白腐菌及黑曲霉所产的纤维素复合酶对稻草秸秆的生物降解

研究了白腐菌及纤维素复合酶对稻草秸秆的协同生物降解。结果表明,利用黄孢原毛平革菌固态发酵稻草秸秆的过程中,LiP和MnP的最大活力可以达到28.3U/g和12.6U/g,同时,秸秆中的木质素能被有效降解,但纤维素、半纤维素降解率较低。添加黑曲霉所产的纤维素复合酶能有效地促进秸秆腐熟程度。在接入白腐菌培养10天后,每克稻草添加3 IU纤维素酶液并酶解48h可以使稻草秸秆中纤维素降解53.8%,半纤维素降解57.8%,木质素降解44.5%,干物质损失46.3%。此时细胞壁出现大范围破损,整个组织变得松散,秸秆完全腐熟。     

应用混料实验设计制备秸秆复合降解菌剂

农业秸秆类废弃物含有大量木质纤维素,该类物质结构稳定,不易降解,为秸秆的合理利用带来诸多困难。本实验尝试利用混料实验设计对筛选出可以共同培养的五种木质纤维素降解菌的配比进行研究,寻求复合发酵降解剂各组分的最佳配比,并分析发酵产品得到适用于不同发酵目的的菌剂。通过对发酵产品中木质素和纤维素降解率及还原糖的含量的分析建立模型,分析预测纤维素降解率最高为35.75%,木质素降解率最高为27%,还原糖含量最高为3.39mg/g。通过优化得出发酵菌剂最优配比为枯草芽胞杆菌12.1%,克鲁斯假丝酵母10%,地衣芽胞杆菌27.2%,变色栓菌10.6%,黄孢原毛平革菌40%。对应三指标的实测值为：35.47%,26.41%和2.37mg/g。     

用cDNA微阵列技术快速筛选粗毛栓菌的表达基因

利用cDNA微阵列技术快速筛选具有较强降解木质纤维素能力的白腐真菌粗毛栓菌(Trametes gallica)的表达基因.利用木质素生物降解模式菌株黄孢原毛平革菌(Phanerochaete chrysosporium)的cDNA制备研究所用微阵列.在含有2 596个cDNA片段的芯片上共检测到172个阳性克隆,其中有165个克隆的荧光信号比值(Cy-5/Cy-3)在0.5和2.0之间,占所检测阳性克隆数的95.9%.对应于在限氮条件下生长5天和12天的粗毛栓菌培养物,分别有3个和4个时序特异性差异表达基因.随机挑取122个克隆进行测序和序列比对,发现所测序列中有118个能够很好地定位于黄孢原毛平革菌的基因组上.结果显示,粗毛栓菌与黄孢原毛平革菌在表达序列上存在较大差异,表明这两种真菌之间存在着较远的亲缘关系.通过同源性比对分析,发现2个令人感兴趣的克隆,一个对应于黄孢原毛平革菌过氧化物酶基因lpoB的部分片段,另一个为编码一种热激蛋白的基因.     

用cDNA微阵列技术快速筛选粗毛栓菌的表达基因(英)

利用cDNA微阵列技术快速筛选具有较强降解木质纤维素能力的白腐真菌粗毛栓菌(Trametes gallica)的表达基因.利用木质素生物降解模式菌株黄孢原毛平革菌（Phanerochaete chrysosporium）的cDNA制备研究所用微阵列.在含有2 596个cDNA片段的芯片上共检测到172个阳性克隆,其中有165个克隆的荧光信号比值（Cy-5/Cy-3）在0.5和2.0之间,占所检测阳性克隆数的95.9％.对应于在限氮条件下生长５天和12天的粗毛栓菌培养物,分别有３个和４个时序特异性差异表达基因.随机挑取122个克隆进行测序和序列比对,发现所测序列中有118个能够很好地定位于黄孢原毛平革菌的基因组上.结果显示,粗毛栓菌与黄孢原毛平革菌在表达序列上存在较大差异,表明这两种真菌之间存在着较远的亲缘关系.通过同源性比对分析,发现２个令人感兴趣的克隆,一个对应于黄孢原毛平革菌过氧化物酶基因lpoB的部分片段,另一个为编码一种热激蛋白的基因.     

黄孢原毛平革菌对黄瓜连作土壤酚酸物质的降解

研究了黄孢原毛平革菌对黄瓜连作土壤中对羟基苯甲酸、香草酸及阿魏酸的降解及连作障碍修复作用.结果表明,在摇瓶条件下,黄孢原毛平革菌在8 d内,对3种酚酸的降解率都达99％以上. 在连续种植7年黄瓜的大棚土壤中,施入黄孢原毛平革菌菌剂后,土壤中3种酚酸的含量都有所降低,降解率为54.46%. 与对照相比,修复土壤真菌数量变化无明显规律. 修复处理后黄瓜株高、茎粗、鲜质量及干质量无明显变化,黄瓜根部病害明显减轻,枯萎病及根结线虫病相对病情指数分别降低10.2%和14.6%.表明施入黄孢原毛平革菌剂对黄瓜连作障碍的解除具有一定的效果.     

黄孢原毛平革茵对黄瓜连作土壤酚酸物质的降解

研究了黄孢原毛平革菌对黄瓜连作土壤中对羟基苯甲酸、香草酸及阿魏酸的降解及连作障碍修复作用.结果表明,在摇瓶条件下,黄孢原毛平革菌在8 d内.对3种酚酸的降解率都达99%以上.在连续种植7年黄瓜的大棚土壤中,施入黄孢原毛平革菌菌剂后,土壤中3种酚酸的含量都有所降低,降解率为54.46%.与对照相比,修复土壤真菌数量变化无明显规律.修复处理后黄瓜株高、茎粗、鲜质量及干质量无明显变化,黄瓜根部病害明显减轻,枯萎病及根结线虫病相对病情指数分别降低10.2%和14.6%.表明施入黄孢原毛平革茵剂对黄瓜连作障碍的解除具有一定的效果.     

黄孢原毛平革菌降解土霉素的影响因素及酶活分析

目的:黄孢原毛平革菌降解土霉素的影响因素及酶活分析。方法:文章使用的方法有制备实验菌种和孢子悬浮液、测定校准曲线及样品与测定酶活性等。结果:培养基中的营养物质真菌降解抗生素和吸附重金属特性减少然后,产生了酶的次生阶段,不利于营养的获得,分泌了锰过氧化物酶,酶活不断增加,达到了最大值。活跃期过后,酶活逐渐降低,在培养后期,产生的代谢物越来越多,堆集了大量的有毒代谢物,降低了锰过氧化物酶活性。讨论:当土壤浓度维持在200mg/L时,采用固化或者是非固化的方式后,黄孢原毛平革菌对土壤的降解度分别为92.44%与67.63%。在实验过程中,使用了两种方式处理,但菌产锰过氧化物酶酶培养96h之后,土霉素达到了最大浓度,为70 mg/L,相应为527.40 U/L与73.90 U/L;使用两种处理方式,菌产木质素过氧化物酶都要先培养96h之后,土霉素浓度变成了70 mg/L,这个时候是最大值,相应为0.384 U/L与O.204 U/L,因此,黄孢原毛平革菌降解土霉素时,起到关键作用的是锰过氧化物酶。文章可行性研究了黄孢原毛平革菌降解土霉素。实验相应地考察了p H值、土霉素初始浓度是如何影响影响黄孢原毛平革菌降解土霉素。实验结果显示,相应地增加土霉素的浓度,能提升酶活,当超过一定限度时,酶的活性会随着增加而降低。     

微生物处理对秸秆结构的影响

使用红外光谱、X射线衍射及扫描电镜对4种经固态培养的微生物降解稻草秸秆的效果进行了研究,并与原稻草粉进行结构对比分析。结果表明：经过高碳低氮培养,秸秆木质素均有明显降解,其中黄孢原毛平革菌达到37.17%。扫描电镜发现秸秆的表面形态和内部结构发生了明显改变,红外光谱中芳香环与醚键吸收明显减少,X射线衍射得到的结晶度最大下降了5.60%。     

固定化白腐真菌降解水溶液中2,4-二氯酚的研究

研究固定化黄孢原毛平革菌对水溶液中2,4-二氯酚（2,4-DCP）的降解效果,探讨固定化黄孢原毛平革菌处理水溶液中氯酚类污染物的可行性．结果表明,采用固定化方法处理的白腐真菌．其产酶稳定性及酶活均比游离态白腐真菌有显著提高．2,4-DCP降解效果受固定化孢子接种量、pH值、摇床转速、2,4-DCP的初始浓度和表面活性剂浓度的影响．当pH为4．5,摇床转速180r／min,培养基含有1％的Tween 80,2,4-DCP初始浓度为40mg／L时,加入10mL固定化白腐真菌孢子,2,4-DCP去除效果最好．     

黄孢原毛平革菌木素降解酶系的研究进展

黄孢原毛平革菌木素降解酶系主要由木素过氧化物酶、锰过氧化物酶和乙二醛氧化酶组成。由于该酶系特殊的降解机制,除了木质素,它能降解许多种类的有机污染物,因此在环保方面有巨大的应用前景。本文主要综述了国内外对该酶系的研究进展。     

Solid-state production of biopulp by Phanerochaete chrysosporium using steam-exploded wheat straw as substrate.

A novel material for biopulp-making, steam-exploded wheat straw (SEWS), was studied. During the steam explosion process, the hemicellulose was partly degraded and became water-soluble sugar as the carbon resource of the chosen microbe growth, and compared with non-SEWS, the degradation amount of cellulose decreased and the degradation amount of lignin increased for the fermented steam-exploded wheat straw (FSEWS) cultured with Phanerochaete chrysosporium ME-446. Under the optimum conditions of solid-state ferrmentation (SSF), the degradation amount of lignin reached 60% on the 5th day and the fermented straw residue could be used directly as the material for pulp making.     

贝壳状革耳菌和黄孢平革菌固体培养酶系比较

白腐菌黄孢平革菌(Phanerochaete chrysosporium) 与贝壳状革耳菌(Panus conchatus)在类似自然状态的固体培养条件下酶的分泌情况有 较大差异。P.conchatus和P.chrysosporium的主要木素降解酶分别是漆酶和锰过氧化物酶 ;两种菌均产生较高水平的木聚糖酶;P.conchatus在整个培养过程中所产生的内切葡 聚糖酶、微晶纤维素酶和纤维二糖酶活力均比P.chrysosporium相应酶的活力低得多, 尤其是内切葡聚糖酶。研究结果初步揭示了P.conchaus降解木素的主要酶系及选择性降 解木素的原因。     

Selective solvent delignification for fermentation enhancement

Cellulose and hemicellulose in renewable biomass resources such as cornstover and wheat straw have been examined as substrates for the production of ethanol. A mixed culture of selected strains of Clostridium thermocellum and Clostridium thermosaccharolyticum are used to accomplish both the hydrolysis and fermentation of these carbohydrates in a single step. However, lignin and related phenolic materials are shown to diminish the rate, extent, and yield at which these carbohydrates can be utilized for ethanol production. In order to overcome this problem, a selective solvent pretreatment with alkaline-ethanol-water mixtures was examined for the delignification of cellulosic biomass under conditions where very little loss of fermentable carbohyrates results. Under optimal conditions, up to 67% of the initial lignin in cornstover can be extracted while 95% of the alpha-cellulose and pentosan carbohydrates remain insoluble. Subsequent mixed culture fermentation of the treated material has shown a 400% increase in the rate of degradation and greater than 85% utilization of the substrate. The effects of various extraction parameters on delignification kinetics and subsequent fermentation performance are discussed.     

Biological pretreatment of wheat straw by Phanerochaete chrysosporium supplemented with inorganic salts

Inorganic salts and tween 80 are known to induce the lignin degrading peroxidase expression of Phanerochaete chrysosporium in submerged culture. In this study, the wheat straw pretreatment supplemented with inorganic salts (salts group), tween 80 (plus) and no supplementation to the biomass (minus) were examined. Among the solid state fermentation groups, salts group resulted in a substantial degradation of wheat straw within one week, along with the highest lignin loss (25%) and ～250% higher efficiency for the total sugar release through enzymatic hydrolysis. The results were correlated with pyrolysis GC-MS (Py-GC-MS), thermogravimetric (TG)/differential thermogravimetric (DTG) and X-ray diffraction (XRD). The results suggested that the supplementation of inorganic salts in the solid state fermentation of wheat straw significantly enhances the degradation rate of the biomass by P. chrysosporium which can be exploited as an alternative means to existing pretreatment technologies.     

不同木质纤维素基质上白腐菌降解特性的研究

通过测定木质素、纤维素、半纤维素和漆酶分泌的变化,研究白腐菌在稻草、木屑、粗纤维素、滤纸、黑液木素基质上的降解特性。结果表明,除黑液木素上白腐菌不能生长外,在前25d,各基质中纤维素、半纤维素和木质素含量呈持续下降趋势,之后,降解速率减少,其中木质素的降解速率大于纤维素和半纤维素的降解速率。漆酶分泌在生长初期呈快速上升趋势,第10d酶活达到最大,第10～20d快速下降,其后基本不变,基质中酶活大小顺序为稻草基质、木屑基质、粗纤维和滤纸基质,显示了木质素存在对漆酶分泌的诱导作用。     

Progress in biopulping of non-woody materials: Chemical, enzymatic and ultrastructural aspects of wheat straw delignification with ligninolytic fungi from the genus Pleurotus

Abstract: During screening of basidiomycetes for wheat straw delignification, considerable lignin degradation with a limited attack to cellulose was attained with Pleurotus eryngii . Straw solid-state fermentation (SSF) was optimized, and the enzymatic mechanisms for lignin degradation were investigated. No lignin peroxidase was detected under liquid or SSF conditions, but high laccase and aryl-alcohol oxidase levels were found. The latter enzyme has been fully characterized in PI. eryngii and it seems to be involved in a cyclic redox system for H₂0₂ generation from aromatic compounds. Results obtained using homoveratric acid suggest that Pleurotus laccase could be involved in degradation of phenolic and non-phenolic lignin moieties. Histological and ultrastructural studies provided some general morphological characteristics of the fungal attack on wheat straw. Whereas a simultaneous degradation pattern was observed in straw treated with Phanerochaete chrysosporium , PI. eryngii caused partial degradation of middle lamella and separation of individual sclerenchymatic fibers. When these straw samples were subjected to refining tests, energy saving after biological treatment was the highest in the case of straw treated with PI. eryngii , which also produced the lowest substrate loss. From these results, a correlation between preferential removal of lignin, separation of sclerenchymatic fibers and pulping properties was provided during fungal treatment of wheat straw.     

Comparative ligninolytic and polysaccharolytic potentials of an alkaliphilic basidiomycete on native ligninocellulose

A comparison of the ligninolytic, cellulolytic and hemicellulolytic abilities of an alkaliphilic white-rot fungus. Coprinus fimetarius, on wheat straw under varying conditions of solid-substrate fermentation is presented. The extent of fractional degradation (percentage of the original dry weight of the fraction) of straw under an optimized set of cultural conditions (pH 9·0, moisture 65%, temperature 37°C, period 21 days) was in the following order: lignin (45%), cellulose (42%), hemicellulose (27%). Urea nitrogen favoured the degradation of lignin as well as cellulose and hemicellulose up to a certain level (1·5% sterile urea or 3% unsterile urea on a dry weight basis) beyond which the degradation of lignin was relatively more adversely affected than cellulose. The addition of phosphorus and sulphur was found essential for selective lignin removal. Increasing the C:N ratio by addition of free carbohydrates resulted in an overall decrease in the degradation wherein cellulose utilization was the most affected event. The pre-treatment (physical or chemical) of the substrate caused a general increase in biodegradation of lignin, cellulose and hemicellulose. The degrading activity of the fungus declined with the scaling-up of the fermentation particularly under non-sterile conditions.     

木质纤维素的定量测定及降解规律的初步研究

为了准确地测定稻草及其发酵物中纤维素、半纤维素、木质素的含量,通过差重法进行定量测定,并以此评价白腐菌株Pleurotus sapidus对稻草秸秆的降解状况,结果表明：利用差重法测定稻草发酵物中纤维素、半纤维索、木质素的百分含量是可行的,并能很好地评价白腐菌对稻草的降解规律,即降解过程中纤维素、半纤维素、本质素在前20d降解的很快,之后降解减缓,在50d内,纤维素被降解34．02％,半纤维素被降解56．29％,木质素被降解61．65％。     

添加剂对水稻秸秆实地混合厌氧发酵产甲烷系统的作用

为提高水稻秸秆利用效率,改进水稻秸秆实地混合厌氧发酵产甲烷技术,本研究开展了添加剂在混合厌氧发酵系统的应用研究。试验选取水稻秸秆和猪粪作为发酵原料,通过分别添加不同的浓度的吐温20和腐植酸,测定甲烷的产气量和浓度、秸秆的降解和土壤肥力的变化,以揭示添加剂类型及其浓度对水稻田实地甲烷生产系统的影响。结果表明:添加剂的掺入并未影响产气的动态变化趋势,但显著地促进产气和提高产气浓度,整体效果由高到低为腐植酸吐温20对照,其中经腐植酸处理的产气量和产气浓度对照相比分别提高了50. 73%和24. 55%。添加剂的掺入有利于水稻秸秆纤维素和半纤维素的降解,但对木质素没有显著影响;其中以0. 15 g/L腐植酸和0. 30 g/L吐温20的降解率最高,相较于对照其纤维素降解率均提高了22. 11%,半纤维素降解率分别提高了107. 13%和98. 39%。添加剂的掺入能显著增加土壤肥力,以0. 15 g/L腐植酸和0. 30 g/L吐温20处理水平的效果最优,相较于对照,其土壤有机质分别增加了29. 63%和23. 72%,全氮分别增加了52. 32%和42. 38%,全磷分别增加了83. 33%和57. 14%。