Enzyme Applications in Fiber Processing - ACS Publications

Chapter 22

Enzymatic Retting of Flax

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 21, 2016 | http://pubs.acs.org Publication Date: March 31, 1998 | doi: 10.1021/bk-1998-0687.ch022

D. E. Akin , G. Henriksson , W. H. Morrison III , and Karl-Erik L . Eriksson

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Russell Research Center, ARS, U.S. Department of Agiculture, P.O. Box 5677, Athens, GA 30604 Department of Biochemistry and Molecular Biology, Center for Biological Resource Recovery, University of Georgia, Athens, GA 30602-7229

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Industrial interest is driving a renewal of a flax and linen industry in the US. A major limitation to production of fiber from flax stems is retting, which is a microbial process involving partial degradation of the plant tissues for isolation of cellulosic fibers. Enzymatic retting of flax has been proposed as a means of replacing the current practice of dewretting, thereby improving the quality and consistency of flax fiber for use in textiles. Since costs were too high for enzymatic retting by the previously proposed procedures, new formulations and methods were tested for improved efficiency of retting. Commercial enzyme mixtures (i.e., Flaxzyme and Ultrazym) at various concentrations and experimental supernatants from fungi isolated from dew-retted plants were tested for retting efficiency by visual (Fried test), analytical, and microscopic methods. Factors that increased retting efficiency at the laboratory level were: specific enzyme mixtures, addition of the chelators oxalic acid and ethylenediamine-tetraacetic acid to the enzymes, increased temperature, and mechanical disruption of flax stems to increase surface area for enzymes. Use of these methods and procedures should increase the efficiency of retting and reduce costs for commercial application. Bast fibers are produced in the cortical regions of plant stems. Examples of bast fiber plants are jute, ramie, hemp, kenaf and flax (1). Flax, which is the source of linen, is perhaps the oldest and best known of the bast fibers used for textiles. Linen and other bast fibers are obtained from the plant stems by a process called retting. Retting, which is usually a microbial process, is the partial degradation of the stems, resulting in the isolation of the cellulosic fibers from the other, non-cellulosic components (2). In past times, flax stems were submerged inriversand lakes, and anaerobic bacteria retted the plants. However, this practice was discontinued in western countries several decades ago because of the pollution from fermentation products and the high cost of drying. Currently, dew-retting is the accepted practice in European countries, which supply

©1998 American Chemical Society

Eriksson and Cavaco-Paulo; Enzyme Applications in Fiber Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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270 much of the linen used in textiles. In dew-retting, flax plants are pulled from the soil and laid out in fields for selective attack by indigenous fungi for several weeks. Disadvantages of dew-retting are: 1) dependence on particular geographical regions that have the appropriate moisture and temperature ranges for retting, 2) coarser and lower quality fiber than with water retting, 3) less consistency in fiber characteristics, and 4) occupation of agricultural fields for several weeks (2). Retting remains the major problem with production of flax fibers for linen. In the 1980's, research was undertaken in Europe to develop enzymatic retting as a method to replace dew-retting. Despite the production of Flaxzyme, a commercial enzyme mixture from Novo Nordisk (Denmark), and several patents (2) no commercial process has been reported and dew-retting is still the practice most widely used in Europe. Cost of the enzymes, and perhaps other less obvious reasons, prevented development of a commercial enzyme retting process. Although the US is the largest per capita user of linen, only small amounts of flax are grown in this country and only one company is currentiy processing (i.e., dew-retting and scutching) flax. The US Flax Initiative, which is a consortium of business and state and federal research entities, was formed in 1996 to promote a flax/linen industry in the US. Part of this effort is to research major problems related to economic production of high and consistent quality flax fiber. We have organized a research effort toward the goal of developing an enzymatic retting process for flax/linen fiber. In this paper, we review results from earlier published studies, with supplementation of unpublished results from new studies. 9

Structure of Flax Stems Bast fibers are located in the cortical regions between the epidermis/cuticle and the core as shown in the scanning electron micrograph of the flax stem cross-section (Figure 1). Bundles are comprised of several ultimate fibers, which have thick secondary walls and small lumens at maturity. Thin-walled parenchyma cells surround the bundles, occupying the inner area (i.e., towards the core), outer area, and regions between bundles. Cambium is located between the bast fibers and the core. Histochemical examination of cross-sections of flax stems stained with ruthenium red, which has been used to locate pectin in plants (3), showed intense positive reactions in epidermis, parenchyma, and cambium cells. A less intense reaction occurred in the secondary walls of the fibers, and middle lamellae, which bind the ultimatefiberstogether, gave stronger reactions. Transmission electron micrographs show structural characteristics of the epidermis and parenchyma cells (Figure 2) and the fibers (Figure 3), which are closely associated and connected by an electron dense middle lamella. Modifications in Structure and Chemistry after Retting Effective retting separatesfiberbundles from the epidermis/cuticle and the core (Figure 4). In addition, fiber bundles are divided into smaller bundles and at times into ultimate fibers. Transmission electron micrographs show attack by indigenous fungi, which primarily carry out dew-retting, on the middle lamella of fiber cells walls (Figure 5). The fibers are released from the non-fiber components, and the middle lamellae are degraded to produce ultimate fibers.



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Figure 1. Scanning electron micrograph of unretted flax stem showing bast fiber bundles (B), epidermis (E) with cuticle (arrow) on the outer side, and core (C). Bar = 50 μπι. From Akin et al. (6).

Figure 2. Transmission electron micrograph of unretted flax stem showing thickwalled epidermis (E) and thin-walled parenchyma (P) cells. Bar = 1 μπι. From Akin etal.ftfj.



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Figure 3. Transmission electron micrograph of unretted flax stem showing thickwalled fiber cells (F) with electron dense middle lamella (arrow). The cell lumen is small in mature fibers. Bar = 1 μπι. From Akin et al. (6).

Figure 4. Scanning electron micrograph of dew-retted flax stem showing separation of fiber bundles from epidermis and cuticle and the core and separation into smaller groups and to ultimate fibers. Bar = 50 μπι. From Akin et al. (6).



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The analysis of structural carbohydrates that comprise plant cell walls in flax stem and modifications after retting are shown in Table I. Components representative of pectins (e.g., uronic acids), arabinose, and xylose were reduced in amounts, while levels of glucose (indicative of cellulose), mannose, and galactose increased after retting. These data, as well as those from other workers (4, 5), suggest that the cellulosic fibers have non-glucose components within the fiber structure. Aromatic compounds are low in amounts in the bast fibers but were localized using microspectroscopy in the middle lamellae and particularly in the cell corners of bast fibers before and after dew-retting/φ. The nature of these aromatic components are not well defined (7; Gamble, personal communication), and some appear to have tannin-like properties. Enzymatic Retting We tested commercial enzymes with high levels of pectinase and which have been previously used (e.g., Ultrazym), or expressly developed (e.g., Flaxzyme), for enzymatic retting of flax (8). We used scanning electron and light microscopies and the Fried test to assess fiber separation from the stems (9). The Fried test, which scores retting efficiency based on standard images, has been used to evaluate enzyme retting (2). For these initial tests, twelve 9 to 10 cm long sections of flax stems were incubated in screwcap vials with 13 ml sodium acetate buffer, pH 5.0, in an end-over-end fashion. Various enzymes and levels, chelators, temperature, and mechanical pretreatments were among the variables tested. The commercial enzyme mixtures are high in pectinolytic activity and also have xylanase and cellulase activities (2,8). We found that experimental cultures with high pectinase activity but without substantial xylanase and cellulase activities were very efficient in retting flax. It is clear from our work as well as others (2) that the presence of pectinases in enzyme mixtures is of paramount importance for effective retting. Scanning electron microscopy had indicated that Ultrazym and Flaxzyme were effective in separating fibers where the plant site was exposed to enzymes but did not ret effectively when stems were more intact. The structural modifications effected by Flaxzyme are shown in Figure 6. Flaxzyme appeared to isolate fibers more effectively than Ultrazym in these studies (9), although other work had indicated that Ultrazym outperformed Flaxzyme with some flax samples (8). Our results documented, as expected, that mechanical pretreatment to fracture and open the stem surface increased the efficiency of enzymatic retting with Flaxzyme also (10). Increased temperature also enhanced the activity of cell wall-degrading enzymes, with retting efficiency at incubation temperatures of 40 to 50 °C about 2 times faster than that at 22 °C (10). A major disadvantage of enzymatic retting is cost of the enzyme required to effectively ret flax stems (2). Therefore, methods to reduce the level of enzyme required would enhance the chances of success of a commercial process. We evaluated a series of chelators added to pectinase mixtures for the increased effectiveness of retting. We are not aware of other work in which chelators have been added to enzyme mixtures for flax retting, although other work (11, 12) indicated that pretreatment with chelators increased retting by fungi. Table II indicates that oxalic acid at 50 m M concentrations substantially increased the effectiveness of retting by Flaxzyme or Ultrazym. This level



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Figure 5. Transmission electron micrograph of dew-retted flax stem showing loss of middle lamella (arrow) and separation into ultimate fibers (F). Bar = 1 μπι. From Akin et al. (6).

Figure 6. Scanning electron micrograph of enzymatically retted flax stem with 4% (dilution of commercial product as supplied by Novo Nordisk) showing separation of fiber bundles from core and cuticle. Bar = 50 μπι. From Akin et al. (9).



0.2 ± 0c

b

Ultrazym 3 %

8.2 ± 1.9a

6.2 ± 0.1b

7.6 ± 2.9a

9.7 ± 0.6a

Rhamnose

7.4 ± 1.4b

5.9 ± 0.4b

5.5 ± 1.2b

15.5 ± 0.6a

Arabinose

13.5 ± 2.6a

8.8 ± 0.9b

7.0 ± 0.2b

15.9 ± 1.7a

Xylose

38.7 ± 2.3b

37.5 ± 1.3b

39.2 ± 2.5b

30.8 ± 0.9a

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-mg g"

Mannose

b

Average and standard deviation of duplicate analyses. Data adapted from Akin et al., (9). Percentages are calculated from dilution of commercial products from Novo Nordisk. a,b,c,d Values within columns with different letters differ at P< 0.05.

a

0.9 ± 0.1b

Flaxzyme l %

0.8 ± 0.1b

Dewretted

b

2.1 ± Oa

Unretted

Treatment

Uronic Acids %

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Carbohydrates

Table I. Carbohydrate Constituents in Flax Bast Before and after Retting

30.6 ± 1.5b

41.4 ± 0.3c

35.0 ± 0.9a

32.5±0.3ab

Galactose


595.0 ± 12.7 b

623.5 ± 17.7 b

649.5 ± 38.9 b

434.0 ±18.3 a

Glucose

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Table II. Enzymatic Retting of Flax with and Without Chelators


b

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0

Treatment

Fried Score

Buffer pH 5.0

0

Oxalic acid (50mM)

2.0 ± 0 . 8

Flaxzyme (0.3%)

1.0 ± 0

Flaxzyme (2.5%)

3.0 ± 0

Flaxzyme (0.05%)+Oxalic acid (30mM)

3.0 ± 0

Ultrazym (5%)

1.3 ± 0 . 5

Ultrazym (5%) + Oxalic acid (30mM)

3.0 ± 0

Ultrazym (5%) + EDTA (30mM)

2.5 ± 0 . 7

Ultrazym (5%) + Citric acid (30mM)

1.0 ± 0

a

Data adapted from Henriksson et al. (10). Percentages are dilutions of commercial product as supplied by Novo Nordisk. Scored by comparison with visual images from 0 (no retting) to 3 (maximum fiber separation from stem). See Henriksson et al., (10) for method. b

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of oxalic acid reduced the amount of Flaxzyme required to give a rating of 3 by the Fried test by 50 fold. Emylenediarnine-tetraacetic acid (EDTA) was comparable to oxalic acid in increasing the efficiency of the enzyme mixtures, whereas sodium citrate was considerably less effective (Table Π; 10). The efficiency of retting by experimental enzyme mixtures from a fungal filtrate that showed primarily pectinase activity was increased with E D T A to a greater extent than Flaxzyme at similar protein concentrations (70;Heniksson, G., 1996, unpublished data). Calcium ions have been reported to act as bridges linking pectin molecules in stem tissues of flax and stabilizing them against pectinase (13, 14). Evaluation of flax hypocotyls indicated that calcium concentrations varied among the tissues, with levels in epidermal cells higher than in other tissues (14). Related to this observation, we found that in lower quality hackled flax fibers large remnants of epidermal/cuticle fragments were still bound with substantial amounts of fiber bundles; the resulting material consisted of coarse fiber strands with considerable cuticle associated. In contrast, the higher quality fiber bundles were finer and had fewer large epidermis/cuticular fragments associated. This result suggests that the recalcitrance of the epidermis/cuticle component to separation from the bast fibers lowers the quality of flax fiber. Further, that calcium is more prevalent in the epidermal tissues and that calcium ions stabilize pectin and inhibit endogalacturonase suggests that this region of the stem is a barrier to retting. Therefore, it is likely that the removal of calcium by



277 chelators would facilitate the action of pectinases within the epidermal wall structure, enhance disorganization of the stem tissues, and increase retting efficiency. Further work is required to confirm this theory and, i f true, to maximize the influence of enzyme/chelator formulations. Preliminary results confirm that retting of mechanically disrupted flax stems with 0.05 % Flaxzyme (% of undiluted commercial product as supplied by Novo) plus 50 m M E D T A produces fibers of similar fineness (based on relative air-flow values) to that with 0.3 % Flaxzyme without chelators. The strength offibersderived by the former method were about 38% higher than those of Flaxzyme without chelators based on stelometer measurements (i.e., g/tex). These results support the contention that chelators enhance the activity of Flaxzyme, thus facilitating flax retting and reducing amounts of enzymes required. Conclusions Enzymatic retting in larger scale is required to assess the commercial feasibility of these procedures discussed above. Further, research on fungal filtrates of higher activity than Flaxzyme in combination with other chelating agents should be continued to acquire enzyme mixtures of maximum retting efficiency. Our preliminary results indicate that the following aspects warrant consideration in any commercial process for enzymatic retting of flax: 1 ) mechanical disruption of the surface (epidermis/cuticle) of the flax stem to increase surface action of the enzyme mixtures (10) is required to obtain optimal retting, 2) the addition of chelators (i.e., oxalic acid and EDTA) to commercial and experimental pectinase mixtures substantially increases the efficiency of enzymatic retting, and 3) increasing the temperature to around 40 °C increases the retting efficiency of Flaxzyme/oxalic acid over that at ambient temperature (10). Literature Cited 1.

2.

3. 4. 5. 6. 7.

van Dam, J.E.G.; van Vilsteren, G.E.T.; Zomers, F.H.A.; Shannon, W.B.; Hamilton, I.T. Industrial Fibre Crops. Increased Application of Domestically Produced Plant Fibres in Textiles, Pulp and Paper Production, and Composite Materials; A T O - D L O : Wageningen, The Netherlands, 1994; 249pp. Van Sumere, C.F. In The Biology and Processing of Flax; Sharma, H.S.S., Van Sumere, C.F., Eds.; M Publications: Belfast, Northern Ireland, U K , 1992; pp 157198. Jensen, W.A. Botanical Histochemistry; W.H. Freeman and Co.: San Francisco, C A , 1962; pp 201-205. Gorshkova, T.A.; Wyatt, S.E.; Salnikov, V . V . ; Gibeaut, D . M . ; Ibragimov, M.R.; Lozovaya, V . V . ; Carpita, N.C. Plant Physiol. 1996, 110, 721-729. Stewart, D.; McDougall, G.J.; Baty, A . J. Agric. Food Chem. 1995, 43, 1853-1858. Akin, D.E.; Gamble, G.R.; Morrison, W.H., III; Rigsby, L . L . ; Dodd, R. B. J. Sci. Food Agric. 1996, 72, 155-165. Love, G.D.; Snape, C.E.; Jarvis, M . C.; Morrison, I.M. Phytochemistry 1994, 35, 489-491.



278 8. Sharma, H.S.S.; Van Sumere, C.F. Genetic Engin. & Biotechnol. 1992, 12, 19-23. 9. Akin, D.E.; Morrison, W.H., III; Gamble, G.R.; Rigsby, L.L.; Henriksson, G.; Eriksson, K.-E.L. Textile Res. J. 1997, 67, 279-287. 10. Henriksson, G.; Akin, D.E.; Rigsby, L.L.; Patel, L.; Eriksson, K.-E.L.; Textile Res. J. 1997, In press. 11. Brown, A.E.; Black, D.L.R. Trans. Br. Mycol. Soc. 1985, 85, 625-630. 12. Bratt, R.P.; Brown, A.E.; Black, D.L.R. Records Agric. Res. 1986, 34, 9-15. 13. Jauneau, Α.; Cabin-Flaman, Α.; Verdus, M.-C.; Ripoll, C; Thellier, M. Plant Physiol. Biochem. 1994, 32, 839-846. 14. Rihouey, C.; Jauneau, Α.; Cabin-Flaman, Α.; Demarty, M.; Lefebvre, F.; Morvan, C. Plant Physiol. Biochem. 1995, 33, 497-508.


Enzyme Applications in Fiber Processing - ACS Publications

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