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。     

亚麻微生物脱胶优势菌的选育及其应用

从亚麻种植土壤与沤麻水中分离出96株能分解果胶的菌株。通过初筛和复筛获得4株果胶酶高产菌株。其中,在果胶平板培养基上生长速度快、产果胶酶活力高的12号菌株被确定为优势菌,经鉴定其为枯草芽孢杆菌。最适脱胶条件为：麻水比1：15,pH7．5,温度32～33℃,预培养的优势菌接种量3％。结果表明,采用优势菌的脱胶周期比对照缩短50％左右,而且麻的纤维质量明显得以改善。     

一株大麻雨露脱胶真菌的分离鉴定及其脱胶性能研究

从大麻根部土壤中分离一株能够以大麻茎粉为唯一碳源生长的真菌。经18S rRNA鉴定此菌为链格孢属。采用GB/T18147.2-2008和DNS法对大麻茎残胶率及真菌果胶酶活性进行分析。将此菌应用于田间试验,对施用本菌的沤麻周期、大麻出麻率及纤维强度进行研究。结果表明此菌具有产果胶酶活性但能力较低仅为150.5 U/m L,能够降解胶质释放大麻纤维,田间雨露沤麻时施用本菌可以加快沤麻速度,出麻率及大麻纤维强度分别提高了1.73%和17.01%。应用此菌沤麻可缩短沤麻周期,提高大麻出麻率及纤维质量,具有较好的应用前景。     

DNA提取方法对苎麻脱胶菌群PCR-DGGE结果偏移的影响

为筛选和优化出较适宜的苎麻脱胶菌群DNA提取方法,本文分别以来自苎麻沤麻环境的6种纯培养菌等丰度混合物和苎麻自然沤麻菌群为材料,研究"溶菌酶-SDS"法、"超声波-溶菌酶-SDS"法、"蛋白酶K-SDS"法以及"冻融-蛋白酶K-SDS"法4种DNA提取方法对菌群16Sr DNA基因PCR-DGGE偏移结果的影响。结果表明,4种方法均能从2类材料中提取出了超过1600ng/μL的DNA,不同方法之间DNA产率略有差异,经过超声波处理或反复冻融处理的DNA有明显的降解,但4种方法提供的DNA模板均扩增出了450bp的16Sr DNA基因片段。4种DNA提取方法对DGGE结果有明显影响,且只有"冻融-蛋白酶K-SDS"法检测到了纯培养菌混合物中的全部6种细菌,4种方法获得的自然沤麻菌群的DGGE指纹图谱也明显不同,最多产生17条带("冻融-蛋白酶K-SDS"法),最少只有9条带("溶菌酶-SDS"法),增加超声波或冻融等物理处理可以使部分弱带变强。因此,组合应用物理、生物和化学等细胞裂解方法可以提供更有代表性的DNA,可减少PCR-DGGE结果的偏移。     

苎麻脱胶菌群RAMCD407中优势菌的分离、鉴定及脱胶能力分析

【目的】以苎麻生物脱胶菌群RAMCD407为研究材料,分析其菌种功能,初步阐明菌群的种间协作机理。【方法】利用4种不同的培养基,对该菌群中的微生物进行分离培养,通过16S r RNA基因序列比对鉴定,将分离得到的菌株进行果胶酶活力、木聚糖酶活力和胶质去除率的测定与筛选。筛选得到的菌株重新组合成为复合菌系JHY,并分析各菌种对JHY的功能影响。【结果】共获得25个菌株,这25个菌株分别属于芽孢杆菌属、假单胞菌属、肠杆菌属、鲁梅利杆菌属、鞘氨醇杆菌属和微小杆菌属。从中筛选出6株细菌,分别为Y1、2H3、Y2、JY31、2H2和2H1,组成复合菌系JHY。经测定,2H2在复合菌系JHY中发挥重要作用,能提高复合菌系的果胶酶活力、木聚糖酶活力和胶质去除率;JY31的存在抑制了其它菌株的生长,降低了复合菌系JHY的酶活力和胶质去除率。【结论】2H2为复合菌系JHY的重要菌种,去除JY31可提高复合菌系JHY的脱胶效率。     

适于罗布麻生物脱胶的果胶酶产生菌分离筛选与表征

在青海柴达木盆地沤制的罗布麻表皮中分离得到两株适于罗布麻脱胶的高产果胶酶菌株,经形态、生理生化指标鉴证和16SrDNA菌种鉴定,确定一株为新的果胶酶产生菌琼氏不动杆菌（Acinetobacter junii）,一株为枯草芽孢杆菌（Bacillus subtilis）。水解圈实验得出前者H／C值（H／C为水解圈直径H与菌落直径C之比）为5,后者为3;经酶活力测定,在37℃下,琼氏不动杆菌（Acinetobacter junii）在11h达到产酶高峰（酶活力为103．2IU／mL）,枯草芽孢杆菌（Bacillus subtilis）培养至9h达到产酶高峰（酶活力为91．6IU／mL）,但前者最高酶活力比后者高12．5％;经脱胶实验得出两菌残胶率分别为18．47％和17．31％,对罗布麻生物脱胶有较好的适用性。     

亚麻纤维脱胶菌的筛选及脱胶效果研究

为筛选亚麻纤维脱胶菌株,从麻脱胶废水、废麻堆积物等7个含麻胶质样品中分离得到39株能分解果胶的菌株.采用水解圈法复筛选出8株果胶酶活性较高的菌株,经果胶酶、纤维素酶活性测定和菌体脱胶试验,最终确定8-1是优良的亚麻脱胶菌株,该菌株果胶酶活可达663.17 u/mL,而纤维素酶活仅为9.13 u/mL.菌株8-1经形态观察、Biolog菌种鉴定系统鉴定以及基于16S rDNA序列构建的系统进化树分析为一株芽孢杆菌.     

脂肪酶假单胞菌的分离培养及最佳产酶条件研究

以麻疯树油为唯一碳源,从以粉碎的麻疯树种子处理过的土壤中分离筛选出1株脂肪酶活性较高的菌株,初步鉴定为假单胞菌属(Pseudomonas).实验观察了碳源、氮源、无机盐及发酵工艺对产酶的影响,摇瓶发酵结果表明.该菌株最适产酶培养基的组成是(%,w/v):橄榄油2,酵母膏0.5,(NH4)2SO4 0.5,MgCl2·6H2O 0.5,最适产酶温度为30℃,最佳产酶pH为6.5,转速180r/min,发酵培养36h酶活达到最高,为14.17U/mL.本研究为以麻疯树油为原料酶法生产生物柴油奠定了一定的基础.     

两株芽孢杆菌在苎麻纤维复合脱胶中的应用

【背景】苎麻纤维细长、强韧、洁白、有光泽,被誉为"天然纤维之王",应用广泛。但其被以半纤维素和果胶为主要成分的胶质所包裹,脱胶是生产精干麻工艺的核心工序。利用单一菌株脱胶,往往因其脱胶酶系不全,存在胶质去除率低的问题,导致后期仍需要大量的碱和漂白剂处理。【目的】丰富苎麻脱胶过程中关键酶系,从而提高苎麻胶质去除率,并降低脱胶后期化学试剂的用量,推进苎麻生物脱胶的工艺应用。【方法】选用2株芽孢杆菌HG-9 (高果胶酶和甘露聚糖酶)和HG-25(高木聚糖酶)建立了复合微生物脱胶技术,并对其进行了优化。【结果】当2株菌接种量均为6%,水料比16:1,初始pH值5.9,在温度37.6°C下脱胶处理14 h时脱胶效果最佳,与菌株HG-9单独脱胶相比,脱胶时间减少2 h,胶质去除率、半纤维素去除率和木质素去除率分别提高9.32%、21.24%和17.93%,次氯酸钠用量减少20%。通过电子显微镜分析其形貌特征发现,混合脱胶获得的纤维表面更加平滑,无明显扭曲和损伤且纤维分散度较高。【结论】通过复合微生物协同作用,丰富脱胶过程中关键酶系,提高了苎麻纤维胶质去除率,缩短了脱胶时间,而且减少了脱胶后期漂白剂的用量,为苎麻生物脱胶工业化应用的进一步发展提供了指导。     

一株果胶酶产生菌HDYM-02的分离鉴定及系统发育分析

对1株从沤麻液中分离的果胶酶产生菌HDYM-02进行了形态、生理生化特征及16S rDNA序列分析,结果表明该菌株为革兰染色阳性,菌体大小为(1~1.2)×(2.7~2.9)μm,最适生长温度为33~35℃之间,最适生长pH为6.5~7.0。以该菌株16S rDNA序列同源性为基础,比对分析发现其与蜡状芽胞杆菌(Bacillus cereus)的同源性高达99.5%。     

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.     

Comparison of subjective and objective methods to assess flax straw cultivars and fibre quality after dew-retting

Subjective and objective methods were compared for the assessment of the quality of unretted and retted straw, and the fibres obtained after dew-retting of four flax cultivars, Ariane, Evelin, Laura and Viola. Maturity of straw, degree of retting, ease of decortication after retting, divisibility, handle, cleanliness and colour were assessed as subjective factors and of these, degree of retting and handle appear to be useful for assessing quality. The samples were also analysed for physical and chemical differences using a range of objective methods. Straw quality could not be graded using height and maturity, although stem diameter and technical length could indicate quality. Changes in N, lipid and ash content, as a result of retting, were observed. The increase in the proportions of fibre fractions present in retted compared with unretted straw, due to microbial degradation of pectin, could be identified by thermal analysis. Analyses of the fibre samples to determine fineness, strength, cellulose, hemicellulose, lignin and minerals were carried out and significant differences in some of the parameters were identified. Cultivar Laura produced the best fibre. Fibre fineness, ash, lipid, caustic weight loss and fibre fractions as determined by thermal analysis were found to be important objective parameters for quality assessment. The assessment of fibre samples performed subjectively by an expert grader and by selected objective methods gave similar quality rankings.     

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 .     

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.     

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’.     

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.     

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.     

Study on microbe retting of kenaf fiber

Retting is the predominant problem in the application of kenaf fiber in high-grade products. While the traditional retting method is water retting, that is, the harvested bast kenaf is immersed in natural water (rivers or tanks) in which indigenous bacteria colonize noncellulosic materials in an anaerobic process resulting in severe environmental problems and low-grade fiber, therefore it is inevitable to seek for a pollution-free or little-pollution retting method. With the more application of biotechnology in textile industry, the more biology-treatments have been researched recently. So microbe retting was employed in this work. The fungus strain was isolated from the river in which kenaf fiber was retted, then microbe retting was performed with this fungus. Substrate species, the initial pH of the culture medium, cultivation temperature, retting time and inoculum size are involved in the experiments and the evaluation of retting is based on the residual gum content in retted kenaf fiber. As a result, the removal of pectin in microbe retting of kenaf is 91.31% under the optimal retting conditions. In addition, the effective retting fungus is also observed with microscope as one kind of filamentous epiphyte.