两株侧耳属真菌对小麦秸秆化学组分及瘤胃消化率的影响

小麦秸秆木质素含量高,蛋白质等营养物质含量低,作为反刍家畜的粗饲料营养价值低,饲料化利用受到限制。通过室内小麦秸秆固态发酵试验,研究了两株侧耳属真菌Tf1（Pleurotussajor—c4u）和JG1（PleurotusCornucopiaeRoll）对小麦秸秆细胞壁化学组分的降解、瘤胃消化率和粗蛋白含量的影响。结果表明,经菌株JG1、Tf1和两菌复合发酵21d的小麦秸秆,木质素降解率分别为28．20％、30．78％和38．41％,纤维素降解率分别为19．26％、19．28％和26．65％;48h干物质瘤胃消化率分别比未发酵秸秆提高了38．62％、44．81％和55．89％,中性洗涤纤维（NDF）消化率分别提高了38．91％、49．00％和63．08％;粗蛋白含量分别比未发酵秸秆提高了58．60％、69．53％和72．22％。表明菌株JG1和Tf1在选择性降解木质素,改善瘤胃消化率,提高粗蛋白含量方面的优势,而且两菌株复合发酵具有协同作用,与单菌发酵相比,木质素降解率、瘤胃消化率和粗蛋白含量都明显提高。     

一株高活性纤维素降解细菌的筛选鉴定及酶学特性

利用小麦/玉米秸秆还田土壤样品,通过富集培养和刚果红平板染色法筛选分离出纤维素降解细菌XWS-12;对分离的菌株进行16S rRNA基因序列系统发育分析,初步鉴定为伯克氏菌属(Burkholderia),定名为Burkholderia sp.XWS-12。以玉米秸秆和麸皮为碳源,研究了氮源、发酵时间、初始发酵温度、培养基初始pH等条件对该伯克氏菌产纤维素酶的影响。结果显示,该菌株产纤维素酶最适氮源为硝酸钠,培养时间为60 h,培养温度为37℃,培养基初始pH为4,该菌株的CMC酶活力最高,可达25 U/ml。其粗酶液的最适反应温度为50℃,最适反应pH为5,在pH 4~8的范围内酶活力较稳定。粗酶液的热稳定较差,当温度超过50℃时,该酶活力显著下降;当温度为50℃时,保温1 h,该酶活力损失53%。     

响应面法优化白腐菌Pleurotus eryngii-Co007产木质素降解酶条件

为提高Pleurotus eryngii-Co007产木质素降解酶能力,考察了初始pH、秸秆浓度、Cu2+浓度、吐温-80含量对其产木质素降解酶的影响.采用单因素试验和响应面分析法的Central Composite进行试验设计,得到Pleurotus eryngii-Co007产木质素降解酶的最佳条件:培养基初始pH...     

秸秆纤维素降解真菌QSH3-3的筛选及其特性研究

为了获得高效降解秸秆纤维素的微生物菌株,采用滤纸降解法和刚果红染色法从含纤维素类物质的土壤中筛选到一株产纤维素酶菌株QSH3-3,通过形态观察和ITS序列分析,鉴定为草酸青霉Penicillium oxalicum QSH3-3。摇瓶产酶试验结果表明,该菌株的最佳产酶条件为:碳源为0.5%的碱处理过的玉米秸秆粉,氮源为0.2% 硫酸铵,起始pH为7,接种量为5%,产酶温度为30℃,培养时间为4 d。最佳产酶条件下,滤纸酶（FPase）、内切酶（CMCase）和木聚糖酶（Xylanase）分别为12 U、33 U、605 U（U为酶活性单位）;在15℃,其残余酶活力可达70%～80%;在pH 4～9 范围内,其残余酶活力可达70%以上。酶学稳定性研究表明,FPase、CMCase和Xylanase在pH 4～9范围残余酶活力达85%以上,具有较强的酸碱适应能力;FPase、CMCase和Xylanase在45℃以上酶活力迅速下降,耐热性较差。该菌株具有较高的木聚糖酶活力以及较强的低温、pH的耐受力,因而该菌株在田间温差大、土壤偏碱性等复杂条件下对秸秆纤维素类物质的降解具有较高的应用潜力。     

侧孢霉利用玉米秸秆固体发酵产生木质纤维素酶的研究

以农作物秸秆为主要原料 ,采用微生物固体发酵方式对侧孢霉 (Sporotrichumsp) .产生木质纤维素酶进行了探讨。研究发现侧孢霉可降解作物秸秆的木质纤维 ,降解作用强 ,同时提高了发酵底物的蛋白含量。玉米秸秆、稻草、麦草及甜菜渣是发酵木质纤维素酶和单细胞蛋白的最适合发酵底物。在以玉米秸秆为原料的发酵过程中 ,发酵到第 4天菌体生长量达到最高值 ,比发酵初期的菌体生长量增加了 8倍 ;发酵第 6天培养物的粗蛋白含量由起初的 1 0 %提高到 1 7 5% ,对底物纤维降解率达到1 3 6%。侧孢霉在发酵过程中产生大量的木质纤维素酶 ,其中羧甲基纤维素酶 (CM Case)、β 葡聚糖苷酶 ( β Gase)、滤纸酶 (FPase)、木质素过氧化物酶 (LiP)和赖锰木质素过氧化物酶 (MnP)在本实验中进行了检测。结果表明 ,最大酶活性的产生时间是在发酵的第 4到第 6天之间 ,其酶活性分别为 :羧甲基纤维素酶 2 40 3 1U/ g ,β 葡聚糖苷酶 86 7U/ g ,滤纸酶 1 3 4 8U/ g ,木质素过氧化物酶 1 4 4U/ g ,赖锰木质素过氧化物酶 1 7 67U/ g。     

不同土著菌及其复合菌对玉米秸秆降解的影响

为研究一种高效的玉米秸秆降解复合菌,选取了木质素降解优势土著菌密黏褶菌、环状芽孢杆菌、铜绿假单胞菌、栗褐链霉菌、黄孢原毛平革菌、杂色云芝、绿色木霉、黑曲霉,对各单一菌种对玉米秸秆的降解能力进行了测定,通过菌种间的拮抗试验,将单一菌种进行组合,初步构建了一组木质纤维素降解复合菌。结果表明：在整个35 d的预处理周期中,黑曲霉、绿色木霉对秸秆中纤维素、半纤维素体现了较高的降解能力,黑曲霉、绿色木霉对半纤维素的降解率分别为47.81%、37.53%,对纤维素的降解率分别为38.96%、46.32%;黄孢原毛平革菌、杂色云芝对玉米秸秆中的木质素体现了较强的降解能力,对木质素的降解率分别为43.56%、39.17%;菌种拮抗试验表明该试验所选用的真菌、放线菌及细菌之间无拮抗反应,可以进行混合培养;对复合菌预处理前后的玉米秸秆微观结构进行扫描电镜分析,发现在降解过程中复合菌对木质纤维素的结构产生了破坏作用,提高了木质纤维素的可及性;木质素、纤维素、半纤维素的含量在整个发酵过程中都在逐渐减少,发酵结束时复合菌对半纤维素的降解率最高达到48.53%,纤维素的降解率为36.38%,木质素的降解率为40.11%,在提高木质素降解率的同时减少了纤维素消耗。该研究为秸秆类生物质降解及利用提供了参考依据。     

多菌种分步降解玉米秸秆生产蛋白饲料的工艺

研究了多菌种分步降解玉米秸秆产蛋白饲料的工艺条件,选用黄孢原毛平革菌固体发酵去除部分木质素,接入木霉菌进一步降解木质纤维素,加入酵母发酵产蛋白饲料。木霉菌最佳接种时间为第10 天,经12 d的共发酵,按秸秆与麸皮配比3.0∶1加入麸皮、酵母接种量8%、固液比1∶3.0、硫酸铵用量2.5%,继续共发酵72 h,粗蛋白质量分数可达25.3%。结果表明,多菌种分步降解产蛋白饲料工艺为玉米秸秆生物利用提供了一种新途径。     

添加木质素降解菌对堆肥中酶活性的影响

以奶牛粪便和稻草为堆腐材料,采用静态好氧堆肥的方式研究接种木质素降解菌对堆肥过程中的温度、pH等理化性质以及木质素降解酶活性动态变化的特征,从生物酶学角度考察人工接入外源菌剂对堆肥的影响。结果表明,接菌后的堆肥处理较CK早2 d进入高温期,并且维持时间多于CK 12 d。发酵前8 d pH值的上升幅度大且高于CK,而且接种处理比对照的C/N提前5 d达到20∶1,提早达到腐熟指标,加快堆肥腐熟化进程。堆肥中酶活分析结果表明,加入菌剂后,β-葡萄糖苷水解酶在堆置第6 d达到第一个峰值14.7μmol,较CK早6 d;羧甲基纤维素钠酶在第12 d达到峰值3 270 U,同比CK高出1 220 U;漆酶酶活峰值高达93.5U,而CK峰值只有82.8 U;锰过氧化物酶进入高温期后酶活最高为75.25 U,CK最高为54.8 U。由此可见,加入微生物菌剂后可使相关酶活性提高并提高堆体温度,加快堆肥腐熟,加速堆料中各种有机质的降解,提高微生物对底物的利用,从而提高好氧堆肥的效率。     

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。     

玉米秸秆低温降解菌的分离筛选及鉴定

利用从渭源县山地采集的30份样品,采用刚果红染色法和液体摇瓶发酵法筛选出了1株能够在15℃条件下降解羧甲基纤维素、玉米秸秆纤维素和高产纤维素酶的真菌菌株D5,同时测定了该菌株纤维素酶活力和对玉米秸秆的降解能力,并通过ITS r DNA序列分析对该真菌进行了初步鉴定。结果表明,菌株D5液体培养7 d的木聚糖酶活为52.7 U·m L~(-1),CMCase酶活为31.5 U·m L~(-1),滤纸酶活为29.6 U·m L~(-1);菌株D5在玉米秸秆为唯一碳源的培养基里发酵10 d,玉米秸秆的失重率为29.8%。菌株D5经ITS r DNA序列分析初步鉴定为Penicillium sp.,在玉米秸秆降解方面具有较大的应用潜力。     

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.     

草浆造纸木质素对土壤性质和小白菜生长的影响

草浆造纸废液是影响秸秆造纸发展的重要因素,利用小白菜盆栽试验研究了施用麦秸造纸废液木质素和麦秸对土壤性质及小白菜生长的影响,以期为造纸废弃物的资源化利用提供依据。结果表明,两茬小白菜收获后,铵木素配施N肥使土壤pH值降低;小白菜两茬连作后铵木质素配施的土壤活性有机碳含量及多酚氧化酶活性分别为2.73g·kg-1和1.77mg·g-1·2h-1,显著高于其他处理;除对照和单施氮肥外,两茬盆栽收获后土壤多酚氧化酶活性均高于第一茬试验。两茬小白菜收获后秸秆配施和碱木质素配施氮肥土壤过氧化物酶活性均高于其他处理。铵木质素配施及秸秆配施的小白菜干重相近,显著高于碱木质素配施。研究表明,与秸秆还田相比,施用铵木质素没有对土壤性状和小白菜生长产生不利影响,故铵法草浆木质素可以作为一种潜在的土壤有机改良剂应用。     

Evolution of Chemistry along the Bagmati Drainage Network in Kathmandu Valley

Two fungal strains producing ligninolytic enzymes and having the potential to decolorize distillery effluent were isolated from the soil of a distillery effluent contaminated site. DNA was isolated from the pure cultures of these fungi and polymerase chain reaction (PCR) amplification of their internal transcribed spacer (ITS) region of nuclear ribosomal DNA was carried out. Further, the DNA was sequenced and the comparison of generated sequence with database led to their identification as Penicillium pinophilum TERI DB1 and Alternaria gaisen TERI DB6 respectively. These two isolates along with one isolate of Pleurotus florida EM 1303 were assessed for their ligninolytic enzyme activity in culture filtrate as well as after solid state fermentation on two substrates wheat straw and corncob powder. Ergosterol was measured to assess the growth of fungi on solid media. Both P. pinophilum TERI DB1 and A. gaisen TERI DB6 were found to produce laccase, manganese-dependent peroxidases (MnP) and lignin peroxidases (LiP). The immobilized fungal biomass was then used for decolorization of the post biomethanated wastewater from the distillery. Reduction in color up to the magnitude of 86, 50 and 47% was observed with P. florida, P. pinophilum and A. gaisen respectively.     

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.     

Breakdown Products in the Metabolic Pathway of Anthracene Degradation by a Ligninolytic Fungus Polyporus sp. S133

Polyporus sp. S133 fungi were selected based on their ability to degrade anthracene in liquid media. The degradation efficiency of anthracene increased by adding 0.5% Tween 80 to reach 71%; agitation at 120 rev/min increased the degradation to 92% after 30?days of incubation. Enzymes such as manganese peroxidase (MnP), lignin peroxidase (LiP), laccase, 1,2-dioxygenase and 2,3-dioxgenase were produced by Polyporus sp. S133 during incubation, and the highest enzyme activity was 182.3 U l^?1 by 1,2-dioxygenase after 20?days of incubation. These results indicate that ligninolytic and dioxygenase enzymes secreted from Polyporus sp. S133 could play an important role in anthracene degradation efficiency. The metabolites detected through the degradation pathway were anthraquinone, phthalic acid, benzoic acid and catechol.     

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.     

酸-超声波预处理及糖化水解稻草研究

采用室内实验方法,研究了酸-超声联合预处理稻草对其化学组成以及糖化效果的影响,并与传统酸预处理法的效果进行了对比。结果表明,与未经处理的稻草相比,经酸-超声波处理的稻草其半纤维素、木质素含量最高分别减少了64．46％、62．19％,纤维素含量最高则上升了73．20％,而酸处理的稻草相应数值只能达到56．72％、59．90％及53．41％。同时分别对两种方法的稻草糖化的工艺条件通过正交试验进行了优化,得出两种方法的稻草最佳糖化条件均为：pH值为4．8,温度为45℃,酶浓度为20mg·g-1。在该条件下,对于酸一超声波预处理稻草,在糖化108h以后还原糖浓度稳定并达到最大值26．4g·L-1,而对于酸预处理稻草,在糖化120h以后还原糖浓度才稳定并达到最大值26．2g·L-1,且前者能比后者产生更多的葡萄糖以及更少的木糖,更有利于提高后续酒精发酵的效率。     

Production of lignocellulose-degrading enzymes and degradation of leaf litter by saprotrophic basidiomycetes isolated from a Quercus petraea forest

Due to the production of lignocellulose-degrading enzymes, saprotrophic basidiomycetes can significantly contribute to the turnover of soil organic matter. The production of lignin- and polysaccharide-degrading enzymes and changes of the chemical composition of litter were studied with three isolates from a Quercus petraea forest. These isolates were capable of fresh litter degradation and were identified as Gymnopus sp., Hypholoma fasciculare and Rhodocollybia butyracea. Within 12 weeks of incubation, H. fasciculare decomposed 23%, R. butyracea 32% and Gymnopus sp. 38% of the substrate dry mass. All fungi produced laccase and Mn-peroxidase (MnP) and none of them produced lignin peroxidase or other Mn-independent peroxidases. There was a clear distinction in the enzyme production pattern between R. butyracea or H. fasciculare compared to Gymnopus sp. The two former species caused the fastest mass loss during the initial phase of litter degradation, accompanied by the temporary production of laccase (and MnP in H. fasciculare) and also high production of hydrolytic enzymes that later decreased. In contrast, Gymnopus sp. showed a stable rate of litter mass loss over the whole incubation period with a later onset of ligninolytic enzyme production and a longer lasting production of both lignin and cellulose-degrading enzymes. The activity of endo-cleaving polysaccharide hydrolases in this fungus was relatively low but it produced the most cellobiose hydrolase. All fungi decreased the C/N ratio of the litter from 24 to 15-19 and Gymnopus sp. also caused a substantial decrease in the lignin content. Analytical pyrolysis mass spectrometry of litter decomposed by this fungus showed changes in the litter composition similar to those caused by white-rot fungi during wood decay. These changes were less pronounced in the case of H. fasciculare and R. butyracea. All fungi also changed the mean masses of humic acid and fulvic acid fractions isolated from degraded litter. The humic acid fraction after degradation by all three fungi contained more lignin and less carbohydrates. Compared to the decomposition by saprotrophic basidiomycetes, litter degradation in situ on the site of fungal isolation resulted in the relative enrichment of lignin and differences in lignin composition revealed by analytical pyrolysis. It can most probably be explained by the participation of non-basidiomycetous fungi and bacteria during natural litter decomposition.     

Adaptation of the biobed composition for chlorpyrifos degradation to Southern Europe conditions

Biobeds developed in Sweden bind and degrade pesticides from point sources. The objective of this work was to adapt the biobed to Italian operating conditions, for example, to identify organic materials as effective as those in the original Swedish composition. The capacity of urban and garden composts alone or mixed with citrus peel or straw to degrade chlorpyrifos and its metabolite TCP was compared to the typical Swedish biomix consisting of straw, peat, and soil. A tendency for higher 14C-chlorpyrifos mineralization and lower TCP levels was observed in the biomixes with garden compost alone or amended with straw. In a second trial, a high correlation of lower TCP with increasing levels of straw in typical Swedish biomixes was observed. Straw stimulates production of lignin-degrading enzymes such as manganese peroxidase (MnP), and further trials with pure MnP showed that this enzyme degrades TCP. Materials with an active lignin-degrading microflora are a prerequisite for effective dissipation of chlorpyrifos and non-accumulation of TCP. Thus, lignocellulosic materials as straw and garden composts should be present in biomixes to be used under Italian conditions.