Enzymatic Scouring of Cotton: Effects on Structure and Properties

By Yonghua Li and Zan R. Hardin, University of Georgia, Athens

7 0 % , and is composed of cellulose. The lumen is composed of protoplasmic residues. The general state of knowledge of the chemical composition of a mature cotton fiber is presented in Table I. Table I shows that noncellulosic materials account for only a very small amount of the fiber weight. These materials are amorphous and are located in Structure and Chemical the cuticle and the lumen. The cuticle Composition of Cotton forms a protective layer to shield the cotton from environmental attacks and The cotton fiber is a single biological water penetration. Waxy materials are cell. The layers in the cell structure are, mainly responsible for the non-absorfrom the outside of the fiber to the inbent characteristics of raw cotton. Pecside, cuticle, primary wall, secondary tins may also have an influence, since wall, and lumen. These layers are dif85% of the carboxyl groups in the pecferent structurally and c h e m i ~ a l l y . ~ - ~ tins are meth~lated.'~Natural pigments The primary and secondary walls have in the cotton and other matter picked LIP different degrees of crystallinity, as by a cotton substrate in yarn or fabric well as different molecular chain orienmaking are responsible for the greyness tations. The cuticle, composed of wax, of the substrate before bleaching. proteins, and pectins, is 2.5% of the Usually, the absorbency of cotton is fiber weight and is amorphous. The improved by alkaline scouring, whiteprimary wall is 2 . 5 % of the fiber ness is increased in oxidative bleachweight, has a crystallinity index of ing, and, at the same time, the cotton 30%, and is composed of cellulose. cellulose is purified to a certain deThe secondary wall is 91.5% of the fiber weight, has a crystallinity index of

reliminary research on enzymatic treatments of cotton indicates that this process is a promising area for an effective alternative to traditional scouring of cotton textiles.'-4 Fundamental research is needed to answer questions related to the enzymatic processes and to explore real possibilities of its industrial application.

gree.14 Motes are swollen in alkaline scouring and removed or rendered colorless in oxidative bleaching.
Enzymesfor Scouring Cotton A study on enzymatic treatments of unscoured cotton fabric conducted by German scientists involved pectinase, cellulase, protease, lipase, and other enzymes.' Significant losses in weight and reduction in tensile strength occurred with pectinase and cellulase only after three hours of treatment. One conclusion was that cellulases are especially suited to scouring of cotton fabrics. In a patented process by Japanese researchers, cotton fibers or their blends with other fibers were treated with aqueous solutions containing protopectinases for 18 hours at 40C to give scoured yarns with good tensile strength r e t e n t i ~ n . ~

Procedure An amount of sample (0.2 g of cotton) was weighed, washed, squeezed with a paper towel to remove excess water, dried in a weighing bottle at 105c for

Table 1. Typical Values for the Composition of a Mature Dry Cotton Fiberlo-12 ABSTRACT In pursuit of effective enzyme systems for scouring of cotton, enzyme activity optima were coupled with an engineering approach. A rapid gain of absorbency by cotton substrates after treatment by engineered enzyme systems was observed. Part of the experimental results are reported herein and followed with an analysis of the structural changes of the cotton caused by the treatment with staining tests and microscopy observations. A discussion on the mechanism of the enzymatic treatments is included. KEYTERMS Cellulase Cotton Enzymes Pectinase Scouring
Constituent Cellulose Protein ("6.25) Pectic substances Wax Mineral matters Maleic, citric, and other organic acids Total sugars Cutin Composition of a Fiber Typical % Low % High % 94.0 1.3 0.9 0.6 1.2 0.8
0.3
88.0

Composition of the Cuticle % 30.4 19.6 17.4 6.5 ?

? 8.7

1.1 0.7 0.4 0.7 0.5

96.0 1.9
1.2 1.o

1.6
1.o

Table II. Cellulases and Pectinases

Enzyme Mutifect cellulase GC Cellulase cl184 Cellulase c8546 Multifect pectinase PL Pectinase p3026 Pectinase p9179 Source Trichoderma longibrachiatum (formerly T. reesi) Aspergillus niger Trichoderma reesi Aspergillus niger Aspergillus japonicus Aspergillus niger Designation'
C1

pH 4.0
5.0

Temperature (C) 50
50 50 50 50 50

c 2 c 3

P1 p2 p3

4.0 4.0 5.5 4.0

and PI from Genencor International: others from Sigma Chemical Co.

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Fabric

Fig. 1. An illustration o f adequate fabric absort ncy, the entire event occurs in one second.

one hour, and then weighed again after cooling overnight in a desiccator. In this way, the dry weight was obtained before a treatment. The sample was treated with one of the systems of the 1 . The control enzymes listed in Table 1 was raw cotton treated with the buffer solution without enzyme, but under the same conditions as in the enzyme treatment. The temperature was raised to 100C to stop the enzyme action. The sample was washed in hot water, squeezed with a paper towel, washed again in hot water, rinsed in cold water, and then squeezed with a paper towel again. The sample was dried in a weighing bottle at 105c for oqe hour and weighed after cooling overnight in a desiccator. The weight loss was calculated. (Note: the weighing processes were only conducted with fiber samples.) Then the following two chemical extractions were conducted on raw cotton for comparisons. A four-hour ethyl alcohol Soxhlet extraction of wax was performed according to the purification process for cotton cellulose proposed by Conrad.j5 A four-hour treatment with an alkaline solution of 1% sodium hydroxide on alcohol-extracted cotton fibers was conducted for purification p ~ r p o s e s . ' ~
Absorbency Testing

Sudan 111, Sudan IV, Congo Red, and Ruthenium Red were conducted when necessary. The former three are solvent dyes for fats. Congo Red is a direct dye for cellulose. Ruthenium Red stains pectins and proteins. Only Congo Red and Ruthenium Red give visual evidence for cotton surface changes. The staining procedure was performed according to Rollins and deGruy.16
Microscopy

A JEOL scanning electron microscope was used for microscopy observations. All the specimens were glued to the stubs by Duco cement. Care was taken not to subject them to mechanical damages when gluing. Then the specimens were coated with palladium by Samsputter-2A Sputter Coater. The thickness was set to level 4. The surface characteristics of cotton samples were determined by summarizing numerous observations of individual fibers from a specimen.
Results
Changes in Surfaces of Cotton The changes in the cotton specimens listed in Table 1 1 1 caused by enzyme action were analyzed with staining tests and scanning electron microscopy observations.

For cotton in fabric form, AATCC Test Method 39-1980 (Evaluation of Wettability) was adopted. A wetting time less than one second was considered to represent adequate absorbency in all practical cases (Fig. For cotton i n fiber form, the standard test method for fabrics is inapplicable. To test for absorbency i n this form, a bundle of cotton fibers was dropped onto the surface of water in a beaker. The immediate sinking of the bundle in the water to the bottom of the beaker was generally considered to represent adequate absorbency (Fig. 2).
Staining Tests

Tables IV-VI show the results of the staining tests and microscopy observations of cotton fibers with different treatments. Raw cotton, controls for the enzymatic treatments, and the cotton from the two chemical extractions were used as reference standards for comparison against the specimens from the pectinase, cellulase, and their mixture treatments. The observations on the absorption of staining solutions by the specimens are also listed in Tables IV and V. Very good means that the specimen absorbed the staining solution immediately, good means that the specinien absorbcd the staining solution gradually but automatically, while poor means that the specimen did not absorb the staining solution automatically and needed to be forced down into the staining solution. These pieces of information were incorporated with the color effects for the analysis of the staining tests, not for the judgement of the absorbency of enzymatic-treated cotton. Ruthenium Red stains pectic substances and proteins by virtue of the carboxyl groups present in the molecules. In Table IV, specimen A (raw cotton) had poor absorbency for the dye solution because of the presence of wax. This resulted in light and uneven stains. The control specimen (AS) also had poor absorbency and floated on top of the dye solution but was stained deeply and unevenly when forced down into the dye solution. This uneven staining may be due to cracks and enlarged micropores in the cuticle created in the treatment. The alcohol-extracted specimen (B) had good absorbency and was stained deeply and evenly, a result of the removal of the wax. Even staining is an indication of a continuous covering of the cotton surface with pectins and proteins. Specimens ASP (pectinase digested), ASC (cellulase digested), ASPC (mixed enzymes digested),and C (alcohol and alkali extracted) had very good absorbency and were stained very faintly,

f?iber bund'e
1Water
Occurs at once

The fiber bundle absorbs water and sinks immediately

Fig. 2. An illustration of adequate fiber absorbency.

F
frr

Staining tests with Sudan Black B,

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Table 111. Specimens Used in Staining Tests and Microscopy Observations

Designation A AS Specimen Raw cotton Control sample for enzymatic treatment Ethylene alcohol extracted sample Additional alkaline extraction on B Pectinase (PI) treated sample Cellulase (C,) treated sample Pi and C, mixture treated sample Treatment Time (hours)
3 4 4 3 3 3

Table IV. Results from Ruthenium Red Staining

Sample A AS Absorption of Staining Solution poor poor good very good very good very good very good Staining Effect Depth Evenness light deep deep very faint very faint very faint very faint uneven uneven even even even even even

B
C ASP ASC ASPC

B
C ASP ASC ASPC

Table V. Results from Congo Red Staining

Sample A AS Absorption of Staining Solution poor poor good very good very good very good very good Staining Effect Depth light light deep deep deep deep deep

Table VI. Microscopical Surface Characteristics of Cotton Fibers with Different Treatments
Specimen A AS

ASP x x

ASC ASPC x x
X X

B
C ASP ASC ASPC

Smooth&convexridges x Flattened ridges andconcavegrooves Protruding micro-fibrillous ends Cavities Polished faces Fuzzy and blurred faces

x
x x

which indicated removal of pectic and proteinaceous substances from cotton. Congo Red is a direct dye and has affinity for cellulose. In Table V, raw cotton (A) and the control (AS] had poor absorbency and were stained lightly, because the cotton cellulose was covered with the cuticle, while the cotton cellulose in specimens ASP (pectinase treated], ASC (cellulase treated), ASPC (enzyme mixture treated], and C (alcohol and alkali extracted) was directly subjected to the stain through removal of the cuticle. When incorporated with staining tests, microscopy observations allow us to see how the surface of a cotton fiber is physically changed in a treatment. Raw mature cotton presents a rather smooth microscopical view with characteristic parallel ridges and gr00ves.l~ These features remained with the control specimen (AS) and the

alcohol-extracted specimen (B) in Table VI, and disappeared in specimens ASP (pectinase treated), ASC (cellulase treated], ASPC (enzyme mixture treated], and C (alcohol and alkali extracted]. New features with enzymetreated specimens were concave groove-dominated surfaces with protruding micro-fibrillous ends, polished faces, and shallow cavities. The alcohol- and alkali-extracted cotton surfaces appeared fuzzy and blurred. Typical micrographs are presented in Figs. 3-8. In the cases involved with cellulases, polished faces and shallow cavities are indicative of heavy hydrolysis of cotton cellulose after removal of the cuticle. This implies that three hours of cellulase enzymatic treatment under the conditions used in this study are long enough not only for the removal of cuticle, but also for causing hydrolysis of the cotton main

body. Hydrolysis of the cotton main body is very undesirable in enzymatic scouring of cotton and can be avoided by reducing the treatment times.
Adequate Absorbency

The ability to create fast enzymatic treatments to improve the absorbency of cotton was a main concern. After staining tests and microscopy observation, research concentrated on designing rapid enzymatic treatment methods for adequate absorbency. The pectinases, cellulases, proteases, and lipases used in these experiments all caused cotton to acquire at least some absorbency in treatment times that varied from a few minutes to hours. The pectinases and cellulases were very effective compared to the proteases and lipases. The most significant results of the experiments are listed in Table VII. The experimental results

Fig. 3.Scanning electron micrograph of the surface of from control specimen AS (Table 111).
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cotton fiber

from

Fig. 4. Scanning electron micrograph o f the surface of a cotton fiber cellulase-treated specimen ASC (Table 111).

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Fig. 5. Scanning electron micrograph o f the surface of a cotton fiber from cellulase-treated specimen ASC (Table Ill).

Fig. 6. Scanning electron micrograph o f the surface o f a cotton fiber from pectinase-treated specimen ASP (Table 111).

were recorded as the lowest amount of an enzyme and shortest treatment time used which caused the specimen to gain adequate absorbency. The results for the fiber substrate give the lowest concentration and times that produced the adequate absorbency. Note the weight losses of less than 4% in every case. For the fabric substrate, the enzyme units and concentrations given are the lowest values run in the experiments. Further work will be needed to establish the lowest values for the fabric form. Both the concentrations of the enzyme proteins and absolute enzyme units used (see Table VII, Note 2) are listed. Figs. 9 and 1 0 show some of the results graphically.
Scouring Cotton with Pectinases

micropores and make contact with the pectic substances in the substrate (Fig. lla). Pectic substances are hydrolyzed

results in the removal or partial removal of the cuticle or breakdown of the continuity of the cuticle (Fig. lib).

From the results of the staining tests, the microscopy observations, and the absorbency tests, it was concluded that pectinases penetrate the cuticle i n

Fig. 7. Scanning electron micrograph of the surface of a Cotton fiber Fig. 8. Scanning electron micrograph of the surface of a cotton fiber from enzyme mixture-treated specimen ASPC (Table Ill). from enzyme mixture-treated specimen ASPC (Table 111).
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3.5 $
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; g

120
80

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0"

2.5

c 1 2

1 :
vy

C1,C2 & C3 - cellulases P1, p2 & P 3 - pectinases (see Table 3)

." 1 3 0.5
0-

,
c1

Fig. 9. Shortest treatment times with different enzymes for substrates to gain adequate absorbency. C,, C p ,and C3 are celluases; P,, P2, and P3 are pectinases (Table Ill).

C1, C2 &C3 -Cellulases PI, P2 &P3

c2

c3

Pl

P2

- Peainases

P3

Fig. 10. Weight losses of fiberspecimens in enzymatic treatments, C,, C,, and C3are celluases;P,, Pp, and P3 are pectinases (Table
111).

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:h
eof

)I.
e

The extent of the reaction depends on the conditions of the treatment. It is not known if a total hydrolysis of pectic substances occurs within a limited treatment time. The break of the linkage between the cuticle and the cellulose body can be assumed to be a direct result of this partial hydrolysis. Fig. 1 l c shows an ideal state of a fiber after a complete pectinase scouring.
Scouring Cotton with Cellulases

loosened and broken down with the help of mechanical forces. The extent of the reaction effect depends on the conditions of the treatment. It is not known if a total hydrolysis of the primary wall occurs within a limited treatment time. A break of the linkage between the cuticle and the cellulose body is assumed to be a direct result of this partial hydrolysis. Fig. 12c shows an ideal state of a fiber after a complete cellulase scouring.
Scouring Cotton with a Mixture of Pectinases and Cellulases At first, the pectinases and cellulases in a mixture act separately according to the models in Figs. 11 and 12. Cellulases break the linkage from the cellulose side and pectinases break the linkage from the cuticle side. The action of pectinases creates more sites in the primary wall layer available for cellulase digestion, while the action of cellulases creates more sites in the pectic substance layer available for pectinase digestion. The result of the synergism is a more effective scouring in both the speed and the evenness of the treatment.

Further Discussion

; ,
it

e e it 5

Cellulases penetrate t h e cuticle i n aqueous solutions through cracks or micropores in the cuticle and make contact with the primary wall (Fig. 12a). The part of the primary wall at the contact point is hydrolyzed by the catalysis of the cellulases (Fig. 12b). After that, both the cellulose of the primary wall and the cellulose of the secondary wall are present for cellulase digestion. The cellulases, however, will catalyze the hydrolysis of the more amorphous primary wall instead of the hydrolysis of the secondary wall because natural crystalline cellulose is very resistant to cellulase digest i ~ n . ' ~The , ' ~ result of this reaction is that the outside layer of the fiber is

In the course of the experimental work, several results were observed but not followed up on in detail. They nevertheless suggest further research areas that need exploring. Cotton was treated with lipases for several hours with resulting improved cotton absorbency, However, the lipases were not as aggressive as pectinases or cellulases in the improvement of cotton absorbency. Proteases were examined because proteins account for the largest proportion in the composition of the cuticle. In these cases, however, proteases were not as effective as pectinases or cellulases in the improvement of cotton absorbency. When mixtures of pectinases and cellulases were used in the experiments, less total amount of enzyme was needed to cause substrates to acquire adequate absorbency. Cavities observed on the surfaces of cellulasedigested cotton were not seen on the surfaces of mixed enzyme-digested cotton. Finally, the work described herein was done on a laboratory scale. The results suggest several benefits if the same or similar results can be obtained

Proteins
&Pectins ,Secondaq I Wall

Secondary , I WaU
Pectinase
OI

,p
Pectinase
ch

econdary Wall

'\Primary Wall Cellulooe

'\PrimaryWaU Cellulose

1
Ctlluldst

Fig. 11. Scouring cotton with pectinases: a. pectinases contacting pectins; b. pectinases digesting pectins; and c. after a complete pectinase scouring.
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Textile Chemist and Colorist

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Proteins
&Pectins Secondary

Proteins Pectins Secondary

Wall

Cellulase
b

0-

TPrimary wan

Cellulose

TPiimary wall Cellulose

Fig. 12. Scouring cotton with cellulases: a. cellulases contacting primary wall cellulose; b. cellulase digesting primary wall cellulase; and c. after a complete cellulase scouring, and ideal situation. after scale-up to pilot plant and industrial scales. Among these possible benefits are: Enzyme working temperatures are about 40-50C, while alkaline scouring is applied at 1OOC or above, which could mean a considerable decrease in the energy requirement when using enzymes for scouring. The wastewater from the enzyme treatment is easily biodegradable. In cotton blends, the other components, such as wool or polyester, would be safe with the enzymatic process because of the specificity of enzyme activities and mild conditions of the treatment. The enzymatic process could be incorporated with the existing amylase desizing process or incorporated into other specific processes if compatible enzymes are used. Work on carrying these laboratory results to a larger scale is proceeding with industry partners.
Conclusions

SEM photographs of the cotton surfaces after enzymatic treatments had different characteristics from ones of raw cotton, control, or chemically extracted cotton. Absorbency tests showed that the enzymatic treatments dramatically changed the water absorption capabilities of raw cotton and unscoured cotton fabrics. The change in the water absorbency of cotton was rapidly catalyzed by pectinases, cellulases, or their mixtures. Pectinases can destroy the cuticle structure by digesting the inner layer pectins in the cuticle of the cotton. Cellulases can destroy the cuticle structure by digesting the primary wall cellulose immediately under the cuticle of cotton.
Acknowledgment

The authors gratefully acknowledge the assistance of Danny Akin and Sandy Silver of the U.S. Department of Agriculture, Hugh McDonald of Genencor International Inc., and Bert Truesdale of Thomaston Mills Inc. in this project.
References
1. Robner, Ulrike, Melliand Textilbericlltc, Vol. 74, No. 2, February 191)3, pEG3. 2. Achwal, W. B., Colourclge. Vol. 39, No. 7, July

Interscience Publishers, New York, N.Y., 1965, p65. 9. deGruy, Inez V., Jarrell 1% Carra, and Wilton R. Goynes, The Fine Structure of Cotton: An Atlas of Cotton Microscopy, Marcel Dekker Inc., New York, N.Y., 1973, p156. 10. Tripp, Verne W., Anna T. Moore, and Mary L. Rollins, Textile Reseorch/ournal,Vol. 21, No. 12, December 1951, p886. 11. Guthrie, John D., Chemistry and Chemical TechnologyofCotton, edited by Kyle Ward Jr., IntersciencePublishers, New York, N.Y.. 1955, P4. 1 2 . McCall, Elizabeth R. and Julian F. Jurgens, Textile Research Journol,Vol. 21, No. 1, January 1951, p19. 13. Freytag, Rene and Jean-JacquesDonze, Fundanientals and Preparation Part A , Handbook of Fiber Science and Technology: Vol,I, Chemical Processing of Fibers and Fabrics, edited by Menachem Lewin and Stephen B. Sello, Marcel Dekker Inc., New York, N.Y. and Basel, Switzerland, 1983, plll. 14. Campbell, K. S., The American CotfonHandbook, Vol. 11, edited by Dame s. Hamby, Interscience Publishers, New York, N.Y., 1965,
p768.

This research has shown that, at the laboratory scale, enzymatic scouring with cellulases and pectinases can be a very effective part of preparing cotton for chemical treatment. The destruction of the cuticle during enzymatic scouring was demonstrated by the results of the Ruthenium Red and Congo Red staining. Ruthenium Red staining clearly showed that proteins and pectins in the surface of cotton were removed in the enzymatic treatments. Physical destruction of the cuticle was also revealed by the scanning electron microscope (SEM) photographs. The

1992, p35.
3. Achwal, W. B.. Colourage. Vol. 40, No. 11,

November 1093,p23. 4. Sakai, T. and A. Masuda,/apcmese Ic~tcnl No. 06220772, August 9,1994. 5. Tripp, Verne W. and Mary L. Rollins, AnolyticaIChemistryVol. 24, No.11,November 1952, ~1721. 6. Boylston. E. K. and J. J. Hebert, Textile Research Journal,Vol. 65, No. 7, July 1995, p4ZcJ. 7. Jeffries,R., et al., Cellulose Chemistry TechnolOgJ3, 1969, ~ 2 5 5 . 8. Rollins, Mary L.. The American Cotton Handbook, Vol. I, edited by Dame s. Hamby,

15. Conrad, C. M., Chemistryand Chemical Technology of Cotton, edited by ]. R. Kyle Ward, IntersciencePublishers, New York, N.Y., 1955, pl9. 16. Rollins. M. L. and I. V. deGruy, Instrumental Analysis of Cotton Cellulose and Modified Cotton Cellulose, edited by Robert T. OConnor, Marcel Dekker Inc., New York, N.Y., 1972, p145. 17. Tripp, Verne W., Anna T. Moore, and Mary L. Rollins, Textile Kcsaarch Journal, Vol. 27, No. G , Junc1057. p419. 18. Walker. L. P. and D. n. Wilson, Enzy~iiofic Hydrolysis of Ccllulose, ediktl by L. 1. Walkor atid U. 13. Wilson, 13lsovicr Science Publishing Co. Inc., New York, N.Y., 1991, p3. 19. Coughlan, M. P.. BiorcsourcelTecl~nology, Vol. 39, No. 2, 1992, p107.

Authors Address

Ian R. Hardin, University of Georgia, 305 Dawson Hall, Athens, Ga. 30602; telephone 706-542-0357; fax 706-5424890.

AATCCs Cumulative Index available on our Web Site http://www.aatcc.org

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