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。     

平板混合培养木质纤维素降解菌研究进展

微生物的混合培养已广泛应用于木质纤维素类物质的转化与降解领域.不同木质纤维素降解菌在混合培养时的相互关系在很大程度上影响混合培养的效果.目前对这种相互关系的研究主要依托平板混合培养展开,所用到的平板主要有基础培养基平板和改进培养基平板两种.其中基础培养基平板法主要根据菌落形态、菌丝体颜色、胞外挥发性有机化合物成分和典型胞外酶活性等进行研究,而改进培养基平板则是将基础培养基平板中的碳源更换为天然木质纤维素类物质进行对比研究.本文综述了采用平板混合培养不同木质纤维素降解菌菌株的研究现状和进展,并对该领域研究应重点关注的问题进行了展望.     

侧耳菌与粗毛栓菌在麦草培养基中产生木质纤维素降解酶的研究

草本植物,包括农作物秸杆的木质素主要是由松柏醇、芥子醇和对香豆醇的脱氢聚合物和对香豆酸组成[1,2],是结构复杂、稳定、多样的生物大分子物质.虽难于被一般微生物降解,但自然界中仍存在一些可降解木质素的微生物种类,白腐真菌是最重要的一类,它们通过分泌漆酶(Laccases,Lac)、木质素过氧化物酶(Lignin peroxidases,LiP)、锰过氧化物酶(Manganese-dependent peroxidases,MnP)、纤维素酶(Cellulas-es,Cel)和半纤维素酶(Hemicellulases,Hcel)等降解植物生物质.由于白腐菌在造纸工业中的生物制浆和纸浆生物漂白、环境保护等方面[4]有着很好的应用前景,因此倍受关注. 本研究选用在液体培养基中产酶能力强且产酶较快的白腐真菌侧耳sp2和粗毛栓菌[5]进行固体培养,研究它们产生木质纤维素降解酶类和降解植物生物质的能力.研究结果报道如下.     

粗毛栓菌降解麦草木质纤维素的试验研究

正交试验结果表明 ,培养基质中葡萄糖的存在 ,抑制粗毛栓菌对麦草木质纤维素的降解作用 ;适量添加酒石酸 ,可提高该菌对木素的降解程度。粗毛栓菌有较强的降解麦草木质纤维素的能力 ,培养 6 0d后 ,原麦草中6 6 .2 1%纤维素、71.96 %半纤维素和 70 .14%木质素将分别消失     

白腐菌液体和固体培养产生木质纤维素降解酶的比较研究

侧耳sp2(Pleurotus sp．2)和粗毛栓菌(Trametes gallica)是产木质纤维素降解酶能力强,且产酶较快的菌株。对其在液体培养基、固体培养基中产生木质纤维素降解酶能力和行为进行了比较分析和研究。结果表明,Pleurotus sp．2在低氮高碳高无机盐培养基中的锰过氧化物酶(Manganese peroxidases, MnPs)、木质素过氧化物酶(Lignin peroxidases．LiPs)、漆酶(laccases,Lacs)和半纤维素酶(Hemicellulases, Hcels)的活性最高。当该菌株培养在含有低氮无碳高无机盐液体培养基的麦草粉中时,MnPs和Lacs的活性峰值均出现在10d,而Hcels的活性在40d时达到峰值。Trametes gallica在高氮低碳高无机盐培养基中的Lacs和LiPs的活性最高,在低氮高碳高无机盐培养基中的MnPs和Hcels的活性最高。当该菌株培养在含有高氮无碳高无机盐和低氮无碳高无机盐液体培养基的麦草粉中时,MnPs存10d、Lacs和Hcels在40d、LiPs存50d,分别达到峰值。Pleurotus sp．2和Trametes gallica在液体培养基中具有很强的木质纤维素降解酶产生能力且产酶速度较快,在固体培养基中具有很强的降解麦秸生物质能力,但这两株菌在液体和固体培养基中,产木质纤维素降解酶的能力和行为都有较大的差异,相关性小。     

基于转录组和加权基因共表达网络的广叶绣球菌木质纤维素降解特性分析

【目的】分析广叶绣球菌（Sparassis latifolia）在不同木质纤维素诱导条件下基因表达差异,为广叶绣球菌木质纤维素降解关键基因和分子机制研究提供参考。【方法】以松木、杉木、甘蔗渣和天然堆积发酵后的杉木和发酵后的甘蔗渣为碳源,在液体培养条件下培养诱导广叶绣球菌,对其转录组进行测序研究,并对不同木质纤维素诱导样本进行加权基因共表达网络分析（weighted gene co-expression network analysis,WGCNA）。【结果】杉木培养与松木培养比较组差异表达基因最少（20个）,蔗渣培养与松木培养比较组差异表达基因最多（486个）。基因本体（gene ontology,GO）富集分析结果表明,差异表达基因主要涉及氧化还原酶活性、单加氧酶活性和铁离子结合活性等,京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）通路富集分析结果表明,差异表达基因主要涉及戊糖和葡萄糖醛酸转换、甲烷代谢和乙醛酸盐和二羧酸盐代谢等通路。发酵甘蔗渣为碳源培养时,纤维素和半纤维素降解相关的糖苷水解酶基因表达量总体上较高,而未发酵的松木、杉木和甘蔗渣为碳源培养时木质素降解或修饰相关的碳水化合物辅助酶基因表达量总体上较高。利用WGCNA共鉴定出10个共表达模块,其中green模块与未发酵蔗渣诱导显著正相关,blue模块与发酵甘蔗渣诱导显著正相关,magenta和turquoise模块与发酵杉木诱导显著正相关。GO富集分析结果表明,turquoise模块内基因显著富集到尿素跨膜转运子活性、甲基转移酶活性和单加酶活性等,blue模块基因显著富集到水解酶活性和β-甘露糖苷酶活性。KEGG通路富集分析结果表明,blue模块内基因显著富集的通路有半乳糖代谢、果糖和甘露糖代谢、苯丙氨酸代谢、精氨酸和脯氨酸代谢等。通过构建互作网络图挖掘到12个核心基因,其可能参与了基质降解及相关基因的表达调控。【结论】不同木质纤维素类型显著影响了广叶绣球菌木质纤维素降解基因的差异表达轮廓,这种差异反映了广叶绣球菌对不同木质纤维素特异的降解策略。     

木质纤维素的微生物降解

木质纤维素广泛存在于自然界中,因结构复杂,其高效降解需要多种微生物的协同互作,由于参与木质纤维素降解的微生物种类繁多,其协同降解机理尚不完全明确。随着微生物分子生物学和组学技术的快速发展,将为微生物协同降解木质纤维素机制的研究提供新的方法和思路。笔者前期研究发现,细菌复合菌系在50℃下表现出强大的木质纤维素降解能力,菌系由可分离培养和暂时不可分离培养细菌组成,但是可分离培养细菌没有降解能力。通过宏基因组和宏转录组研究表明,与木质纤维素降解相关的某些基因表达量发生显著变化,通过组学方法有可能更加深入解释微生物协同降解木质纤维素的微生物学和酶学机理。文中从酶、纯培养菌株和复合菌群三个方面综述了木质纤维素微生物降解研究进展,着重介绍了组学技术在解析复合菌群作用机理方面的现状和应用前景,以期为探索微生物群落协同降解木质纤维素的机理提供借鉴。     

青霉生产木质纤维素降解酶系的研究进展

木质纤维素降解酶系的高效生产是实现植物生物质大规模生物炼制的重要支撑。就地生产木质纤维素降解酶,有助于降低其使用成本,提高技术经济效益。青霉是自然界常见的木质纤维素降解真菌,可以合成分泌种类多样、组分齐全的木质纤维素降解酶系,已被应用于纤维素酶制剂的工业生产。文中从就地生产降解酶,为木质纤维素生物炼制构建“糖平台”的角度,综述了青霉木质纤维素降解酶系的性质、菌株遗传改造及发酵工艺的研究进展。     

鸡菌的液体发酵与毒理研究

鸡菌Termitomycesalbuminosus（Berk）．Heim采自四川西昌野生子实体,经分离、纯化、鉴定、保存于PDA琼脂斜面培养基。报道鸡菌菌丝体深层发酵在500L发酵罐中,温度28～30℃,pH6.0～7．0,接种量1％～1．5％,通气量1：1～1：1．2（体积比）,可在30h获得鸡菌菌丝体24．5g/L（干重）。还对鸡菌菌丝体的毒性进行了探讨,选用Ames试验（TA97,TA98,TA100,TA1024个标准菌株）、小鼠骨髓微核试验,小鼠睾丸初级精母细胞染色体畸变分析,小鼠精子畸形试验,试验结果为阴性。说明鸡菌菌丝体基本安全,无毒。     

抗凝活性蛋白产生菌Termitomyces albuminosus的诱变育种

真菌Termitomyces albuminosusTA-SD能产生具有抗凝活性的蛋白质TA-P。本实验采用紫外诱变法对TA-SD菌株进行诱变育种,筛选高活性的突变株。出发菌株TA-SD紫外照射40s时,正突变率和抗凝活性提高的幅度最大,分别为33.3%和104.7%。N型突变株和放线菌酮抗性突变株的正突变率和活性提高的幅度均大于S型突变株和非抗性突变株。经筛选突变株SD-A-8的抗凝活性比出发菌株提高了2.3倍。该突变株传代稳定。     

富铬蚁巢伞（鸡菌）液体培养(英文)

A species of mushroom, Termitomyces albuminosus, was cultured in liquid medium for production of chromium-enriched mycelium. The influence of chromium (Ⅲ ) on mycelial growth of T. albuminosus was investigated. An optimum medium composed of 5.6g/L yeast extract, 51.6g/L hydrolyzed rice, 2g/L KH2PO4, and 20mg/L chromium(Ⅲ ) with initial pH of 4.5 was obtained by using method of central composite design (CCD). After incubation of 84h, the maximal biomass of chromium-enriched mycelia reached 24.23g DMW(dried mycelial weight)/L with 272μg/g DMW chromium content in 500mL flasks containing 100mL medium with an inoculum of 8% on a shaker of 100r/min under an optimized cultivation condition at 28℃.     

Metagenomic analysis of the Rhinopithecus bieti fecal microbiome reveals a broad diversity of bacterial and glycoside hydrolase profiles related to lignocellulose degradation

Background

The animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host and the diet adopted by the host. Although the importance of gut microbiota of humans has been well demonstrated, there is a paucity of research regarding non-human primates (NHPs), especially herbivorous NHPs.

Results

In this study, an analysis of 97,942 pyrosequencing reads generated from Rhinopithecus bieti fecal DNA extracts was performed to help better understanding of the microbial diversity and functional capacity of the R. bieti gut microbiome. The taxonomic analysis of the metagenomic reads indicated that R. bieti fecal microbiomes were dominated by Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria phyla. The comparative analysis of taxonomic classification revealed that the metagenome of R. bieti was characterized by an overrepresentation of bacteria of phylum Fibrobacteres and Spirochaetes as compared with other animals. Primary functional categories were associated mainly with protein, carbohydrates, amino acids, DNA and RNA metabolism, cofactors, cell wall and capsule and membrane transport. Comparing glycoside hydrolase profiles of R. bieti with those of other animal revealed that the R. bieti microbiome was most closely related to cow rumen.

Conclusions

Metagenomic and functional analysis demonstrated that R. bieti possesses a broad diversity of bacteria and numerous glycoside hydrolases responsible for lignocellulosic biomass degradation which might reflect the adaptations associated with a diet rich in fibrous matter. These results would contribute to the limited body of NHPs metagenome studies and provide a unique genetic resource of plant cell wall degrading microbial enzymes. However, future studies on the metagenome sequencing of R. bieti regarding the effects of age, genetics, diet and environment on the composition and activity of the metagenomes are required.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1378-7) contains supplementary material, which is available to authorized users.     

Submerged culture of chromium-enriched Termitomyces albuminosus

A species of mushroom, Termitomyces albuminosus, was cultured in liquid medium for production of chromium-enriched mycelium. The influence of chromium （Ⅲ） on mycelial growth of T. albuminosus was investigated. An optimum medium composed of 5.6g/L yeast extract, 51.6g/L hydrolyzed rice, 2g/L KH2PO4, and 20mg/L chromium（Ⅲ） with initial pH of 4.5 was obtained by using method of central composite design （CCD）. After incubation of 84h, the maximal biomass of chromium-enriched mycelia reached 24.23g DMW（dried mycelial weight）/L with 272μg/g DMW chromium content in 500mL flasks containing 100mL medium with an inoculum of 8% on a shaker of 100r/min under an optimized cultivation condition at 28 ℃.     

In vitro Studies on lignocellulose degradation by microbial strains isolated from composting processes

An in vitro study of different strains isolated from composting piles in relation to their capacity to biodegrade lignocellulose was achieved. Thirteen microorganisms (five bacteria, one actinomycete, and seven fungi) isolated from compost windrows were grown on agricultural wastes and analyzed for cellulose, hemicellulose, and lignin degradation. Hemicellulose fraction was degraded to a lesser extent because only two of the isolates, B122 and B541, identified as Bacillus licheniformis and Brevibacillus parabrevis, respectively, were able to decrease the concentration of this polymer. On the contrary, most of the isolates were capable of reducing cellulose and lignin concentrations; strain B541 was the most active cellulose degrader (51%), while isolate B122 showed higher lignin degradation activity (68%). Consequently, an increase in humification indices was detected, especially with respect to humification index (HI) for both bacteria and CAH/AF in the case of strain B122. According to these data, the use of microbial inoculants as a tool to improve organic matter biodegradation processes (i.e., composting) may become important if microorganisms’ capabilities are in accordance with the final characteristics required in the product (high humic content, lignin content decrease, cellulose concentration decrease, etc.).     

Characterization of a microbial consortium capable of degrading lignocellulose

A microbial consortium, designated WCS-6, was established by successive subcultivation in the presence of rice straw under static conditions. The degradation efficiencies of WSC-6 for 0.5 g filter paper, cotton and rice straw after 3 days of cultivation were 99.0±0.7%, 76.9±1.5% and 81.3±0.8%, respectively as determined by gravimetrical methods. Nine bacterial isolates were obtained from WCS-6 plated under aerobic conditions, and sequencing of their 16S rDNA indicated that these bacteria were related to Bacillus thermoamylovorans BTa, Paenibacillus barengoltzii SAFN-016, Proteobacterium S072, Pseudoxanthomonas taiwanensis CB-226, Rhizobiaceae str. M100, Bacillus sp. E53-10, Beta proteobacterium HMD444, Petrobacter succinimandens 4BON, and Tepidiphilus margaritifer N2-214. DGGE (denaturing gradient gel electrophoresis) and sequencing of 16S rDNA sequences amplified from total consortium DNA revealed the presence of sequences related to those of Ureibacillus thermosphaericus, uncultured bacterium clone GC3, uncultured Clostridium sp. clone A1-3, Clostridium thermobutyricum, and Clostridium thermosuccinogenes in addition to the sequences identified from the cultured bacteria. The microbial community identified herein is a potential candidate consortium for the degradation of waste lignocellulosic biomass.     

白蚁共生真菌——蚁巢伞属研究概况

蚁巢伞属真菌是一类极具市场开发价值的野生食用真菌。本文对蚁巢伞属真菌的分类、与白蚁的共生关系、活性成分和作用、人工培养研究以及产生的相关木质纤维素降解酶类等方面进行综述,总结了近年来该属真菌的研究现状,指出了研究过程中出现的分类混乱、重名异名现象严重等问题,展望了未来有望开发菌丝体相关产品及深化出菇机制以逐步实现蚁巢伞人工栽培的研究方向,以期为未来的研究提供依据。     

蚁巢伞属真菌研究进展

从蚁巢伞属真菌的分类鉴定、活性成分、生长培养及与宿主白蚁共生关系等方面进行综述。其中包括对近年来相关研究成果的总结,指出其中存在的问题,对蚁巢伞属真菌的研究前景进行展望。     

Enzymes and small molecular mass agents involved with lignocellulose degradation

Abstract: Electron microscopic and biochemical studies of lignocellulose degradation by wood-rotting fungi have shown that enzymes such as lignin peroxidases, manganese-dependent peroxidases, laccases and cellulases are too large to penetrate undegraded secondary wood cell walls. Degradation occurs by surface interaction between cell wall and enzymes, but initiation of decay at a distance from the fungal hyphae must involve diffusible low-molecular mass agents. The roles of hydrogen peroxide, veratryl alcohol, oxalate, Fe²⁺-Fe³⁺ and Mn²⁺-Mn³⁺, as such agents in lignocellulose degradation are discussed.