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。     

Importance of galacturonic Acid in controlling the retting of flax

The empirical test was compared with the determination of pH, total acidity, and galacturonic acid (GA) as methods for establishing the termination time of the retting process in flax. The GA dosage was the best method for determining the end point of this process by distinctly dividing the phases of retting and over-retting in the bacterial loosening of flax fiber. This end point is critical in the production of standard fibers for the weaving industry.     

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.     

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.     

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’.     

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。     

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.     

Characterization of bacterial pectinolytic strains involved in the water retting process

Pectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilisT and B. pumilusT.     

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.     

Effects of selected pectinolytic bacterial strains on water-retting of hemp and fibre properties

Aims:  To study the effect of selected bacterial strains on hemp water-retting and properties of retted fibre.
Methods and Results:  The trials were performed in laboratory tanks. The traditional water-retting process, without inoculum addition, was compared to a process modified by inoculating water tanks with two selected pectinolytic bacteria: the anaerobic strain Clostridium sp. L1/6 and the aerobic strain Bacillus sp. ROO40B. Six different incubation times were compared. Half the fibre obtained from each tank was combed. Micromorphological analyses were performed by scanning electron microscopy on uncombed and combed fibres. Moreover, organoleptic and chemical analyses of uncombed fibres were performed.
Conclusions:  The inoculum , besides speeding up the process, significantly improved the fibre quality. The fibre was not damaged by mechanical hackling, thanks to the good retting level obtained by the addition of selected strains, differently to what happened with the traditionally retted fibre. The best fibre quality was obtained after 3–4 days of retting with the addition of the bacterial inoculum .
Significance and Impact of the Study:  Retting is the major limitation to an efficient production of high-quality hemp fibres. The water-retting process and fibre quality were substantially improved by simultaneously inoculating water tanks with two selected pectinolytic strains.     

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.     

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.     

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.     

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.     

Investigation of the bacterial retting community of kenaf (Hibiscus cannabinus) under different conditions using next-generation semiconductor sequencing

The microbial communities associated with kenaf (Hibiscus cannabinus) plant fibers during retting were determined in an effort to identify possible means of accelerating this process for industrial scale-up. Microbial communities were identified by semiconductor sequencing of 16S rRNA gene amplicons from DNA harvested from plant-surface associated samples and analyzed using an Ion Torrent PGM. The communities were sampled after 96 h from each of three different conditions, including amendments with pond water, sterilized pond water, or with a mixture of pectinolytic bacterial isolates. Additionally, plants from two different sources and having different pretreatment conditions were compared. We report that the best retting communities are dominated by members of the order Clostridiales. These bacteria appear to be naturally associated with the plant material, although slight variations between source materials were found. Additionally, heavy inoculations of pectinolytic bacteria established themselves and in addition their presence facilitated the rapid dominance of the original plant-associated Clostridiales. These data suggest that members of the order Clostridiales dominate the community and are most closely associated with efficient and effective retting. The results further suggest that establishment of the community structure is first driven by the switch to anaerobic conditions, and subsequently by possible competition for nitrogen. These findings reveal important bacterial groups involved in fiber retting, and suggest mechanisms for the manipulation of the community and retting efficiency by modifying nutrient availability.     

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.     

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.     

The influence of the pH on the pectinolytic activity of flax-retting bacteria

The pectinase produced during the anaerobic retting of flax appeared to have a pH optimum between 5.4 and 5.6. Continuous neutralization during the fermentation, however, induced the production of a pectinase with a pH optimum of 7.1.The majority of the pectin splitting bacteria in the acid and in the neutral retting liquors belonged to the speciesClostridium pectinovorum.Pure culture studies withCl. pectinovorum at fixed pH levels showed that the pectinase produced is adapted to the pH of the environment. No differences in pH dependency could be established between the hemicellulases from the acid and the neutral retting liquor.Although a pH of 6.5 is far from optimal for the hemicellulase, the actual hemicellulolytic activity in the neutral pH retting method is greater than the activity of the normal, acid retting liquor. This fact supports the theory that hemicellulase is responsible for losses in fibre quality and quantity in the neutral pH retting method.     

Pre-harvest retting of flax with glyphosate: effects of novel methods of spray application on penetration of the canopy and uniformity of deposition

Four methods of spray application were compared in 1986 with the recommended method of glyphosate application for pre-harvest retting of flax (Linum usitatissimum). Sampling individual plants immediately after application of glyphosate and dye demonstrated that spray penetration through the crop canopy, measured as dye deposition on the lower stem as a percentage of total deposition per plant, was the same for all methods of application. None of the methods of application improved the uniformity of dye deposition.
The following year, effects of deseeding prior to spraying and of using an air-assisted sprayer were investigated. Deseeding increased dye deposition on the lower stem but did not improve the efficacy of glyphosate. Application with an air-assisted sprayer increased the proportion of spray deposited on the lower stem but not the absolute amount deposited or the efficacy of glyphosate. Dye deposition on individual plants varied greatly for all methods of application and, when glyphosate was applied using the same methods, undesiccated plants occurred in all plots.
Considerable improvements in spray penetration and uniformity of spray deposition appear necessary for successful pre-harvest retting of flax with glyphosate.