MAIJALA, Pekka (Ulvilantie 27 e A 5, Helsinki, FI-00350, FI)
GALKIN, Sari (Valkoinen linja 11, Tolkkinen, FI-06750, FI)
HATAKKA, Annele (Tulustie 16 B 7, Helsinki, FI-00670, FI)
NYKTER, Minna (Nurmilinnuntie 3 C 24, Espoo, FI-02620, FI)
MAIJALA, Pekka (Ulvilantie 27 e A 5, Helsinki, FI-00350, FI)
GALKIN, Sari (Valkoinen linja 11, Tolkkinen, FI-06750, FI)
HATAKKA, Annele (Tulustie 16 B 7, Helsinki, FI-00670, FI)
NYKTER, Minna (Nurmilinnuntie 3 C 24, Espoo, FI-02620, FI)
CLAIMS
1. Method for retting bast fibers comprising the step of adding to retting water a culture of fungus Fusarium sambucinum or a lysate thereof, or culture medium used for cultivation of said fungus, or a purified fraction of said culture or culture medium.
2. The method according to claim 1, characterized in that the proteins have been removed from the culture medium before it is added to the retting water.
3. The method according to claim 1 or 2, characterized in that the bast fibers are cottonized during retting.
4. The method according to claim 1 or 2, characterized in that the bast fibers are softened during retting.
5. The method according to any of the previous claims, characterized in that the bast fiber is flax, hemp, jute, gensitra, ramie, nettle or sunn.
6. The method according to claim 1, characterized in that the bast fiber is from linseed or fiber variety of fiber plant.
7. The method according to claim 1, characterized in that the fungus is Fusarium sambucinum Fuckel var. sambucinum.
8. The method according to claim 1, characterized in that the fungus is Fusarium deposited with accession number DSM 16924.
9. The method according to claim 1, characterized in that retting takes 2-24 hours.
10. The method according to claim 1, characterized in that it is performed in temperature +20 - +60 0 C.
11. The method according to claim 1, characterized in that during retting the retting water is stirred mechanically.
12. The method according to claim 1, characterized in that the wood- like material is mechanically removed from the fiber raw material before retting.
13. The method according to claim 1, characterized in that at least one of the j following is performed during the retting: washing of fibers, bleaching or dyeing.
14. The method according to claim 1, characterized in that retting is performed during fire dying process.
)
15. Use of Fusarium sambucinum for retting of bast fibers.
16. Use of Fusarium sambucinum for softening of bast fibers.
17. Use of Fusarium sambucinum for cottonization of bast fibers.
18. Use of Fusarium sambucinum for removal of lignin from cellulose based plant fibers.
19. The use according to any of claims 15-18, characterized in that the fungus used is Fusarium sambucinum Fuckel var. sambucinum.
20. The use according to any of claims 15-18, characterized in that the fungus is Fusarium deposited with accession number DSM 16924. |
Method for retting, smoothening and cottonizing bast fibers, and for removal of lignin of plant origin
FIELD OF THE INVENTION
This invention takes advantage of living biological microbes to modify plant fibers. Especially, this invention focuses on fungus Fusarium sambucinum and its metabolites that can be used for retting, smoothening and cottonizing bast fibers, and for removal of lignin of plant origin.
BACKGROUND OF THE INVENTION
Plant cells are surrounded by a cell wall, which gives strong protection and rigidity to the plant. Typically, it is composed of cellulose, hemicelluloses and lignin, and it can also contain various fats and waxes. Lignified cells are usually called fibers. Fibers are used in many industrial processes as in the pulp and paper industry to produce paper and in the textile industry to produce fiber. In the process of making paper the cellulose fibers are separated from the other structural materials of wood either mechanically or chemically. Cotton is the most widely used and produced cellulose-based textile fiber in the world. Cellulose is the compound that is desired in textile fibers. Other industrially important cellulose-based bast fibers are flax (Linum usitatissimum L), hemp (Cannabis Sativa L), jute (Corchoruscapsularis L. and Corchorus olitorius L. ), gensitra, ramie (Boehmerica nivea H etA) and sunn (Crotalaria juncea L). Of these flax is the most important textile fiber. Flax is used as material for decorative textiles and garments as such or blended together with other fibers. Flax fiber is also used in technical textiles as in hoisting belts and fire hoses. Short fibers of flax are suitable for insulation, chipboards, fiberboards, to reinforce plastics, to replace asbestos, to strengthen paper, non-woven products and as absorption medium. Flax is also used in composite materials. Hemp is mainly used for coarse clothing fabrics and because of its strength also in ropes, sails, covers and straps. Other bast fibers include e.g. kenaf (Hibiscus cannabinus L), urena (Urena lobata L), rosella (Hibiscus sabdariffa L) and nettle (Urtico dioica L).
Bast fibers are located in the stem of the fiber plant underneath the bark, where they provide mechanical support for the plant. Fiber plants are annual plants in which the
fibers grow in between the outermost bark and wood-like core layer to form fiber bundles. Fiber bundles consist of elementary fibers (single cells) which are held together by a gluing substance called pectin. Pectin A which surrounds fiber bundles, and pectin B, found in between elementary fibers, differ slightly from each other. In the pre-stage of bast fiber refining, called retting, microbes degrade pectin A and fiber bundles are separated from the surrounding straw. Pectin B, which holds the fibers together, is not degraded during normal duration of retting and the structure of fiber bundles is preserved. During excessive retting also pectin B will be degraded and elementary fibers are separated from each other. This is called cottonization. In other words, the purpose of retting is to remove the pectin that glues the fiber bundles together. This makes it easier to separate the fiber bundles from the wood-like parts of the stem mechanically in such a way that the fiber bundles stay intact.
Refining of bast fibers to be suitable for industrial use is a multistage process. In the traditional method of producing textile fibers from flax, the stems with their roots are harvested in the autumn with pulling machines and baled with special baling machines. After this, stalks are retted usually by dew retting, water retting or enzyme retting.
For the dew retting the pulled straw are kept parallel on the field in a swath, to undergo microbiological attack on the pectin bonds. The stems are spread as uniform layers on the field for at least 10 to 30 days. During retting the stems have to be turned in order for retting to take place uniformly. Naturally occurring bacteria and fungi are utilized in dew retting. Many species of fungi belonging to genera Aspergillus, Penicillium and Fusarium have been isolated from dew retted stems. (FiIa et αZ.,2001, Annals of Applied Biology 138(3):343-351; Henriksson et ah, 1997, Applied and Environmental Microbiology 63(10):3950-3956; Sharma, 1986, Annals of Applied Biology 109(3):605-612; Brown and Sharma, 1984, Annals of Applied Biology 105(l):65-74; Akin et al., 1998, Textile Research J. 68(7):515-519; Ulbricht and Gregor, 1963, Zeitschrift fur Allgemeine Mikrobiologie 3(4):297-307; Marsh and Bollenbacher, 1949, Textile Research J. 19:313-324; Fuller and Norman, 1946, Iowa Agr. Expt. Sta. Research Bull. 344:928-944). The problem with dew retting is that it cannot be controlled, e.g. weather causes unevenness in the quality. Especially, high relative humidity during retting leads to notable increase in the cost of drying.
Water retting is traditionally performed in natural waters by steeping the straw in water. In this process anaerobic bacteria degrade pectin A and the end result depends on the quality, flow rate and temperature of the water. Cold water retting in natural waters takes 6 to 9 days. As a drawback traditional water retting causes negative environmental effects. Water retting can be done under more controlled conditions in tanks with warm or cold water. Warm water retting aided by bacteria can also be used.
Enzyme retting means speeding up of natural retting process by adding industrially produced enzyme to the retting water (WO9516808, EP0220913). This way retting can be carried out very rapidly and it can be much better controlled than in the traditional dew retting. Short affined fibers, the so called cottonized fibers, can be obtained by increasing duration of the enzyme retting. Although only 20 to 30 % of the harvest is fibers, all of the harvest has to be dried. Even though there are many patents and commercially available enzymes (e.g. Viscozyme L and Flaxzyme for bast fiber treatment) there are no enzyme retting methods used in industrial scale (Koslowki et al. 2006, Biotechnology Letters 28:761-765; Wang et al. 2003, Textile Research Journal 73(8):664-669). Other retting methods include steaming, steam explosion and chemical retting by using solvents or tensides. It is also possible to obtain the so-called green fiber by omitting the retting stage.
Besides using enzymes it is possible to add microbes to the retting water to enhance the separation of fibers. In patent RU2181797, a method is described for retting flax on a slowly moving convey belt and at the same time adding spores of pectinase secreting fungi, and nitrogen nutrients. Similar microbiological, aerobic process is described in patent RU2124591. Processing of flax fibers by microbes has emphasized the better removal of pectin either by enzymes or by pectinase producing microbes. In more general, extensive research has been carried out to study enzymatic or microbiological modification of plant fibers. Especially, white-rot fungi belonging to the class Basidiomycetes are known for their ability to degrade lignin and cellulose as well as hemicelluloses. There are many patents related to this, e.g. in pulp and paper industry. Fungi belonging to the class Ascomycetes are known for their substantial ability to produce enzymes. For instance, fungi Aspergillus, Penicillium, Trichoderma
(Emericella) and Fusarium (Gibberella) belong to the class Ascomycetes. A method for hydrolyzing wood by anthraquinone secreting ascomyceteous fungi such as Fusarium tricenetum has been patented (DE4103983). Microbes can be separated from the
culture medium by filtration and use only the culture filtrate. The culture filtrate contains e.g. the enzymes secreted by the microbe. Culture filtrate can be further fractionated and/or filtrated in order to remove the proteins from it. Nevertheless, using protein free culture filtrates is not common practice in similar applications.
In order to stop the retting process and for storage purposes the bast fiber stems have to be dried after all of the previously mentioned retting methods. The problem with this is that the entire crop has to be dried for storage and processing although the amount of useful fiber in it is relatively low. Energy consumption of drying (kWh/kg evaporated water) differs depending on drying method. For flax it is 0.91-2.7 kWh/kg and for hemp it is slightly higher. In order to obtain industrially useful raw fiber material from the dried stems they have to be broken, scutched and hackled.
When using the long fiber processing methods the yield of yarn from the original flax crop is approximately 8.4 %. Fibers produced with the fore mentioned method require special spinning methods (e.g. Gill, dry-, or wet-spinning). By cottonization flax can be broken down to short single fibers. These short fibers can be spun with the same machinery as cotton. End products made of flax are nowadays expensive because of the multistage processing of raw material. The methods described above cannot be directly used for processing of hemp because hemp is sturdier and contains more lignin than flax.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Cultivation of fungus F.sambucinum in pectin containing culture medium. During the three to five week cultivation time the fungus raises the pH of the culture liquid to over 9.
Figure 2. Production of hydrogen peroxide by the fungus F.sambucinum when grown in pectin containing culture medium. Hydrogen peroxide is produced during the first week of cultivation after which it is scarcely found.
Figure 3. Lignin content of linseed fibers after treatment with protein free culture filtrate.
Figure 4. Amount of extractives in linseed fibers, cultivar Laser.
DETAILED DESCRIPTION OF THE INVENTION
The fungus used in this invention, Fusarium sambucinum var. sambucinum (Gibberella pulicaris), is common all around the world, and G. pulicaris is the type species of the genus Gibberella. Gibberella are the sexual forms of Fusarium. The fungus can infect great number of host plants, but is especially harmful for potato in which it is a causal agent of potato dry rot. G. pulicaris, like many other fungi belonging to the family Gibberella can be toxic and secrete besides different toxins also many others so called secondary metabolites, including colored pigments. Some fungi belonging to Fusarium have been found to be able to degrade to some extent environmentally harmful PAH- compounds and lignin model compounds. It is well known that many environmental factors like the nutrients in the culture medium, temperature, amount of light, etc. can have effect on the production of secondary metabolites by fungi.
There is very little research results published on the fungus Fusarium sambucinum. Instead many other Fusarium-species have been studied to unravel the characteristics of this genus. Benzo(a)pyrene degrading activity has been found from Fusarium solani (Veignie et al. 2004, Environ. Pollution 129:1-4). This was considered to be the result of hydrogen peroxide produced by the fungus and the reactions of the resulting reactive oxygen species (ROS). In the example 4 we have studied the production of hydrogen peroxide by F. sambucinum. The fungus, when grown on pectin containing growth medium, produces hydrogen peroxide during the first week of cultivation. But after the first week the amount of hydrogen peroxide decreases sharply and hydrogen peroxide is scarcely found in older cultivations. There is a correlation between production of hydrogen peroxide and production of an enzyme called glucose oxidase which suggests that this enzyme is involved in the production of hydrogen peroxide.
Cottonization of bast fibers has previously been done by long-lasting retting after which the fiber that is obtained is dark and stiff. It has required vigorous bleaching to obtain light colored cottonized fiber. This invention in hand solves the problems involved with retting and cottonization of flax and hemp by describing the use of microbe, namely Fusarium sambucinum, for retting of bast fiber raw materials. The aim of the retting method described in this invention is, besides to shorten the production line, especially to change the way the material feels and to change the visual characteristics and thus expand the use of fibers. The most notable profits of the invention are the rapidity of
the retting and the obtained light color and better spinning qualities of the cottonized fiber.
The advantageous method of this invention corresponds most closely to the existing enzyme retting methods of bast fibers. According to the invention, instead of an enzyme, fungal culture, lysate, cultivation medium, protein free culture filtrate or other purified fraction of the culture medium after cultivation of fungus Fusarium sambucinum that has the bast fiber cottonizing properties as mentioned in the invention will be used. Favorably the retting described by the invention is done in water- containing liquid (so called retting water) into which the fungus Fusarium sambucinum and the fiber to be retted will be added. Also salt can be added (e.g. 9 mg NaCl/ml) to the retting water. Retting or dyeing will favorably take 2-24 hours and it will be done favorably in +20-+60 0 C, most favorably in room temperature. Mechanical stirring and raising the temperature (40-60 0 C) during the retting will significantly shorten the time needed for the treatment.
Straw of various fiber plants and also from plants that are used for linseed production can be used as raw material for the method of this invention. Large amounts of linseed flax straw occur as a by-product of the linseed industry. The straw from linseed is nowadays mainly unexploited. Alone in Western Canada over million metric tons of straw is left unused annually and constitute a major environmental problem for disposal, with most of this residue burned. (Akin et al. 2000, Journal of Polymers and the
Environment, 8(3), 103-109). After harvesting the straw must be disposed of before the field can be ploughed. This is done either by burning or by removing the straw from the field and handling it as waste. Linseed fiber is generally considered coarser than required for high quality textiles, but is an option for production of technical-grade fiber for composites, (van Dam et al. 1994; Industrial fiber crops. Study on increased application of domestically produced plant fibers in textiles, pulp and paper production and composite materials. European Commission: EC DGXII-EUR 16101 EN). Further it has been proposed that the fiber to be used for cottonization should be as fine as possible so that the technical quality and appearance of the yarn would be satisfactory (Salmon & Minotte 2005, in the book Bast and other plant fibers, ed. Robert R. Franck. Cambridge: Woodhead Publishing Limited, 94-175). Cottonized fiber flax fibers have been used blended together with e.g. cotton. The percentage of flax in ring spun yarn (50 tex) is 40 % and in 22-66 tex rotor spun yarns 25-50 %. According to a study done
in the University of Helsinki, Department of Agrotechnology, the baling and removal of straw from the field increases the required work time two hours per hectare.
The straws of the linseed are harvested when the fiber crop is mature and thus the fibers are easily mechanically separated. Cottonization method is based on short fiber methods in which all the fibers are treated together without separating the long fiber bundles from the short tow fibers. Thus the harvesting can be done with normal farm machinery and by baling all the straw together. If the straw is decorticate during the harvest part of the shives are left on the field to loosen the ground. At the same time the fraction of fiber in the bale increases and the cubic weight of the bale grows since the cut up straw can be baled more tightly. Thus, the volume of the cut straw material to be transported is markedly less than the volume of the uncut straw material. The volume of the decorticate straw material was 7.3 m 3 per hectare while the volume of uncut straw material was 18 m 3 per hectare.
First the wood like material is cost efficiently mechanically removed from the straw after which the fungus Fusarium sambucinum as such or its lysate or its culture medium or its protein free culture filtrate will be used for retting. This way the expenses of drying will be directed only to the fiber. Fiber that has been treated as described in this invention will be bleached and cottonized. That is to say that the elementary fibers are separated from each other, undamaged, and the raw material is soft (rigidity and strength of the fiber are lower) and the fiber is shiny, cotton like. These elementary fibers (fiber length app. 25-50 mm), unlike long flax fibers, can be spun with same spinning machines as cotton, and many different varieties of yarn can be obtained. Linseed fibers are cottonized faster than green (unripe) fiber flax fibers.
Cleaned and carded fiber will be degraded to elementary fibers with the method described in this invention and thus washing, cottonization, bleaching and dyeing of fibers can all be done in the same wet-process. At the same time the loss of the mass reduces the need for respooling of yarn common in traditional methods of flax yarn manufacturing. When this treatment is combined with dyeing of fibers novel ingrain yarns of flax and hemp will be obtained (usually bast fibers are dyed as yarn or fabric). This whole process can be performed with normal fiber dyeing techniques. Fiber that has been cottonized this way is raw material, whose composition and degree of purity can be defined, and is ready for yarn making with cotton spinning methods, non woven
industry, or other technical applications. Recently, the use of bast fibers as an alternative material in composites has been considered, but the problem with it is the high cost of refining, fiber stiffness, and the high percentage of unnecessary particles (e.g. pectin and lignin), which restrict the adhesion of fibers to the plastic or other matrices. Fiber material that has been treated according to this invention is suitable for use as textile fiber and as raw material for composites.
Method of this invention is also suitable for traditional long fiber flax and hemp refining. This treatment can also be done to raw fiber, line flax sliver, roving, yarn or fabric to make them softer, shinier and less stiff. Linen yarns treated this way are suitable for knitwear. At present, most flax and hemp yarns are too stiff to be knitted, and also the dust from linen yarns create problems by causing interruptions in production, straining the machinery and clogging the washing machines during the first few washes.
EXAMPLES
Example 1
Isolation and cultivation of the microbe
Samples of the microbe were collected during the spring 2001 in Siuntio, research farm of the University of Helsinki, Siggans, from a fiber hemp field, where the vegetation had been left over winter for thermal retting. The barks of the stems infected by the microbe were red/orange, but the fiber material was almost white and cottonized. Pigmented fiber was mixed with salt water (9 mg NaCl/ml) for 5 minutes in homogenizer. Hygicult Yeast and Fungi splints (Orion Diagnostica) were dipped in the liquid. A few days later mycelium was scraped from the splints and transferred to new splints and to malt agar plates. Fungal monoculture was obtained by cultivating it twice through dilution series on potato dextrose agar plates. Fungal monoculture was identified preliminarily by light microscope and was sent to CBS (Centralbureau voor Schimmelcultures, Utrecht) for further identification of the exact species.
According to the preliminary microscopic results the microbe was found out to belong to the genus Fusarium. CBS further identified the fungus to be Fusarium sambucinum Fuckel var. sambucinum based on the morphology of the fungus. This species is common all around the world and it grows on grain, potato, tree bark and gramineous
plants (Samson, R.A. (ed.) Introduction to food- and airborne fungi, 6 th edition, p. 144, CBS, Utrecht, Holland).
Fusarium sambucinum ARTl strain has been deposited to the DSMZ microbe collection (Deutsche Sammlung von Mikroorganismen und Zellculturen GmbH) according to the treaty of Budapest with accession number DSM 16924.
Example 2
The fungus is grown to produce asexual spores in order to start liquid cultivations from spores stored in deep freezer. As cultivation medium 2 % malt agar plates are used. The fungus is left to grow on plates e.g. in room temperature for a few weeks. During this time the fungus will start to produce asexual spores that can be collected from plates using a small amount (app. 3-4 ml) of sterilized water with the help of a sterilized glass rod and pipette tip. The spores containing water solution will be stored. The spore suspension obtained this way from F. sambucinum plates typically contains 10 000-50 000 spores/ml. The spore suspension is mixed well with 40 % glycerol and frozen. Stored this way the spores stay vital for very long period of time, practically forever.
Example 3
Mycelia are grown in flasks into which sterilized culture liquid has been added. Flasks can be normal laboratory flasks and the ratio of the volume of the liquid and volume of the flask should preferably be app. 1:10, e.g. 20 ml culture liquid in 200 ml flask. For instance 50 μl of spore suspension will be added to 200 ml of culture liquid. The fungus will be grown in constant light without shaking to produce the highest amount of desired compound/compounds. The light used can be artificial obtained e.g.by fluorescent light or sunlight. Various culture media designed for growing moulds can be used. A good culture medium contains e.g. 1-2 % (w/v) pectin, together with 0.6 g/1 trypton, 0.04 g/1 yeast extract, 0.1 g/1 magnesium sulfate (MgSO 4 -7H 2 O) and vitamin solution that will be added through sterile filter to culture medium sterilized by autoclaving. Carboxymethyl cellulose or other plant cell wall based carbon sources can be used instead of pectin so that the fungus will get the carbon it needs for growth. More simple carbon sources as polygalacturonic acid or malt extract can also be used. During the cultivation the pH of the culture medium rises, at 28 days it should be app.
8.5. By following the changes in pH and changes in the color of mycelium from fair to dark red, the proper cultivation time as to when there is the ideal amount of the active compound/compounds produced, can be determined. A culture medium that has been let to grow for too long period of time does not act as well as 28 day old fungal pectin culture medium in which the pH has risen over 8.5. After this the fungal culture is filtrated e.g. through Miracloth in order to separate the mycelium from the culture liquid. Ammonium sulfate is added to the culture liquid gradually until the percentage of ammonium sulfate is 70. Ammonium sulfate lowers the pH of the liquid by 1.5 units. The solution will be mixed for one hour in +4 0 C after which it will be centrifuged for 30 minutes in +5 0 C with 10 000 .rpm. The precipitate formed contains the proteins of the culture liquid. Supernatant is collected and used for fiber treatments. Alternatively, the proteins can be removed by ultrafiltrating the culture liquid through 10 kDa membrane under nitrogen atmosphere.
Example 4
In this example, the ability of fungus F.sambucinum to secrete hydrogen peroxide to the culture medium was examined. The culture medium that was used contained either pectin or carboxymethyl cellulose. Hydrogen peroxide was measured by using mixture containing 125 μM xylenol orange and 100 μM sorbitol, which was mixed with 25 mM FeSO 4 -ammoniumsulfate solution (prepared in 2.5 M sulfuric acid) in 100:1 ratio just prior the measurement. Glucose oxidase activity was measured by using ABTS. The reaction mixture contained 1 μM ABTS, 100 μM sodium phosphate buffer, pH 6.5, 2 U horseradish peroxidase (type II, Sigma) and 50 μmol D-glucose. 100 μl of sample was mixed with reaction mixture and incubated in 30 0 C. The reaction was followed by spectrophotometer at 420 nm. Laccase activity was measured by using 1 mM 2,6- dimethoxyphenol as substrate for laccase. Reaction mixture contained also 100 μl of sample and 850 μl Na-malonate buffer, pH 4.5. Progress of the reaction was followed at 476 nm. F.sambucinum secretes hydrogen peroxide especially during the first week of cultivation when grown on pectin containing medium, as shown in figure 2. Production of hydrogen peroxide is scarce when fungus is grown on cellulose containing medium. Glucose oxidase is the most likely enzyme produced by the fungus to take part in the hydrogen peroxide production in the medium. Catalase activity was also measured from the media. Catalase activity was measured at 240 nm in a reaction mixture that contained 100 μl of sample together with 800 μl 100 mM Na-phosphate
buffer, pH 6.0 and 100 μl 100 rnM hydrogen peroxide. The decrease in absorbance was followed in 25 0 C for three minutes. F.sambucinum secretes also catalase. Catalase activity increases after the first week of cultivation and this could explain why hydrogen peroxide disappears from the culture medium. Low laccase activity can be detected only in the later stages of cultivation after three weeks of cultivation. The fungus secretes both hydrogen peroxide and the fore mentioned enzymes independent of light.
Example 5
The pretreatment of fibers
As raw material linseed Laser mowed in 2003 or mowed and cut up in 2005 was used. The fibers were carded to remove the shives. Because a lot of dust and other microparticles and water-soluble material was separated already during the rinsing of flax and hemp the fibers were thoroughly washed. The fibers were soaked in bleach containing detergent liquid for about one hour after which they were rinsed. (3-5 ml detergent/1 water, fiber-liquid ratio app. 1/10: detergent: 15-39 % zeolite, oxygen based bleach, 5-15 % anionic and nonionic tensides, > 5 % soap, phosphate, polycarboxylate, enzymes, pH 10.4). During washing much dust and cuticle parts were removed. Rinsing had only slight effect on the color of the fibers, but washing with bleach containing detergent made the fibers lighter in color and increased the loss of mass. Regardless of temperature (20-60 0 C) the washing with detergent (1 hour) increased the loss of mass of linseed by 12.5-13.7 %. But with hemp the loss of mass was 15.3 % when washing was done in 60 0 C, but only 8.3 % when washing was done in 40 0 C.
Washing also had an effect on the amount of extractives compared to untreated raw material. Fiber (app. 2 g abs. dry) was extracted for five hours with pure acetone in Soxhlet- apparatus and the acetone soluble material was weighted and the percentage of abs. dry material was calculated. The proportion of extractives in the raw fiber material was 1.164116 % . During washing almost half of the extractives were removed and the amount was 0.658938 %.
Example 6
Determination of treatment conditions
Only the microbial cultivation and the isolation of the compound have been done in sterile laboratory conditions. All other treatments were done in normal conditions; room temperature, tab water and unsterilized vessels.
To find the optimal treatment conditions different amounts in the range 1-30 ml of culture liquid were tested per one gram of fiber in steps of 0.5 ml. Linitest-testdyeing apparatus was used for the mechanical stirring. Linitest-apparatus consists of eight steel 500 ml vessels, which can be filled up to 400 ml. The vessels were placed in waterbath where temperature could be controlled with +/- 1 0 C accuracy. The rotation rate of the apparatus was 36 rpm. The samples together with all the other needed substances were added to the vessels in room temperature and the final temperature of the treatment 57 0 C, (set for 60 0 C) was reached in 30 minutes. After several trials the suitable amount of culture liquid was found to be 14 ml/gram fiber. Already in the early stages of trials addition of salt to the mixture was found to be beneficial. In all trials the fibers treated in salt containing liquid were softer and lighter colored than the ones treated in plain tab water. The idea of addition of salt to the treatment water was based on the idea of swelling the fibers and making it easier for the microbes to penetrate. E.g. salt adds the substantivity (the attraction between a substrate and a dye or other substance under the precise conditions of test whereby the latter is selectively extracted from the application medium by the substrate of the cellulose fiber during reactive dyeing, which causes the dye to seek towards the textile material). (Forss, Maija 2000. Varimenetelmat. Varjays-maalaus-kankaanpainanta. Taideteollisen korkeakoulun julkaisu B 60. University of Art and Design Helsinki). It was also assumed that by adding salt the percentage of salt in the liquid would be the same as salt content in cell which would reduce the osmosis from the cell to the water. Proper percentage of salt was estimated by series of trials using 10, 20, 30, 40, and 50 g of salt per liter of water. Proper liquid to fiber ratio was estimated by series of trials using ratios 1/10, 1/20, 1/30, 1/40, and 1/50. Also the possible effects of pH to the treatment were estimated. Salt water acidified with acetic acid to pH 5 and salt water alkalized with sodium carbonate to pH 9 were compared. After these initial trials the actual experiments were done in water with 10 g/1 added salt and the liquid to fiber ratio was 1/20 and the pH of the liquid was not changed. When using mechanical stirring the temperature was kept app. 57 0 C and during the static retting experiments the temperature was room temperature app. 20 0 C.
The effect of cultivation time to the fairness and softness of the fibers obtained with the treatment was also studied. Best results were obtained when the pH of the liquid used in the treatment was app. 7. When using culture liquid without removing the proteins the optimum cultivation time was 18 days. The removal of proteins by ammonium sulfate lowered the pH by 1.5 units which prolonged the cultivation time to app. 28 days.
Example 7
The static retting of fibers
As raw material prewashed, carded with no shives, linseed Laser. Treatment liquid (pH 7) culture liquid (pH 8.5) cultivated for 28 days in light, after removal of proteins. To the retting water (10 g NaCl/ 1 H 2 O, fiber to liquid ratio 1/20, app. 20 0 C) is added: 1) every 6 hours twice 0.5 ml/g fiber. Total treatment time 12 h and the volume of culture liquid 1 ml/g fiber. 2) every 6 hours three times 0.5 ml/g (total treatment time 18 h; 1,5 ml/g). 3) 8 h retting, during which culture liquid added twice 0.5 ml/g (16 h; 1.0 ml/g) and 4) every 8 hours three times 0.5 ml/g (total treatment time 24 h; 1,5 ml/g). Finally washing and rinsing. Three 3 g parallels of all were performed, which were repeated at least for three times. In static retting and final washing the loss of mass was 1.4-4.4 % depending on time.
Example 8
Mechanical stirring of the fibers
As raw material prewashed, carded with no shives, linseed Laser and hemp. Culture liquid (pH 8.5) cultivated for 28 days in light after removal of proteins (pH 7) and culture with mycelia cultivated for 18 days in light (pH 7.1), homogenized to a suspension in a blender. Retting water contained 1O g NaCl/ 1 H 2 O, fiber to liquid ratio 1/20. For mechanical stirring washing machine programs at 40 and 60 0 C, 2.15 h were used and also Linitest-apparatus was used.
Series of trials were done for oil hemp Finola in which 5 ml/g culture medium with mycelium was added to the salt water and packed tightly in plastic bags and run in the washing machine using 40 °C/2.15 h program. The experiment was repeated 1-5 times
using 5, 10, 15, 20 and 25 ml of culture medium per gram and the treatment time was 2.15-11.15 h. Best result was obtained by using 5x2.15 h.
Linseed in washing machine with culture liquid without mycelium: 1) 3x2.15 h, 3 x 1 ml/g fiber (6 h 45 min; 3 ml/g); 2) 3x2.15 h, 3 x 3 ml/g (6 h 45 min, 9 ml/g). Experiments with protein free culture liquid were carried out with Linitest-apparatus 57 0 C (set for 60 0 C) 1) 2 h and 2) 4 h. Both batches 14 ml of culture liquid per gram fiber.
After the treatments the fibers were washed with detergent and rinsed. The loss of mass was weighted from air-dried samples and average for three 3 g samples was calculated. The loss of mass in both 2 h and 4 h Linitest-treatments were the same, being 4.4 % in both.
Example 9
Parallel experiments
To compare with F.sambucinum (Gibber ella pulicaris) experiments were carried out with two closely related species F.poae (strain 93146) and F.langsethiae (strain 113). Taxonomy of fungi placed in the family Fusarium is problematic. The family contains many species whose sexual form is unknown. Both F.poae and F.langsethiae belong to these fungi. F.langsethiae is closely related to Fusarium sporotrichioides (Yli-Mattila et al. 2004, Journal of Food Microbiology 95:267-285), which is also called Dactylium fusarioides. It is noteworthy, that only the asexual form of G. pulicaris is found in the continent of America, but in Europe G.pulicaris is found in both sexual and asexual forms, which makes it very heterogenic species (Desjardins, A. 2003: Gibberella from A (venaceae) to Z (eae). Annual review of phytopathology 41: 177-198). In the parallel experiments also differently isolated and/or maintained strains of the fungus of this invention, Fusarium sambucinum ART- 1 were studied. Four treatment liquids were prepared from each of the fungi: 1) original culture liquid with the mycelium, 2) protein free culture liquid 3) water extract of the culture liquid and 4) ethyl acetate extract of the culture liquid. The proteins were removed in order to find out if enzymes were involved in the reaction. Experiments were carried out as two or four hour treatments in Linitest apparatus in 37 0 C and 57 0 C using 14 ml of liquid per 1 gram of fiber. Three 3 gram replicates were done of each sample. Further, extraction of active compounds was
tested with benzyl alcohol, after which the water phase was salted out by ammonium sulfate and collected. Controls were done in tab water and saltwater.
Based on these experiments the lightest colored and softest fibers were obtained with the culture liquid containing the mycelium or with the protein free culture liquid of Fusarium sambucinum ART-I ("Fusart") grown in light.
Example 10
Linear density, breaking tenacity and elongation at break
Linear density [dtex] was determined of the elementary fibers that were separated by hand with a vibroscope (Lenzing AG, Austria). Breaking tenacity [cN/tex] and elongation at break [%] were measured from elementary fibers according to standard SFS-EN ISO 5079, 1995(E) using Alwertron TCT 10 device (SFS 4639). The initial gauge length was 20 mm and constant rate of extension (CRE) that was applied 20 mm/min. For the preliminary tension 200 or 300 mg weight was used. Measurements were done under standard conditions (20.0 +1-2 0 C) when the humidity of the samples was 65.0 %+/-4.0 % (SFS EN ISO 20139, confirmed 2005-8-15; SFS 2600).
As comparison raw fiber which was carded by hand and washed linseed were used. In all of the treatments linear density increased compared to the control samples (table 1). The mean of the linear density for the linseed control fiber was 4.57 dtex. With the Fusart-treatment the linear density of oil hemp was markedly reduced and the elongation at break was increased to 2.7 %, while the breaking tenacity was lowered down to 47.5 cN/tex. The effect of the treatments for the decrease in the linear density of the linseed was 0.26-1.1 dtex, for the breaking tenacity -10.6-31.7 cN/tex and the effect of the treatments on the elongation at break was -0.4-+0.4 %. The elongation at break for the Fusart I linseed were 12.4 % in average, which seems quite unrealistic. Most likely, the numbers are distorted by the wax that was seen by microscope. The wax could cause the fibers to glide between the clamps of the apparatus used for measurements.
The treatments affected the Linear density and breaking tenacity in such a way that they resemble principally the average values obtained for cotton. The effect on elongation at break is clearly less, but the treatment increased the elongation at break of the best samples app. 0.4 %.
Table 1.
Example 11
The effect of treatments on the lignin content
The lignin content of the fiber was measured using standard method TAPPI T222 om- 88. In the method extractive-free fiber is hydrolyzed with 13.5 M sulphuric acid. Polysaccharides are hydrolyzed and the lignin, so-called Klason-lignin is measured as the unhydrolyzed residue. In order to measure the total lignin content also the so-called soluble lignin was measured spectrophotometrically at 203 nm as described by Dence, CW (1992), The determination of lignin, in: Lin, SY and Dence, CW (Eds.); Springer
Series in Wood Science, Methods in Lignin Chemistry, Springer, Berlin, p. 33-61 (Chapter 2.2).
In table 2 are the total lignin contents for the following fiber samples: 1) retting with stirring 2 h: Linitest 60 0 C, 14 ml/g; 2) retting with stirring 4 h: Linitest 60 0 C, 14 ml/g; 3) static retting 2 x 6 h; 2 x 0.5 ml/g (12 h; 1.0 ml/g); 4) static retting 3 x 6 h; 3 x 0.5 ml/g (18 h; 1,5 ml/g); 5) static retting 2 x 8 h; 2 x 0.5 ml/g (16 h; 1.0 ml/g); 6) static retting 3 x 8 h; 3 x 0.5 ml/g (24 h; 1.5 ml/g); 7) soaking app. 1 h in detergent water (3-5 ml detergent/ 1 water, fiber to liquid ratio app. 1/10: detergent: 15-39 % zeolite, oxygen based bleach, 5-15 %; anionic and non-ionic tensides, > 5 % soap, phosphate, polycarboxylates, enzymes, pH 10.4 ) and rinsing and 8) raw fiber material.
Table 2. Total lignin content of fiber samples.
sample treatment total lignin standard content% deviation
1 retting with stirring 2 h: linitest 60 0 C, 14 ml/g; 2,89 0,09
2 retting with stirring 4 h Linitest 60 0 C, 14 ml/g 5,13 0,16
3 static retting 2 x 6 h; 2 x 0,5 ml/g (12 h; 1,0 ml/g) 2,82 0,04
4 static retting 3 x 6 h; 3 x 0,5 ml/g (18 h; 1,5 ml/g) 3,40 0,09
5 static retting 2 x 8 h; 2 x 0,5 ml/g (16 h; 1,0 ml/g) 4,04 0,68
6 static retting 3 x 8 h; 3 x 0,5 ml/g (24 h; 1,5 ml/g) 3,15 0,13
7 soaking app. 1 h in detergent water and rinsing 4,17 0,35
8 raw fiber 6,48 0,20
Addition of culture liquid to the treatment can reduce the lignin content of the fiber almost 50 % compared to unwashed reference fiber and almost 20 % compared to the washed fiber. The Klason-lignin method most likely also measures other compounds besides lignin which would explain the effect of washing on the measured lignin contents.
The best results for lignin removal were obtained by 2 h Linitest treatment (60 0 C, 14 ml/g) and by long-lasting static retting 2 x 6 h; 2 x 0.5 ml/g (12 h; 1 ml/g). By the treatment with culture liquid (mycelium removed) (washing machine 3 x 2.15 h, 40 0 C):
"Fusart" I (3 x 1 ml/g) 29.6 % of lignin was removed and by "Fusart" II (3 x 3 ml/g) 44.6 %.
Figure 3 shows the lignin content of linseed after treatment with Fusarium protein free liquid.
Example 12
Effect of treatments on the extractives
The amount of extractives in the fibers was measured by using standard method TAPPI T 204 om-88 modified so that the solvent used was acetone. Table 3 shows the results of the analyses. From the results can be concluded that the washing of flax fibers reduced the amount of extractives considerably app. 50 % but the addition of culture liquid of the fungus Fusarium in various treatment sequences had no effect on the amount of acetone extractives.
The results in the table 3 are for the following fiber samples: 1) retting with stirring 2 h: Linitest 60 0 C, 14 ml/g; 2) retting with stirring 4 h: Linitest 60 0 C, 14 ml/g; 3) static retting 2 x 6 h; 2 x 0.5 ml/g (12 h; 1.0 ml/g); 4) static retting 3 x 6 h; 3 x 0.5 ml/g (18 h; 1,5 ml/g); 5) static retting 2 x 8 h; 2 x 0.5 ml/g (16 h; 1.0 ml/g); 6) static retting 3 x 8 h; 3 x 0.5 ml/g (24 h; 1.5 ml/g); 7) soaking app. 1 h in detergent water (3-5 ml detergent/ 1 water, fiber to liquid ratio app. 1/10: detergent: 15-39 % zeolite, oxygen based bleach, 5-15 %; anionic and non-ionic tensides, > 5 % soap, phosphate, polycarboxylates, enzymes, pH 10.4 ) and rinsing and 8) raw fiber material.
Table 3. Extractive content of treated samples.
treatment extractives %
1 retting with stirring 2 h Linitest 60 0 C, 14 ml/g 0,721094
2 retting with stirring 4 h Linitest 60 0 C, 14 ml/g 0,718219
3 static retting 20 0 C 2 x 6 h; 2 x 0,5 ml/g (12 h; 1,0 ml/g) 0,636081
4 static retting20 0 C 3 x 6 h; 3 x 0,5 ml/g (18 h; 1,5 ml/g) 0,711726
5 static retting 20 0 C 2 x 8 h; 2 x 0,5 ml/g (16 h; 1,0 ml/g) 0,646088
6 static retting 20 o C 3 χ 8 h; 3 χ 0,5 ml/g (24 h; l,5 ml/g) 0,780521
7 soaking app. 1 h in detergent water and rinsing 0,658938
8 raw fiber 1,164116
Figure 4 shows the amount of extractives of linseed Laser after the above mentioned treatments.
Example 13
Fabric hand of the fibers
The comfort sensation of a raw material has multidimensional attributes and is impossible to quantify through a single physical property. In order to find a method for the comfort evaluation of textiles, the concept "fabric hand/handle" is commonly used to assess textile materials. Fabric hand is a generic term for tactile sensations associated with fabrics that influence consumer preferences. (Makinen et al.2005, HAP05, Workshop on Haptic and Tactile Perception of Deformable Objects 1.12.2005, 8-16. ftp://ftp.gdv.uni-hannover.de/papers/haptex05/12.pdf; Bishop, D.P. 1996, Textile Progress 26(3), 1-62; Hui, CL. - Lau, T.W. - Ng, S. F. & Chan, K.C.C. 2004. Neural Network Prediction of Human Psychological Perceptions of Fabric Hand. Textile Research Journal 74(5), 375-383. Subjective handle evaluation of fibers were made by five textile design expert judges. The assessment was done by sensory evalution with touch and sight together. Seven samples were to be put in order according to opposite characteristics: soft-hard; smooth-coarse; glossy-dull; light-dark and general impression of the best sample was given in words. Six of the samples were treated with the protein free culture liquid and one sample was washed raw fiber. Samples of linseed that had been retted 3 x 6 h/3 x 0.5 ml/g and 2 x 6 h/3 x 0.5 ml/g in room temperature or treated for 2 hours in Linitest apparatus (2h/14 ml/g, 57 0 C) were evaluated to be the softest and best samples. These samples were characterized as being silky, warm, glossy, and because of their pleasing touch suitable for clothing. The washed raw fiber was distinguished from the other samples for being the coarsest, hardest, darkest and dullest.
Example 14
Rotor spinning
The spinning experiments were carried out at the Tampere University of Technology, Department of Materials Science, The Institute of Fibre Materials Science. The treatment included carding the lap and sliver and rotor spinning. The carding machines were not set specifically for flax instead the settings for cotton were used. The spinning was done to the linseed flax that had been retted in room temperature with protein free culture liquid (3 x 8 h/0.5 ml x 3 per gram of fiber) and to the washing machine treated (mycelium removed) linseed (3 x 2.15 h/3 x 1 ml/g). 20 % of pretreated cotton was added to both batches, so that the portion of flax was 80 %. During the spinning the rotor speed of rotation was 23-30 U/min x 1000, withdrawal speed 30-50 m/min, delivery speed 0.6-0.9 m/min.