Professional Documents
Culture Documents
ISRN Biotechnology
Volume 2013, Article ID 186534, 23 pages
http://dx.doi.org/10.5402/2013/186534
Review Article
Linen Most Useful: Perspectives on Structure, Chemistry, and
Enzymes for Retting Flax
Danny E. Akin
Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA 30606, USA
Copyright © 2013 Danny E. Akin. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
e components of �ax �Linum usitatissimum) stems are described and illustrated, with reference to the anatomy and chemical
makeup and to applications in processing and products. Bast �ber, which is a ma�or economic product of �ax along with linseed
and linseed oil, is described with particular reference to its application in textiles, composites, and specialty papers. A short history
of retting methods, which is the separation of bast �ber from non�ber components, is presented with emphasis on water retting,
�eld retting �dew retting), and experimental methods. Past research on enzyme retting, particularly by the use of pectinases as a
potential replacement for the current commercial practice of �eld retting, is reviewed. e importance and mechanism of Ca2+
chelators with pectinases in retting are described. Protocols are provided for retting of both �ber-type and linseed-type �ax stems
with different types of pectinases. Current and future applications are listed for use of a wide array of enzymes to improve processed
�bers and blended yarns. Finally, potential lipid and aromatic coproducts derived from the dust and shive waste streams of �ber
processing are indicated.
1. Introduction of Flax and Linen Fiber Russia produced about 80% of the world’s �ber �ax crop and
before 1936 was the greatest exporter of �ax.
e history of �ax �Linum usitatissimum L.) is long and Fiber �ax came to North America by European colonists.
important. e translation of its scienti�c name, �linen most Production of �ax in Connecticut was reported as early
useful” [1, 2], aptly describes its versatility and importance as 1640 [6]. While �ax was grown in several regions of
to world economy. Linen, the long, strong �bers from �ax the United States, particular states developed well-organized
stems, is considered one of the earliest successes in textiles commercial efforts, particularly Michigan and the Willamette
[3]. While evidence does not exist on how early people Valley in Oregon [7]. e production of �ax �ber in Oregon
learned to separate �bers from the stems, �ax as a ma�or in the early 1900s, led by efforts of the US Department of
textile in ancient Egypt is well documented in depictions of Agriculture and the Oregon Agricultural Experiment Station,
its cultivation and processing [4]. Linen samples have been was remarkably well documented and included production
reported in the remains of Swiss lake dwellings dating back yields, processing and mills, and other advancements [5, 8].
some 10,000 years [3]. Production and use expanded beyond As in Europe, specially designed equipment to pull, turn,
the Mediterranean countries to central and northern Europe, deseed, and scutch �ax was developed to increase agricultural
making its way to Great Britain about 2,000 years ago from e�ciency. Oregon’s commercial enterprise for �ax �ber,
the Middle East by Phoenician traders [3]. Linen, as one of the however, ended in the 1950s due to introduction of synthetic
primary �bers for Europe throughout the Middle Ages and �bers and loss of government subsidies.
the Renaissance period, was used extensively for clothing. e linen industry also declined in Europe due to the
Linen was important to Russia and its economy through coming of synthetic �bers, such as nylon and polyester for
various stages of its political history [5]. Flax became the apparel [3, 4]. Cotton before then, however, had overtaken
greatest export item and the basis of economic life in Russia the high position of linen and industrial �ax �bers, which
in the late 1800s and into the twentieth century. At one time, had existed for millennia, due to large amounts of inexpensive
2 ISRN Biotechnology
cotton and its improved mechanical processing. Generally, sources, likely markets will exist for the long, strong, and
cotton has led as the natural �ber of choice since this time, clean linen �ber in speci�c, high-value applications along
with only short times of reversals such as blockades during with cottonized �ax �bers for blends [10].
the American Civil War (1861–1865) and disruptions caused e ma�or source for �ax �ber in North America, how-
by World War II (1939–1945). Aer the war, the lower ever, is straw from the linseed industry. Linseed provides an
production levels returned. industrial oil widely used in paints, varnishes, cosmetics, and
In traditional linen-producing areas, promotional pro- linoleum [15]. In the past and even more recently, �ax seeds
grams by linen industries in Northern Ireland and western are being recognized as a health food, with the intact seeds
European countries led to a strategic organization to promote providing a laxative effect and linseed products providing
linen in the 1960s [4]. In the 1980s, the FAO (Food and lignans and omega-3 fatty acids [16, 17]. It is likely, then, that
Agriculture Organization of the United Nations) sponsored production of linseed will continue and provide a consistent
workshops on �ax, and in 1993, the “FAO Flax Group” source of stems for �ax �ber production.
became the “European Cooperative Research Network on In contrast to agronomic systems to optimize �ber pro-
Flax,” with coordination through the Institute of Natural duction in �ax, linseed production seeks to optimize seed
Fibres, Poznan, Poland [9]. is program continues to com- yield [18]. Short branching plants are grown until seeds are
pile data on crop production, facilitates interaction of several fully matured. Stems are thicker in these plants than in �ber-
working groups, and sponsors numerous workshops thus type �ax plants. In North America, as well as Europe and
promoting global interests in �ax �ber [10]. Two publications Russia, linseed production is in colder climates, where �ber
per year still appear under its auspices. extraction from the stems is very difficult due to reduced
Recently, however, production of textile �ax �ber has microbial activities and poor retting (see later). Most of the
decreased in most of the reporting European countries North American �ax �ber is extracted from linseed stalks by
and including the Baltic states and the Russian Federation hammer-milling to obtain all the �ber, regardless of length,
[10]. In the European Union (EU) countries, production to be used in specialty papers. e quality of linseed �bers
of �ax declined from 122,379 ha in 2005/2006 to 73,029 ha processed in this manner is considered too low for apparel
in 2010/2011. Declining EU subsidies since the 1990s have or other high-value uses under the current commercial
reduced production levels and regions. Production in China, production and cleaning operations. Even with this hammer
which has varied over the last two decades, has occupied a milled product for paper and pulp, however, there is currently
prominent position for the last several years [11]. However, interest in producing a cleaner �ber product, that is, less
the quality of Chinese-produced linen is not high enough shive or non�ber fractions, in order to reduce the amount of
for textiles, and China imports considerable amounts of long chemicals needed to remove lignin from the �ax in pulping.
�ber from France and Belgium. e Belgium market for the Several research programs are in place to promote the
prices of long �ber, which varies based on quality, recently use of �ax and other natural �bers from various sources,
was reported to range from 130 to 210€/100 kg, with prices including linseed stalks, in new products and in particular
for unscutched short �ber of 30 to 50€/100 kg [10]. biocomposites. Efforts are currently under way to improve
Aer �ax production ended in Oregon and effectively processing methods and �nd more uses for value-added
ended �ax �ber production in the US, efforts to renew �bers in industrial, for example, biocomposite, applications.
a �ax �ber industry in the United States have occurred In fact, industrial applications with composites and nonwo-
over the years. Most have not been successful. In 1998, ven materials may provide the greatest potential for expanded
Naturally Advanced Technologies (NAT) and the Alberta use of �ax �bers in the future [19–21]. Canada, which
Research Council in Canada developed Crailar �ax. NAT and is the world’s leading producer of linseed [22], currently
the Hanes clothing company promoted the development of promotes expanding the �ax industry, both for �ber and
this product for complementary use with cotton in textiles. seed, through genetics research programs and organizations
While the method is proprietary, popular news stories [12] such as the Saskatchewan Flax Development Commission,
and promotional websites [13] indicated that Crailar �ax the Composites Innovation Centre, and Biolin Research,
increased performance characteristics in textiles. Inc. in Saskatchewan, Canada [19]. To this end, Canadian
Despite the reduction in production and usage from research into �ax has recently increased, particularly towards
previous times, linen imparts characteristics of comfort, improving �ber quality of high-value applications such as
drape, and a distinctive appearance that have maintained a biocomposites. North Dakota, the main producer of linseed
share of the market, particularly the luxury market for textiles in the US, follows the trend of the linseed industry in Canada.
[4, 10, 11]. Blending of cottonized �ax, that is, short, re�ned About 20% of the straw from the linseed industry satis�es
�ax �bers, with cotton and other �bers offers a potential the specialty paper (mostly cigarette) industry in North
for nontraditional, �ax �bers to impart distinctive properties America. e remainder of this straw by-product (greater
in textiles. For example, blending cotton with increasing than 1 million tons from western Canada) is now burned
levels of cottonized �ax �ber for rotor spinning improved or chopped to spread on �elds [22]. With the closing of
air permeability and wicking rates for moisture and modi�ed the Ecusta operation, only one large processor for �ax �ber
the fabric structure [14]. e �ne linen used for painting from linseed straw, that is, Schweitzer Mouduit International,
canvasses also requires strong and clean �bers from linen- Winkler, Canada, still produces �ber and mostly for paper
type production systems, such as that from Belgium. Despite and pulp and lower-end uses. A very great opportunity
the reduced production of traditional linen and move to other exists with linseed straw to improve farm economy, replace
ISRN Biotechnology 3
low versus high �uality �ber and yarn, likely because of the about 1.5%, with a higher level of guaiacyl to syringyl units
contamination by cuticle [25]. (Table 3). e low S/G ratio of lignin components was also
Cuticle, short �bers broken during processing, and woody shown by others [49]. Of the low molecular weight aromatics,
core fragments comprise the dust particles that are emitted only small amounts of ferulic acid were found (Table 3)
during �ber processing. e dust so generated is considerable, [24, 49]. Nuclear magnetic resonance (NMR) spectrometry
and dust bags or other suitable collection containers are used con�rmed that the aromatics were present as lignin [24].
to maintain a clean environment. Potential lipid coproducts Further, ultraviolet (uv) absorption microspectrophotometry
from the dust fraction, as well as from a purer cuticle fraction of thin sections also showed a strong absorbance near
removed during experimental enzyme retting, have been 280 nm in the core cell walls and no absorbance beyond, but
evaluated [46, 47]. A hot alcoholic extraction was effective absorbance maxima shied slightly in cells more towards the
in removing the wax components, which could then be stem center [24]. e presence of a single strong absorption
separated by controlled cooling [47]. ese waxes and other near 280 nm, as well as lack of absorbance near 320 nm,
lipids have potential as commercial products from this waste suggests a complex structure of the lignin similar to that in
material of �ax �ber processing. woody plants [50].
During �eld retting by indigenous fungi, the core cell
walls remain intact and are virtually impervious to fungal
2.2. Inner Core Cells. e central woody core tissues are attack, showing a typically high resistance to degradation for
the primary xylem and other structural cells, which provide this type of lignocellulose [24]. ese lignin-rich core cells
support and water conduction for the plant (Figures 1 and 4). make up a large portion of the “shive” waste fraction gen-
e core cells are about 65%–75% of stem material. e main erated during �ber processing. �ith such a high proportion
sugars are glucose, representative of cellulose, and xylose, of stem material, shive waste is a huge by-product of com-
representative of hemicelluloses (Table 2) [24]. Other car- mercial �ber production. Unless there are uses for it, shive
bohydrate components are lower in amounts and represent becomes an economic de�cit in processing, and its disposal is
hemicelluloses and pectins. necessary. Shive, however, has economic value in the overall
Almost all of the lignin in �ax stems is present in the core �ax processing system, generally now �nding application
cells. e histochemical stain for lignin, acid phloroglucinol in low-value uses typical of lignocelluloses, such as animal
[48], stains the entire core cell walls a bright red color, bedding, mulch, particle boards, and thermal energy from
indicating coniferyl-rich constituents throughout this tissue burning. ese additional sources of revenue provided by
(Figures 4 and 5). Positive reactions with acid phloroglucinol selling the shive, 9.00€/100 kg [10], are essential to guarantee
for coniferyl lignin (monomethoxylated aromatic rings) and a positive economic position of the �ax processing facility.
chlorine-sul�te for syringyl lignin (dimethoxylated aromatic More recently, the lignocellulosic nature of shive has shown
rings) [48] indicated that both lignin types are present in to be exceptional as activated carbon for heavy metal absorp-
core cells. Lignin values for core cells have been reported tion [51] and for removal of chlorinated hydrocarbon [52],
at 25%–30% [49]. Results, however, vary with the analytical outperforming selected commercial products. Extraction and
method used, such as Klason lignin with 72% sulfuric acid separation of cellulose, hemicellulose, and lignin from �ax
or that derived from permanganate oxidation. Oen, Klason shives by various methods, notably pressurized low polarity
lignin gives higher values because components other than water extraction and under different conditions of pH and
aromatics are in the residue. By alkaline extraction and sub- temperature, are reported as effective means of obtaining
se�uent summation of similar structures, speci�c lignin types aromatic feedstocks from this by-product of �ber processing
were determined. Using this method, aromatic levels totaled [49, 53].
ISRN Biotechnology 5
e fact that the core cells, that is, shive, contain by far the liquids preferentially react there �rst [61–63]. Cellulases also
most aromatics in the plant stem while �ber contains very attack preferentially at the �bernodes, even across bundles
little gave rise to a rapid method to assess �ber cleanliness. (Figure 7). Breaks during stress and compression tests occur
Near infrared re�ectance (NIR) spectroscopy coupled with preferentially at �bernodes and kink bands [64, 65].
chemometric models for ratios of shive : �ber from 0% to Cellulose is the main component in bast �bers, with
100% was used to develop a method to predict shive content values of 65%–80% of the dry weight reported [63]. In
in cleaned �ber [24, 54–57]. is method has been further addition to cellulose, bast �bers contain pectins, hemicel-
developed and employed for at-line assessment of quality in lulose, and aromatic compounds in small amounts (Tables
which commercial bales were assessed over several weeks 4 and 5). Field-retted �ber showed an expected increase in
[58]. In converse, the cleanliness of shive from residual glucose (by weight) indicative of cellulose, while increases
�ber le� from processing can be similarly predicted in also occurred in mannose and galactose. ese noncellulosic
materials sought for clean woody lignocellulosic materials sugars appear to be inherently part of the �ber [66]. e
[59]. fact that hemicelluloses, such as galactoglucomannans and
xylans are substantial components in �ax �bers has been
2.3. Bast Fibers. e industrially important bast �bers are shown by considerable research [63, 66–68]. Distinguish-
long, slender, and strong specialized cells that develop in ing characteristics of linen, such as high moisture regain,
bundles in the cortex region, located between the cuti- may be in�uenced by the presence of these noncellulosic
cle/epidermis layer and the innermost woody cells (Figure carbohydrates within the cellulosic structure. Proteins and
1). ese cellulose-rich cells are the source of linen and other proteoglycans are also associated with secondary walls of �ax
commercial �bers. Based on quality properties such as length, �ber and possibly provide structure [69].
strength, and cleanliness, the bast �bers are sought for high- Data on lipids associated with bast �bers were collected
value apparel and other textiles, natural �ber composite rein- from several studies and presented in Table 5. Fibers that
forcement, and specialty papers such as cigarette, currency, had been manually separated and cleaned of all other visible
and Bible sheets [15, 18, 19]. materials showed the presence of low levels of waxes, cutins,
In nature, these �bers exist in bundles of individual (or and sterols, with amounts of about 0.2% of �ber dry weight
ultimate) �bers encircling the ligni�ed core tissues (Figure 1). and 1/20th or less of levels in the cuticularized epidermis.
Ten to 40 spindle-shaped ultimate �bers, each 2 to 3 cm long Other work, however, on a series of dew-retted and water-
and 15 to 20 𝜇𝜇m in diameter, form in 20 to 50 discrete bundles retted �ax �bers from �urope [27] showed that lipids were
[3, 37]. Nodes or ��bernodes� [60], which are dislocations present on �bers, with higher levels on the better quality
perpendicular to the axis, occur in �bers and entire bundles, (i.e., stronger, and �ner) water-retted ones (Table 5). With
as clearly shown by microscopy (Figure 6). What creates these latter samples, it was not clear if the lipids represented
�bernodes is not clear, and kink bands are similar in structure residual cuticle or if these �bers had more inherent lipids
and apparently give similar responses. Possibly, pressure on the surfaces. Chemical analyses of highly cleaned �bers
exerted during mechanical handling or even through growth from linseed �ax and mature plants (i.e., grown for seed) had
and expansion of tissues may create their presence. ese higher lipid levels than did bast �bers of the �ber-type plants
�bernodes are important, because dyes, enzymes, and other (Table 5). ese compounds in linseed may re�ect a higher
6 ISRN Biotechnology
1.5
1.2 CC
Absorbance
0.9
0.6
0.3 ML
NF S
0
230 254 278 302 326 350
F 4: Digital photograph of the stem fraction separated from
(nm)
�bers during processing and stained with acid phloroglucinol. e
F 3: Ultraviolet microspectrophotometry of thin section stems are bright red and well differentiated from �bers.
of unretted �ax stem showing the absorption over a range of
230�350 nm. Spectra CC is of selected cell corners of �ber bundles,
ML is middle lamellae, NF is non�ber region, and S is secondary
wall of bast �ber. From [24].
compared to cotton �bers. While �ax �ber is primarily dry climates for an extended time. Proper retting also releases
a cellulosic �ber, its chemistry and characteristics provide the cuticle/epidermis layer from the �ber bundles. It is the
speci�c properties that di�er from cotton and many other pectin-rich regions that are of prime importance in retting,
natural �bers. and considerable work has been done on the pectinases and
ways to degrade the pectin in �ax.
2.4. Other Cell Types and Structures. Parenchyma, cambium,
and the middle lamellae, which bind ultimate �bers in the 2.5. Pectin. Pectin is a complex polysaccharide of many plant
bundles, are particularly rich in pectins, hemicelluloses, and cell walls and plant tissues [76]. Pectin, while oen low in
other matrix polysaccharides as shown by response of these amounts, is strategically located and binds cell walls within
tissues to pectinolytic enzymes [26]. Lignin is lacking in plants [66]. While pectin is, therefore, particularly important
these structures for the most part. �e separation of �bers in maintaining the structure of �ax stems, its degradation is of
from the woody core occurs at the outer surface of the fundamental importance for retting and the resulting quality
cambium and is facilitated when stems have been stored in of �ax �bers [37, 73].
8 ISRN Biotechnology
shive in the hammer milled �ber material in order to reduce production are unable to ret because of the noncompliant
chemicals used for deligni�cation in making pulp. e fact climates, such as England, Scandinavia, and Ireland. Field
that linseed straw is produced in regions unsuitable for dew retting only works with appropriate moisture and tempera-
retting is problematic in having high �ber yields and clean ture for fungal activity. Another disadvantage of �eld retting
�ber. is that large tracts of land are tied up for weeks until �ax
Two main methods have been historically employed is suitably retted. In intensive agricultural areas (e.g., for
commercially to ret �ax for textile-grade �bers, namely, multiple cropping), farmers are disadvantaged by having land
water retting and dew (or �eld) retting [3, 37]. In traditional occupied by retting �ax. Dew-retted �ber is dirty due to the
water retting, as practiced in western Europe when the fungi and soil. e vagaries of weather constantly threaten
linen industry was �ourishing, �ax stems were pulled and the harvest. Too dry weather results in poor fungal growth
submerged in bodies of water, for example, lakes, rivers, and lack of proper retting� wet weather delays �eld harvest
and ponds, for �ve to seven days. Aerwards, retted stems and also interferes with fungal growth, resulting in pockets
were dried and sun bleached in the �eld. Anaerobic bacteria, of anaerobic degradation. Over-retting occurs with excessive
primarily pectin degraders, were the main organisms in water growth of cellulolytic fungi in the retting consortium and
retting. Because the process was understood to some extent, results in weakened �ber. So, even in the best regions for �eld
technological methods were adapted to improve the process. retting, crop losses of about one-third are expectedly for one
Retting pits or tanks were constructed where temperature reason or another.
could be controlled. Selected microorganisms were chosen From the early 1900s, technological efforts have been
to improve water retting. At times, tanks were aerated to attempted to improve retting [37]. is subject has been
modify the bacterial consortium and thereby the bacterial brie�y and recently reviewed by this author [18, 38]. Methods
metabolism, reducing the problems of pollution and stench for improved retting included modifying water retting or
from anaerobic metabolism that caused widespread concern dew retting to remove the inherent problems and to select
in areas of western Europe [37]. speci�c retting microorganisms. Stand retting, where stand-
Water retting resulted in long, strong, and �ne �bers ing plants are dried with a herbicide, notably glyphosate (N-
of excellent quality for apparel and other textiles. e high phosphonomethyl glycine), and allowed to ret by indigenous
cost of this method and resulting pollution, with the stench microorganisms in a modi�ed form of �eld retting, has
residing in water-retted �bers, however, resulted in water been tried [93, 94] and is still being developed [95]. Oen
retting being abandoned for the most part in the mid 1950s the �ber properties were shown to be improved over dew
in western Europe [37]. Water retting has been mostly retting, although dry weather interfered with retting and was
replaced by dew retting, although some water retted �ax problematic with this herbicide [96]. is method is still
was commercially available in the early 2000s [91]. Reports promising, particularly with new forms of the herbicide, and
indicate that China, the largest producer of �ax, may still has been used to produce test plots of �ax for cottonized �ber
produce water retted �ber in aerated tanks, but the �ber in England [97].
quality is not reported to be of good quality for textiles In addition to modi�cation of the traditional water and
[11]. �eld retting methods, much research has focused on chemical
Dew (or �eld) retting is the method used in western retting approaches, sometimes with and sometimes without
Europe for obtaining high quality �bers for textiles. Field ret-
microorganisms or enzymes [80, 98–106]. As mentioned
ting, however, is reported to be the oldest method of retting
previously [80], the use of chelators, notably EDTA, has
�ax, practiced thousands of years ago by the Egyptians [37].
been pursued, and its value is shown by strong sequestration
Field retting is carried out by pulling �ax stems and laying
them in even layers of rows for the moisture to encourage of Ca2+ at various pHs [98] to disrupt the pectin linkages.
indigenous fungi to colonize and grow on the stem. e Autoclaving �ax straw with the chelators EDTA and oxalate
farmers of western Europe reportedly produced the best dew- has been used with breeding programs to effectively extract
retted �ber because of the climate and their knowledge of unretted �ax �bers [101]. A patent exists for a mechan-
when to turn and harvest the �ax for uniform retting. In areas ical process to produce �ber strips followed by a chemi-
of proper climate and expertise, commercially dew retting cal/cooking process under pressure [102]. Flash hydrolysis
works and has been the method of choice for linen and other or steam explosion treatment, with or without impregnation
�ax �ber production. Most of the world�s textile �ax �ber is before steam treatment, has been used to remove pectins
produced by �eld retting [10]. and hemicelluloses from decorticated �ax to produce small
e quality of �ax �ber has declined over the years since bundles and ultimate �bers [104–106]. Ultrasonic treatment,
dew retting replaced water retting as the main method for following decortication and opening of green �ax or hemp
getting textile grade �bers in western Europe [92]. In addition stalks, has been used to obtain �bers from diverse sources
to lower quality, �eld retting results in an inconsistent without the use of chemicals [103]. e use of low energy,
quality �ber. Dew retting continues to be the main retting uniform ultrasonic treatment, combined with enzymes, has
method over water retting because production costs are shown increased activity of various enzymes for cotton
lower, however, and �ber yields are higher and there is no fabrics [107] and could be a useful method to improve the
stench. Dew retting, though, has a number of disadvantages e�ciency in retting �ax. Chemical separation has resulted in
other than poor and inconsistent quality compared to water- successful laboratory results, but at times, �ber properties are
retted �ber. Certain areas formerly known for their linen less satisfactory than those from other methods. Efforts are
10 ISRN Biotechnology
reported to be still underway to assess physical and chemical 3.2. Further Research on Enzyme Retting. In the 1990s,
methods to separate �ber. e use of enzymes, focusing on the United States was the largest per capita user of �ax-
pectinases, also has been researched for some time [37, 108– containing textiles, but no linen or �ax �ber for textiles was
110]. None of these methods has replaced �eld retting as a produced domestically. e only �ax grown was for linseed.
commercial practice. Enzyme retting, however, has proven to is statistic prompted the Agricultural Research Service
offer promise as a biotechnological improvement and is still (ARS) of the US Department of Agriculture to begin research
undergoing research and development for improving �ber toward developing a �ax �ber program for textiles. e �rst
quality. goal in this initiative was to improve retting emphasizing
enzymes. Results from the work in Europe, particularly with
pectinases, were the basis of research. ere were other
3.1. Enzyme Retting. Sharma and his colleagues in Europe considerations, however, that became apparent for this work.
and the United Kingdom in the 1980s carried out a major Canada and the US northern plains states had a thriving
effort on enzyme retting, primarily attempting to mimic linseed industry, with tons of waste straw available. Linseed
water retting with a consortium of plant cell wall-degrading straw removal aer harvest presented a problem for farmers.
enzymes. ese efforts, as well as other topics related to �ax Producers wished to remove the straw from their �elds
production for textiles, have been documented in numerous soon aer harvest, and the straw did not degrade readily.
papers and in the book e Biology and Processing of Flax A small portion of this crop, estimated around 25%, was
[111]. Since the plant cell wall is a complex lignocellulosic used for hammer milling and pulp for specialty papers,
material, it was believed that a mixture of cellulases, hemicel- such as cigarette, currency, and Bible sheets. Most of the
lulases, and most notably pectinases was required to ret �ax linseed straw was not used and was (and still is) burned to
as occurred with the plant-associated natural microbial con- remove it from the �elds. Further, the US textile industry was
sortia in water or dew retting [110]. In hindsight, part of this tied to cotton �ber processing and not the long-line linen
approach could have been due to the lack or cost of speci�c processing of Europe. In fact, no specialized wet-spinning
enzymes available at the time, as shown by attempts to use equipment required for long-line linen yarn existed in the
highly pectinolytic microorganisms [110]. Fungal cultures US. e textile spinning technology was based on short staple
contained this mixture of cell wall-degrading enzymes but �bers like cotton and synthetic blends. A �ax tow product,
with some different pro�les and activities. Microorganisms which is short �ber as a by-product of long-line linen,
with high levels of pectinases were chosen, but most culture was used in blends with cotton, but that product, too, was
�ltrates also contained some cellulases. imported.
Sharma and colleagues enjoyed success in their work. So, quickly, the ARS research effort incorporated linseed
Several commercial enzyme mixtures containing plant cell stems as a source of �bers, with attempts to improve retting
wall-degrading enzymes were tested. A product from Novo and processing for higher, quality in the �bers. Further, since
Nordisk (Copenhagen, Denmark) called SP 249 was used in the �bers desired were short-staple like cotton, “total �ber
a side-by-side test of enzyme versus water retting [108]. SP production,” with collection of all bast �ber regardless of
249 was used at 3 g/L in an 11 : 1 liquid-to-solid ratio at 45∘ C length, was employed with later “cottonizing” to shorten and
for 24 hours. Eighty kg of �ax stalks were submerged for re�ne the �bers.
each of the two test methods. Aer retting, �ber yield and e ARS �ax research focused on lowering enzyme
quality were equal for the two retting procedures. Oxidizing amount, �nding purer and more active enzymes, and devel-
agents, however, were required to denature the enzymes oping a protocol for enzyme retting. Earlier research indi-
and stop the continuing action of cellulases in the enzyme cated the value of calcium chelators for disrupting pectins
mixture. e study showed the successful application of in retting (see ealier). Tests with oxalic acid showed that
enzyme retting in pilot plant scale, promising that retting chelators could greatly reduce the amount of enzyme needed
of �ax could occur with enzymes and with a reduced time for retting [45]. So, with following tests, the retting mixture
of retting [108]. e liquid method also ensured a more was almost always an enzyme/chelator mix.
consistent product and could be carried out in any location Excellent �eld retting of �ax had been noted in research
with proper equipment. From the work of Sharma and experiments at Clemson University, South Carolina, for
colleagues, Flaxzyme, which was a patented liquid prepa- winter production of �ax. From this material, the major fungi
ration of balanced cellulases, pectinases, and hemicellulases colonizing the stems were cultured and isolated in a search
from Aspergillus species, was developed by Novo Nordisk for more active retting enzymes. One fungus stood out from
(Copenhagen, Denmark) for retting �ax. is enzyme also the others as the most active retter of �ax [43]. is fungus,
resulted in �ber yield and properties equal to or better than identi�ed eventually as Rhizopus oryzae sb NRRL 29086,
�ber from water retting [37]. Later, Lyvelin (Lyven, Caen, produced a potent endopolygalacturonase (EPG) and few
France), a pectinase, but not pure, from Aspergillus niger other enzymes in the �ltrate [30, 112]. is enzyme was puri-
was marketed speci�cally for retting of �ax. Despite these �ed and tested for its ability to separate �bers in stems and
positive results and developments, an enzyme retting method compared in mixtures with potentially complementary cell
did not replace dew retting. While the reasons are complex, wall-degrading enzymes. In these early tests, �ber separation
likely enzyme costs and lack of industry support prevented was judged by light microscopy and the Fried’s Test, which
further development. Flaxzyme is no longer sold under this is an in vitro test to judge �ber separation from stems by
name. comparing visual images [37].
ISRN Biotechnology 11
Results indicated that this puri�ed EP� with oxalic acid barrier improved enzyme retting over that by increased or
alone was sufficient to separate �ax �bers (Table 6). e other reduced atmospheric pressures [120]. Although inhibitory,
potentially complementary enzymes tested, namely, pectin aromatic compounds had been shown to be released by
methyl esterase, xylanase, and cellulases, did not improve chemical treatments [74], presoaking of �ber with water
�ber separation [30]. Data, therefore, indicated that enzymes to remove these compounds showed no clear bene�t with
other than EP� were not required to separate �ber from enzyme retting and was not included as part of the enzyme
non�ber fractions. retting protocol [121].
Based on these results, a search for a potential commercial e enzyme retting method, termed SER, was tested on
enzyme, with high pectinase and low cellulase activity, seed- and �ber-type plants, with various levels of enzyme
indicated that Viscozyme L (Novozymes North America) had and chelator. Ultimately, enzyme retting would have to
similarities to SP 249 and Flaxzyme. Oxalic acid in later be integrated to a commercial cleaning system for �ber
tests proved not to be suitable as a chelator, as a precipitate production, but such cleaning systems did not exist in the US.
formed and remained on the �bers. �hile tests continued Accordingly, arrangements were made to produce pilot scale
on a series of chelators of different types and under different amounts of enzyme-retted �ax for commercial processing
conditions [113, 114], EDTA replaced oxalic acid because of in Europe. About 12 kg of retted �ax for each formulation
efficiency at pH 5 to 10 and its commercial availability for was commercially cleaned by Ceskomoravsky len (CML) in
the textile market. e retting test method of choice, then, Humpolec, Czech Republic, using a Uni�ed Line scutching
was a combination of the commercial products Viscozyme L system and La Roche cottonizing system [33]. ese �bers
and EDTA (later using Mayoquest, a 36%–38% EDTA com- were tested for quality parameters and then spun into blended
mercial product) for other comparisons and modi�cations. yarns with cotton at 50 : 50 and at 10 : 90 �ax : cotton amounts.
A series of Viscozyme/EDTA formulations, with increases in Fibers were tested with modi�ed cotton testing equipment
each of the enzyme and chelator, was used to ret a mature, (Table 8) and the yarn properties by commercial testing
�ber-type �ax. Rather than the Fried�s test, cotton �ber tests equipment and methods (Table 9).
were used for strength by Stelometer [115] and �neness Results indicated that the enzyme mixtures retted both
[116] using a modi�ed microaire system equilibrated with �ber- and seed-type plants (Table 8). Higher levels of
a series of �neness standards �bers [117]. An estimate of enzyme reduced �ber strength but produced �ner and higher
the percentage of �ne �ber, collected by passing retted and amounts of �ne �ber. Other than strength, �neness and �ne
mechanically cleaned �ber through the Shirley Analyzer, was �ber yield were better than these characteristics in dew-retted
calculated from the starting material. e Shirley Analyzer, �bers. Chelator levels did not seem to vary in their impact,
which is an instrument to separate and collect trash in cotton, with 25 mM amounts equal to the 50 mM levels. e seed �ax
provided a percent of �ne, cleaned �ber as an additional �bers were of less quality than �ber-�ax �bers with similar
statistic to judge quality. formulations. Retesting of the �bers 30 months later showed
Experiments with a series of retting combinations [32] no further loss in strength, indicating the washing step aer
indicated that increasing Viscozyme levels increased �ne �ber enzyme retting was sufficient to stop further enzyme activity.
yield but reduced strength, regardless of chelator levels (Table Yarn properties were compared favorably between dew retted
7). Increasing levels of chelator, within each enzyme series, and enzyme retted at the higher level of enzyme (Table 9).
increased �ne �ber yield and resulted in �ner �bers up to Seed-�ax �bers processed into blends much like the �ber-
18 mM. Chelator level alone did not affect strength. Fibers �ax �bers, even the dew-retted sample. Speci�c areas for
from this study were blended with cotton (50 : 50), spun as improvement were identi�ed. Of concern was the loss of
yarn in a miniature spinning system, and the yarn properties strength with increasing level of enzyme.
determined [118]. Results indicated that enzyme treatments Since retting, processing, and yarn construction are inter-
affected yarn properties, with the highest enzyme level related, a �ax pilot plant was constructed based on the Uni�ed
producing �ner �bers, easier yarn construction, and better Line at CML in order to have a commercial method for
quality yarns. Assessments are difficult to compare, however, cleaning retted �ax stems [122]. is system was developed
because, for example, the highest level of enzyme produced a by engineers at CML but reduced in size and with each of
�ner but weaker �ber. Possibly, �ner but weaker �ber was also four parts separately positioned for research. e parts were:
less stiff and brittle and therefore more amenable for blending 9-roller calender for breaking stems, 5-roller calender for
with cotton. Both of these characteristics are important in further crushing shive, scutching wheel to produce a total
yarn construction. e clearest result from this work was that �ber from the stems, and an upper pinned shaker to remove
the miniature spinning system has value in predicting the loose shive and straighten �bers.
optimal retting formulations for yarn quality. Further, a series of test protocols was developed for
In order to develop a new pilot plant method for enzyme objective test results of �bers. Fibers were tested using cotton
retting, a spray enzyme method, or a brief (2 min) soaking, equipment and protocols where applicable, such as strength
to deliver the enzyme/chelator mixture was used to reduce and elongation by Stelometer [115]. Further test standards
liquid : solid ratio compared to the former method of retting were developed for �ax, including color, �neness, and pre-
�ax in submerged tests [119]. Since the cuticle/epidermis dicted shive through the Flax and Linen subcommittee of
layer protected the internal stem tissues [45], methods ASTM International [56, 57]. e following tests methods
were explored to facilitate the entry of enzymes into stems. adopted were (1) percent of �ne �ber yield produced by
Physically crushing stems with �uted rollers to breach this passing through a Shirley Analyzer, (2) tensile strength
12 ISRN Biotechnology
T 7: Effect of enzyme and chelator amounts on properties of mature Ariane �ax stems.
Viscozyme/Mayoquest 200 (%)a Fine �ber yieldb (%) Strengthc (g/tex) Finenessd (air�ow)
0/0 4.3 ± 1.7f 26.9 ± 0.8a 8.0 ± 0a
0.05/0.4 5.4 ± 2.2ef 24.0 ± 1.4abc 7.7 ± 0.1abc
0.05/0.7 7.0 ± 1.8bcde 23.9 ± 5.5abc 7.9 ± 0ab
0.05/1.8 8.5 ± 0.6abc 24.6 ± 2.3ab 7.7 ± 0.1abc
0.1/0.4 6.2 ± 1.3def 20.3 ± 2.5bcd 7.6 ± 0.1abc
0.1/0.7 7.3 ± 1.8bcde 17.9 ± 2.3de 7.6 ± 0.1abc
0.1/1.8 7.9 ± 1.2abcd 20.3 ± 1.8bcd 7.1 ± 0cde
0.2/0.4 6.7 ± 0.9cde 18.1 ± 0.6de 7.4 ± 0.1bcde
0.2/0.7 7.9 ± 1.3abcd 17.6 ± 0de 7.0 ± 0.5de
0.2/1.8 8.9 ± 2.1ab 17.7 ± 1.4de 6.9 ± 0.4e
0.3/0.4 5.5 ± 0.9ef 15.3 ± 0.5e 7.5 ± 0.7abcd
0.3/0.7 7.3 ± 1.1bcde 18.1 ± 1.3de 6.9 ± 0.1e
0.3/1.8 9.8 ± 0.8a 19.5 ± 0.7cde 6.9 ± 0e
a
Viscozyme L (Novozymes) added as percentage of product as supplied. EDTA was supplied as Mayoquest 200 with 38% EDTA and used to enzyme ret mature
Ariane stems.
b
Enzyme-retted straw passed through hand-card 2X and passed 1X through a Shirley Analyzer.
c
Average and standard deviation of 2 replicates of Shirley-cleaned �ber, with each replicate an average of 6 tests by Stelometer (force at break divided by weight
by standard cotton testing).
d
Average and standard deviation of 2 replicates of Shirley-cleaned �ber, with each replicate an average of 2 tests by air �ow using approximately 5 g based and
IFC �ax �ber standards for �neness (similar to methods used for cotton �neness).
a,b,c,d,e,f
Within columns, values with different lower case letters differ at 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃.
Methods in [31]. Data from [32].
and elongation by Stelometer, (3) �neness based �rst on a work (Table 8). Purer enzymes were becoming more readily
modi�ed cotton air�ow method and later re�ned for a new available at this time, and research had showed that puri�ed
ASTM test method [57], and (4) the percent shive in cleaned EPG alone could separate �bers from core without the
�ber using near infrared spectroscopy and chemometric other cell wall-degrading enzymes. Further, results indicated
models from �ber : shive combinations [54, 55, 59]. is latter these methods worked on both �ber-type and seed-type �ax
method was accepted as a new ASTM test method in 2005 cultivars, indicating that these enzymes should be applicable
[57]. For certain assessments, the Fried’s Test and light and to linseed straw. Commercial enzyme products, developed
scanning electron microscopy were used when appropriate. A for various applications, were tested with the intent of �nding
color test method (D-6961-03) was approved as a new ASTM purer pectinases (i.e., low or no cellulases) that effectively
test method in 2003 [57]. Enzyme retting results in a lighter retted �ax without loss of �ber strength. As these further
�ber color than that by �eld retting, and various enzyme tests were being carried out, Novozymes North America, Inc.
retting formulations resulted in different color characteristics (Franklinton, NC, USA) released a commercial pectate lyase
based on the CIELAB L∗ , a∗ , and B∗ values [123, 124]. (PL) product for removing the cuticle of cotton �bers as
ese results suggest methods to tailor color properties for an environmentally friendlier way of scouring cotton, which
applications. traditionally used high levels of NaOH [125, 126]. BioPrep
Enzyme mixtures that included cellulases, such as Vis- 3000L is a liquid commercial PL produced by multiplying the
cozyme L, weakened �bers (Figure 7), as shown by previous native gene for alkaline PL in Bacillus lichniformis, placing
ISRN Biotechnology 13
T 8: �roperties of enzyme-retted and commercially cleaned and cottonized �ax �ber.
Fiber samplea Viscozyme/EDTA (%/mM) Fineness (air �ow) Strength (g tex−1 )b Fine �ber yield (%)
5.9 ± 0.1b,c 25.9 ± 2.9b,c
Seed �ax straw 0.05/50 25.3 ± 1.0e,f
6.1 ± 0.1 24.7 ± 1.9
6.0 ± 0.2b 19.6 ± 1.1d,e
Seed �ax straw 0.05/25 23.6 ± 1.0f
6.1 ± 0.1 25.3 ± 3.0∗
5.8 ± 0.1c,d 24.0 ± 2.0c
Ariane (early) 0.05/50 30.7 ± 8.8d,e
5.5 ± 0.1 25.0 ± 4.5
5.7 ± 0.1d 20.9 ± 1.3d
Ariane (early) 0.05/25 37.9 ± 0.2b,c
6.1 ± 0.1 25.3 ± 3.0∗
3.9 ± 0.1g 13.0 ± 1.3g
Ariane (early) 0.3/50 61.4 ± 0.7a
4.1 ± 0 16.3 ± 1.9∗
4.6 ± 0.1f 15.8 ± 1.8f
Ariane (early) 0.3/25 58.7 ± 1.1a
4.5 ± 0.2 15.6 ± 2.1
5.3 ± 0.1e 36.2 ± 2.3a
Ariane (early) dew-retted 43.0 ± 1.1b
5.3 ± 0.2 32.5 ± 2.2∗
6.7 ± 0.1a 26.8 ± 3.4b
Ariane (late) 0.05/50 32.3 ± 0.3c,d
7.1 ± 0.1 28.6 ± 3.7
a
Seed �ax straw was from North Da�ota, USA and grown in 1999. Ariane was either grown optimally as a winter crop in South Carolina, USA, 1999, for �ber
(early) or to maturity for seed (late). Flax stems were enzyme retted by soa�ing for 2 min and washing with water. �e �ber was cleaned in a commercial system
in �umpolec, Czech Republic through the Uni�ed �ine followed by cottonizing by the �a Roche system.
b
�e �rst number in each column is for samples tested in August, 1999 (2�4 months a�er retting), and the second number is for the same samples tested April,
2002 (30 month later).
a−g
Within columns and for samples tested August, 1999, values with different lower case letters differ, 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃.
∗
Within columns and for a particular sample, values for two test dates differ, 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃, using the 𝑡𝑡 test.
Data from [33].
T 9: �roperties of yarns made with enzyme-retted �ax �bers and cotton.
a
Fiber sample Viscozyme/EDTA (%/mM) Single end strength (g/tex)b Mass evenness (CV)b Nep imperfections/1000 yards b
10.6 ± 2.0 35.9 2659
Seed �ax straw 0.05/50
14.7 ± 3.1 25.5 628
8.7 ± 2.2 38.7 3658
Seed �ax straw 0.05/25
13.0 ± 3.2 27.1 647
11.2 ± 2.0 38.3 3373
Ariane (early) 0.05/50
13.8 ± 3.3 28.9 736
9.3 ± 1.8 39.9 3250
Ariane (early) 0.05/25
13.9 ± 4.0 25.2 597
9.7 ± 1.6 36.7 2961
Ariane (early) 0.3/50
11.4 ± 3.1 24.6 572
10.0 ± 1.7 35.1 2312
Ariane (early) 0.3/25
13.9 ± 2.5 26.1 571
9.8 ± 1.7 38.7 2811
Ariane (early) Dew-retted
13.7 ± 2.4 24.6 555
9.3 ± 1.9 43.5 3205
Ariane (late) 0.05/50
14.3 ± 2.3 25.2 665
Upland cotton NA 17.4 ± 1.8 19.4 461
a
Seed �ax straw was grown in North Da�ota, USA in 1999. Ariane was grown optimally as a winter crop in South Carolina, USA, 1999, for �ber (early) or to
maturity for seed (late).
b
�e top value in each column is a 50/50 blend of �ax and cotton. �e bottom number in each column is a 90/10 cotton/�ax blended yarn. �e �ax was
commercially cleaned through the Uni�ed �ine and �aRoche cottonization systems.
Data adapted from [33].
14 ISRN Biotechnology
T 10: Properties of mature Ariane �ax �ber enzyme retted with various commercial products.
1
Retting formulation Fine �ber yield (%)2 Strength (g/tex)3 Fineness (SSI)4 Predicted shive (%)5
2.0% Texazym BFE + M 7.0 ± 1.2a 36.7 ± 1.5ab 4.7 ± 0.1ab 2.7 ± 0.8bc
5.0% Texazym BFE 7.2 ± 0.6a 34.6 ± 2.0b 4.3 ± 0.3bcd 3.3 ± 0.3b
0.2% Multifect Pectinase + M 2.2 ± 0.2c 17.8 ± 2.2d 4.1 ± 0.1bcde 1.2 ± 0.7c
0.1% Bioprep 6.0 ± 1.1ab 33.2 ± 2.4bc 3.8 ± 0.1cde 4.1 ± 0.2b
0.1% Bioprep + M 7.7 ± 1.4a 34.9 ± 2.0b 3.0 ± 0.6f 3.7 ± 1.0b
0.1% Bioprep + Barapon + Clavodene 5.7 ± 1.3ab 34.8 ± 4.8b 3.6 ± 0.4ef 3.2 ± 1.1bc
0.05% Viscozyme + M 5.2 ± 2.3ab 27.6 ± 3.8c 3.6 ± 0.7def 2.9 ± 2.1bc
Untreated 3.2 ± 1.5bc 42.0 ± 5.5a 5.0 ± 0a 6.2 ± 1.0a
1
Texazyme BFE from Inotex Ltd., Dvűr Královi, Czech Republic; Multifect Pectinase FE is from Genencor International, Inc., Rochester, NY; Bioprep and
Viscozyme L from Novozymes North America, Inc., Franklinton, NC; M is Mayoquest 200 used to provide 18 mM EDTA as chelator.
B + C is Barapon C-108, an amino polycarboxylic acid salt mixture, and Clavodene CIU, a mixture of surfactants (Dexter Chemical L.L.C., Bronx, NY)
recommended in cotton scouring. Enzymes and chemicals are used as provided by suppliers and under optimal conditions for activity.
2
Percent of �ber a�er passing cleaned �ber through the Shirley Analyzer �ax stem. 3 Modi�ed method ASTM D1445-95, 1999. 4 ASTM D7025-04a, 2005.
5
ASTM D7076-05, 2005.
a,b,c,d,e,f
Values followed by different letters differ at 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃.
Data modi�ed from [34].
Bioprep and Viscozyme to ret two linseed varieties, and retting, Hermes was retted with a range of Bioprep levels
�ber properties were determined. Hermes and Omega were from 0.1% to 5% and followed by chelator or combined
grown to full seed maturity under production conditions in with chelator in the formulation. e higher levels of “�ber”
North Dakota. Stems were enzyme retted using formulations with the lower enzyme levels arise from �ber plus shive in
with Bioprep or Viscozyme in side-by-side tests (Table 11). varying amounts, as shown by predicted shive amounts. For
e Omega sample had rain prior to baling, and substantial Shirley-cleaned �ne �ber, Bioprep at 1.0% to 5.0% followed by
weathering had occurred as indicated by darkening of the chelator produced the highest �ber yields and the lowest shive
straw. Hermes, in contrast, was light and showed no effects of contents, ranging from 1.5% to 2.3%. Bioprep at 5% did not
weathering prior to enzyme retting. Bioprep effectively retted produce higher yields or cleaner �bers than 1% or 2% levels.
both cultivars and resulted in higher �ber yield and �ber Shirley-cleaned �bers do not represent all the �bers that could
strength than Viscozyme. Hermes was �ner a�er retting with be extracted in commercial, cottonizing systems. erefore,
Viscozyme plus chelator (Table 11). In this test, the chelator �ber yields from a single pass through the Shirley Analyzer
was used subsequent to the soaking with Bioprep but with the were used only to rank enzyme formulations.
Viscozyme in a single solution. Fiber strength was maintained at all levels of Bioprep
Tests for incubation times with Bioprep, level of Bio- (Table 12), showing a signi�cantly greater strength than for
prep (without chelator), and levels and incubation times of �bers retted with Viscozyme (Table 11). erefore, a major
chelator were further assessed [35]. Based on �ne �ber yield objective of enzyme retting with increased �ber strength
and percentage shive content, incubation with Bioprep for was reached with pectate lyase followed by EDTA. e
1 h followed by incubation with 18 mM EDTA for 24 h was commercial enzymes used in the present study represented
equal or better than other conditions. Retting effectiveness, a mixture of polysaccharidases, for example, cellulases and
however, improved with increased amounts of Bioprep up to hemicellulases in some, as well as different types of pecti-
0.5%, which was the highest level tested in this experiment nases. Viscozyme, or EPG, and pectate lyase were effective in
and suggested that further increases in enzyme level may attacking pectin and retting �ax, but the two enzymes have
improve �ber separation. Furthermore, scanning electron different optimal conditions for activity and different modes
microscopy of retted �bers indicated that Bioprep levels of of action.
5% appeared to remove more contaminants than 0.1%. Advances for �ax �ber processing could occur with
Based on earlier results and general recommendations for speci�c enzymes and systems. Polygalacturonase (PG) and
bioscouring cotton with Bioprep (personal communication, pectate lyase (PL) are both depolymerizing enzymes for
S. Salmon, Novozymes North America, Inc.), a series of pectin but work in different ways and under different con-
evaluations was carried out to optimize the use of Bioprep and ditions. PG is reported to catalyze random hydrolysis of
EDTA for retting �ax (Table 12). Linseed variety Hermes was 𝛼𝛼-1,4 polygalacturonic acid, and PL carries out a nonhy-
selected for these tests. e recommendation for bioscouring drolytic breakdown of pectates and pectinates by a trans-
cotton was to treat with Bioprep about 15 min prior to adding elimination split of the pectic polymer [77]. PL is activated
chelators (S. Salmon, personal communication). e use by Ca2+ and usually is active at higher pHs (e.g., 8–10)
of Mayoquest 200 to supply EDTA as chelator at 18 mM and temperatures (55–60∘ C) than PG. Research has been
concentration, which had been determined from use with carried out to correlate �ber separation with the degradation
Viscozyme, appeared to work adequately with Bioprep. To of different “pectins,” that is, various functional groups and
further optimize the formulation and method for enzyme linkages, using a series of commercial PGs and PL with
16 ISRN Biotechnology
Retting formulation1 Fine �ber yield (%)3 Strength (g/tex)4 Fineness5 Predicted shive (%)6
0.1% followed by M 10.0 ± 2.9bcd 34.7 ± 2.1a 4.5 ± 0.1ab 5.1 ± 1.9bc
0.1% containing M 6.3 ± 0.2ef 31.8 ± 1.5a 4.5 ± 0.1ab 4.6 ± 0.9bc
0.1% no chelator 5.6 ± 0.4f ND ND 11.4 ± 2.6a
0.5% followed by M 10.7 ± 2.5bcd 36.1 ± 3.6a 4.5 ± 0.1ab 2.0 ± 1.1def
0.5% containing M 9.3 ± 2.7bcdef 33.3 ± 1.4a 4.5 ± 0.1a 3.9 ± 1.9bcd
0.5% no chelator 8.5 ± 1.0cdef ND ND 5.7 ± 1.7b
1.0.% followed by M 11.8 ± 2.2abc 32.1 ± 0.7a 4.3 ± 0.1c 1.7 ± 0.9ef
1.0% containing M 9.0 ± 1.3bcdef 30.6 ± 1.1a 4.4 ± 0.1abc 3.6 ± 1.4bcdef
1.0% no chelator 9.5 ± 0.3bcde ND ND 3.7 ± 1.5bcde
1.5% followed by M 11.8 ± 2.1abc 29.8 ± 6.8a 4.1 ± 0.1d 1.5 ± 0.2f
1.5% containing M 8.1 ± 0.1def 33.2 ± 0.6a 4.4 ± 0.1abc 3.0 ± 1.1cdef
1.5% no chelator ND ND ND ND
2.0% followed by M 10.2 ± 2.0bcd 32.6 ± 0.9a 4.1 ± 0.1d 2.3 ± 1.2def
2.0% containing M 7.4 ± 1.1def 31.6 ± 0.6a 4.4 ± 0.1bc 2.9 ± 1.3cdef
2.0% no chelator ND ND ND ND
5.0% followed by M2 11.7 ± 2.9abc 32.6 ± 1.3a 4.2 ± 0.1de 1.6 ± 0.5ef
5.0% containing M2 13.2 ± 5.2a 33.9 ± 0.8a 4.1 ± 0.1d 2.0 ± 0.4def
5.0% no chelator 12.7 ± 2.4ab 29.8 ± 3.6a 4.2 ± 0.1de 2.3 ± 0.8def
1
Hermes �ax was dried at 55∘ C before crimping through the 9-roller calender. M is 1.83% Mayoquest 200. Triplicate samples of 150 g were tested.
2
Mayoquest used at 3.0% (30 mM EDTA) as chelator (C).
3
Shirley-cleaned �ber yield aer 1 pass.3 Modi�ed test method ASTM D1445-95, 1999.
4
Stelometer. 5 Air�ow. 6 ASTM D7076-05, 2005.
a,b,c,d,e,f
Values followed by different letters differ at 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃.
Data from [35].
low xylanase and cellulase activities [130]. Retting efficiency �bers with reduced enzyme levels, likely by removing the
was highly correlated (correlation coefficient of 0.99) with Ca2+ in pectin.
sparsely esteri�ed pectin, but correlations between retting Work with commercial PL has indeed shown efficiency
and activities against other pectins were low. Since Ca2+ in separating bast �ber from stems (Figure 8, Tables 10–12).
binds acidic groups of pectin molecules and various types Bioprep levels around 2% with 18 mM EDTA were optimal
of pectin are in different regions of the bast [66, 81], these with the �ax samples used and conditions tested (Table
data further reveal a coordinated mechanism for degradation 12). Fiber yield, �neness, and cleanliness were not improved
of nonesteri�ed �ax pectins with chelators and pectinases. with higher Bioprep levels. Sequential treatment of Bioprep
Further, these and other spectroscopic data [131] suggest followed by EDTA was the most effective for retting, but
that the pectins in the middle lamella and those binding the combining both enzyme and EDTA also retted �ax. �e
cuticle/epidermis to �ber bundles in �ax stems are targeted procedure most effective for producing �ne, clean �ber was
by this mechanism. Towards a more cost-effective enzyme as follows: (a) saturate crimped �ax stems with Bioprep at
retting system along these lines, other work [132] indicated 2%, (b) incubate for 1 h at 55∘ C, (c) without washing, resoak
that weak acid with enzymes was effective in separating bast with 18 mM EDTA at pH 12, (d) continue incubation at
ISRN Biotechnology 17
55∘ C for about 24 h total time, and (e) wash and dry �ber in to economically extract �bers of high and consistent quality
preparation for mechanical cleaning. and for directed purposes.
Other work has shown the potential of alkaline pectinases
such as PL to ret �ax and the bast plant ramie [127, 3.3. Enzymes for Postharvest Treatment of Flax Fiber and
133]. Bioprep-treated �ber was not tested in the minia- Yarns. Perhaps one of the most effective uses of enzymes may
ture spinning system or for biocomposites to this author’s be in postharvest treatments of �ax �bers to impart speci�c
knowledge. An engineered pectate lyase from Xanthomonas properties. Fiber-modifying enzymes are marketed for a
campestris, however, was developed and used at 37∘ C and variety of purposes, including enrichment of dew retting,
pH 8.5 to “bioscour” commercially grown and decorticated repair of poor quality �ax (such as underretted material),
linseed �ax [134–136]. Matched PL-treated and untreated cottonization of bast �ber tows for textiles or biocomposites,
�bers were then used in manufactured biocomposites. PL- tailored �ber length, and processing of rovings to reduce
treated �bers were cleaner and �ner than untreated �bers. noncellulosic content [128]. e use of enzymes pertinent to
In some biocomposites, PL-treated �bers performed better �eld retting includes spray applications a�er stalk pulling to
than similar but untreated �bers [134]. Other linseed straw minimize the effect of inclimate weather and to better utilize
samples, which had been le� in the �eld for a few weeks linseed stalks in Europe (J. Marek, Inotex, Czech Republic,
and then decorticated and treated with X. campestris PL personal communication). Weak cellulases may have appli-
for various times up to 46 hr followed by chelator, did not cations where precision in limited attack on cellulose may
result in improved biocomposites [135]. Still further tests be bene�cial, such as for cottonization or shortening of
of commercially decorticated linseed straw showed that PL- �ber. To this purpose, laboratory tests of �ax pulp treated
retted �bers, although �ner and cleaner than untreated ones, with commercial pectinases and cellulases showed improved
performed better in tensile strength tests but not in interfacial characteristics of hand sheets compared to those prepared
shear strength tests [136]. Further assessment is required to by traditional (nonenzymatic) methods [137]. For pulping,
optimize use of Bioprep and other pectate lyases as a retting the breakdown of the �ber bundles by pectinases and the
enzyme for �ax. It is clear that many factors in�uence the shortening of the cellulosic �bers by attack of cellulases at
successful production and processing of �bers for textiles and the �bernodes improved some paper properties in laboratory
biocomposites. Further, while weak cellulases, such as those studies. e nature of �ax �bers, that is, the lack of limiting
in �iscozyme, reduced �ber strength, the resulting �ner �bers lignin in bundles and presence of susceptible �bernodes,
o�en spun better than �bers produced by lower �iscozyme provided opportunities for use of these enzymes not possible
levels in blends with cotton. Further assessment is required in highly ligni�ed, woody sources of pulp. e authors further
on �ber characteristics for speci�c applications (e.g., blended suggested that a more precise attack by speci�c enzymes may
textiles or biocomposites), as well as the economics of enzyme provide additional attributes in the pulp.
retting. It is clear, however, that pectate lyases can separate Research with an atomized enzyme delivery system
�ber of non�ber components and retain �ber strength. e showed that endoglucanases could be effectively delivered
designed purpose of Bioprep as a cotton scouring agent, in small amounts onto �eld-retted �bers, likely resulting in
which acts by removing the cotton �ber cuticle, has shown attack at the �bernodes to reduce �ber length, strength, and
to be effective in several large tests [125, 126]. elongation [138]. Application of the atomized method with
e ARS research on enzyme retting of �ax had ended by endoglucanase and extended to other enzymes [36] modi�ed
2012, with the retirement of key individuals and closing of the the properties in �ax �ber and in �ax�cotton blended (50�50)
USDA pilot plant. Research and development continues for yarns (Table 13). ese enzymes were used as supplied,
retting and other �ber applications with enzymes [127, 134]. and such mixtures usually have multiple enzyme pro�les
Inotex (Dvur Kralove n.L., Czech Republic) has developed against �bers [34, 109, 129]. While further work is needed
enzymes to assist with �eld retting [128], particularly towards to assess speci�c activities, data suggest that all enzyme types
producing consistent �bers in varying weather conditions were active in atomization, and various properties could be
and including use of oilseed straw (J. Marek, Inotex, Czech modi�ed. For example, lipase and arabinase improved certain
Republic, personal communication). yarn properties, such as increased strength and elongation
Genetics for plant modi�cation of �ax to improve and reduced neps and thick and thin places. Results further
�ber properties for linen and biocomposites are active suggest that precise enzyme activities could tailor �ber and
areas of research. Related to the idea of improved retting, yarn properties.
some research is focused on genetically modifying �ax
for improved �ber extraction from linseed stems (Michael
Deyholos, University of Alberta, Canada, personal com- 3.4. Enzyme Retting of Other Bast Plants. is paper has
munication). One goal of another program, FIBRAGEN, focused on �ax structure and composition, with potential for
is to identify genetic markers, including those determining enzyme retting. Emphasis has been placed on the nature of
anatomy and physical properties, for expanding �ax markets the �ax bast �ber and bundle, speci�cally the lack of high
in textiles and biocomposites (Jörg Müssig, Hochschule levels of lignin, the binding of Ca2+ in pectin molecules in the
Bremen, University of Applied Sciences, Department for cuticle, and the presence of the more susceptible �bernodes
Biometrics, personal communication). Advances in plant and kink bands within the �bers and bundles. Pectinases,
modi�cation coupled with knowledge of speci�c action of either polygalacturonases or pectate lyases, alone are able
enzyme systems for extracting �ber bode well for new systems to separate �bers from cuticle and core. Flax is �ust one
18 ISRN Biotechnology
T 13: Properties of 50 : 50 �ax : cotton blended yarns a�er atomized enzyme treatment.
of many bast plants that are economically important for Woven �ax �bers as insets with resins particularly provide
myriad uses throughout the world. Would the same enzyme good strength and rigidity in composites. Substantial savings
work for other bast plants as for �ax� Research suggested in energy costs are possible with natural �ber mats, which
that bast �bers with other characteristics may require other reportedly require about 80% less energy than those made
types of enzymes. Ramie, which is a nonligni�ed, cellulose- with glass. Flax �ber provides a low cost alternative for
rich bast �ber-like �ax (unpublished data), has been retted glass �ber in reinforced composites. e replacement of glass
with pectin lyase [133]. Enzyme retting of hemp, which is �bers with �ax for this application, even with its important
more heavily ligni�ed than �ax, showed some success, but advantages, is nonetheless a considerable challenge.
different enzymes or protocols from those with �ax were Consistency in supply and in �ber characteristics is
needed [139]. �enaf is highly ligni�ed in the secondary required for �ax �ber to expand further into markets,
walls and middle lamella of the bast �bers and bundles and especially higher value-added products. Reportedly, the best
has been retted by chemical means [140, 141]. Use of a �ax �ber is still produced in western Europe, where climate
commercial enzyme, having cellulase and xylanase activities, and grower experience provide quality �bers for textiles.
with chelators and a crimping pretreatment separated the e drive for �bers in biocomposites and other industries
bast tissue to �ber bundles [139]. is process only produced has focused on getting �ax �ber from nontraditional linen
coarse �ber bundles, and a delignifying process seems to plants, namely, linseed straw. Field retting, which is the
be required for effective retting of kenaf. To this end, primary method of �ax production, is problematic in that the
enzymes from noncellulolytic, lignin-degrading white rot �bers are o�en poor and inconsistent in quality. Climate is
fungi to remove aromatics and leave cellulose [142, 143] may a major factor in quality, and outside western Europe, the
�nd applications. Successful, cost-effective, and commercial major areas of �ax production are o�en in harsh climates
technologies will have requirements such as the following: for �eld retting. It is in regard to all these factors that
selected �ax material, enzyme formulations and conditions research has focused on other methods, including enzyme
to tailor �bers with speci�c properties, integrated cleaning retting, to improve retting and thereby �ber processing and
procedures, objective assessment methods to assure high and quality.
consistent quality, and directed applications. Replacement methods for �eld retting have been sought
for a long time, but currently, there are no such methods
4. Summary and Conclusions used commercially. Enzyme retting has been researched for
several years and is still undergoing development. ere have
Flax has had a long and illustrious impact on human been positive results, and there is considerable interest in
development for millennia. e long, �ne, and strong bast postretting and treatment of roving and yarn to improve
�bers provide apparel and other textiles, and other varieties of their properties. New developments in enzyme production
�ax provide linseed and its oil. e textile industry that once by commercial companies have provided purer and more
�ourished in western Europe has declined, but the desire for active pectinases that have promise in enzyme retting. Our
�ax and linen is still strong. �uality �bers are still marketed in work examined several commercial enzymes for retting and
Europe, and China and other regions desire more quality �ax focused on the endopolygalacturonase-rich, mixed product
�bers for products. Even the paper and pulp industries desire Viscozyme plus EDTA and the purer alkaline pectate lyase
cleaner �bers to reduce the amount of chemicals required product Bioprep followed by a commercial EDTA chelator.
for deligni�cation. Biocomposites and nonwoven materials Protocols were developed on enzyme concentrations and
are predominant areas of interest, with the automotive conditions to separate �bers, which were then cleaned in a
industry continuing to focus on natural �ber composites. �ber processing pilot plant and characterized by objective test
In particular, biocomposites are sought for weight and cost methods. While Bioprep-retted �bers had good properties
savings, improved structural properties, processing bene�ts, of �neness, strength, and cleanliness, tests in textiles or
and design �exibility and ease. Compared to glass, �ax �bers composites have not been carried out. e vast amount
are lower in cost, lower in density, biodegradable, and similar of research on enzyme retting indicates that pectinases
in elongation at break� tensile strength is lower for �ax. without the need of complementary enzymes are effective
ISRN Biotechnology 19
in separating �ber from �ax straw, even linseed straw. e [4] R. R. Franck, “e history and present position of linen,” in
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for some products, the requirements of cleanliness and pro-
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