Retting of Flax by Aspergillus niger

In this study, retting was carried out by Aspergillus niger. The pH, galacturonic acid (GA), and total reducing sugar were determined; the end point was identified by the classic empirical processes and by the maximal GA content of the retting water. The process gave clear and resistent fibers, and the retting time was similar to that of current industrial processes with bacterial enzymes. Control of total acidity was not required, since the pH remained close to neutrality throughout the entire process.     

亚麻脱胶菌种的选育及脱胶过程的初步研究

从沤麻主生物期的水中分离产果胶酶的菌株经初筛、复筛获得了三株专性厌氧细菌,初步鉴定为费氏芽孢杆菌,对其亚麻脱胶性能进行了初步研究,确定人工加菌沤麻的最适工艺条件为：加菌量2％,加菌时间：沤麻进入主生物期零时,菌株A优于其它菌株．结果表明：采用上述工艺进行沤麻实验,可缩短沤麻时间30％,并可提高麻纤维质量。     

The retting of flax after preservation with sulphur dioxide

Experiments were carried out to compare the retting of moist flax preserved with sulphur dioxide with that of green dried flax, using whole straw samples. When retted in water at either a constant 20°C or 28°C dried flax was fully retted after 15 and 10 days respectively whereas the sulphur dioxide treated flax (20 g sulphur dioxide kg“¹ flax DM) had undergone almost no retting after 20 days at 20dC or 10 days at 28°C. Pre-soaking the treated flax for 24 h in water and changing the acidified water, raised the pH of the retting liquor to a more normal value but did not significantly increase the rate of retting. Addition of the pectinase enzyme preparation ‘Flaxzyme’ to retting liquor at the rate of either 1.5 g kg^-1 or 3.0 g kg^-1 water, and at a constant temperature of 20°C, substantially increased the rate of retting of both sulphur dioxide treated and dried flax. Optimum degree of retting was achieved at 24 h with the treated flax and at 97 h with the dried flax. Pre-rinsing of the sulphur dioxide treated straw only served to reduce the rate of retting. It was concluded that natural water retting of sulphur dioxide treated flax is retarded by the presence of acidic residues of sulphur dioxide, while enzyme retting is enhanced by these. In further smaller scale experiments using bundles of cut flax straw Flaxzyme was added at concentrations ranging from 0–8.0 ml litre ¹ to containers containing flax in water at ratios from 1:10 to 1: 600 flax:water and the producion of galacturonic acid was used as an indicator of retting progress. Retting took place more rapidly at higher flax to water ratios for a given enzyme concentration. This effect was attributed to the lower pH of higher flax to water ratios which created pH conditions closer to the pH optimum for the retting enzymes. When enzyme retting was compared at a range of buffered pH's the optimum was pH 4.0. At a buffered pH of 4.0 and a temperature of 19°C, retting of sulphur dioxide treated moist flax (flax to water ratio of 1:10) was achieved with Flaxzyme concentrations as low as 0.5 ml litre”‘,much lower than the previously reported minimum of 3.0 ml litre’.     

The active component in the flax-retting system of the zygomycete Rhizopus oryzae sb is a family 28 polygalacturonase

The zygomycete Rhizopus oryzae sb is a very efficient organism for retting of flax, the initial microbiological step in the process of making linen. An extracellular polygalacturonase, when isolated could perform retting, and therefore probably is the key component in the retting system of R. oryzae. This was purified and characterized. The purified enzyme has a molecular mass of 37,436 Da from mass spectrometric determination, an isoelectric point of 8.4, and has non-methylated polygalacturonic acid as its preferred substrate. Peptide sequences indicate that the enzyme belongs to family 28, in similarity with other polygalacturonases (EC. 3.2.1.15). It contains, however an N-terminal sequence absent in other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium Ralstonia solanacearum. The biochemical background for the superior retting efficiency of R. oryzae sb is discussed.     

Drastic changes in lake ecosystem development as a consequence of flax retting: a multiproxy palaeolimnological study of Lake Kooraste Linajärv,Estonia

This study demonstrates the power of multiproxy palaeolimnological analyses in investigating environmental changes in the Lake Kooraste Linajärv ecosystem through historical time in response to flax retting. Flax retting history was proven by applying pollen and macrofossil evidence and by using several biotic and geochemical proxies on a sediment core. Continuous findings of flax pollen and macrofossil remains in lake sediments were considered as strong evidence for the occurrence of retting. Analyses of the well-dated sediment core show the consequences of flax retting in the lake. As a result, the once clear soft water oligotrophic endorheic lake with limited sedimentation has turned into a hypertrophic high-sedimentation lake with anoxic bottom water, strong stratification and intense water blooms. Despite the fact that flax retting was forbidden in Estonia around ad 1950s and retting has not occurred over the last six decades, anthropogenic alterations were so pervasive in the past, that they have prevented any lake water improvements until the present-day.     

亚麻微生物脱胶菌种的筛选与鉴定

在研究天然水沤法脱胶的过程中,通过初筛、复筛,从沤麻主生物期的沤麻液中筛选出两株茵落周围产生透明圈较大、脱胶酶活较高的菌株。通过形态观察,并对其多项生理、生化指标进行了分析研究,初步鉴定并命名为枯草芽孢杆菌A1和B1。初步加茵脱胶实验表明：枯草芽孢杆菌A1产生果胶酶、木聚糖酶,而不产生纤维素酶,脱胶周期为72小时;枯草芽孢杆茵B1产生果胶酶、木聚糖酶和纤维素酶,脱胶周期为50小时。     

Studies on chemical and enzyme retting of flax on a semi-industrial scale and analysis of the effluents for their physico-chemical components

Retting trials carried out in this study have shown that chemical and enzyme retting could be carried out at a semi-industrial scale. The yield from enzyme retted fibres was higher than chemical or water retted flax fibres. The variation in fibre fineness, strength, fluidity and moisture regain was not significantly different, thereby proving that chemicals and enzymes do not degrade or depolymerise the cellulose chains. The activity of the enzymes at the end of retting, when investigated, remained quite high, suggesting that the waste liquor could be recycled after removing the suspended solids present in the liquor. Biochemical oxygen demand (BOD) and chemical oxygen demand (COD) were highest in effluent from enzyme retting compared to effluents from chemical or water retting. All three effluents were analysed for N₂, PO₄, Cl, SO₄, Na and K. Aeration of the effluents reduced the level of BOD and COD by more than 50%.     

Polygalacturonase is the key component in enzymatic retting of flax

Seven commercial enzyme mixtures were tested for their ability to perform retting of flax (i.e. to separate flax fibers by partly removal of middle lamella) and were assayed for hydrolysis of xylan, cellulose and four kinds of pectin. The only activity that showed correlation to the ability to perform retting was the degradation of low esterfied pectin. A purified Aspergillus niger polygalacturonase was also shown to be able to perform retting. From this data it is hypothesized that degradation of the smooth regions (i.e. non-methylated polygalacturonase) in the middle lamella pectin is the most important step in enzymatic retting.     

Production of polysaccharide-degrading enzymes by saprophytic fungi from glyphosate-treated flax and their involvement in retting

The fungi present on glyphosate-treated flax plants were isolated. Cladosporium herbarum, Epicoccum nigrum, Botrytis cinerea and yeasts occurred most frequently immediately after glyphosate treatment but as retting progressed the frequency of occurrence of Fusarium culmorum, Alternaria alternata and a Phoma sp. increased. Many of the fungi isolated from retting flax were also present as epiphytes on healthy flax stems. Glyphosate was shown to be fungitoxic in vitro but it had only a very slight effect on fungi colonising the flax. The application of sucrose and urea to flax 1 wk after glyphosate treatment resulted in more rapid fungal colonisation of the stems, but did not significantly enhance retting. When grown on sterilised flax stem sections, fungi known to be saprophytic on flax produced polysaccharide-degrading enzymes. All seven fungi tested produced polygalacturonase, pectin-lyase and xylanase. The greatest cellulase activity was present in stem tissues inoculated with F. culmorum and the Phoma sp. while no cellulase was detected in tissue inoculated with B. cinerea, a Mucor sp. or a Penicillium sp. Extracts from flax inoculated with the cellulolytic fungi caused the solubilisation of native cellulose. Pectinases, xylanase and cellulase were also detected in naturally-colonised senescing and dead flax stems. Stems which had been treated with a sucrose solution tended to contain the greatest enzyme activity.     

Identification and Retting Efficiencies of Fungi Isolated from Dew-Retted Flax in the United States and Europe

Seven strains of filamentous fungi and one yeast were isolated from flax that was dew retted in the United States. These filamentous fungi were subcultured to purity and identified, and six appear not to have been reported earlier as isolates from dew-retted flax. Five of the purified U.S. strains, two fungi isolated from flax that was dew retted in Europe, and a laboratory culture of Aspergillus sojae were tested for their ability to ret flax stems. The monocultures were evaluated for the degree of retting, fiber strength, dry weight loss, and tactile response (i.e., feel of softness) as reflected in the retted fiber. Structural modifications of representative samples of the retted flax were assessed by scanning electron microscopy. All of the filamentous fungi were able to carry out some retting, whereas the isolated yeast could not. All organisms produced pectinases when they were cultivated in shake flasks on ball-milled flax as the sole carbon source. Some fungi also produced cellulases, mannanases, and xylanases. Rhizomucor pusillus and Fusarium lateritium were noteworthy as retting organisms by their high level of pectinase activity, ability to attack noncellulosic cell types without attacking cellulose, capacity to penetrate the cuticular surface of the stem, and efficient fiber release from the core. The results indicated that these organisms deserve further study as potential organisms for retting of bast fibers in industrial applications.     

Pre-harvest retting of flax with glyphosate — Effects of moisture and microbial colonisation on retting

Increasing the frequency of misting of flax plants which had been treated with the herbicide glyphosate also tended to increase the numbers of microbial isolates and increase retting. Fungicides, applied at weekly intervals, suppressed the numbers of fungi but increased the numbers of bacterial isolates; they also decreased retting. Numbers and proportions of fungal isolates varied to some extent with treatment but the spectrum of isolates was relatively constant and appeared little influenced by artificial inoculation. Alternaria alternata was the dominant fungal isolate.     

亚麻脱胶新工艺的初步研究

研究了温水浸渍亚麻脱胶过程中的产果胶酶的微生物数量、种类和果胶酶活力变化规律,分离筛选出了产果胶酶活力较高的厌氧和兼性厌氧菌各l株,研究了这2个菌株的种子培养条件,用正交实验法优化了接入厌氧和兼性厌氧菌的亚麻脱胶工艺.实验结果表明亚麻脱胶周期缩短35%,可改善麻纤维质量.     

In vitro evaluation of dew-retting of flax by fungi from southern Europe

Flax dew-retting is widely adopted in most flax-growing countries, but it does not represent a practical solution where dry weather conditions occur after harvest. A study of the local microbiological aspects was undertaken as a contribution to improve field-retting of flax under southern European climates. Fungi were isolated from soil and dew-retted flax in northern Italy, and 23 representative strains were chosen to test their ability to ret flax stems. Experiments were performed in vitro on flax stem pieces artificially inoculated with single fungal strains. Retting degree was assessed with a mechanical test, to evaluate the ease with which the bast was detached from the wood core, and by the analysis of the residual fibre pectins using uronic acid. Uronic acid dosage provided a better differentiation of the strains than the mechanical test. There was a large variability in retting ability among the species assayed and even among strains of the same species. The best results were obtained with all Aspergillus and Penicillium strains, while Mucor and Rhizopus strains showed a variable retting ability. Fusarium, Trichoderma strains and Epicoccum nigrum had the poorest retting abilities among all the fungal strains assayed.     

Improving retting of fibre through genetic modification of flax to express pectinases

Flax (Linum usitatissimum L.) is a raw material used for important industrial products. Linen has very high quality textile properties, such as its strength, water absorption, comfort and feel. However, it occupies less than 1% of the total textile market. The major reason for this is the long and difficult retting process by which linen fibres are obtained. In retting, bast fibre bundles are separated from the core, the epidermis and the cuticle. This is accomplished by the cleavage of pectins and hemicellulose in the flax cell wall, a process mainly carried out by plant pathogens like filamentous fungi. The remaining bast fibres are mainly composed of cellulose and lignin. The aim of this study was to generate plants that could be retted more efficiently. To accomplish this, we employed the novel approach of transgenic flax plant generation with increased polygalacturonase (PGI ) and rhamnogalacturonase (RHA) activities. The constitutive expression of Aspergillus aculeatus genes resulted in a significant reduction in the pectin content in tissue-cultured and field-grown plants. This pectin content reduction was accompanied by a significantly higher (more than 2-fold) retting efficiency of the transgenic plant fibres as measured by a modified Fried’s test. No alteration in the lignin or cellulose content was observed in the transgenic plants relative to the control. This indicates that the over-expression of the two enzymes does not affect flax fibre composition. The growth rate and soluble sugar and starch contents were in the range of the control levels. It is interesting to note that the RHA and PGI plants showed higher resistance to Fusarium culmorum and F. oxysporum attack, which correlates with the increased phenolic acid level. In this report, we demonstrate for the first time that over-expression of the A. aculeatus genes results in flax plants more readily usable for fibre production. The biochemical parameters of the cell wall components indicated that the fibre quality remains similar to that of wild-type plants, which is an important pre-requisite for industrial applications. Magdalena Musialak and Magdalena Wróbel-Kwiatkowska participated equally in the preparation of this paper     

Retting pits for textile fibre plants at Danish prehistoric sites dated between 800 b.c. and a.d. 1050

During the last decade, a new type of structure has been found at several archaeological sites in Denmark. These structures can be interpreted as having been used for retting the stems of textile plants such as Linum usitatissimum L. (flax), Cannabis sativa L. (hemp) and Urtica dioica L. (nettle). In order to obtain fine threads for textile production, these plants need to pass through several biological and technical processes. The first process is the retting of the plant stems to dissolve the pectin which fixes the fibres to the stalk. This can either be done by water retting, where the plant stems are soaked in lakes, rivers or waterlogged pits, or by field retting, where the stems are laid out in a field in order to absorb dew. The first method is shorter in time and the process is easier to control. In this article, details of archaeological structures are presented from eight sites in southern Scandinavia that can be interpreted as textile plant retting pits. The constructions of the pits are described, as well as the archaeological contexts and the relevant associated archaeobotanical records. Some of the presented sites, of which the oldest are dated to the late Bronze Age and early pre-Roman Iron Age (800–250 b.c.) and the youngest to the Viking Age (a.d. 750–1050), indicate a large-scale production of flax that had been underestimated up to now.     

Development of a hand tool to test the degree of retting of flax straw

To determine the optimum conditions for inducing retting of flax, by the application of glyphosate herbicide, it is necessary to have an easily portable tool which will give a rapid, accurate assessment of the stage that retting has reached. Such a tool, which can be used to test individual stems, is described and the estimates of retting obtained compared with those from other methods.     

Determining the physical properties of flax fibre for industrial applications: the influence of agronomic practice

Field trials of fibre flax were sown in 2002 and 2003 comprising 29 and 26 flax varieties, respectively. In 2002, two nitrogen treatments of 40 and 80 kg ha^?1 were imposed. The trials were sprayed with a desiccating herbicide prior to retting at 35 and 15 days after midpoint of flowering in 2002 and 2003, respectively, and were harvested once retting had completed. Fibre was extracted from the flax stems using a laboratory‐scale scutcher and hackling pins, and long and total fibre yields were determined. The breaking load of flax technical fibres was assessed using a novel technique employing ‘zero twist’ yarns. Fineness was assessed using the Wool Industries Research Association airflow method. The tenacity of a single technical fibre was then derived from the average breaking load of the yarn and knowledge of its fineness. Both flax variety and season were found to contribute to variations in fibre fineness and strength, with the dry season in 2003 leading to less variation across the varieties and much finer fibres. Few of the varieties were found to be stable across seasons, and environmental variation accounted for 96% of the variation in fibre fineness between years, and 69% of the variation in tensile strength and breaking load. Fibre yield was found to be related to fibre fineness, with the coarser fibres corresponding to higher yields. The new yarn test presented herein has been shown to be a valid and useful method for determining the tensile properties of technical flax. The varieties and the sample sizes required to adequately measure variation in fibre properties are discussed.     

Engineering of PHB synthesis causes improved elastic properties of flax fibers

Flax stem is a source of fiber used by the textile industry. Flax fibers are separated from other parts of stems in the process called retting and are probably the first plant fibers used by man for textile purposes (1). Nowadays flax cultivation is often limited because of its lower elastic property compared to cotton fibers. Thus the goal of this study was to increase the flax fiber quality using a transgenic approach. Expression of three bacterial genes coding for beta-ketothiolase (phb A), acetoacetyl-CoA reductase (phb B), and PHB synthase (phb C) resulted in poly-beta-hydroxybutyrate (PHB) accumulation in the plant stem. PHB is known as a biodegradable thermoplastic displaying chemical and physical properties similar to those of conventional plastics (i.e., polypropylene). The fibers isolated from transgenic flax plants cultivated in the field and synthesizing PHB were then studied for biomechanical properties. All measured parameters, strength, Young's modulus, and energy for failure of flax fibers, were significantly increased. Thus the substantial improvement in elastic properties of fibers from the transgenic line has been achieved. Since the acetyl CoA, substrate for PHB synthesis, is involved not only for energy production but also for synthesis of many cellular constituents, the goal of this study was also the analysis of those metabolites, which interfere with plant physiology and thus fiber quality. The analyzed plants showed that reduction in lignin, pectin, and hemicellulose levels resulted in increased retting efficiency. A significant increase in phenolic acids was also detected, and this was the reason for improved plant resistance to pathogen infection. However, a slight decrease in crop production was detected.     

Changes in microbial populations during anaerobic flax retting

D onaghy , J.A., L evett , P.N. & H aylock , R.W. 1990. Changes in microbial populations during anaerobic flax retting. Journal of Applied Bacteriology 69 , 634–641.
The bacterial flora of industrial and laboratory scale anaerobic flax rets were determined at intervals throughout the rets. Although after an initial lag period total bacterial numbers remained roughly constant there were fluctuations in the bacterial species constituting the total. Pure culture rets and enzyme assays were used to determine which strains had retting potential. Of the strains demonstrated to have retting ability Bacillus licheniformis and B. subtilis were numerically dominant from 10 to 40 h and were succeeded in dominance by Clostridium acetobutylicum and Cl. felsineum .     

Construction of a pilot plant for producing fine linen fibers for textiles

A newly built 200-L-pilot plant was used for testing a novel chemo-enzymatic process for producing fine flax fibers without the weather-associated risks of dew retting. Raw, green and decorticated flax fibers were placed inside trays in the tempered main tank of the pilot plant, where a vertically acting mechanism gently moved the parallel fiber bundles. The fibers were incubated in an alkaline bath, in a pectinolytic culture of Geobacillus thermoglucosidasius PB94A and in a peroxide-softener bath. Finally the fibers were dried, combed and hackled into a sliver that is ready for wet-spinning of linen yarns. A total of 140 kg of raw fibers were treated in 40 different experiments in the pilot plant. The resolution of the raw fibers improved from 7.3 to 2.7 ± 0.3 after the treatment. The fineness was enhanced from 37.4 dtex to 11.1 ± 1.2 dtex. The proposed pilot-plant process produced constant-quality fibers and could be easily up-scaled.