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Enzyme-retting of flax and characterization of processed fibers
Journal of Biotechnology 89 (2001) 193– 203 www.elsevier.com/locate/jbiotec
Enzyme-retting of flax and characterization of processed fibers Danny E. Akin a,*, Jonn A. Foulk a, Roy B. Dodd b, David D. McAlister III c a
US Department of Agriculture, Agricultural Research Ser6ice, Russell Research Center, PO Box 5677, Athens, GA 30604, USA b Department of Agricultural and Biological Engineering, Clemson Uni6ersity, Clemson, SC 29634, USA c US Department of Agriculture, Agricultural Research Ser6ice, Cotton Quality Research Station, Clemson, SC 29633, USA Received 26 June 2000; received in revised form 21 November 2000; accepted 1 December 2000
Abstract Enzyme-retting formulations consisting of Viscozyme L, a pectinase-rich commercial enzyme product, and ethylenediaminetetraacetic acid (EDTA) were tested on Ariane fiber flax and North Dakota seed flax straw residue. Flax stems that were crimped to disrupt the outer layers were soaked with various proportions of Viscozyme-EDTA solutions, retted, and then cleaned and cottonized with commercial processing equipment. Fiber properties were determined and crude test yarns were made of raw and Shirley cleaned flax fibers and cotton in various blend levels. Cleaned fibers were obtained from both seed and fiber flax types, but with variations due to treatment. Retting formulations produced fibers having different properties, with enzyme levels of 0.3% (v/v as supplied) giving finer but weaker fibers than 0.05% regardless of EDTA level. Experimental yarns of blended flax and cotton fibers varied in mass coefficient of variation, single end strength, and nep imperfections due to sample and formulation. With cost and fiber and yarn quality as criteria, results established a range in the amounts of components comprising retting formulations as a basis for further studies to optimize enzyme-retting formulations for flax. Under conditions examined herein, Viscozyme L at 0.3% (v/v) plus 25 mM EDTA produced the best test yarns and, therefore, established a base for future studies to develop commercial-grade, short staple flax fibers for use in textiles. © 2001 Published by Elsevier Science B.V. Keywords: Pectinase; Chelators; Crimping; Strength; Fineness; Flax; Yarn
1. Introduction Flax (Linum usitatissimum L.) is likely the oldest textile fiber known, with evidence of production dating back 7000 years or more (Franck, 1992; Stephens, 1996). European colonists brought the skills for flax production to North * Corresponding author. Fax: + 1-706-546-3607.
America very early in its development (Stephens, 1996). While many parts of the world have continued to produce fiber flax, the United States produces no flax for textiles. Flax is grown as an oilseed crop with Canada as the major supplier, and a small percentage of the straw is used for speciality paper and pulp (Domier, 1997). However, considerable interest exists now in the US for production of flax fiber for use in textiles and
0168-1656/01/$ - see front matter © 2001 Published by Elsevier Science B.V. PII: S 0 1 6 8 - 1 6 5 6 ( 0 1 ) 0 0 2 9 8 - X
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a variety of high value products. While seed flax straw is an option for a lower grade of fiber for certain composites, fiber flax could supply the textile industry with a natural fiber for blending with cotton to enhance the properties of the world’s foremost natural fiber. In the US, industry analysts indicate that the major application for flax fiber in textiles will be in ‘cottonized’ form (i.e. cleaned and cut to staple length) for blending with other fibers on high efficiency cotton equipment. Retting, which is the separation of bast fiber from non-fiber tissues in the stems, is the major problem in processing flax (Pallesen, 1996). Water-retting, which was formerly the method of choice because of high quality fiber, produces environmentally unacceptable fermentation waste (Sharma and Van Sumere, 1992a,b). This practice was mostly discontinued in western countries several decades ago because of the pollution from fermentation products and the high cost of drying (Brown, 1984). Currently, dew-retting, which depends on indigenous, aerobic fungi to colonize pulled plants in the fields, is the accepted practice in European countries and accounts for much of the linen used in textiles. Disadvantages of dewretting are (1) dependence on particular geographical regions that have the appropriate moisture and temperature ranges for retting, (2) coarser and lower quality fiber than with water-retting, (3) less consistency in fiber characteristics, and (4) occupation of agricultural fields for several weeks (Van Sumere, 1992). Dew-retting further results in a heavily contaminated fiber that is particularly disadvantageous in cotton textile mills. An alternative that has had long-term consideration is enzyme-retting (Sharma and Van Sumere, 1992a; Schunke et al., 1995). In the 1980s, research was undertaken in Europe to develop enzyme-retting as a method to replace dew-retting. Flaxzyme, a commercial enzyme mixture from Novo Nordisk (Denmark), and several patents (Van Sumere, 1992) resulted from this research. Van Sumere and Sharma (1991) evaluated Flaxzyme, which consists of pectinases, hemicellulases, and cellulases, at a concentration of 3 g l − 1 and a liquor to stem ratio of 10:1 and reported that enzyme-retting produced fiber fineness,
strength, color and waxiness comparable to the best water-retted fiber. Sharma (1987) further used commercial enzymes to remove non-cellulosic components from dew-retted fibers. Despite the apparent success of Flaxzyme, no commercial process was developed. Cost of the enzymes, and perhaps other less obvious reasons, prevented development of a commercial enzyme-retting process. Dew-retting remains the practice most widely used in Europe to obtain fibers commercially for industrial use, despite continuing research on other methods (Sharma et al., 1999). Research was initiated about 5 years ago in the US to evaluate enzyme-retting for potential in supplying a short staple flax fiber suitable to blending with cotton in textile mills. Progress reported to date includes: good production yields in various regions including the southeastern US as a winter crop (Foulk et al., 2000), experimental enzyme-retting method using commercial enzymes with chelators sprayed onto mechanically crimped stems (Akin et al., 2000), and initiation of standards development for textile and industrial applications. The development of a cost effective, efficient enzyme-retting system requires additional work. This report provides recent data on pilot scale studies of enzyme-retting of fiber and seed flax plants that have been commercially cleaned, cottonized, and tested in experimental yarn blends.
2. Materials and methods
2.1. Samples Flax samples used included both seed flax and fiber flax types. Seed flax fiber was derived from an unknown variety produced under commercial conditions in North Dakota, USA. The seed was removed by combining, and the straw residue was collected and baled without dew-retting. During storage and before tests of enzyme-retting, the samples became dark indicating weathering and likely colonization by indigenous microorganisms. For fiber flax, the cultivar ‘Ariane’ was produced as a winter crop in the coastal plain region of South Carolina from December 1998 to May
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1999. Production, harvesting, and yields have been provided in detail (Foulk et al., 2000). Two harvests of Ariane were collected and included an early harvest (May 5, 1999) for high quality fiber and a late harvest (May 27, 1999) for both seed and fiber. At late harvest, seeds were collected by a stripper header attached to a combine, and both early and late stems were cut by a drum-mower about 5 –7 cm above the soil line. Plants for enzyme-retting were then either dried in an enclosed shed or field dew-retted for 3 weeks from the early harvest or field-dried and baled without any dew-retting from the late harvest. Prior to enzyme-retting, monolayers of dried flax stems were crimped through fluted steel rollers at :80 N in order to disrupt inherent stem barriers (i.e. cuticle/parenchyma) to enzyme penetration into the tissues (Akin et al., 2000).
2.2. Enzyme-retting formulations A series of formulations based on earlier laboratory work (Henriksson et al., 1997; Akin et al., 1999, 2000) was used to ret both seed flax and fiber flax. The multi-enzyme complex Viscozyme L, which is commercially available for use in breakdown of plant cell walls for extraction of components from tissues, was used in all enzymeretting formulations. Viscozyme L, which is prepared from a selected strain of Aspergillus, has activity against branched pectin-like compounds and also contains a wide range of carbohydrases including arabanase, cellulase, b-glucanase, hemicellulase, and xylanase (Product Sheet, Novo Nordisk, Franklinton, NC). In this regard, Viscozyme L is similar to Flaxzyme, which was developed specifically for enzyme-retting (Van Sumere, 1992). The retting formulation included ethylenediaminetetraacetic acid (EDTA) at 25 or 50 mM levels in water and adjusted with NaOH to pH 5.0. The enzyme was added to the EDTA solution at either 0.3 or 0.05% (v/v) as supplied.
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of formulation required in tank retting. About 12 kg of crimped flax stems were soaked with : 40 l of retting formulation and rotated in a sealed container for 2 min. Soaked flax was then placed in an insulated chamber, covered with wet burlap to retain humidity, and incubated at 40 °C for 24 h. Retted flax was then washed with 2 changes of : 110 l of tap water for 5 min each and dried in the chamber at 50 °C. For one lot of seed flax straw, : 12 kg was sprayed with : 40 l of enzyme formulation rather than soaking.
2.4. Microscopy Ariane stems were excised about midway of the plant. Sections 2–3 mm long were incubated with 0.05% Viscozyme L in sodium acetate buffer at pH 5 without chelator for 2, 4, and 8 h and buffered Viscozyme L plus 50 mM EDTA for 8 h. Control, unretted sections incubated in buffer alone were also selected for comparison. Sections were prepared for scanning electron microscopy by methods described (Akin et al., 1996).
2.5. Commercial cleaning and cottonizing Retted flax was shipped to the flax company Ceskomoravsky len, Humpolec, Czech Republic, to be cleaned through the Unified cleaner. This device uses a series of crushing rollers, shaker prongs, and a scutching wheel to commercially clean large amounts of flax. Residual fiber was removed between runs of each of the treatments so that contamination was kept to a minimum. The Ariane fiber samples were cleaned twice, but the seed flax straw samples were cleaned only once due to their brittleness. The Unified cleanedflax was then cottonized by the LaRoche system, in which flax fibers were chopped to : 5 cm and then blown through a series of ducts and beaters to separate and further clean the staple length fibers.
2.3. Application of retting formulation
2.6. Determination of fiber properties
The enzyme formulation was applied as a modification of the spray enzyme-retting method (Akin et al., 2000) designed to reduce the amount
Cottonized flax fiber was analyzed for fineness by micronaire (ASTM, 1997a), which was modified to use 5.0 g fiber based on calibration
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al., 2000). This flax fiber was blended with raw cotton at 10:90 (flax:cotton) and yarns spun as above. Finally, fibers from two retting treatments, namely Ariane flax retted with either 0.05 or 0.3% Viscozyme L and each with 25 mM EDTA, were cleaned twice through the Shirley Analyzer for blending with cotton, which was Shirley cleaned once, in 10:90 and 30:70 levels. Yarns were evaluated using the Uster II Evenness Tester or the ILE DS-65 Evenness Tester for mass coefficient of variation (CV) of yarn width and nep imperfections (200+ ) per 1000 yards. Strength was evaluated by the Statimat-M single end yarn strength tester (LawsonHemphill, Central Falls, RI.)
Fig. 1. Scanning electron micrograph of control, unretted Ariane fiber flax stem showing fiber bundles (F) between shive (S) and the cuticle/parenchyma barrier (arrows). Bar = 50 mm.
with flax fineness standards (Institut Textile de France, Lille, France) as reported (Akin et al., 1999). Strength and elongation were determined by the flax bundle method using the Stelometer (ASTM, 1997b) and length parameters determined using the array method (ASTM, 1997c).
2.7. Production and e6aluation of experimental yarn blends Hand-blended flax and commercial cotton samples were processed through a card to remove fiber impurities and short fibers and to align fibers. After carding, the fibers were drawn into a sliver and draw frame to further align and help blend fibers. Crude test yarns were then ring spun from the sliver at the Cotton Quality Research Station, ARS-USDA. In one test, cottonized flax, without further cleaning, and raw cotton were hand-blended in a ratio of 50:50. In another test, cottonized flax was further cleaned by passing one time through a Shirley Analyzer, which separates the finer fibers from coarse fibers and residual shive (Akin et
3. Results and discussion Viscozyme L is a commercial product containing the following activities of major polysaccharidases: 10 700 PSU g − 1 pectinase, 108 FBG g − 1 b-glucanase, 725 NCU g − 1 cellulase, and 31 200 VHCU g − 1 hemicellulase (data supplied by Novozymes, Franklinton, NC). Therefore, it is rich in pectinase but low in cellulase activities and effected structural modifications of flax stalks required for retting, including the separation of fiber bundles from the shive and epidermis/cuticle and also separation into fibers and bundles of smaller divisions (Figs. 1–5). Longer incubation times increased separation of bast fibers, with 8 h (the longest time in these studies) resulting in considerable modification of the bast region (Figs. 2–4). However, addition to the enzyme mixture of EDTA at 50 mM further improved retting, with a greater apparent separation of cuticle/epidermis from the fibers (Fig. 5). These results confirm those of other studies (Akin et al., 1997; Henriksson et al., 1997), in which commercial pectinase-rich enzyme mixtures, with the addition of chelators, efficiently retted flax without destruction of the cellulosic fibers. Further, results indicate that Viscozyme L could provide a readily available ‘off-the-shelf’ retting enzyme.
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Fig. 2. Scanning electron micrograph of Ariane fiber flax stem retted in Viscozyme L at pH 5.0 for 2 h showing slight modification of bast fiber bundles. Bar = 50 mm.
Fig. 4. Scanning electron micrograph of Ariane fiber flax stem retted in Viscozyme L at pH 5.0 for 8 h showing considerable separation of fibers within the bast region. Bar =50 mm.
The production of Ariane flax for staple length fibers in this study has been reported (Foulk et al., 2000). The dry weight yield from early plots (average of 4) was 4075 kg ha − 1 and mature plots
averaged 5075 kg ha − 1. Using Viscozyme L in the Fried test for in vitro retting as described by Henriksson et al. (1997), fiber content in the stem was estimated to be :33%. Fiber yields of the
Fig. 3. Scanning electron micrograph of Ariane fiber flax stem retted in Viscozyme L at pH 5.0 for 4 h showing initial separation of fibers from shive and cuticle and slight modification of bast fiber bundles. Bar =50 mm.
Fig. 5. Scanning electron micrograph of Ariane fiber flax stem retted in Viscozyme L plus 50 mM EDTA at pH 5.0 for 8 h showing significant separation from the epidermis/cuticle (arrows) and loosening of fibers and fiber bundles within the bast region. Bar = 50 mm.
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Table 1 Properties of enzyme-retted and commercially cleaned and cottonized flax fiber Sample
Seed flax straw Seed flax straw Ariane (early) Ariane (early) Ariane (early) Ariane (early) Ariane (early) Ariane (late) a–g
Retting treatment EDTA (mM)
Viscozyme (%)
50 25 50 25 50 25 dew-retted 50
0.05 0.05 0.05 0.05 0.3 0.3 3 weeks 0.05
Micronaire
5.9 9 0.1b,c 6.0 90.2b 5.8 9 0.1c,d 5.7 9 0.1d 3.9 90.1g 4.6 90.1f 5.3 90.1e 6.7 90.1a
Strength (g tex−1)a
25.9 92.9b,c 19.6 9 1.1d,e 24.0 9 2.0c 20.9 9 1.3d 13.0 9 1.3g 15.8 91.8f 36.2 9 2.3a 26.8 93.4b
Length
Shirley cleaned yield (%)
UQL
Uniformity
1.355 9 0.009 1.200 9 0.075 1.346 90.114 1.428 90.024 1.247 9 0.025 1.247 9 0.037 1.311 9 0.082 1.428 90.065
78.3 9 0.6 73.7 9 1.6 74.6 99.7 78.4 92.1 71.39 1.9 73.0 9 2.0 73.89 0.5 79.9 92.5
25.3 9 1.0e,f 23.6 9 1.0f 30.7 9 8.8d,e 37.9 9 0.3b,c 61.4 9 0.7a 58.7 9 1.1a 43.0 9 1.1b 32.3 9 0.3c,d
Within columns, values with different letters differ at PB0.05.
enzyme-retted samples could not be accurately determined after Unified cleaning, but approximate yields ranged at least from 20 to 29% based on original straw weights. Properties of the fibers from the various samples and treatments showed significant differences(Table 1). Enzyme-retting of early-harvested Ariane with 0.3% Viscozyme L plus chelator resulted in finer (P00.05) fibers based on the modified micronaire method, with addition of 50 mM EDTA in the formulation producing the finest fibers of any treatment. The coarsest fiber was from late-harvested Ariane enzyme-retted with 0.05% Viscozyme L+50 mM EDTA. Within sample type, the lower levels of chelator resulted in coarser fibers. Based on comparisons with calibrated flax standards, the micronaire method using air flow to determine fiber fineness effectively differentiated treatments, but the values at present time do not indicate performance properties as they do for cotton. Cleaning through the Shirley Analyzer indicated that enzyme-retting with 0.3% Viscozyme L gave finer fibers as indicated by significantly (P 0 0.05) greater yields (Table 1). In this regard, micronaire and Shirley-cleaning both indicated that the 0.3% Viscozyme L plus chelator resulted in finer fibers. The Shirley Analyzer, which cleans
the flax by carding action (Van Langenhove and Bruggeman, 1992), separates the finer fibers from the coarser fibers and shive with little breakdown of bundles to smaller divisions during cleaning (Akin et al., 2000). Within enzyme levels, 25 mM EDTA at times tended to produce lower yields after Shirley-cleaning, but differences were not significant with either flax type. Fiber elongation was lower after enzyme-retting with 0.3% Viscozyme L than with other treatments (Table 1). However, elongation was low overall compared to cotton, as is typical for flax fibers. Fiber strength was lowest with 0.3% Viscozyme L treatments (Table 1). This reduction in fiber strength with higher enzyme levels occurred in other retting tests (Akin et al., 1999). Enzyme treatments with 0.05% produced fibers ranging from : 20 to 25 g tex − 1, and dewretted fibers were significantly stronger than all other treatments. Fiber length as indicated by the upper quartile and uniformity was similar for all treatments (Table 1). This parameter for 0.3% Viscozyme L was 1.247 in. regardless of chelator level. Commercial cleaning and cottonizing appeared to have the greatest influence on fiber length in these samples.
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Blended yarns with cotton and flax fibers from the various treatments and not further cleaned in proportions of 50:50 gave low quality yarns with a broad range in quality parameters useful in ranking the retting formulations (Table 2). However, the card removed a substantial portion of the flax fiber and likely resulted in an actual flax proportion lower than 50%. For these comparisons, treatment with 0.3% Viscozyme L+ 25 mM EDTA had the lowest mass CV and the fewest neps of the blends but had strength almost as high as any other blended yarn. Further, fewer breakages during spinning occurred with this formulation compared with others. Further tests were made of yarns constructed with 90:10 cotton:flax blends (Table 3). In these yarns, the flax fiber was cleaned through the Shirley Analyzer one time prior to blending with raw cotton. Flax levels on carding flats was lower after flax was processed in this manner. The fewest neps occurred in yarns from dew-retted flax, while those from flax enzyme-retted with 0.3% Viscozyme L had fewer neps than other enzymeretted fibers. Strength and mass CV were similar for all blends, and the use of 25 mM EDTA did not seem to impair yarn quality in these tests.
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Other yarns were spun with fibers from 0.05 or 0.3% Viscozyme L, both with 25 mM EDTA, that had been Shirley cleaned twice (Table 4). Twicecleaned flax fiber blended with once-cleaned cotton fibers improved parameters of 10:90 flax:cotton yarns over those, in which flax fibers only cleaned once with the Shirley Analyzer (Table 3). Yarns from fibers retted with 0.3% Viscozyme L plus 25 mM EDTA were particularly improved in strength, mass CV, and neps. Twice-cleaned flax fiber blends described above were also spun at 30% flax, with parameters similar for both enzyme levels and lower in quality than 10:90 blended yarns (Table 4). Results from the combination of tests indicated that the enzyme treatments produced fibers with a range of properties. The use of 0.3% Viscozyme L plus EDTA was superior to other enzyme treatments in producing fine fibers and yarns with fewer neps. The significantly higher fiber yield from Shirley cleaning of fine fibers from both 0.3% enzyme treatments indicated that an enzyme level higher than 0.05%, which was identified as a level that worked well at laboratory levels, would produce a greater yield of finer fibers. This enzyme level adds considerably greater expense, but
Table 2 Yarn properties of cotton/flax (50/50) blends Fiber source–rettinga or preparation
Single end strength (g tex−1)
Mass evenness (CV)
Nep imperfections/1000 yards
Seed flax strawb–0.05% Viscozyme L+50 mM EDTA Seed flax straw–0.05% Viscozyme L+50 mM EDTA Seed flax straw–0.05% Viscozyme L+25 mM EDTA Arianec (early)–0.05% Viscozyme L+50 mM EDTA Ariane (early)–0.05% Viscozyme L+25 mM EDTA Ariane (early)–0.3% Viscozyme+50 mM EDTA Ariane (early)–0.3% Viscozyme+25 mM EDTA Ariane (early)–dew-retted 3 weeks Ariane (late)–0.05% Viscozyme L+50 mM EDTA Upland cotton (deltapine acala 90) 100%
10.6 9 2.0
35.9
2659
10.59 1.6 8.79 2.2 11.29 2.0 9.39 1.8 9.791.6 10.0 91.7 9.891.7 9.391.9 17.49 1.8
37.7 38.7 38.3 39.9 36.7 35.1 38.7 43.5 19.4
3398 3658 3373 3250 2961 2312 2811 3305 461
Hand-blended (50:50 original weight basis) and carded to form sliver and yarn on traditional cotton equipment. Actual proportion likely less than 50% flax due to high losses during processing. a Crimped at 80 N, : 12 kg immersed in retting formulation in water, pH 5.0, for 2 min , incubated at 40 °C for 24 h, washed, dried. b Unknown commercially grown variety in North Dakota and combined for seed; second sample in list sprayed to soaking. c Grown as a winter crop in 1998/1999 in coastal plain region of South Carolina. Early harvest for fiber and late harvest for seed and fiber.
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Retting formulationa
90:10 yarn Single end strength (g tex−1)
0.05% Viscozyme L + 25 mM EDTA 13.39 2.0 0.3% Viscozyme L + 25 mM EDTA 14.5 9 2.4
70:30 yarn Mass evenness (CV)
Nep imperfections /1000 yards
Single end strength (g tex−1)
Mass evenness (CV)
Nep imperfections /1000 yards
24.0 24.2
530 490
10.8 9 2.3 10.19 1.9
30.4 30.1
1125 1240
Once Shirley cleaned cotton and twice Shirley cleaned Ariane flax fiber hand-blended and carded to align fibers to form sliver and yarn with traditional cotton equipment. a In water at pH 5.0 for 2 min, incubated at 40 °C for 24 h, washed, dried.
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Table 4 Yarn properties of cotton/flax blends from Ariane flax fiber twice cleaned through the Shirley Analyzer
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Table 3 Yarn properties of cotton/flax (90/10) blends through the Shirley Analyzer with flax fiber cleaned once Flax–retting treatmenta
Single end strength (g tex−1)
Mass evenness (CV)
Nep imperfections/1000 yards
Seed flax strawb–0.05% Viscozyme L+50 mM EDTA Seed flax straw–0.05% Viscozyme L+50 mM EDTA Seed flax straw–0.05% Viscozyme L+25 mM EDTA Arianec (early)–0.05% Viscozyme L+50 mM EDTA Ariane (early)–0.05% Viscozyme L+25 mM EDTA Ariane (early)–0.3% Viscozyme L+50 mM EDTA Ariane (early)–0.3% Viscozyme L+25 mM EDTA Ariane (early)–dew-retted 3 weeks Ariane (late)–0.05% Viscozyme+L 50 mM EDTA
14.793.1
25.5 9 3.5
628
14.39 2.7 13.09 3.2 13.89 3.3 13.99 4.0 11.49 3.1 13.99 2.5 13.79 2.4 14.3 92.3
23.4 9 4.3 27.19 6.9 28.9 9 8.5 25.2 93.4 24.6 95.8 26.195.5 24.6 96.4 25.29 7.8
509 647 736 597 572 571 555 665
Cleaned fiber samples hand-blended and carded to form sliver and yarn with traditional cotton equipment. a Crimped at 80 N, :12 kg immersed in retting formulation in water, pH 5.0, for 2 min, incubated at 40 °C for 24 h, washed, dried. b Unknown commercially grown variety in North Dakota and combined for seed; second sample in list sprayed to soaking. c Grown as a winter crop in 1998/1999 in coastal plain region of South Carolina. Early harvest for fiber and late harvest for seed and fiber.
zyme level adds considerably greater expense, but reducing chelator levels in half from the 50 mM level used earlier (Akin et al., 2000) reduced some costs and appeared to produce fibers of equal or even superior properties in fiber and yarns tests. While fiber strength was somewhat related to yarn strength, other factors obviously influence yarn strength, and lower strength obtained from 0.3% enzyme level with 25 mM EDTA in retting formulations did not appear to be detrimental to yarn strength. Properties of flax fibers are a result of the actions of retting and subsequent mechanical processing for cleaning of shive. Fineness is among the foremost factors affecting fiber quality for spinning into yarns (Sharma et al., 1999). Along with fiber type (Sharma et al., 1999), the degrees of retting and cleaning affect the breakdown of bundles to smaller units and ultimate fibers, and therefore determine fineness. Fiber strength, which has been related to fineness in enzymeretted flax (Akin et al., 2000), and length are other important fiber parameters that, along with fineness, influence quality and production efficiency of yarns. In earlier laboratory studies with other flax samples, we identified enzyme types and formulations that gave good retting criteria in the
laboratory (Akin et al., 1997, 1999, 2000; Henriksson et al., 1997). Henriksson et al. (1997) reported that addition of chelators to Flaxzyme in laboratory trials lowered considerably the recommended 0.3% enzyme level (Sharma and Van Sumere, 1992a,b). Therefore, pectinase-rich commercial products similar to Flaxzyme at 0.05% plus 30–50 mM EDTA or oxalic acid were tested and appeared to ret flax efficiently. The current work on flax samples, which had been commercially cleaned and tested in spinning trials for crude cotton:flax blended yarns, suggested that a higher enzyme level (0.3%) produced significantly higher yields of finer fibers for the Ariane fiber flax and North Dakota seed flax used herein. Since cost is a primary concern in commercial applications, reducing the components of the formulation to the least amounts for efficient retting is a priority of research. In the present study, use of 25 mM EDTA, in combination with 0.3% Viscozyme L, resulted in similar fiber yield and quality to 50 mM EDTA and gave superior overall properties for the test yarns in our treatments. With cost and fiber and crude yarn quality as criteria, our data established a range for retting formulations, which form the basis for further studies to optimize methods for enzyme-retting of flax.
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This report provides the first results from pilot plant studies using flax fibers produced by our enzyme-retting method, which were subsequently cleaned and cottonized by commercial equipment. Use of a chelator, which likely removed the Ca2 + that binds and stabilizes epidermal cells (Rihouey et al., 1995), improves activity of the pectinases (Henriksson et al., 1997). In laboratory tests of our method, the liquid to fiber ratio was less than 2 ml g − 1 (Akin et al., 2000) and considerably lower than that the 10:1 ratio reported for tank methods (Sharma and Van Sumere, 1992b). Residual activity of enzymes not absorbed during the 2-min soaking (Foulk and Akin, unpublished data) could further improve cost effectiveness. Superior yarn results with 0.3% Viscozyme and 25 mM EDTA identified a point for further research to optimize methods and formulations with particular sample types to produce commercial quality fibers and yarns. Specifically, research should further investigate various combinations of enzymes and chelators within these established ranges on various flax types for optimal efficiency, cost effectiveness, and resultant fibers with tailored properties. Towards this goal, preliminary work in our laboratory has shown that endopolygalacturonase (EPG) from a dew-retting fungus was as effective as commercial enzyme mixtures in retting flax (Akin et al., 1998; Henriksson et al., 1999). The data indicated the paramount importance of EPG to retting and suggested a future opportunity, perhaps through cloning technology, to produce a consistently effective but simplified enzyme formulation for flax retting. Determination of retting efficiencies and fiber properties of particular flax samples and types, in conjunction with specific formulations, could establish important criteria for expanding the application of enzymatic retting to produce flax fibers for a variety of industrial applications.
References Akin, D.E., Dodd, R.B., Perkins, W., Henriksson, G., Eriksson, K.-E.L., 2000. Spray enzymatic retting: a new method for processing flax fibers. Textile Res. J. 70, 486 – 494.
Akin, D.E., Gamble, G.R., Morrison, W.H. III, Rigsby, L.L., Dodd, R.B., 1996. Chemical and structural analysis of fibre and core tissues from flax. J. Sci. Food Agric. 72, 155 – 165. Akin, D.E., Henriksson, G., Slomczynski, D., Eriksson, K.E.L., 1998. Rhizomucor pusillus: pectinolytic fungus with industrial applications for retting flax. Abstracts of the 98th General Meeting of the American Society for Microbiology, vol. 0 – 5, p. 394. Akin, D.E., Morrison, W.H. III, Gamble, G.R., Rigsby, L.L., Henriksson, G., Eriksson, K.-E.L., 1997. Effect of retting enzymes on the structure and composition of flax cell walls. Textile Res. J. 67, 279 – 287. Akin, D.E., Rigsby, L.L., Perkins, W., 1999. Quality properties of flax fibers retted with enzymes. Textile Res. J. 69, 747 – 753. ASTM D 1448 – 90, 1997a. Standard test method for micronaire reading of cotton fibers, Annual Book of Standards, Sec 7 Textiles, ASTM, West Conshohocken, PA. ASTM D 1445 – 95, 1997b. Standard test method for breaking strength and elongation of cotton fibers (flat bundle method), Annual Book of Standards, Sec 7 Textiles, ASTM, West Conshohocken, PA. ASTM D 1440 – 90, 1997c. Standard test method of length and length distribution of cotton fibers (array method), Annual Book of Standards, Sec 7 Textiles, ASTM, West Conshohocken, PA. Brown, A.E., 1984. Epicoccum nigrum, a primary saprophyte involved in the retting of flax. Trans. Br. Mycol. Soc. 83, 29 – 35. Domier, K.W. 1997. Fibre industry in Canada. Euroflax Newsletter No. 2(8), December, pp. 8 – 10. Foulk, J.A., Dodd, R.B., Akin, D.E., 2000. New low cost flax fibers for composites. In: Proceedings of the 2000 Society of Automotive Engineers World Congress and Exposition, Paper no. 2000-01-1133. Franck, R.R., 1992. The history and present position of linen. In: Sharma, H.S.S., Van Sumere, C.F. (Eds.), The Biology and Processing of Flax. M. Publications, Belfast, Northern Ireland, pp. 1 – 9. Henriksson, G., Akin, D.E., Rigsby, L.L., Patel, N., Eriksson, K.-E.L, 1997. Influence of chelating agents and mechanical pretreatment on enzymatic retting of flax. Textile Res. J. 67, 829 – 836. Henriksson, G., Akin, D.E., Slomczynski, D., Eriksson, K.E.L., 1999. Production of highly efficient enzymes for flax retting by Rhizomucar pusillus. J. Biotechnol. 68, 115 – 123. Pallesen, B.E., 1996. The quality of combine-harvested fibre flax for industrials purposes depends on the degree of retting. Ind. Crops Prod. 5, 65 – 78. Rihouey, C., Jauneau, A., Cabin-Flaman, A., Demarty, M., Lefebvre, F., Morvan, C., 1995. Calcium and acidic pectin distribution in flax cell walls: evidence for different kinds of linkages in the cell junction and middle lamella of the cortical parenchyma of flax hypocotyl. Plant Physiol. Biochem. 33, 497 – 508. Schunke, H., Sanio, C., Pape, H., Schunke, U., Matz, C.,
D.E. Akin et al. / Journal of Biotechnology 89 (2001) 193–203 1995. Reduction of time required for dew retting of flax: influence of agricultural, mechanical and microbiological techniques on fibre processing. Melliand Textilberichte (Eng. Transl.) 76, E101 –104. Sharma, H.S.S., 1987. Enzymatic degradation of residual noncellulosic polysaccharides present on dew-retted flax fibres. Appl. Microbiol. Biotechnol. 26, 358 –362. Sharma, H.S.S., Faughey, G., Lyons, G., 1999. Comparison of physical, chemical, and thermal characteristics of water-, dew-, and enzyme-retted flax fibers. J. Appl. Polym. Sci. 74, 139 – 143. Sharma, H.S.S., Van Sumere, C.F., Eds., 1992a. The Biology and Processing of Flax. M. Publications. Belfast, Northern Ireland. Sharma, H.S.S., Van Sumere, C.F., 1992b. Enzyme treatment
.
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of flax. Genetic Eng. Biotechnol. 12, 19 – 23. Stephens, G.R., 1996. Connecticut fiber flax trials 1992 – 93. Bulletin 932, The Connecticut Agric. Exp. Sta., New Haven, CT. Van Langenhove, L., Bruggeman, J.P., 1992. Methods of fibre analysis. In: Sharma, H.S.S., Van Sumere, C.F. (Eds.), The Biology and Processing of Flax. M. Publications, Belfast, Northern Ireland, pp. 311 – 327. Van Sumere, C.F., 1992. Retting of flax with special reference to enzyme-retting. In: Sharma, H.S.S., Van Sumere, C.F. (Eds.), The Biology and Processing of Flax. M. Publications, Belfast, Northern Ireland, pp. 157 – 198. Van Sumere, C.F., Sharma, H.S.S., 1991. Analyses of fine flax fibre produced by enzymatic retting. Aspects Appl. Biol. 28, 15 – 20.
Enzyme-retting of flax and characterization of processed fibers Danny E. Akin a,*, Jonn A. Foulk a, Roy B. Dodd b, David D. McAlister III c a
US Department of Agriculture, Agricultural Research Ser6ice, Russell Research Center, PO Box 5677, Athens, GA 30604, USA b Department of Agricultural and Biological Engineering, Clemson Uni6ersity, Clemson, SC 29634, USA c US Department of Agriculture, Agricultural Research Ser6ice, Cotton Quality Research Station, Clemson, SC 29633, USA Received 26 June 2000; received in revised form 21 November 2000; accepted 1 December 2000
Abstract Enzyme-retting formulations consisting of Viscozyme L, a pectinase-rich commercial enzyme product, and ethylenediaminetetraacetic acid (EDTA) were tested on Ariane fiber flax and North Dakota seed flax straw residue. Flax stems that were crimped to disrupt the outer layers were soaked with various proportions of Viscozyme-EDTA solutions, retted, and then cleaned and cottonized with commercial processing equipment. Fiber properties were determined and crude test yarns were made of raw and Shirley cleaned flax fibers and cotton in various blend levels. Cleaned fibers were obtained from both seed and fiber flax types, but with variations due to treatment. Retting formulations produced fibers having different properties, with enzyme levels of 0.3% (v/v as supplied) giving finer but weaker fibers than 0.05% regardless of EDTA level. Experimental yarns of blended flax and cotton fibers varied in mass coefficient of variation, single end strength, and nep imperfections due to sample and formulation. With cost and fiber and yarn quality as criteria, results established a range in the amounts of components comprising retting formulations as a basis for further studies to optimize enzyme-retting formulations for flax. Under conditions examined herein, Viscozyme L at 0.3% (v/v) plus 25 mM EDTA produced the best test yarns and, therefore, established a base for future studies to develop commercial-grade, short staple flax fibers for use in textiles. © 2001 Published by Elsevier Science B.V. Keywords: Pectinase; Chelators; Crimping; Strength; Fineness; Flax; Yarn
1. Introduction Flax (Linum usitatissimum L.) is likely the oldest textile fiber known, with evidence of production dating back 7000 years or more (Franck, 1992; Stephens, 1996). European colonists brought the skills for flax production to North * Corresponding author. Fax: + 1-706-546-3607.
America very early in its development (Stephens, 1996). While many parts of the world have continued to produce fiber flax, the United States produces no flax for textiles. Flax is grown as an oilseed crop with Canada as the major supplier, and a small percentage of the straw is used for speciality paper and pulp (Domier, 1997). However, considerable interest exists now in the US for production of flax fiber for use in textiles and
0168-1656/01/$ - see front matter © 2001 Published by Elsevier Science B.V. PII: S 0 1 6 8 - 1 6 5 6 ( 0 1 ) 0 0 2 9 8 - X
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a variety of high value products. While seed flax straw is an option for a lower grade of fiber for certain composites, fiber flax could supply the textile industry with a natural fiber for blending with cotton to enhance the properties of the world’s foremost natural fiber. In the US, industry analysts indicate that the major application for flax fiber in textiles will be in ‘cottonized’ form (i.e. cleaned and cut to staple length) for blending with other fibers on high efficiency cotton equipment. Retting, which is the separation of bast fiber from non-fiber tissues in the stems, is the major problem in processing flax (Pallesen, 1996). Water-retting, which was formerly the method of choice because of high quality fiber, produces environmentally unacceptable fermentation waste (Sharma and Van Sumere, 1992a,b). This practice was mostly discontinued in western countries several decades ago because of the pollution from fermentation products and the high cost of drying (Brown, 1984). Currently, dew-retting, which depends on indigenous, aerobic fungi to colonize pulled plants in the fields, is the accepted practice in European countries and accounts for much of the linen used in textiles. Disadvantages of dewretting are (1) dependence on particular geographical regions that have the appropriate moisture and temperature ranges for retting, (2) coarser and lower quality fiber than with water-retting, (3) less consistency in fiber characteristics, and (4) occupation of agricultural fields for several weeks (Van Sumere, 1992). Dew-retting further results in a heavily contaminated fiber that is particularly disadvantageous in cotton textile mills. An alternative that has had long-term consideration is enzyme-retting (Sharma and Van Sumere, 1992a; Schunke et al., 1995). In the 1980s, research was undertaken in Europe to develop enzyme-retting as a method to replace dew-retting. Flaxzyme, a commercial enzyme mixture from Novo Nordisk (Denmark), and several patents (Van Sumere, 1992) resulted from this research. Van Sumere and Sharma (1991) evaluated Flaxzyme, which consists of pectinases, hemicellulases, and cellulases, at a concentration of 3 g l − 1 and a liquor to stem ratio of 10:1 and reported that enzyme-retting produced fiber fineness,
strength, color and waxiness comparable to the best water-retted fiber. Sharma (1987) further used commercial enzymes to remove non-cellulosic components from dew-retted fibers. Despite the apparent success of Flaxzyme, no commercial process was developed. Cost of the enzymes, and perhaps other less obvious reasons, prevented development of a commercial enzyme-retting process. Dew-retting remains the practice most widely used in Europe to obtain fibers commercially for industrial use, despite continuing research on other methods (Sharma et al., 1999). Research was initiated about 5 years ago in the US to evaluate enzyme-retting for potential in supplying a short staple flax fiber suitable to blending with cotton in textile mills. Progress reported to date includes: good production yields in various regions including the southeastern US as a winter crop (Foulk et al., 2000), experimental enzyme-retting method using commercial enzymes with chelators sprayed onto mechanically crimped stems (Akin et al., 2000), and initiation of standards development for textile and industrial applications. The development of a cost effective, efficient enzyme-retting system requires additional work. This report provides recent data on pilot scale studies of enzyme-retting of fiber and seed flax plants that have been commercially cleaned, cottonized, and tested in experimental yarn blends.
2. Materials and methods
2.1. Samples Flax samples used included both seed flax and fiber flax types. Seed flax fiber was derived from an unknown variety produced under commercial conditions in North Dakota, USA. The seed was removed by combining, and the straw residue was collected and baled without dew-retting. During storage and before tests of enzyme-retting, the samples became dark indicating weathering and likely colonization by indigenous microorganisms. For fiber flax, the cultivar ‘Ariane’ was produced as a winter crop in the coastal plain region of South Carolina from December 1998 to May
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1999. Production, harvesting, and yields have been provided in detail (Foulk et al., 2000). Two harvests of Ariane were collected and included an early harvest (May 5, 1999) for high quality fiber and a late harvest (May 27, 1999) for both seed and fiber. At late harvest, seeds were collected by a stripper header attached to a combine, and both early and late stems were cut by a drum-mower about 5 –7 cm above the soil line. Plants for enzyme-retting were then either dried in an enclosed shed or field dew-retted for 3 weeks from the early harvest or field-dried and baled without any dew-retting from the late harvest. Prior to enzyme-retting, monolayers of dried flax stems were crimped through fluted steel rollers at :80 N in order to disrupt inherent stem barriers (i.e. cuticle/parenchyma) to enzyme penetration into the tissues (Akin et al., 2000).
2.2. Enzyme-retting formulations A series of formulations based on earlier laboratory work (Henriksson et al., 1997; Akin et al., 1999, 2000) was used to ret both seed flax and fiber flax. The multi-enzyme complex Viscozyme L, which is commercially available for use in breakdown of plant cell walls for extraction of components from tissues, was used in all enzymeretting formulations. Viscozyme L, which is prepared from a selected strain of Aspergillus, has activity against branched pectin-like compounds and also contains a wide range of carbohydrases including arabanase, cellulase, b-glucanase, hemicellulase, and xylanase (Product Sheet, Novo Nordisk, Franklinton, NC). In this regard, Viscozyme L is similar to Flaxzyme, which was developed specifically for enzyme-retting (Van Sumere, 1992). The retting formulation included ethylenediaminetetraacetic acid (EDTA) at 25 or 50 mM levels in water and adjusted with NaOH to pH 5.0. The enzyme was added to the EDTA solution at either 0.3 or 0.05% (v/v) as supplied.
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of formulation required in tank retting. About 12 kg of crimped flax stems were soaked with : 40 l of retting formulation and rotated in a sealed container for 2 min. Soaked flax was then placed in an insulated chamber, covered with wet burlap to retain humidity, and incubated at 40 °C for 24 h. Retted flax was then washed with 2 changes of : 110 l of tap water for 5 min each and dried in the chamber at 50 °C. For one lot of seed flax straw, : 12 kg was sprayed with : 40 l of enzyme formulation rather than soaking.
2.4. Microscopy Ariane stems were excised about midway of the plant. Sections 2–3 mm long were incubated with 0.05% Viscozyme L in sodium acetate buffer at pH 5 without chelator for 2, 4, and 8 h and buffered Viscozyme L plus 50 mM EDTA for 8 h. Control, unretted sections incubated in buffer alone were also selected for comparison. Sections were prepared for scanning electron microscopy by methods described (Akin et al., 1996).
2.5. Commercial cleaning and cottonizing Retted flax was shipped to the flax company Ceskomoravsky len, Humpolec, Czech Republic, to be cleaned through the Unified cleaner. This device uses a series of crushing rollers, shaker prongs, and a scutching wheel to commercially clean large amounts of flax. Residual fiber was removed between runs of each of the treatments so that contamination was kept to a minimum. The Ariane fiber samples were cleaned twice, but the seed flax straw samples were cleaned only once due to their brittleness. The Unified cleanedflax was then cottonized by the LaRoche system, in which flax fibers were chopped to : 5 cm and then blown through a series of ducts and beaters to separate and further clean the staple length fibers.
2.3. Application of retting formulation
2.6. Determination of fiber properties
The enzyme formulation was applied as a modification of the spray enzyme-retting method (Akin et al., 2000) designed to reduce the amount
Cottonized flax fiber was analyzed for fineness by micronaire (ASTM, 1997a), which was modified to use 5.0 g fiber based on calibration
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al., 2000). This flax fiber was blended with raw cotton at 10:90 (flax:cotton) and yarns spun as above. Finally, fibers from two retting treatments, namely Ariane flax retted with either 0.05 or 0.3% Viscozyme L and each with 25 mM EDTA, were cleaned twice through the Shirley Analyzer for blending with cotton, which was Shirley cleaned once, in 10:90 and 30:70 levels. Yarns were evaluated using the Uster II Evenness Tester or the ILE DS-65 Evenness Tester for mass coefficient of variation (CV) of yarn width and nep imperfections (200+ ) per 1000 yards. Strength was evaluated by the Statimat-M single end yarn strength tester (LawsonHemphill, Central Falls, RI.)
Fig. 1. Scanning electron micrograph of control, unretted Ariane fiber flax stem showing fiber bundles (F) between shive (S) and the cuticle/parenchyma barrier (arrows). Bar = 50 mm.
with flax fineness standards (Institut Textile de France, Lille, France) as reported (Akin et al., 1999). Strength and elongation were determined by the flax bundle method using the Stelometer (ASTM, 1997b) and length parameters determined using the array method (ASTM, 1997c).
2.7. Production and e6aluation of experimental yarn blends Hand-blended flax and commercial cotton samples were processed through a card to remove fiber impurities and short fibers and to align fibers. After carding, the fibers were drawn into a sliver and draw frame to further align and help blend fibers. Crude test yarns were then ring spun from the sliver at the Cotton Quality Research Station, ARS-USDA. In one test, cottonized flax, without further cleaning, and raw cotton were hand-blended in a ratio of 50:50. In another test, cottonized flax was further cleaned by passing one time through a Shirley Analyzer, which separates the finer fibers from coarse fibers and residual shive (Akin et
3. Results and discussion Viscozyme L is a commercial product containing the following activities of major polysaccharidases: 10 700 PSU g − 1 pectinase, 108 FBG g − 1 b-glucanase, 725 NCU g − 1 cellulase, and 31 200 VHCU g − 1 hemicellulase (data supplied by Novozymes, Franklinton, NC). Therefore, it is rich in pectinase but low in cellulase activities and effected structural modifications of flax stalks required for retting, including the separation of fiber bundles from the shive and epidermis/cuticle and also separation into fibers and bundles of smaller divisions (Figs. 1–5). Longer incubation times increased separation of bast fibers, with 8 h (the longest time in these studies) resulting in considerable modification of the bast region (Figs. 2–4). However, addition to the enzyme mixture of EDTA at 50 mM further improved retting, with a greater apparent separation of cuticle/epidermis from the fibers (Fig. 5). These results confirm those of other studies (Akin et al., 1997; Henriksson et al., 1997), in which commercial pectinase-rich enzyme mixtures, with the addition of chelators, efficiently retted flax without destruction of the cellulosic fibers. Further, results indicate that Viscozyme L could provide a readily available ‘off-the-shelf’ retting enzyme.
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Fig. 2. Scanning electron micrograph of Ariane fiber flax stem retted in Viscozyme L at pH 5.0 for 2 h showing slight modification of bast fiber bundles. Bar = 50 mm.
Fig. 4. Scanning electron micrograph of Ariane fiber flax stem retted in Viscozyme L at pH 5.0 for 8 h showing considerable separation of fibers within the bast region. Bar =50 mm.
The production of Ariane flax for staple length fibers in this study has been reported (Foulk et al., 2000). The dry weight yield from early plots (average of 4) was 4075 kg ha − 1 and mature plots
averaged 5075 kg ha − 1. Using Viscozyme L in the Fried test for in vitro retting as described by Henriksson et al. (1997), fiber content in the stem was estimated to be :33%. Fiber yields of the
Fig. 3. Scanning electron micrograph of Ariane fiber flax stem retted in Viscozyme L at pH 5.0 for 4 h showing initial separation of fibers from shive and cuticle and slight modification of bast fiber bundles. Bar =50 mm.
Fig. 5. Scanning electron micrograph of Ariane fiber flax stem retted in Viscozyme L plus 50 mM EDTA at pH 5.0 for 8 h showing significant separation from the epidermis/cuticle (arrows) and loosening of fibers and fiber bundles within the bast region. Bar = 50 mm.
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Table 1 Properties of enzyme-retted and commercially cleaned and cottonized flax fiber Sample
Seed flax straw Seed flax straw Ariane (early) Ariane (early) Ariane (early) Ariane (early) Ariane (early) Ariane (late) a–g
Retting treatment EDTA (mM)
Viscozyme (%)
50 25 50 25 50 25 dew-retted 50
0.05 0.05 0.05 0.05 0.3 0.3 3 weeks 0.05
Micronaire
5.9 9 0.1b,c 6.0 90.2b 5.8 9 0.1c,d 5.7 9 0.1d 3.9 90.1g 4.6 90.1f 5.3 90.1e 6.7 90.1a
Strength (g tex−1)a
25.9 92.9b,c 19.6 9 1.1d,e 24.0 9 2.0c 20.9 9 1.3d 13.0 9 1.3g 15.8 91.8f 36.2 9 2.3a 26.8 93.4b
Length
Shirley cleaned yield (%)
UQL
Uniformity
1.355 9 0.009 1.200 9 0.075 1.346 90.114 1.428 90.024 1.247 9 0.025 1.247 9 0.037 1.311 9 0.082 1.428 90.065
78.3 9 0.6 73.7 9 1.6 74.6 99.7 78.4 92.1 71.39 1.9 73.0 9 2.0 73.89 0.5 79.9 92.5
25.3 9 1.0e,f 23.6 9 1.0f 30.7 9 8.8d,e 37.9 9 0.3b,c 61.4 9 0.7a 58.7 9 1.1a 43.0 9 1.1b 32.3 9 0.3c,d
Within columns, values with different letters differ at PB0.05.
enzyme-retted samples could not be accurately determined after Unified cleaning, but approximate yields ranged at least from 20 to 29% based on original straw weights. Properties of the fibers from the various samples and treatments showed significant differences(Table 1). Enzyme-retting of early-harvested Ariane with 0.3% Viscozyme L plus chelator resulted in finer (P00.05) fibers based on the modified micronaire method, with addition of 50 mM EDTA in the formulation producing the finest fibers of any treatment. The coarsest fiber was from late-harvested Ariane enzyme-retted with 0.05% Viscozyme L+50 mM EDTA. Within sample type, the lower levels of chelator resulted in coarser fibers. Based on comparisons with calibrated flax standards, the micronaire method using air flow to determine fiber fineness effectively differentiated treatments, but the values at present time do not indicate performance properties as they do for cotton. Cleaning through the Shirley Analyzer indicated that enzyme-retting with 0.3% Viscozyme L gave finer fibers as indicated by significantly (P 0 0.05) greater yields (Table 1). In this regard, micronaire and Shirley-cleaning both indicated that the 0.3% Viscozyme L plus chelator resulted in finer fibers. The Shirley Analyzer, which cleans
the flax by carding action (Van Langenhove and Bruggeman, 1992), separates the finer fibers from the coarser fibers and shive with little breakdown of bundles to smaller divisions during cleaning (Akin et al., 2000). Within enzyme levels, 25 mM EDTA at times tended to produce lower yields after Shirley-cleaning, but differences were not significant with either flax type. Fiber elongation was lower after enzyme-retting with 0.3% Viscozyme L than with other treatments (Table 1). However, elongation was low overall compared to cotton, as is typical for flax fibers. Fiber strength was lowest with 0.3% Viscozyme L treatments (Table 1). This reduction in fiber strength with higher enzyme levels occurred in other retting tests (Akin et al., 1999). Enzyme treatments with 0.05% produced fibers ranging from : 20 to 25 g tex − 1, and dewretted fibers were significantly stronger than all other treatments. Fiber length as indicated by the upper quartile and uniformity was similar for all treatments (Table 1). This parameter for 0.3% Viscozyme L was 1.247 in. regardless of chelator level. Commercial cleaning and cottonizing appeared to have the greatest influence on fiber length in these samples.
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Blended yarns with cotton and flax fibers from the various treatments and not further cleaned in proportions of 50:50 gave low quality yarns with a broad range in quality parameters useful in ranking the retting formulations (Table 2). However, the card removed a substantial portion of the flax fiber and likely resulted in an actual flax proportion lower than 50%. For these comparisons, treatment with 0.3% Viscozyme L+ 25 mM EDTA had the lowest mass CV and the fewest neps of the blends but had strength almost as high as any other blended yarn. Further, fewer breakages during spinning occurred with this formulation compared with others. Further tests were made of yarns constructed with 90:10 cotton:flax blends (Table 3). In these yarns, the flax fiber was cleaned through the Shirley Analyzer one time prior to blending with raw cotton. Flax levels on carding flats was lower after flax was processed in this manner. The fewest neps occurred in yarns from dew-retted flax, while those from flax enzyme-retted with 0.3% Viscozyme L had fewer neps than other enzymeretted fibers. Strength and mass CV were similar for all blends, and the use of 25 mM EDTA did not seem to impair yarn quality in these tests.
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Other yarns were spun with fibers from 0.05 or 0.3% Viscozyme L, both with 25 mM EDTA, that had been Shirley cleaned twice (Table 4). Twicecleaned flax fiber blended with once-cleaned cotton fibers improved parameters of 10:90 flax:cotton yarns over those, in which flax fibers only cleaned once with the Shirley Analyzer (Table 3). Yarns from fibers retted with 0.3% Viscozyme L plus 25 mM EDTA were particularly improved in strength, mass CV, and neps. Twice-cleaned flax fiber blends described above were also spun at 30% flax, with parameters similar for both enzyme levels and lower in quality than 10:90 blended yarns (Table 4). Results from the combination of tests indicated that the enzyme treatments produced fibers with a range of properties. The use of 0.3% Viscozyme L plus EDTA was superior to other enzyme treatments in producing fine fibers and yarns with fewer neps. The significantly higher fiber yield from Shirley cleaning of fine fibers from both 0.3% enzyme treatments indicated that an enzyme level higher than 0.05%, which was identified as a level that worked well at laboratory levels, would produce a greater yield of finer fibers. This enzyme level adds considerably greater expense, but
Table 2 Yarn properties of cotton/flax (50/50) blends Fiber source–rettinga or preparation
Single end strength (g tex−1)
Mass evenness (CV)
Nep imperfections/1000 yards
Seed flax strawb–0.05% Viscozyme L+50 mM EDTA Seed flax straw–0.05% Viscozyme L+50 mM EDTA Seed flax straw–0.05% Viscozyme L+25 mM EDTA Arianec (early)–0.05% Viscozyme L+50 mM EDTA Ariane (early)–0.05% Viscozyme L+25 mM EDTA Ariane (early)–0.3% Viscozyme+50 mM EDTA Ariane (early)–0.3% Viscozyme+25 mM EDTA Ariane (early)–dew-retted 3 weeks Ariane (late)–0.05% Viscozyme L+50 mM EDTA Upland cotton (deltapine acala 90) 100%
10.6 9 2.0
35.9
2659
10.59 1.6 8.79 2.2 11.29 2.0 9.39 1.8 9.791.6 10.0 91.7 9.891.7 9.391.9 17.49 1.8
37.7 38.7 38.3 39.9 36.7 35.1 38.7 43.5 19.4
3398 3658 3373 3250 2961 2312 2811 3305 461
Hand-blended (50:50 original weight basis) and carded to form sliver and yarn on traditional cotton equipment. Actual proportion likely less than 50% flax due to high losses during processing. a Crimped at 80 N, : 12 kg immersed in retting formulation in water, pH 5.0, for 2 min , incubated at 40 °C for 24 h, washed, dried. b Unknown commercially grown variety in North Dakota and combined for seed; second sample in list sprayed to soaking. c Grown as a winter crop in 1998/1999 in coastal plain region of South Carolina. Early harvest for fiber and late harvest for seed and fiber.
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Retting formulationa
90:10 yarn Single end strength (g tex−1)
0.05% Viscozyme L + 25 mM EDTA 13.39 2.0 0.3% Viscozyme L + 25 mM EDTA 14.5 9 2.4
70:30 yarn Mass evenness (CV)
Nep imperfections /1000 yards
Single end strength (g tex−1)
Mass evenness (CV)
Nep imperfections /1000 yards
24.0 24.2
530 490
10.8 9 2.3 10.19 1.9
30.4 30.1
1125 1240
Once Shirley cleaned cotton and twice Shirley cleaned Ariane flax fiber hand-blended and carded to align fibers to form sliver and yarn with traditional cotton equipment. a In water at pH 5.0 for 2 min, incubated at 40 °C for 24 h, washed, dried.
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Table 4 Yarn properties of cotton/flax blends from Ariane flax fiber twice cleaned through the Shirley Analyzer
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Table 3 Yarn properties of cotton/flax (90/10) blends through the Shirley Analyzer with flax fiber cleaned once Flax–retting treatmenta
Single end strength (g tex−1)
Mass evenness (CV)
Nep imperfections/1000 yards
Seed flax strawb–0.05% Viscozyme L+50 mM EDTA Seed flax straw–0.05% Viscozyme L+50 mM EDTA Seed flax straw–0.05% Viscozyme L+25 mM EDTA Arianec (early)–0.05% Viscozyme L+50 mM EDTA Ariane (early)–0.05% Viscozyme L+25 mM EDTA Ariane (early)–0.3% Viscozyme L+50 mM EDTA Ariane (early)–0.3% Viscozyme L+25 mM EDTA Ariane (early)–dew-retted 3 weeks Ariane (late)–0.05% Viscozyme+L 50 mM EDTA
14.793.1
25.5 9 3.5
628
14.39 2.7 13.09 3.2 13.89 3.3 13.99 4.0 11.49 3.1 13.99 2.5 13.79 2.4 14.3 92.3
23.4 9 4.3 27.19 6.9 28.9 9 8.5 25.2 93.4 24.6 95.8 26.195.5 24.6 96.4 25.29 7.8
509 647 736 597 572 571 555 665
Cleaned fiber samples hand-blended and carded to form sliver and yarn with traditional cotton equipment. a Crimped at 80 N, :12 kg immersed in retting formulation in water, pH 5.0, for 2 min, incubated at 40 °C for 24 h, washed, dried. b Unknown commercially grown variety in North Dakota and combined for seed; second sample in list sprayed to soaking. c Grown as a winter crop in 1998/1999 in coastal plain region of South Carolina. Early harvest for fiber and late harvest for seed and fiber.
zyme level adds considerably greater expense, but reducing chelator levels in half from the 50 mM level used earlier (Akin et al., 2000) reduced some costs and appeared to produce fibers of equal or even superior properties in fiber and yarns tests. While fiber strength was somewhat related to yarn strength, other factors obviously influence yarn strength, and lower strength obtained from 0.3% enzyme level with 25 mM EDTA in retting formulations did not appear to be detrimental to yarn strength. Properties of flax fibers are a result of the actions of retting and subsequent mechanical processing for cleaning of shive. Fineness is among the foremost factors affecting fiber quality for spinning into yarns (Sharma et al., 1999). Along with fiber type (Sharma et al., 1999), the degrees of retting and cleaning affect the breakdown of bundles to smaller units and ultimate fibers, and therefore determine fineness. Fiber strength, which has been related to fineness in enzymeretted flax (Akin et al., 2000), and length are other important fiber parameters that, along with fineness, influence quality and production efficiency of yarns. In earlier laboratory studies with other flax samples, we identified enzyme types and formulations that gave good retting criteria in the
laboratory (Akin et al., 1997, 1999, 2000; Henriksson et al., 1997). Henriksson et al. (1997) reported that addition of chelators to Flaxzyme in laboratory trials lowered considerably the recommended 0.3% enzyme level (Sharma and Van Sumere, 1992a,b). Therefore, pectinase-rich commercial products similar to Flaxzyme at 0.05% plus 30–50 mM EDTA or oxalic acid were tested and appeared to ret flax efficiently. The current work on flax samples, which had been commercially cleaned and tested in spinning trials for crude cotton:flax blended yarns, suggested that a higher enzyme level (0.3%) produced significantly higher yields of finer fibers for the Ariane fiber flax and North Dakota seed flax used herein. Since cost is a primary concern in commercial applications, reducing the components of the formulation to the least amounts for efficient retting is a priority of research. In the present study, use of 25 mM EDTA, in combination with 0.3% Viscozyme L, resulted in similar fiber yield and quality to 50 mM EDTA and gave superior overall properties for the test yarns in our treatments. With cost and fiber and crude yarn quality as criteria, our data established a range for retting formulations, which form the basis for further studies to optimize methods for enzyme-retting of flax.
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This report provides the first results from pilot plant studies using flax fibers produced by our enzyme-retting method, which were subsequently cleaned and cottonized by commercial equipment. Use of a chelator, which likely removed the Ca2 + that binds and stabilizes epidermal cells (Rihouey et al., 1995), improves activity of the pectinases (Henriksson et al., 1997). In laboratory tests of our method, the liquid to fiber ratio was less than 2 ml g − 1 (Akin et al., 2000) and considerably lower than that the 10:1 ratio reported for tank methods (Sharma and Van Sumere, 1992b). Residual activity of enzymes not absorbed during the 2-min soaking (Foulk and Akin, unpublished data) could further improve cost effectiveness. Superior yarn results with 0.3% Viscozyme and 25 mM EDTA identified a point for further research to optimize methods and formulations with particular sample types to produce commercial quality fibers and yarns. Specifically, research should further investigate various combinations of enzymes and chelators within these established ranges on various flax types for optimal efficiency, cost effectiveness, and resultant fibers with tailored properties. Towards this goal, preliminary work in our laboratory has shown that endopolygalacturonase (EPG) from a dew-retting fungus was as effective as commercial enzyme mixtures in retting flax (Akin et al., 1998; Henriksson et al., 1999). The data indicated the paramount importance of EPG to retting and suggested a future opportunity, perhaps through cloning technology, to produce a consistently effective but simplified enzyme formulation for flax retting. Determination of retting efficiencies and fiber properties of particular flax samples and types, in conjunction with specific formulations, could establish important criteria for expanding the application of enzymatic retting to produce flax fibers for a variety of industrial applications.
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D.E. Akin et al. / Journal of Biotechnology 89 (2001) 193–203 1995. Reduction of time required for dew retting of flax: influence of agricultural, mechanical and microbiological techniques on fibre processing. Melliand Textilberichte (Eng. Transl.) 76, E101 –104. Sharma, H.S.S., 1987. Enzymatic degradation of residual noncellulosic polysaccharides present on dew-retted flax fibres. Appl. Microbiol. Biotechnol. 26, 358 –362. Sharma, H.S.S., Faughey, G., Lyons, G., 1999. Comparison of physical, chemical, and thermal characteristics of water-, dew-, and enzyme-retted flax fibers. J. Appl. Polym. Sci. 74, 139 – 143. Sharma, H.S.S., Van Sumere, C.F., Eds., 1992a. The Biology and Processing of Flax. M. Publications. Belfast, Northern Ireland. Sharma, H.S.S., Van Sumere, C.F., 1992b. Enzyme treatment
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