木质素酶及其生产菌的筛选育种

木质素酶降解木质纤维素材料中的木质素,使木质素-半纤维素-纤维素结构解体,纤维素得以暴露出来供后续步骤处理。它广泛应用于生物制浆、生物漂白、废水处理等工业过程中。由于近年利用可再生木质纤维素材料用酶法水解生产酒精成了研究热点,因而作为纤维素材料生物转化工艺预处理过程中的关键角色,木质素酶也极大地唤起人们的研究兴趣。本文介绍了木质素与白腐真菌（phanerochaete chrysosporium）木质素降解酶系的特征以及锰过氧化物酶、木质素过氧化物酶、漆酶等3种木质素酶的催化作用机理,归纳了目前流行的木质素酶产生菌的筛选方法及近年来从自然界筛选木质素酶高产菌的种类,并对产木质素酶野生菌株的诱变育种与基因工程改造的进展进行了阐述。     

高效纤维素优势分解菌的筛选和鉴定

本试验基于获得高效纤维素优势分解菌的目的,通过分离纯化初步得到30株菌株,利用刚果红染色法初筛共得到14株纤维素分解菌,并通过滤纸条崩解实验进一步进行筛选得到5株效果较好的纤维素分解菌,通过发酵产酶利用DNS显色法测定CMC酶活力和FPA酶活力最终确定了4株优势纤维素分解菌,通过测定4株菌株的羧甲基纤维素酶(CMCase)、滤纸酶(FPA)以及β-葡萄糖苷酶(β-Gase)活,验证4株纤维素优势分解菌的产酶能力,并分别命名为X-1、X-6、X-7和X-11,并将该4株优势纤维素分解菌应用于秸秆的液态发酵,其对秸秆的降解率较自然降解相比,降解率分别提高了31.92%、40.15%、35.29%和39.98%。对4株优势菌株进行了分子鉴定,根据16S r DNA序列比对结果表明,菌株X-1、X-7和X-11均为粪产碱杆菌;菌株X-6属于解糖假苍白杆菌。     

降解玉米秸秆真菌复合菌系的构建及其降解效果评价

  【目的】  秸秆的木质纤维素含量丰富、结构复杂，在自然界中降解较慢，增加秸秆降解菌剂中菌株的多样性有利于提升还田秸秆的降解效果。探究菌株多样性水平和组成影响复合菌系秸秆降解的效果及原因，为复合菌系在秸秆降解中的应用提供理论支撑。  【方法】  通过富集驯化培养，从玉米秸秆还田土壤中筛选具有秸秆降解能力的真菌，从中挑选5株高效秸秆降解真菌进行基因间隔区序列 (ITS) 测定和物种鉴定，明确其分类地位。通过全组合构建菌株多样性为1～5的复合菌系，分别检测复合菌系的秸秆相对降解率及其滤纸酶、纤维素内切酶和木聚糖酶活性，利用方差分析和相关性分析等方法研究菌株多样性和组成对复合菌系玉米秸秆降解效果及其纤维素酶活性的影响。  【结果】  共筛选获得了15株具有秸秆降解能力的真菌，其中5株真菌的秸秆降解效果好、纤维素水解能力强。经ITS序列鉴定和系统发育分析，发现5株降解真菌的遗传差异较大，Z7-6、F7-5、F4-3、L1-1和J2-5分别与草酸青霉 (Z7-6: Penicillium oxalicum)、烟曲霉 (F7-5: Aspergillus fumigatus)、哈茨木霉 (F4-3: Trichoderma harzianum)、白囊耙齿菌 (L1-1: Irpex lacteus) 和木贼镰刀菌 (J2-5: Fusarium equiseti) 的ITS序列相似度均超过99.95%。全组合复配结果表明，复合菌系的秸秆降解能力和纤维素酶活力均高于各单一菌株，且随着菌株多样性水平的增加而提高。滤纸酶、纤维素内切酶和木聚糖酶的活力越强，复合菌系对玉米秸秆的降解效果越好，而其秸秆相对降解率主要取决于滤纸酶和纤维素内切酶的活性。抽样效应分析发现，不同菌株对复合菌系的秸秆降解效果、滤纸酶和纤维素内切酶活性的影响不同。不含菌株F7-5的复合菌系降解效果显著优于含有该菌株的组合，以Z7-6 (P. oxalicum)、F4-3 (T. harzianum)、L1-1 (I. lacteus) 和J2-5 (F. equiseti) 组合F1的玉米秸秆降解效果最佳、酶活性最高。  【结论】  秸秆降解复合菌系的构建过程需要同时考虑多样性效应和抽样效应，增加降解菌的多样性有助于增强秸秆的降解效果。本研究筛选获得的复合菌系F1在玉米秸秆降解中具有潜在的应用前景。     

高效纤维素降解菌的筛选及复合菌系的构建

本研究旨在筛选高效纤维素降解菌,组建复合菌系,用于堆肥接种剂的开发。通过纤维素刚果红选择性培养基,从腐殖土、堆肥样品中分离高效纤维素降解常温菌和耐高温菌。经过羧甲基纤维素酶活(carboxymethyl cellulase,CMCase)、滤纸条崩解能力、对稻杆及苦参(Sophora flavescens Ait.)残渣在液态和固态发酵条件下的降解能力测定逐级淘汰,筛选高效菌株组建复合菌系。研究结果表明,通过拮抗作用、单一菌株在复合菌系中作用测定,最终构建了由3株细菌(弗留明拜叶林克氏菌(Beijerinckia fluminensis)、微杆菌(Microbacterium sp.)和芽胞杆菌(Bacillus sp.)),1株链霉菌(Streptomyces sp.)和1株毛壳菌(Chaetomium sp.)组成的木质纤维素降解高效复合菌系。供试菌株在液态发酵条件下对稻杆和苦参残渣的降解效果明显强于固态发酵,稻杆比苦参残渣更容易被大多数微生物降解,供试真菌在稻杆和苦参残渣的降解中普遍表现出较高的降解效果。筛选得到弗留明拜叶林克氏菌XM-3可以在48 h内将滤纸条完全崩解,复合菌系在固态发酵20 d后对苦参残渣和稻秆的降解率分别达到31.4%和63.1%。本研究对堆肥接种剂的开发具有参考和借鉴意义。     

采用白腐菌对难降解有机污染物的生物净化

白腐菌对难降解污染物的生物降解作用已引起世界范围内的普遍关注，它可通过其分泌的特殊的降解酶系或其他机制将各种难降解的有机污染物彻底降解为CO₂和H₂O。该文介绍了白腐真菌的生物学特性及其分泌的酶系，阐述了白腐菌所分泌的酶在降解各种难降解有机物中的作用机制，分析了提高白腐菌降解能力的方法，总结了白腐菌在治理环境污染方面的应用现状与研究进展，探讨了白腐菌在实际应用方面的不足以及今后的研究方向，对白腐菌在解决环境污染方面的问题具有实际意义。     

菌酶共降解玉米秸秆的工艺研究

该文研究玉米秸秆菌酶共降解工艺,利用黄孢原毛平革菌固体发酵去除部分木质素,再添加外源纤维素酶、木聚糖酶降解纤维素和半纤维素.黄孢原毛平革菌培养12 d时木素过氧化物酶(LiP)酶活达到最大值11.3 u/g,15d时漆酶(Lac)酶活达到最大值0.0992 u/g,秸秆降解集中在10～20 d,第25d时菌解结束,干基总损失率为18.94%;再经过6 d的酶解,纤维素、半纤维素的相对含量分别从27.1%、20.3%下降到13.1%、11.9%.玉米秸秆经菌酶共降解,木质素、纤维素、半纤维素的降解率分别达到67.0%、60.4%、33.0%,每克秸秆还原糖含量达0.507 g.结果表明,菌酶共降解为玉米秸秆的生物利用提供了一种新方法.     

稻草秸秆纤维素分解菌的分离筛选

本研究基于获得高效木质纤维素分解菌的目的,以刚果红纤维素琼脂和滤纸条培养基为初筛培养基,从分离获得的124株真菌中筛选出透明圈与菌落直径比值较大、滤纸条分解能力较强的11个菌株。经液体发酵,测定其酶活力,复筛得到羧甲基纤维素酶活和滤纸酶活均较高的4个菌株;并进行了不同碳源和不同pH对筛选菌株产酶能力的影响试验,发现不同菌株对不同纤维素物质的分解能力不一样,同一菌株对不同纤维素碳源的利用能力也不相同。     

碳氮源对复合木质素降解菌木质素降解能力及相关酶活的影响

白腐真菌所具有的降解木质素能力源于其所产生的酶系统,碳源和氮源是其降解木质素和产酶的一个极为重要的影响因素。通过室内小麦秸秆固态发酵试验,研究了不同的碳、氮源对两株侧耳属真菌Tf1（P.pulmonarius）和JG1（P.cornucopiae）产酶活力、木质素降解和粗蛋白含量的影响。结果表明,Lip和MnP是参与复合木质素降解菌Tf1＋JG1降解小麦秸秆重要的木质素降解酶。以葡萄糖为碳源,酒石酸铵为氮源能显著提高复合木质素降解菌对木质素的降解能力,发酵9 d后小麦秸秆的失重率为14.87%,木质素含量为8.68%,木质素降解率为22.95%;粗蛋白含量为7.28%,比未发酵麦秸提高了36.84%（P〈0.05）;Lip和MnP活力分别为629.11 U.g-1和622.22 U.g-1。     

堆肥中纤维素降解菌的筛选及复配菌降解性能研究

为加快堆肥过程中秸秆纤维素的降解速率,本研究从玉米秸秆堆肥中分离纤维素分解菌,并通过测定羧甲基纤维素酶(CMCase)活力、滤纸条崩解能力及兼容性,筛选出优良菌株,进而构建复合菌系,并对降解性能进行评价。结果共获得29株纤维素分解菌,对其中的高效菌株进行配伍,构建了6组复合菌系。除复合菌系F外,其他复合菌系的滤纸酶活力均显著高于单一菌株(P<0.05),尤以复合菌系B(xw1、xw3、xw8)、D(xw16、xw21、xw31)的酶活力最高,分别为22.8、20.4 U·mL^-1,比其中的最强单菌株xw3、xw21高出58.3%、68.6%,且所产酶具有耐高温(40～55℃)性。复合菌系B、D培养5 d可将滤纸条崩解为糊状,10 d内对秸秆的降解率达24.5%、21.9%,较单菌株xw8、xw31增加9.4和4.7个百分点。经16S rDNA分子鉴定,复合菌系B由微杆菌属(Microbacterium sp.)、类芽孢杆菌属(Paenibacillus sp.)组成,复合菌系D由芽孢杆菌属(Bacillus sp.)、类芽孢杆菌属(Paenibacillu ...     

一株降解水稻秸秆的丝状真菌降解特性的研究

研究了高效降解纤维素的丝状真菌菌株藤仓赤霉对天然水稻秸秆的降解特性,并对该菌株所产的羧甲基纤维素酶的酶学性质进行了初步研究.结果显示,该菌对未处理的水稻秸秆作用8d,水稻秸秆失重率可达53％以上,纤维素、半纤维素和木质素的降解率分别可达38.9％、57.9％和26.5％;扫描式电子显微镜分析表明,该菌作用水稻秸秆是从内...     

Degradation of isoproturon by the white rot fungus Phanerochaete chrysosporium

The ability of the white rot fungus Phanerochaete chrysosporium to degrade isoproturon was tested in solid substrate fermentation (SSF) cultures using straw as substrate/carrier material. The role of the lignin degrading enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP), in the degradation of the herbicide was also studied. Isoproturon concentration and LiP and MnP activities were followed in sterile straw cultures of the fungus. In vitro degradation tests with pure LiP and MnP were performed. P. chrysosporium in straw cultures was able to degrade 91% of the herbicide isoproturon in 14 days of incubation. A sharp decrease of isoproturon coincided with the largest MnP activity. Although LiP activity was also present, its role in SSF is unclear. The in vitro tests showed a strong isoproturon oxidation by LiP and a slower oxidation by MnP in the presence of Tween 80 probably by a lipid peroxidation process. Two N-demethylated metabolites were identified in pure enzyme tests and in SSF cultures. Several unidentified isoproturon derivatives, most likely hydroxylated, were also formed in both systems. The different pattern of derivatives detected in pure LiP and MnP tests showed a completely different metabolism by these two enzymes.     

瓦克青霉F10-2对川楝树皮残渣纤维素酶解研究

以川楝树皮残渣为原料进行纤维素酶解研究,测定了不同培养时间培养基质中主要胞外酶活性的变化,并对发酵前后残渣结构进行扫描电镜观察和红外光谱分析。结果表明:瓦克青霉F10-2具有木质纤维素降解能力,酶解液中纤维素酶、半纤维素酶、木质素过氧化物酶和锰过氧化物酶活性在发酵的8～16d内分别达到最大值6.42U·g-1、7.62U·g-1、6.55U·g-1和3.33U·g-1。利用扫描电镜对降解后底物表面结构进行观察,可看到残渣表面变得疏松、柔软,且具有部分微孔。底物残渣傅立叶红外光谱分析表明,该菌株对残渣中各组分均有一定降解。固态发酵培养试验表明,培养24d后残渣中纤维素、半纤维素和木质素的降解率分别达到42.70%、33.96%和24.62%。     

Ligninolytic fungi in bioremediation: extracellular enzyme production and degradation rate

Ligninolytic fungi can be used for remediation of pollutants in water and soil. Extracellular peroxidases and laccases have been shown to oxidize recalcitrant compounds in vitro but the likely significance of individual enzyme levels in vivo remains unclear. This study documents the amounts and activities of Mn-dependent peroxidase (MnP), lignin peroxidase and laccase (LAC) in various species of ligninolytic fungi grown in liquid medium and soil and their effect on degradation of polycyclic aromatic hydrocarbons (anthracene and pyrene), a polychlorinated biphenyl mixture (Delor 106) and a number of synthetic dyes. Stationary cultures of a highly degradative strain Irpex lacteus exhibited 380-fold and 2-fold increase in production of MnP and LAC, respectively, compared to submerged cultures. Addition of Tween 80 to the submerged culture increased MnP levels 260-fold. High levels of MnP correlated with efficient decolorization of Reactive Orange 16 azo dye but not of Remazol Brilliant Blue R anthraquinone dye. Degradation of anthracene and pyrene in spiked soil by straw-grown explorative mycelium of Phanerochaete chrysosporium, Trametes versicolor and Pleurotus ostreatus showed the importance of MnP and LAC levels secreted into the soil. The importance of high fungal enzyme levels for efficient degradation of recalcitrant compounds was better demonstrated in liquid media compared to the same strains growing in soil.     

Dynamics and availability of phosphorus in the rhizosphere of a temperate silvopastoral system

A field rhizosphere study was carried out over a period of 12 months on a 6-year-old silvopastoral trial in New Zealand. The trial comprised radiata pine (Pinus radiata) with lucerne (Medicago sativa) and perennial ryegrass (Lolium perenne) understoreys. The study was initiated because of the unique interrelationships between roots in silvopastoral systems and a paucity of understanding about the processes involved in phosphorus (P) dynamics in temperate silvopastoral systems. Improving our understanding in this area has important implications for nutrient management in silvopastoral systems. Rhizosphere soils were analysed to determine inorganic (Pi) and organic (Po) P fractions, macroporous resin Pi and Po, phosphatase enzyme activity, microbial biomass carbon and pH. Concentrations of labile Pi were consistently greater and Po lower in tree rhizosphere soil compared to the companion understorey, indicating that radiata pine when grown with a productive understorey mineralised Po to a greater extent than either understorey species. Tree rhizosphere soil from under lucerne and lucerne rhizosphere soil contained the lowest concentrations of total Pi and Po compared with tree under ryegrass and ryegrass rhizosphere soils. This was partly attributed to higher levels of phosphatase enzyme activity in the lucerne rhizosphere soils. The results suggest the combination of lucerne with radiata pine may enhance greater utilisation of soil P, although this requires further investigation. Lower levels of labile Po, and higher levels of labile Pi and phosphatase enzyme activity, were determined in tree and understorey lucerne and ryegrass rhizosphere soils in spring compared with autumn. This data confirmed that overall rates of soil organic P mineralisation are greatest in spring.     

Effect of environmental C/N ratio on activities of lignin-degrading enzymes produced by Phanerochaete chrysosporium

Lignin-degrading enzymes secreted by white rot fungi play an important role in the degradation of lignin and persistent organic pollutants(POPs).In this study,effect of environmental C/N ratio on the activities of lignin-degrading enzymes,lignin peroxide(Li P)and manganese peroxidase(Mn P),produced by Phanerochaete chrysosporium,a white rot fungus,was investigated.Glucose was used as C source,and ammonium tartrate of different concentrations was used as N source to provide different C/N ratios.Relationships between Li P and Mn P activities and environmental C/N ratio were explored.The results showed that the higher the N source concentration,the faster the mycelium pellets aged.The faster the mycelium dry weight increased,the higher the Li P and Mn P activities.A high C/N ratio was a necessary condition for the secretion of Li P or Mn P.In addition,mycelium dry weight essentially affected enzyme activities.In the 122 C/N ratio and 50 C/N ratio treatments,mycelium dry weight essentially affected Mn P activity and both Li P and Mn P activities,respectively.     

复合菌剂对小麦秸秆降解速率、土壤酶和土壤 微生物类群的影响

秸秆在农业上的有效利用日益受到关注。针对秸秆还田后腐解难的问题,本文选择7株纤维素降解菌种进行复配:拟茎点霉B3、茎点霉YY11、绿色木霉、里氏木霉、根霉、黑曲霉、蜂房芽孢杆菌。最终采用两种复配液体菌剂A(B3、YY11、绿色木霉和蜂房芽孢杆菌)、C(上述7个菌种),同时制作与液体菌剂A、C分别对应的固体菌剂B、D。探讨了复合菌剂对秸秆降解速率的促进作用和对土壤质量的提升效果。结果表明,4种菌剂均可促进小麦秸秆降解,改善土壤生物学特性,增加土壤养分,改良玉米农艺学性状。综合评价各个指标,菌剂B(B3、YY11、绿色木霉、蜂房芽孢杆菌;固体剂型)的表现最好,且玉米总产量比市售秸秆腐熟剂处理高20.72%。总之,本研究表明小麦秸秆还田后和联合菌剂使用能明显提高土壤降解功能,并且为实现经济可持续的农业管理方式提供了一种有应用前景的秸秆腐熟剂组合方式。     

甘肃省兴隆山森林主要树种凋落叶分解速率与初始质量的关系

[目的]开展凋落叶分解速率研究,探讨凋落叶分解速率与初始质量的关系,为甘肃省兴隆山森林生态系统物质循环研究提供依据。[方法]采用凋落物分解袋法,以兴隆山青杄、山杨和白桦3种主要树种的凋落叶为研究对象,进行凋落叶分解速率及凋落叶初始质量的研究,明确凋落叶分解速率与初始质量的关系。[结果]青杄中龄林针叶分解速率为0.16,95%分解期为19.08a;青杄近熟林针叶分解速率为0.13,95%分解期为23.70a;山杨和白桦凋落叶分解速率均为0.11,95%分解期分别为28.57a和27.27a;山杨和白桦凋落叶分解速率明显要小于青杄针叶,这很可能是凋落叶分解主场效应和分解袋孔径较小所致。凋落叶分解速率与氮含量呈显著线性正相关,与木质素含量、碳/氮值、木质素/氮值和钾含量呈显著线性负相关,特别是与木质素含量、氮含量和木质素/氮值,相关系数均达0.700 0以上;钾含量、木质素含量、木质素/氮、碳/磷和纤维素含量是影响兴隆山森林凋落叶分解速率的重要指标。[结论]木质素/氮值是影响凋落叶分解速率的关键质量指标,凋落叶初始木质素/氮值越高,分解速率越低。     

Characterization of a Mediterranean litter by 13C CPMAS NMR: relationships between litter depth, enzyme activities and temperature

Organic matter mineralization of forest litter is catalysed by the action of different extracellular enzymes produced by microorganisms. Coupling enzyme activities with data on the general macromolecular structure of organic matter, provided by cross‐polarization magic angle spinning ¹³C nuclear magnetic resonance (¹³C CPMAS NMR), allows researchers new insights into organic matter degradation processes. In this paper, the effect of the temperature of incubation on the degradation processes was evaluated in three distinct layers (OhLn, OhLv and OhLf) of an evergreen oak litter (Quercus ilex L.), located in the Mediterranean area of south‐eastern France. We studied degradation phenomena by a combination of ¹³C CPMAS NMR and microbiological analysis. In order to determine the microbial activity of litter layers, three enzyme activities (laccase, cellulase and butyrate esterase) were measured in a 6‐month mesocosm study. Results showed an increase in the alkyl C to O‐alkyl‐C ratio and an increase of the phenolic C and carboxyl C regions, indicating a preferential degradation of polysaccharides. The aromaticity also increased with litter depth and degradation, and humification processes were more elevated at 30°C. anova showed significant effects (P < 0.001) of increased temperature, depth and time of degradation on microbiological variables. Further information is needed about the variations in temperature and temperature‐litter response and soil functions to link fundamental understanding of carbon stabilization, climate change and global C cycling.     

Decomposition of lignin in soil microcompartments: A methodical study with three different C-14-labelled lignin substrates

Summary C mineralization of three different C-14-labelled lignin substrates (Klason-lignin, dioxane-lignin, lignocellulose) was investigated in four microcompartments, consisting of white-rotted beechwood (Fagus sylvatica L)., brown-rotted beechwood, earthworm [Octolasion lacteum (Örley)] faecal particles mixed with white-rotted beechwood, and earthworm feacal particles mixed with brown-rotted beechwood, incubated at 10°C for 193 days. Conversion of the labelled substrates to ¹⁴CO₂ was low in both white-rotted and brown-rotted wood without faecal particles. Overall C mineralization followed the order Klason-lignin > dioxane-lignin > lignocellulose, indicating that there were different amounts of labelled contaminants in the lignin substrates. Lignin degradation was more pronounced in earthworm faeces mixed with wood materials, and overall C mineralization ranged between 4.4% and 6.3% of the inital C content. C mineralization of the lignin substrates increased considerably in faecal particles after about 90 days, presumably due to nutrient immobilization and microbial succession. The usefulness of the three lignin substrates in monitoring the time-course and extent of lignin degradation in soil microcompartments is discussed.