The white-rot fungi Phlebia radiata and Dichomitus squalens in wood-based cultures: expression of laccases, lignin peroxidases, and oxalate decarboxylase

Basidiomycetous white-rot fungi are the only organisms that can efficiently decompose all the components of wood. Moreover, white-rot fungi possess the ability to mineralize recalcitrant lignin polymer with their extracellular, oxidative lignin-modifying enzymes (LMEs), i.e. laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Within one white-rot fungal species LMEs are typically present as several isozymes encoded by multiple genes. This study focused on two effi cient lignin-degrading white-rot fungal species, Phlebia radiata and Dichomitus squalens. Molecular level knowledge of the LMEs of the Finnish isolate P. radiata FBCC43 (79, ATCC 64658) was complemented with cloning and characterization of a new laccase (Pr-lac2), two new LiP-encoding genes (Pr-lip1, Pr-lip4), and Pr-lip3 gene that has been previously described only at cDNAlevel. Also, two laccase-encoding genes (Ds-lac3, Ds-lac4) of D. squalens were cloned and characterized for the first time. Phylogenetic analysis revealed close evolutionary relationships between the P. radiata LiP isozymes. Distinct protein phylogeny for both P. radiata and D. squalens laccases suggested different physiological functions for the corresponding enzymes. Supplementation of P. radiata liquid culture medium with excess Cu2+ notably increased laccase activity and good fungal growth was achieved in complex medium rich with organic nitrogen. Wood is the natural substrate of lignin-degrading white-rot fungi, supporting production of enzymes and metabolites needed for fungal growth and the breakdown of lignocellulose. In this work, emphasis was on solid-state wood or wood-containing cultures that mimic the natural growth conditions of white-rot fungi. Transcript analyses showed that wood promoted expression of all the presently known LME-encoding genes of P. radiata and laccase-encoding genes of D. squalens. Expression of the studied individual LME-encoding genes of P. radiata and D. squalens was unequal in transcript quantities and apparently time-dependent, thus suggesting the importance of several distinct LMEs within one fungal species. In addition to LMEs, white-rot fungi secrete other compounds that are important in decomposition of wood and lignin. One of these compounds is oxalic acid, which is a common metabolite of wood-rotting fungi. Fungi produce also oxalic-acid degrading enzymes of which the most widespread is oxalate decarboxylase (ODC). However, the role of ODC in fungi is still ambiguous with propositions from regulation of intra and extracellular oxalic acid levels to a function in primary growth and concomitant production of ATP. In this study, intracellular ODC activity was detected in four white-rot fungal species, and D. squalens showed the highest ODC activity upon exposure to oxalic acid. Oxalic acid was the most common organic acid secreted by the ODC-positive white-rot fungi and the only organic acid detected in wood cultures. The ODC-encoding gene Ds-odc was cloned from two strains of D. squalens showing the first characterization of an odc-gene from a white-rot polypore species. Biochemical properties of the D. squalens ODC resembled those described for other basidiomycete ODCs. However, the translated amino acid sequence of Ds-odc has a novel N-terminal primary structure with a repetitive Ala-Ser-rich region of ca 60 amino acid residues in length. Expression of the Ds-odc transcripts suggested a constitutive metabolic role for the corresponding ODC enzyme. According to the results, it is proposed that ODC may have an essential implication for the growth and basic metabolism of wood-decaying fungi.

Characterization of lignin-modifying enzymes of the selective white-rot fungus Physisporinus rivulosus

White-rot fungi are wood degrading organisms that are able to decompose all wood polymers; lignin, cellulose and hemicellulose. Especially the selective white-rot fungi that decompose preferentially wood lignin are promising for biopulping applications. In biopulping the pretreatment of wood chips with white-rot fungi enhances the subsequent pulping step and substantially reduces the refining energy consumption in mechanical pulping. Because it is not possible to carry out biopulping in industrial scale as a closed process it has been necessary to search for new selective strains of white-rot fungi which naturally occur in Finland and cause selective white-rot of Finnish wood raw-material. In a screening of 300 fungal strains a rare polypore, Physisporinus rivulosus strain T241i isolated from a forest burn research site, was found to be a selective lignin degrader and promising for the use in biopulping. Since selective lignin degradation is apparently essential for biopulping, knowledge on lignin-modifying enzymes and the regulation of their production by a biopulping fungus is needed. White-rot fungal enzymes that participate in lignin degradation are laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP) and hydrogen peroxide forming enzymes. In this study, P. rivulosus was observed to produce MnP, laccase and oxalic acid during growth on wood chips. In liquid cultures manganese and veratryl alcohol increased the production of acidic MnP isoforms detected also in wood chip cultures. Laccase production by P. rivulosus was low unless the cultures were supplemented with sawdust and charred wood, the components of natural growth environment of the fungus. In white-rot fungi the lignin-modifying enzymes are typically present as multiple isoforms. In this study, two MnP encoding genes, mnpA and mnpB, were cloned and characterized from P. rivulosus T241i. Analysis of the N-terminal amino acid sequences of two purified MnPs and putative amino acid sequence of the two cloned mnp genes suggested that P. rivulosus possesses at least four mnp genes. The genes mnpA and mnpB markedly differ from each other by the gene length, sequence and intron-exon structure. In addition, their expression is differentially affected by the addition of manganese and veratryl alcohol. P. rivulosus produced laccase as at least two isoforms. The results of this study revealed that the production of MnP and laccase was differentially regulated in P. rivulosus, which ensures the efficient lignin degradation under a variety of environmental conditions.

Lignocellulose degradation and humus modification by the fungus Paecilomyces inflatus

Composting is the biological conversion of solid organic waste into usable end products such as fertilizers, substrates for mushroom production and biogas. Although composts are highly variable in their bulk composition, composting material is generally based on lignocellulose compounds derived from agricultural, forestry, fruit and vegetable processing, household and municipal wastes. Lignocellulose is very recalcitrant; however it is rich and abundant source of carbon and energy. Therefore lignocellulose degradation is essential for maintaining the global carbon cycle. In compost, the active component involved in the biodegradation and conversion processes is the resident microbial population, among which microfungi play a very important role. In composting pile the warm, humid, and aerobic environment provides the optimal conditions for their development. Microfungi use many carbon sources, including lignocellulosic polymers and can survive in extreme conditions. Typically microfungi are responsible for compost maturation. In order to improve the composting process, more information is needed about the microbial degradation process. Better knowledge on the lignocellulose degradation by microfungi could be used to optimize the composting process. Thus, this thesis focused on lignocellulose and humic compounds degradation by a microfungus Paecilomyces inflatus, which belongs to a flora of common microbial compost, soil and decaying plant remains. It is a very common species in Europe, North America and Asia. The lignocellulose and humic compounds degradation was studied using several methods including measurements of carbon release from 14C-labelled compounds, such as synthetic lignin (dehydrogenative polymer, DHP) and humic acids, as well as by determination of fibre composition using chemical detergents and sulphuric acid. Spectrophotometric enzyme assays were conducted to detect extracellular lignocellulose-degrading hydrolytic and oxidative enzymes. Paecilomyces inflatus secreted clearly extracellular laccase to the culture media. Laccase was involved in the degradation process of lignin and humic acids. In compost P. inflatus mineralised 6-10% of 14C-labelled DHP into carbon dioxide. About 15% of labelled DHP was converted into water-soluble compounds. Also humic acids were partly mineralised and converted into water-soluble material, such as low-molecular mass fulvic acid-like compounds. Although laccase activity in aromatics-rich compost media clearly is connected with the degradation process of lignin and lignin-like compounds, it may preferentially effect the polymerisation and/or detoxification of such aromatic compounds. P. inflatus can degrade lignin and carbohydrates also while growing in straw and in wood. The cellulolytic enzyme system includes endoglucanase and β-glucosidase. In P. inflatus the secretion of these enzymes was stimulated by low-molecular-weight aromatics, such as soil humic acid and veratric acid. When strains of P. inflatus from different ecophysiological origins were compared, indications were found that specific adaptation strategies needed for lignocellulosics degradation may operate in P. inflatus. The degradative features of these microfungi are on relevance for lignocellulose decomposition in nature, especially in soil and compost environments, where basidiomycetes are not established. The results of this study may help to understand, control and better design the process of plant polymer conversion in compost environment, with a special emphasis on the role of ubiquitous microfungi.

Composicion e identificacion de especies forrajeras y no forrajeras en las Fincas Santa Rosa y Las Mercedes de la Universidad Nacional Agraria, Managua

Con el objetivo de caracterizar la composición botánica de especies forrajera y no forrajera en sistemas de pastura de pasto Brachiaria (Brachiaria brizantha), en la Hacienda Las Mercedes se ubicada en el km 10 de la carretera norte, contiguo al CARNIC, y Santa Rosa, ubicada de la Entrada a la Zona Franca 4 Km sur, del desvío a Sabana Grande 200 m Norte, 100 m Oeste. Se realizó el presente trabajo. Para la ejecución del mismo y dada las posibilidades se eleccionaron dos escenarios ambientales, una con árboles y otra sin árboles, donde se realizó primeramente la determinación de la composición botánica, utilizando para ello el método del doble muestreo, simultáneamente se realizó la primera colecta de material vegetativo, el cual posteriormente fue identificado en el herbario de la UNA, herbario Nacional y a través de consultas con expertos en la materia (Botánicos). A cada una de las especies identificadas se les realizó fichas botánicas, que comprendieron nombre común, nombre científico, origen y distribución, características de las plantas, aptitudes forrajera y cuando fue posible si presentaba Características anticualutativas o antinutricionales. Posteriormente se realizó una valoración técnica, para considerar el estado de dichas pasturas y las implicaciones económicas que estas podrían tener en manejo y recuperación. Registrándose coberturas vegetales que fluctuaron de 30 a 70%, y composición de las especies forrajeras en rangos similares. Se identificaron 23 familias, 43 géneros y 43 especies, dentro de los cuales destacaron por su mayor presencia las especies de escoba lisa (Sida cuta), bledo espinoso (Amaranthus espinosus, Melón amargo (Momordica charanthia, Cinco negritos ( Lantana camara), Flor amarilla (Baltimora recta), Añil Forrajero (Indigofera hirsuta), Grama de conejo (Oplismenus burmani)dormilona (Mimosa pudica), entre otras, también se considera como forrajera a algunas especies leguminosa herbáceas como patito (Centrocema plumieri), centro Chorreque (Vigna Vexillata), Zorrillo (Petiveria alliacea), Flor amarilla (Melampodium divaricatum) pega pega (priva lappulacea), chilillo de gato (Achyranthes aspera), se observó una diversidad de especies, cuando existían árboles en el área de pasturas, minimizándose estas cuando no existían árboles. A través del análisis técnico–económico se determinó que los factores cobertura vegetal y Porcentaje de malezas son de mucha importancia, en el manejo y productividad de las pasturas y que aun cuando se tenga pasturas de buen comportamiento productivo, se registran perdidas, lo cual indica que en las pasturas no todo es forrajero.

Development of Metalloenzyme Dioxygen Reduction Cathodes

The prime thrust of this dissertation is to advance the development of fuel cell dioxygen reduction cathodes that employ some variant of multicopper oxidase enzymes as the catalyst. The low earth-abundance of platinum metal and its correspondingly high market cost has prompted a general search amongst chemists and materials scientists for reasonable alternatives to this metal for facilitating catalytic dioxygen reduction chemistry. The multicopper oxidases (MCOs), which constitute a class of enzyme that naturally catalyze the reaction O₂ + 4H⁺ + 4e^- → 2H₂O, provide a promising set of biochemical contenders for fuel cell cathode catalysts. In MCOs, a substrate reduces a copper atom at the type 1 site, where charge is then transferred to a trinuclear copper cluster consisting of a mononuclear type 2 or “normal copper” site and a binuclear type 3 copper site. Following the reduction of all four copper atoms in the enzyme, dioxygen is then reduced to water in two two-electron steps, upon binding to the trinuclear copper cluster. We identified an MCO, a laccase from the hyperthermophilic bacterium Thermus thermophilus strain HB27, as a promising candidate for cathodic fuel cell catalysis. This protein demonstrates resilience at high temperatures, exhibiting no denaturing transition at temperatures high as 95°C, conditions relevant to typical polymer electrolyte fuel cell operation.

In Chapter I of this thesis, we discuss initial efforts to physically characterize the enzyme when operating as a heterogeneous cathode catalyst. Following this, in Chapter II we then outline the development of a model capable of describing the observed electrochemical behavior of this enzyme when operating on porous carbon electrodes. Developing a rigorous mathematical framework with which to describe this system had the potential to improve our understanding of MCO electrokinetics, while also providing a level of predictive power that might guide any future efforts to fabricate MCO cathodes with optimized electrochemical performance. In Chapter III we detail efforts to reduce electrode overpotentials through site-directed mutagenesis of the inner and outer-sphere ligands of the Cu sites in laccase, using electrochemical methods and electronic spectroscopy to try and understand the resultant behavior of our mutant constructs. Finally, in Chapter IV, we examine future work concerning the fabrication of enhanced MCO cathodes, exploring the possibility of new cathode materials and advanced enzyme deposition techniques.

Cloning and analysis of laccases from acinetobacter baumannii isolates

Laccases (benzenediol : oxygen oxi doreductases; EC 1.10.3.2) are wide spread i n nature. They are usually found in higher plants and fungi (Thurston 19 94; Mayer and Staples 2002), but recently some bacterial laccases have also been found . The first laccase studied was from Rhus vernicifera in 1883, a Japanese lacquer tree, fr om which the name laccase was derived (Yoshida , 1883). These enzymes belong to the group of bl ue multi - copper oxidases (MCOs) . They usually contain four copper atoms located in three distinct sites. Each site reacts differently to light. The Type 1 (T1) site copper atom absorbs intensely at 600 nm and emits the blue light , the Type 2 (T2) site copper atom is not visible in the absorption spectr um and last, the Type 3 (T3) site has two c opper atoms and absorbs at 330 nm ( Santhanam et al . , 2011; Quintanar et al . , 2007 ) . The protei n structure acts as a complex ligand for the catalytic coppers, providing them the right structure where changes between the reduction states are thermodynamically possible (Dub é , 2008 ) . These enzymes oxidize a surprisingly wide variety of organic and inorganic compounds like, diphenols, polyphenols, substituted phenols, diamines and a romatic amines, with concomitant reduction of molecular oxygen to water (Thurston , 1

Análisis bioinformático de los genes de lacasas YfiH en clones virulentos de Acinetobacter baumannii

[ES] Acinetobacter baumannii es una bacteria Gram negativa, patógena y multirresistente. Su alta capacidad de supervivencia en hospitales y su resistencia a químicos puede deberse a la producción de lacasas. Estas enzimas son capaces de oxidar un sinfín de compuestos como los fenoles utilizados en hospitales para la desinfección de superficies. En este estudio se ha realizado un análisis de actividad lacasa en aislamientos altamente virulentos de los clones internaciones I y II, observando que estas cepas presentan actividad lacasa. Paralelamente, se ha realizado un análisis bioinformático con el que se ha determinado la similitud de los genes de estas lacasas con las ya descritas de la familia “YfiH” y con otras enzimas procedentes de otras especies, demostrando su similitud de secuencia con la lacasa RL5, procedente de una muestra de rumen bovino. Estos hechos suponen un avance en el estudio de lacasas bacterianas en Acinetobacter baumannii cuya caracterización podría desembocar en nuevas líneas de lucha contra dicho patógeno.

山黑豆属(豆科)的分类学修订

山黑豆属Dumasia DC.隶属于豆科、菜豆族、大豆亚族，广泛分布于亚洲、非洲东南部的热带、亚热带地区以及大洋洲的巴布亚新几内亚地区，其中大多数种在我国均有分布。该属形态变异复杂，命名混乱，缺乏世界性的系统分类学研究基础。基于这样的背景，本论文通过开展形态学、解剖学、细胞学及孢粉学等方面的综合研究工作，对该属进行了全面的分类修订，对属内种间关系及地理分布格局进行了分析和探讨，结果如下： 1、形态学 在研究了大量腊叶标本和进行了野外考察的基础上，对山黑豆属植物的形态性状的变异式样进行了分析。该属植物的根系形态较为多样，可以作为划分种的辅助形状，分为有主根类型 (2个种，心叶山黑豆D. cordifolia Benth. ex Baker和柔毛山黑豆D. villosa DC.) 和无主根类型 (5个种，云南山黑豆D. yunnanensis Y. T. Wei & S. K. Lee、小鸡藤D. forrestii Diels、硬毛山黑豆D. hirsuta Craib、山黑豆D. truncata Siebold & Zucc.和长圆叶山黑豆D. henryi (Hemsl.) B. Pan & X. Y. Zhu)；该属几乎全为多年生缠绕草本，只有柔毛山黑豆为半灌木，因此可能是该属中较原始的种类；该属植物的萼筒管状、筒口斜截形及花柱细长且弯曲处膨大等独特的解剖结构是区别该属其它近缘属的重要性状；花序和复叶的长度及荚果内种子数目变异幅度极大，缺乏稳定性，并非好的分类性状。 2、解剖学 应用光学显微镜，首次对山黑豆属7种植物 (心叶山黑豆、柔毛山黑豆、云南山黑豆、小鸡藤、硬毛山黑豆、山黑豆和长圆叶山黑豆) 的叶表皮进行了观察。结果表明，该属植物叶表皮细胞均呈不规则形，垂周壁为波状，气孔器类型以平列型为主。心叶山黑豆、柔毛山黑豆和云南山黑豆上表皮细胞垂周壁呈深波状；小鸡藤、长圆叶山黑豆、山黑豆和硬毛山黑豆上表皮细胞垂周壁呈浅波状。在一定程度上，上表皮细胞垂周壁的形态反映了山黑豆属种间的近缘关系。 3、细胞学 首次报道了山黑豆属3个种 (云南山黑豆、小鸡藤和硬毛山黑豆) 的染 色体数目，它们均为2n=20。在已报道染色体的植物种类中 (共计6种)，仅心叶山黑豆染色体数目为2n=22，其余5种 (柔毛山黑豆、山黑豆、云南山黑豆、小鸡藤和硬毛山黑豆) 染色体数目均为2n=20。与大豆亚族大多数属染色体数目为2n=22相比较，心叶山黑豆可能是该属中较为原始的种。 4、孢粉学 在光学显微镜下，对该属7种植物 (心叶山黑豆、柔毛山黑豆、云南山黑豆、小鸡藤、硬毛山黑豆、山黑豆和长圆叶山黑豆) 的花粉进行了观察，结果表明：该属植物为三角形6孔花粉，极轴短，每个角的萌发孔由一对小孔组成；外壁具网纹，网脊粗，网眼不规则。柔毛山黑豆和小鸡藤极面的网脊几乎不断裂；心叶山黑豆、云南山黑豆、硬毛山黑豆、山黑豆和长圆叶山黑豆极面的网脊断裂较多。外壁的纹饰在一定程度上反映了该属植物的近缘关系。 5、地理分布格局 该属植物主要分布在中国南部和西南，只有柔毛山黑豆分布到非洲和大洋洲。研究结果表明，具主根的2个种 (心叶山黑豆和柔毛山黑豆) 主要分布在中国西南部，其分布区域相互重叠；而无主根的5个种 (云南山黑豆、小鸡藤、硬毛山黑豆、山黑豆和长圆叶山黑豆) 由中国西南部向东到日本、韩国都有分布，分布区域几乎不重叠。本研究认为：有主根的心叶山黑豆和柔毛山黑豆可能代表了该属的原始类群；有主根类群衍生出其余无主根类型的各种，并由中国西南扩散到其他地区。 综合以上资料，本研究对山黑豆属进行了分类修订，认为该属含8种、1亚种及2变种，其中建立新组合1个，归并6个类群，并指定了4个后选模式。另外，本研究同时提供了该属植物的检索表、种的形态描述和插图以及地理分布图等信息。

Purification and characterization of hydrosoluble components from the sap of Chinese lacquer tree Rhus vernicifera

Continuous gradient elution chromatography (CGEC) was employed to purify and separate enzymes and polysaccharides from the sap of Rhus vernicifera Chinese lacquer tree. There are three different molecules with laccase enzyme activity. Two are enzymes of each other (L1, and L2), whereas the third (RL) is an entirely separate entity. Two polysaccharides (GP1 and GP2) were also found. The Rhus laccase (RL), and isoenzymes L1 and L2, have peak molecular masses of 109,100, 120,000, 103,000 respectively; each has four copper atoms per molecule, and the pI values were 8.2, 8.6, and 9.1, respectively. The structure of the laccases was studied by Fourier-transform infrared (FT-IR) and Matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry. The typical amide I (1646 cm(-1)) and amide II (1545 cm(-1)) bands were observed. The results from MALDI-TOF were similar to those from CGEC, but the molecular mass from the MALDI-TOF was significantly different from that obtained from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (c) 2006 Elsevier B.V. All rights reserved.

新疆主要林区多孔菌研究

新疆地区的多孔菌研究开始于邓叔群（1963），有51个多孔菌名称被报道过，但是这些报道都零星的分布于各种真菌志书和杂志中，而且先前报道的多孔菌很多是同物异名，也有一些标本的鉴定有疑问。本论文是首次对新疆的多孔菌展开系统研究。 通过对新疆喀纳斯自然保护区、天池自然保护区、西天山自然保护区、果子沟以及那拉提森林公园等新疆地区的主要林区进行考察采样，共采集多孔菌标本356号，用形态学和解剖学进行了分类学研究，共鉴定出3科、48属、100种，其中中国新记录种3种，新疆新记录种70个。本文对山梅花锐孔菌（Oxyporus philadelphi (Parmasto) Ryvarden）、桃泊氏孔菌（Postia persinica Niemelä & Y.C. Dai）和柳氏栓孔菌（Trametes ljubarskyi Pilát）三种中国新记录种的担子果形态和显微结构特征进行了详细的描述，并对其显微结构进行了绘图。 在鉴定结果的基础之上，对新疆主要林区多孔菌区系成分进行了初步的分析。结果表明新疆主要林区多孔菌的优势属为栓孔菌属，有8种（占总种数的8%），其次是薄孔菌属，有7种（占总种数的7%）。在属的区系成分上，新疆地区的多孔菌以广布成分为主（占58.3%），其次是北温带成分（占35.4%）；在种的区系成分上，以北温带成分（占49%）为主，其次是世界广布种成分（占24％），表明新疆主要林区的多孔菌的温带区系特征。 关键词：多孔菌，名录，分类学，区系

华东地区木材腐朽菌多样性与资源研究

木材腐朽菌是生物多样性的重要组成部分，在森林生态系统中起着关键的降解还原作用；同时，木材腐朽菌还是重要的生物资源，与人类的生产与生活密切相关，具有重要的经济价值。因此，木腐菌一直是真菌研究的重点领域之一，然而过去对华东地区木腐菌的研究还非常薄弱，对华东地区木腐菌的种类和资源还缺乏清晰的认识。本文对华东地区不同森林生态类型中的木腐菌进行了广泛的调查采样，通过形态学、分子系统学和单孢融合交配实验，对华东地区的木腐菌多样性与资源进行了系统的研究，同时对具有重要经济意义和潜在应用价值的种类进行了菌种的分离保藏，并初步分析了华东地区木腐菌的区系特点。通过本论文的研究，提高了对华东地区木腐菌资源与多样性的认识，丰富了我国木材腐朽菌的种类，为控制林木病原菌、开发利用有益真菌资源提供了基本信息和科学依据。本论文的主要研究结论如下： 1．华东地区的木腐菌有3目13科92属270种，其中真菌新种6个，分别是：铁杉集毛孔菌Coltricia tsugicola Y.C. Dai & B.K. Cui，香榧嗜蓝孢孔菌Fomitiporia torreyae Y.C. Dai & B.K. Cui，拟囊状体大孔菌Megasporoporia cystidiolophora B.K. Cui & Y.C. Dai，菌索多年卧孔菌Perenniporia rhizomorpha B.K. Cui, Y.C. Dai & Decock，微小硬孔菌Rigidoporus minutus B.K. Cui & Y.C. Dai，浅黄芮氏孔菌Wrightoporia luteola B.K. Cui & Y.C. Dai；中国新记录种9个，分别是：紫多孢孔菌Abundisporus violaceus (Wakef.) Ryvarden，卡玛蜡孔菌Ceriporia camaresiana (Bourdot & Galzin) Bondartsev & Singer，撕裂蜡孔菌Ceriporia lacerata N. Maek., Suhara & R. Kondo，塔斯马尼亚集毛孔菌Coltriciella tasmanica (Cleland & Rodway) D.A. Reid，瑞克纤孔菌Inonotus rickii (Pat.) D.A. Reid，骨质多年卧孔菌Perenniporia minutissima (Yasuda) T. Hatt. & Ryvarden，灰硬孔菌Rigidoporus cinereus Núñez & Ryvarden，凹形栓孔菌Trametes ectypus (Berk. & M.A. Curtis) Gilb. & Ryvarden，变形干酪菌Tyromyces transformatus Núñez & Ryvarden。 2．利用分子系统学方法，确定了一些通过形态学研究难以鉴定或容易混淆的种类的分类地位，并对嗜蓝孢孔菌属、彩孔菌属和多年卧孔菌属进行了系统发育分析，发现木腐菌新种3个：Fomitiporia sp.、Hapalopilus sp. 和Perenniporia sp.。 3．华东地区的异担子菌为岛生异担子菌，并且存在两个生物种：T生物种和Y生物种。T生物种为腐生菌，Y生物种为兼性腐生菌，华东地区不存在多年异担子菌这一严重的森林病原菌。 4．华东地区的木腐菌种类丰富，组成复杂多样。优势科为多孔菌科，其次为锈革孔菌科，这两科的种类构成了华东地区木腐菌类区系的主体；优势属主要有多年卧孔菌属、多孔菌属、针层孔菌属和泊氏孔菌属。 4．华东地区木腐菌科的地理成分分为3类：热带亚热带成分，北温带成分和世界广布成分，以世界广布成分为主。属的地理成分分为5类：世界广布属，北温带分布属，热带–亚热带分布属，东亚–北美分布属，大洋洲–北温带分布属，以世界广布属和北温带分布属为主。种的地理成分分为8类：世界广布种，北温带分布成分，泛热带分布成分，亚–欧共有成分，东亚–北美共有成分，东亚–澳大利亚共有成分，东亚成分，中国特有种，以北温带分布种和世界广布种为主。 5．华东地区木腐菌的各主要区系成分均有分布，以世界广布成分和北温带成分为主，表现出明显的北温带性质，可能起源于北半球，华东地区木腐菌类的区系地理成分与其所处的地理位置、气候特点与植物区系的组成密切相关。此外，华东地区的中国特有种类也较为丰富，说明了华东地区木腐菌类的区系具有一定的独特性，也在一定程度上反映了华东地区生态环境条件的特殊性，而木腐菌的生长与环境条件和寄主树木种类有很大的关系。 6．华东地区的木腐菌资源非常丰富，林木病原菌有36种，其中新发现的病原菌有3种；野生的食用木腐菌有11种，野生的药用木腐菌64种，工业用木腐菌有28种。对其中的一种重要经济真菌——黄白多年卧孔菌进行了分离培养，找出了最佳生长条件：最适生长温度为25℃左右，相对最适pH值为5.5，相对最好的碳源为可溶性淀粉和葡萄糖，相对最好的氮源为酵母汁。

植物根瘤内Frankia生物多样性的研究

建立了一种简捷、快速植物根瘤内痕量FrankiaDNA的提取方法，经过东北的沙棘、色赤杨、毛赤杨和云南的冬赤杨根瘤的瘤内痕量FrankiaDNA及体外培养的Frankia菌株DNA提纯，并用于酶切和PCR，获满意结果.首次应用RAPD（RandomAmplifiedPolymorphicDNA）与ARDRA（AmplifiedRibosomalDNARestrictionAnalysis）方法，直接扩增中国云南、东北地区赤杨属三种植物和沙棘属一种植物根瘤内的FrankiaDNA，所获指纹图谱揭示了其丰富的生物多样性.研究结果显示，与同属不同种植物（Alnus.tinctoriaSarg和Alnus.hirsuta）结瘤的Frankia，可有相同指纹图谱;同种植物（Alnus.nepalensisDon或Hippophae.rhamnoides）瘤内的Frankia指纹图谱可明显不同.瘤内Frankia有丰富的生物多样性，并且这种Frankia多样性可能与样品采集地点、地势、海拔高度等有关.

白腐菌选择性降解木质素的研究

对15株白腐真菌进行了以玉米秸秆为基质的初步筛选，从中获得一株选择性系数较高的菌株Y10，并对其降解玉米秸秆的情况进行了研究。结果表明，在30天的培养过程中菌株Y10对玉米秸秆降解的选择性系数都大于1，第15天选择性系数最高为3.88。对未经降解和降解过的玉米秸秆分别作了紫外光谱和红外光谱分析，结果表明，经该菌降解后玉米秸秆的化学成分发生了很大变化，且木质素的降解程度要大于纤维素的降解程度。对菌株Y10进行了ITS-5.8S rDNA序列鉴定，初步判定其为Cerrena sp.。 为了考查不同的外源添加物对菌株Y10降解玉米秸秆的影响，在以玉米秸秆为基质的固态发酵培养基中分别添加了7种金属离子、8种碳源、6种氮源。结果显示，这7种金属离子均能促进木质素的降解，并且一定浓度的某些离子明显抑制纤维素的降解；其中添加0.036%的MnSO4·H2O和0.36%的MgSO4·7H2O对纤维素降解的抑制作用比较强，降解率分别为0.96%和1.31%，木质素的选择性系数分别达到了34.40和20.17。8种碳源中除麦芽糖外都能促进木质素的降解，除微晶纤维素外都明显促进纤维素的降解。6种氮源中酒石酸铵、硫酸铵、草酸铵和氯化铵的添加都会使该菌生长变慢，而且氮源浓度越高菌丝生长越慢。外加碳源和金属离子对半纤维素降解和选择性系数的影响不大。 同时对菌株Y10在液态培养下产木质素降解酶的条件和培养基做了优化。结果表明，在初始产酶培养基中，菌株Y10的漆酶酶活在第10d达到最高，锰过氧化物酶酶活在第11d达到最高，基本上检测不到木质素过氧化物酶。菌株Y10产漆酶的最适温度为32℃，最适PH为6.0；产锰过氧化物酶的最适温度为32℃，最适PH为6.5。菌株Y10产漆酶的最佳碳源为甘露糖，最佳氮源为酒石酸铵，最适诱导剂VA浓度为3 mmol/L，最适表面活性剂TW-80浓度为1%。 利用响应面法对其产漆酶的培养基进行优化，优化后的培养基配方为葡萄糖10.00 g/L，酒石酸铵0.50 g/L，大量元素296.50 ml/L，微量元素100.00 ml/L，NTA 1.40 g/L，VA 5.00 mmol/L，吐温-80加入量为0.10%。进行了菌株Y10产漆酶的验证实验，实测酶活为5282.56 U/L，与预测酶活5162.73 U/L接近。在优化后培养基中，菌株Y10在第14 d达到生长的最高峰，第20 d时，漆酶酶活最高，为11325.00 U/L；第16 d时，锰过氧化物酶酶活最高，为30.77 U/L。 对菌株Y10的漆酶酶学性质做了初步的研究，结果显示，酶反应的最适温度为40℃-65℃，最适PH为3.0。在40℃，PH=3.0时，漆酶催化ABTS反应的米氏方程为 。 Fifteen white-rot fungi based on corn stalk were screened. One white-rot fungus Y10 with high selectivity value was obtained. The degradation of corn stalk was initially studied. The results indicated that the selectivity value was above 1 during the 30 day-cultivation and the highest was 3.88 after 15 days. The composition of untreated and treated stalk was analyzed through ultraviolet spectroscopy and infrared spectroscopy. It was found that the composition of treated stalk was greatly altered and the degree of the degradation of lignin is greater than the cellulose. Y10 was identified as Cerrena sp. by ITS -5.8S rDNA sequence analysis. The influence of metal ions, carbon sources and nitrogen sources on corn stalk degradation by white-rot fungus was studied. While all seven metal ions could promote lignin degradation, the cellulose degradation was best inhibited at certain ion concentrations. Notably, when 0.036% MnSO4·H2O and 0.36% MgSO4·7H2O were added into the medium, the cellulose degradation was restrained to the extents that the coefficients of lignin selectivity rose to 34.40 and 20.17 respectively. It was also found that all carbon sources except maltose can promote lignin degradation. The addition of carbon sources other than microcrystalline cellulose significantly promoted cellulose degradation. The addition of the nitrogen sources, ammonium tartrate, ammonium sulfate, oxalate, ammonium chloride, resulted in remarkable inhibition to mycelium growth; the larger the concentrations of nitrogen sources are, the slower the mycelium grew. The addition of carbon sources and metal ions had less impact on the degradation of hemicellulose and selectivity value. Meanwhile, we optimized the conditions and culture medium of the lignin-degrading enzyme production of strain Y10. The results showed that in the initial culture medium, the Lac activity was highest at the 10th day, the MnP activity was highest at the 11th day and the LiP could not be detected. The optimum condition of Lac was at temperature 32 and PH =6.0 and the optimum condition of MnP was at temperature 32 and PH =6.5. The optimum carbon source for Lac was seminose, the optimum nitrogen source was ammonium tartrate, the optimum content of VA was 3 mmol/L, the optimum content of TW-80 was 1%. PB and RSM were used to optimize the culture medium of laccase by white-rot fungus Y10. The optimum culture medium was consist of glucose 10.00 g/L, ammonium tartrate 0.50 g/L, macro elements 296.50 ml/L, trace elements 100.00 ml/L, NTA 1.40 g/L, VA 5.00 mmol/L, TW-80 0.10%. Under the optimal conditions, the activity of laccase was 5282.56 U/L and the experimental value agreed with the predicted value 5162.73 U/L. The biomass was highest at the 14th day, the Lac activity was highest at the 20th day, the MnP activity was highest at the 16th day. The results of the studies on the characteristics of Lac showed that the optimum temperature for Lac activity is 40℃-65℃ ; the optimum PH for Lac activity is 3.0 and under 40℃，PH=3.0, the Michaelis-menten equation of Lac catalized ABTS oxidation was .

产于天目山的中国多孔菌二新记录种(英文)

报道了产自浙江省天目山自然保护区的2种多孔菌:塔斯马尼亚小集毛孔菌Coltriciella tasmanica和凹形栓孔菌Trametes ectypus,2种多孔菌均为中国新记录种,根据所采集的材料对这2种真菌进行了详细描述和绘图。

Highly ordered mesoporous carbons-based glucose/O-2 biofuel cell

This study demonstrates a novel compartment-less glucose/O-2 biofuel cell (BFC) based on highly ordered mesoporous carbons (OMCs) with three-dimensionally (3D) interconnected and ordered pore structures. OMCs are used as supports for both stably confining the electrocatalyst (i.e., meldola's blue, MDB) for NADH oxidation and the anodic biocatalyst (i.e., NAD(+)-dependent glucose dehydrogenase, GDH) for glucose oxidation, and for facilitating direct electrochemistry of the cathodic biocatalyst (i.e., laccase, LAC) for O-2 electroreduction. In 0.10 M pH 6.0 PBS containing 20 mM NAD(+) and 60 mM glucose under the air-saturated atmosphere, the open circuit voltage (0.82 V) and the maximum power output (38.7 mu W cm(-2) (at 0.54V)) of the assembled compartment-less OMCs-based BFC are both higher than those of carbon nanotubes (CNTs)-based BFC (0.75 V and 2.1 mu W cm(-2) (at 0.46 V)).