cations would thereby prevent them from

forming oxyhydroxide with higher catalytic ability.

The combined biopolishing and bleaching

procedure

First, the biopolishing step was carried out in solutions

containing cellulase and nonionic surfactant at the

optimum hydrolytic condition for enzymatic action. In

this step, the cellulase could cause mild and effective

modification of the cotton substrate for surface

polishing without severely affecting its mechanical

properties. The enzymatic polishing involves the

following stages: (1) transfer of cellulase molecules

from the aqueous phase to the fabric surface through

the boundary layer, (2) binding of cellulase molecules

by the cellulose bulk, (3) catalysis of the hydrolytic

reaction of cellulose chains, and (4) transport of the

products of the hydrolytic reaction to the aqueous

phase (Yachmenev et al. 2004). Noncellulosic impu-

rities in raw cotton, especially concomitant hydropho-

bic lipids and waxes, undermine the wettability of the

cotton substrate and hinder close contact between the

cellulase protein and cellulose molecules, thus

severely reducing the enzymatic effect (Dourado et al.

1999; Ogeda et al. 2012). Nonionic surfactant based

on polyoxyethylene can be safely selected to remove

these hydrophobic substances in order to enhance the

enzymatic hydrolysis, because it has no negative

impact on cellulase activity or can even improve

saccharification of highly crystalline cellulose within a

broad range of concentrations (Mizutani et al. 2002).

It was also suggested by Castanon and Wilke (1981)

that nonionic surfactants could render cellulose read-

ily wettable by cellulase solution, bring the substrate

into intimate contact with the enzymes, and allow the

enzymes to reach otherwise inaccessible places.

The amount of cellulase adsorbed over treatment

time is characterized in Fig. 2. Cellulase adsorption onto

cotton cellulose is necessarily the first step of the

hydrolytic process, being very quick and reaching

equilibrium within 60 min, consistent with many other

reports (Banka 2002; Du et al. 2012). With extension of

the incubation time, more reducing sugars are released

from the cotton bulk. At the early stage, the hydrolytic

rate is fast, then the pace of hydrolysis gradually

declines with elongation of the treatment time. In

biochemical systems, accumulation of end products

such as soluble oligomers, cellobiose, and glucose

exercises an inhibitory effect on the rate of the onward

enzymatic reaction (Ghose and Das 1971). As stated

above, the current work targets the establishment of a

combined biopolishing and peroxide bleaching proce-

dure for lower water and energy consumption. With this

in mind, hydrogen peroxide was supplemented into the

biopolishing bath to afford bleaching using the cellulase

as a stabilizer. Figure 3 shows the effect of the

concentration of peroxide incorporated into the reaction

medium on the whiteness of the cotton substrate. The

obtained results reveal that increasing the amount of

peroxide from 4 to 12 mL/L favors enhanced whiteness.

Obviously, the presence of cellulase in the bleaching

bath improves the whiteness of the bleached samples

due to its prominent stabilizing effect. Clearly, the

cellulase is still effective as a peroxide stabilizer in the

real bleaching process, implying that the combined

biopolishing and peroxide bleaching procedure is

practically feasible.

Characterization of cotton cellulose treated

by the combined procedure

The physicochemical properties of cotton processed

by the combined operation are summarized and listed

in Table 1. The weight loss during the processing was

nearly 7 % relative to raw cotton. The weight reduc-

tion results from: (1) the reducing sugars released from

the hydrolytic scission of cellulose chains by the

cellulase, (2) the hydrophobic waxes and lipids

782 Cellulose (2014) 21:777–789

123

removed by the nonionic surfactant during the biopo-

lishing and bleaching processes, and (3) other impu-

rities eliminated by the alkaline bleaching at high

temperature. The water absorbency time was reduced

from 186 to 2.6 s, indicating that the combined

procedure allows the cotton to achieve superior

hydrophilicity without severe fiber deterioration. It is

now generally accepted that the pectin and wax and

their distribution on the cotton surface are responsible

for the nonwetting behavior of greige cotton by water.

These hydrophobic impurities can be efficiently

removed by the comprehensive actions of cellulase,

surfactant, hydrogen peroxide, and caustic soda. The

strength loss of cotton during the combined treatment

was around 8 %, indicating that no severe damage

occurred to its intrinsic structure (Buschle-Diller et al.

1999). Cellulase could lead to certain strength loss

because of its enzymatic hydrolysis of cellulose

chains, but at a low level. Relative to acid hydrolysis,

enzymatic hydrolysis of cellulose causes a slower

reduction because the cellulase molecules are too large

to permeate the fine structure of cellulose. Upon

cellulase action, both amorphous and crystalline

cellulose structures will be attacked, but at different

levels, and the degradation of crystalline cellulose is

responsible for the decrease in strength (Lenting and

0

0.2

0.4

0.6

0.8

1

0 5 10 20 30 40 50 60

Time (min)

Red

uci

ng

su

gar

as

glu

cose

(mg

/ml)

0

1

2

3

4

5

6

Pro

tein

ad

sorb

ed (

mg

/g)

glucose

protein

Fig. 2 Adsorptive and

hydrolytic properties of

cellulase on cotton cellulose

20

40

60

80

4 6 8 10 12

Hydrogen peroxide conc.(ml/l)

CIE

WI

Without cellulase

With cellulase

Fig. 3 Effect of hydrogen

peroxide concentration on

whiteness of cotton

cellulose

Cellulose (2014) 21:777–789 783

123

Warmoeskerken 2001). According to the specification

information from the supplier, the commercial cellu-

lase used in the experiment mainly contains the

endoglucanase (EG) type of enzyme along with an

amount of cellobiohydrolase (CBH). As is well

known, the EG type of enzyme mainly focuses its

attack on the amorphous structure by random breakage

of cellulose chains, without apparently undermining

the strength of cotton. Besides, some strength loss of

cotton likely results from oxidative degradation of

cellulose chains by the hydrogen peroxide in the

alkaline condition. During the bleaching process,

oxidation of some alcohol groups would readily

undergo aldol dehydration or b-alkoxycarbonyl elim-

ination, cleaving the glucosidic bond to reduce the

molecular weight of cellulose and thus lower the

strength of the cotton (Haskins and Hogsed 1950;

Knill and Kennedy 2003). On the whole, it is possible

to keep the balance between improvement in fabric

appearance (biopolishing) and strength loss during the

combined procedure in a real industrial application.

By measuring the carboxyl and carbonyl contents of

raw and treated cotton, it is observed that the cotton

cellulose after treatment is characterized by higher

carbonyl and carboxyl group contents than for the raw

material. This means that oxidation by hydrogen

peroxide creates new carbonyl and carboxyl groups in

the cellulose molecules. Generally, carbonyl groups

are predominantly formed at the C3 carbon of the

anhydroglucose units of cellulose and do not cause the

peeling-off reaction (Lewin and Ettinger 1969).

FT-IR and XRD analyses

Mid-infrared spectroscopy measures the absorption of

radiation in the wavenumber range from about 4,000 to

400 cm-1. Such absorption involves transitions

between vibrational energy states and rotational sub-

states of the molecule. It is possible to assign absorp-

tions to specific functional groups, making IR

spectroscopy very useful in structural elucidation

(Kacurakova et al. 2000). The infrared spectra,

measured in transmission mode, of raw and treated

cotton cellulose are presented in Fig. 4. The two

spectra are basically the same due to the small changes

between the tested samples. The obvious absorption

bands at 3,400 and 2,900 cm-1 can be assigned to O–H

stretching and C–H stretching, respectively. A peak at

around 1,640 cm-1 is due to adsorbed water molecules

(Chung et al. 2004). Absorption bands at 1,429 and

1,372 cm-1 can be assigned to CH2 scissoring motion

and C–H bending, respectively. The bands at 1,163 and

1,060 cm-1 are dominated by C–O–C bond vibration

and in-plane ring stretching, respectively (Chung et al.

2004). Since the transmission spectra were taken from

KBr pellets made of finely ground cotton fabric, they

can only reveal the characteristic peaks of cellulose

because the quantity of impurities such as wax, pectin,

and lignin in the cotton is too small to appear in the

spectra. Only by FT-IR attenuated total reflection

(ATR) measurements with suitable treatment of the

sample by HCl could some differences between raw

and treated cotton be demonstrated (Chung et al. 2004;

Wang et al. 2006).

FT-IR spectroscopy is a powerful tool for investi-

gating cellulose crystalline structure and can definitely

exhibit some structural changes of cotton during the

combined biopolishing and bleaching process.

Although the crystal structure of cellulose has been

studied for almost a century, some details remain to be

unraveled (Nishiyama 2009). Cellulose Ib is the

dominant crystalline structure of natural cotton, hav-

ing a monoclinic unit cell containing two parallel

chains (Nishiyama 2009; Sugiyama et al. 1991).

Cellulose crystallites are thought to be imperfect,

and thus a significant portion is often referred to as

amorphous. The ratio of the absorptive intensity at

1,372 and 2,900 cm-1 (A1372/A2900) was proposed to

measure the crystallinity of natural cellulosic materi-

als (Akerholm et al. 2004; Nelson and O’Connor

1964). This ratio could be applied to celluloses I and

II, and samples containing a mixed lattice. The values

of A1372/A2900 for raw and treated cotton were

calculated as 0.966 and 0.983, respectively, indicating

Table 1 Physicochemical properties of raw and treated cotton cellulose

Sample Weight loss (%) Water absorbency (s) Strength

loss (%)

Carboxyl content

(mM/100 g)

Carbonyl content

(mM/100 g)

Raw cotton – 186 ± 27 – 4.5 ± 0.3 6.6 ± 0.7

Treated cotton 6.5 ± 0.4 2.6 ± 0.2 8.2 ± 0.9 14.4 ± 1.2 18.7 ± 1.6

784 Cellulose (2014) 21:777–789

123

that the crystallinity of cotton is increased by the

combined treatment.

Compared with FT-IR spectroscopy, XRD analysis

is more widely utilized for calculating the crystallinity

of cellulose (French and Cintron 2013; Langan et al.

2001; Nishiyama et al. 2002, 2003, 2012; Sugiyama

et al. 1991; Wada et al. 1997; Yue et al. 2012). A

parameter termed the crystallinity index (CrI) has been

proposed to depict the relative amount of the crystal-

line portion in cellulose, being calculated as the ratio

of the area arising from the crystalline phase to the

total area. The XRD spectra of raw and treated cotton

cellulose, with labels to indicate their crystal lattice

assignments according to the monoclinic indexation

(Elazzouzi-Hafraoui et al. 2008; French and Cintron

2013; Nishiyama et al. 2012), are presented in Fig. 5.

Individual crystalline and amorphous peaks were

separated from the diffraction intensity profiles using

a curve-fitting process. It is observed from Fig. 5 that

the CrI of cotton is increased by the combined

procedure. The bleaching step would not result in

detectable change to the crystallinity of cellulose

(Buschle-Diller et al. 1999), so the enzymatic hydro-

lysis in the biopolishing step is the sole reason for this

phenomenon. Cellulase can more easily digest amor-

phous cellulose due to its higher enzyme-accessible

surface area, while crystalline cellulose is recalcitrant

to enzymatic attack due to the presence of strong

intracrystalline (O3–H���O5 or O2–H���O6) and inter-

molecular (O6–H���O3) hydrogen bonding and stack-

ing forces (Cao and Tan 2005; Chen et al. 2007;

Chundawat et al. 2011; Yue et al. 2012). Moreover,

the change in crystallinity of the samples can be

expressed in terms of the change in crystallite size.

Based on the peak width at half-maximum height, the

crystallite sizes can be estimated using the Scherrer

equation. The crystal sizes perpendicular to the (200)

planes were calculated to be 6.11 and 6.65 nm for

original and treated cellulose cotton, respectively.

This trend is consistent with previous reports by Wang

et al. (2008) and Cao and Tan (2005). The slight

3500 3000 2500 2000 1500 1000 500

1163

Tra

nsm

itta

nce

(%

)

Wavenumber (cm-1)

2900 1640 1372 106014293400

Raw cotton

Treated cotton

Fig. 4 FT-IR spectra of raw and treated cotton cellulose

5 10 15 20 25 30 35 40

Treated cotton

(004)

(200)

(012/102)

2 Theta (degrees)

(110)(110)

Raw cotton

CrI=63.06%

CrI=68.26%

Fig. 5 XRD spectra of raw and treated cotton cellulose

Cellulose (2014) 21:777–789 785

123

Fig. 6 Fuzz fiber of a raw and b treated cotton

Fig. 7 Surface of a raw and b treated cotton; SEM image of c seed coat

Fig. 8 SEM images of a raw and b treated cotton

786 Cellulose (2014) 21:777–789

123

increase in crystallite size of cellulose also indicates

that the amorphous portion of the cellulose is more

readily hydrolyzed than the crystalline portion.

Morphological structure of cotton cellulose

Light and electron microscopes were used to reveal the

changes in the morphological structure of the cotton

fabric after the combined biopolishing and bleaching

procedure. By light microscopy (Fig. 6), it was

observed that fuzz fibers protruding from the cotton

surface were obviously removed by the enzymatic

hydrolysis during the biopolishing process, resulting in

an apparent improvement in fabric appearance (Cavac-

o-Paulo 1998). Cellulase can lead to systematic removal

of primary and secondary walls of cotton progressively,

and thus cause scission and rupture of fuzz fibers

(Ibrahim et al. 2011; Paralikar and Bhatawdekar 1984;

Saravanan et al. 2009). It is also noted from Fig. 7 that

the cotton seed coats are completely eliminated after the

treatment. As is known, cotton seed coat has a complex

composition and five-layer structure: epidermal layer,

outer pigment layer, colorless layer, palisade layer, and

inner pigment layer, making it the most resistant

impurity to be completely removed from cotton by

wet preparation (Yan et al. 2009). Cellulase can also

degrade the lignocellulosic structure of cotton seed,

making it easier to be totally removed in the subsequent

peroxide bleaching stage (Csiszar et al. 1998). In this

regard, the combined biopolishing and bleaching pro-

cedure is considerably recommendable.

The raw and treated cotton were both characterized

by SEM (Fig. 8). Examination of raw cotton under

2,0009 magnification showed typical cotton fibers with

parallel ridges, grooves, wrinkled surfaces, and occa-

sional breaks (Hebeish et al. 2009b; Li and Hardin

1997; Wang et al. 2006). The treated sample showed

flatter and smoother surface, resulting from the nonse-

lective impact of cellulase, surfactant, and alkali. This

is consistent with previous analysis reported by Wang

et al. (2006), who concluded by atomic force micros-

copy (AFM) that greige cotton has a rougher surface

than bioscoured and alkali-treated cotton.

Conclusions

It was found that cellulase protein could greatly inhibit

hydrogen peroxide decomposition in alkaline condition.

In the absence of metal ions, this effect stems from the

formation of molecular hydrogen bonding between

peroxide and amino acids in the cellulase protein. In the

presence of metal ions, this result derives from direct

sequestering of metal ions by amino acids to form stable

complexes. Based on the stabilization effect on hydro-

gen peroxide, a combined biopolishing and bleaching

method was successfully afforded for more efficient wet

preparation of cotton cellulose. By measuring the

weight loss, water absorbency, strength loss, and

carbonyl and carboxyl contents of the treated cotton, it

was concluded that this combined procedure was

feasible in a real preparation. Based on FT-IR, XRD,

and SEM analyses, it was noted that the crystallinity of

cotton cellulose was increased and the appearance of

cotton cellulose was greatly improved by the combined

procedure.

Acknowledgments This work was financially supported by

the National Natural Science Foundation of China (21303092,

51173086), Science and Technology Plan of Qingdao (13-1-4-

247-jch), Shandong Provincial Natural Science Foundation

(ZR2010EQ034), and Taishan Scholars Program of Shandong

Province.

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