复合菌系降解纤维素过程中微生物群落结构的变化

为明确高效纤维素降解复合菌系降解过程中微生物群落结构的变化规律及关键的降解功能菌,利用该复合菌系对滤纸和稻秆进行生物处理,通过底物降解、微生物生长量、发酵液pH的变化情况,选择不同降解时期复合菌系提取的总DNA进行细菌16S rRNA基因扩增子高通量测序。通过分解特性试验确定在接种后培养第12、72、168 h分别作为降解初期、高峰期、末期。该复合菌系分别主要由1个门、2个纲、2个目、7个科、11个属组成。随着降解的进行,短芽胞杆菌属Brevibacillus、喜热菌属Caloramator的相对丰度逐渐降低;梭菌属Clostridium、芽胞杆菌属Bacillus、地芽胞杆菌属Geobacillus、柯恩氏菌属Cohnella的相对丰度逐渐升高;解脲芽胞杆菌属Ureibacillus、泰氏菌属Tissierella、刺尾鱼菌属Epulopiscium在降解高峰期时相对丰度最高;各时期类芽胞杆菌属Paenibacillus、瘤胃球菌属Ruminococcus的相对丰度无明显变化。上述11个主要菌属均属于厚壁菌门,具有嗜热、耐热、适应广泛pH、降解纤维素或半纤维素的特性。好氧型细菌是降解初期的主要优势功能菌,到中后期厌氧型细菌逐渐增多,并逐步取代好氧型细菌成为降解纤维素的主要细菌。     

Function of a low molecular peptide generated by cellulolytic fungi for the degradation of native cellulose

Peptides (MW < 5 kDa) produced by 57 cellulolytic fungi can form free radicals. Scanning tunneling microscopy (STM) and IR spectroscopy showed that the peptide produced by Trichoderma pseudokoningii can break the hydrogen bond network of cellulose. The synergic action of these peptides and cellulases increased production of reducing sugars during degradation of native cellulose.     

裂解性多糖单加氧酶HcLPMO与纤维素酶协同降解纤维素

【目的】为研究HcLPMO的活性测定方法及其与纤维素酶的协同降解特性。【方法】利用大肠杆菌表达系统进行HcLPMO异源表达,研究以AmplexTM Ultra Red为荧光底物的LPMOs活性检测条件;研究HcLPMO与纤维素酶最优配比协同降解微晶纤维素及其他多种生物质底物的能力。【结果】表达条件确定最适装液量为20%,最适诱导温度为20°C。活性测定研究结果表明HcLPMO需先与铜离子结合才具有活性,电子供体抗坏血酸钠(ASC)最适浓度为10–4 mol/L,并发现AmplexTM Ultra Red浓度以及辣根过氧化物酶浓度对酶活的检测影响较小。HcLPMO与纤维素酶协同降解微晶纤维素研究确定HcLPMO与纤维素酶最优配比为2:3,葡萄糖产量相较纤维素酶单独作用提高了99.48%。此外,针对多种生物质底物,发现该酶与纤维素酶的复配体系对汽爆玉米秸秆和微晶纤维素的协同降解效果较好,相较于单独用纤维素水解酶,葡萄糖产量分别提高了63.81%和59.43%,而对碱处理玉米芯和木薯渣降解效果次之,葡萄糖产量仅分别提高35.41%和11.06%。【结论】HcLPMO与纤维素酶复配能够有效提高酶法降解纤维素效率;而底物前处理如蒸汽爆破或碱处理对于HcLPMO与纤维素酶协同降解木质纤维素影响较大。     

Thermal luminescence spectra of polyamides and their Maillard reaction with reducing sugars

Thermal luminescence (TL) spectra of polyamides were measured with a Fourier‐transform chemiluminescence spectrometer to elucidate the emission mechanism. A TL band of ε‐polylysine with a peak at 542 nm observed at 403 K was assigned to the emission due to the interaction of the –CO–NH– group with oxygen molecules by comparison with nylon‐6, polyglycine, and polyalanine. When the sample was kept at 453 K, the intensity of the TL band decreased and the wavelength of the peak shifted to 602 nm, which was assigned to the emission due to the interaction of the NH₂ group on the side chain with oxygen molecules by comparison with monomeric lysine. A weak emission with a peak at 668 nm was assigned to the advanced glycosylation end products (AGEs) yielded by the Maillard reaction with a catalytic amount of water. To understand this reaction and to examine the TL emission of AGEs, we measured TL spectra of mixtures of polylysine and reducing sugars such as glucose, maltose, lactose, and dextrin. The minimum temperature for TL emission, wavelength of the peak and the relative intensities of the TL emission were found to depend on the size of the sugars. Copyright © 2011 John Wiley & Sons, Ltd.     

Lignocellulose degradation during the life cycle of Agaricus bisporus

Abstract Lignocellulose degradation was monitored during the complete life cycle of the edible mushroom Agaricus bisporus grown on composted straw. Lignin and cellulose degradation were assayed by the use of ¹⁴C-labelled lignin and cellulose. Agaricus degraded both polymers but cellulose degradation was more extensive. Cellulolysis increased markedly at fruit body production and was greater still in non-axenically fruited cultures. No large change in the rate of lignin degradation occurred during the life cycle.     

Thermal degradation studies and kinetic modeling of cardoon (Cynara cardunculus) pyrolysis using thermogravimetric analysis (TGA)

A key element in the design of sustainable pyrolysis processes is the thermal degradation kinetics of biomass. In this work, pyrolysis tests for cardoon (Cynara carduculus) stems and leaves were performed in a non-isothermal thermogravimetric analyzer (TGA) in order to determine the thermal degradation behavior of both stems and leaves. The kinetic parameters of the process were evaluated using three different kinetic models, the independent parallel reaction model, KAS and OFW iso-conversional model. Good agreement with the experimental TGA data was observed for all models, the best being with the independent parallel reaction model. A variance in the activation energy with conversion was observed when the KAS and OFW models were employed, which reveals that the pyrolysis of cardoon progresses through more complex and multi-step kinetics.     

Zinc chloride mediated degradation of cellulose at 200 °C and identification of the products

The effect of ZnCl₂ on the degradation of cellulose was studied to develop conditions to produce useful feedstock chemicals directly from cellulosic biomass. Cellulose containing 0.5 mol of ZnCl₂/mol of glucose unit of cellulose was found to degrade at 200 °C when heated for more than 60 s in air. The major non-gaseous products of the degradation were identified as furfural, 5-hydroxymethylfurfural and levulinic acid. The maximum yields for furfural and 5-hydroxymethylfurfural are 8% and 9%, respectively, based on glucose unit of cellulose. These yields are reached after 150 s of heating at 200 °C. A cellulose sample containing 0.5 mol of ZnCl₂/mol of glucose unit of cellulose and 5.6 equivalents of water when heated for 150 s at 200 °C produced levulinic acid as the only product in 6% yield. The ZnCl₂ mediated controlled degradation of cellulose at 200 °C is shown to produce useful feedstock chemicals in low yield.     

Cumulative cellulolytic enzyme activities and initial litter quality in prediction of cellulose degradation in an alpine meadow of the eastern Tibetan Plateau

植物凋落物分解是决定陆地生态系统碳和养分循环的关键生态系统过程。作为凋落物的主要组成部分,纤维素是与凋落物分解相关的微生物的重要能量来源。纤维素酶在凋落物纤维素降解过程中的重要作用已为人们所熟知,然而纤维素降解的季节模式、累积酶活性和凋落物质量是否能预测高寒草甸的纤维素降解仍是一个未解之谜,这限制了我们对草本植物凋落物纤维素降解的认识。 为了探究纤维素降解的季节性模式以及累积纤维素分解酶活性和凋落叶初始质量对纤维素降解的影响,我们在青藏高原东部的高山草甸选取了三种优势种[圆叶筋骨草(Ajuga ovalifolia)、藏羊茅(Festuca wallichanica)和草甸马先蒿(Pedicularis roylei)],进行了为期两年的凋落物网袋分解实验。 我们的研究发现,纤维素在第一年中迅速降解且降解率超过50%,而且主要发生在第一个生长季(31.9%–43.3%)在两年的分解过程中,纤维素降解由累积内切葡聚糖酶(R²= 0.70),累积纤维二糖水解酶(R²= 0.59)和累积1,4-β-葡糖苷酶(R²=       0.57)共同驱动。此外,在这两年的分解过程中,纤维素、可溶性有机碳、总酚、木质素的浓度和木质素/N可以解释纤 维素降解变异的52%–78%)。用初始纤维素浓度模型预测纤维素降解效果最佳(R² = 0.78)。在凋落物的分解过程中,酶效率和微生物对纤维素分解酶的分配因物种而异。藏羊茅凋落物中纤维素酶效率较高,但质量相对较低。与草甸马先蒿相比,完全降解圆叶筋骨草和藏羊茅的纤维素需要4倍和6.7倍的内切葡聚糖酶、3倍和4.5倍的纤维二糖水解酶、1.2倍和1.4倍的1,4-β-葡糖苷酶。我们的研究结果表明,虽然微生物酶活性和凋落物初始质量都对高山草甸纤维素降解有显著影响,但使用纤维素浓度来预测纤维素降解是简化凋落物分解过程中纤维素降解和C循环模型的好方法。     

The roles of the cytoskeleton during cellulose deposition at the secondary cell wall

During secondary cell wall formation, developing xylem vessels deposit cellulose at specific sites on the plasma membrane. Bands of cortical microtubules mark these sites and are believed to somehow orientate the cellulose synthase complexes. We have used live cell imaging on intact roots of Arabidopsis to explore the relationship between the microtubules, actin and the cellulose synthase complex during secondary cell wall formation. The cellulose synthase complexes are seen to form bands beneath sites of secondary wall synthesis. We find that their maintenance at these sites is dependent upon underlying bundles of microtubules which localize the cellulose synthase complex (CSC) to the edges of developing cell wall thickenings. Thick actin cables run along the long axis of the cells. These cables are essential for the rapid trafficking of complex-containing organelles around the cell. The CSCs appear to be delivered directly to sites of secondary cell wall synthesis and it is likely that transverse actin may mark these sites.     

Quantitative analysis of cellulose degradation and growth of cellulolytic bacteria in the rumen

Ruminant animals digest cellulose via a symbiotic relationship with ruminal microorganisms. Because feedstuffs only remain in the rumen for a short time, the rate of cellulose digestion must be very rapid. This speed is facilitated by rumination, a process that returns food to the mouth to be rechewed. By decreasing particle size, the cellulose surface area can be increased by up to 10⁶-fold. The amount of cellulose digested is then a function of two competing rates, namely the digestion rate ( K _d) and the rate of passage of solids from the rumen ( K _p). Estimation of bacterial growth on cellulose is complicated by several factors: (1) energy must be expended for maintenance and growth of the cells, (2) only adherent cells are capable of degrading cellulose and (3) adherent cells can provide nonadherent cells with cellodextrins. Additionally, when ruminants are fed large amounts of cereal grain along with fiber, ruminal pH can decrease to a point where cellulolytic bacteria no longer grow. A dynamic model based on stella ^® software is presented. This model evaluates all of the major aspects of ruminal cellulose degradation: (1) ingestion, digestion and passage of feed particles, (2) maintenance and growth of cellulolytic bacteria and (3) pH effects.     

蛋白激酶C在晚期糖基化终产物促进人腹膜间皮细胞分泌纤维连接蛋白中的作用

目的:观察晚期糖基化终末产物(AGEs)对人腹膜间皮细胞合成和分泌纤维连接蛋白(FN)的影响及细胞内蛋白激酶C(PKC)在其中的作用。方法:在体外制备晚期糖基化人血清白蛋白(AGE-HSA),分别用不同浓度的(0,100,500,1 000μg/ml)AGE-HSA作用于人腹膜间皮细胞,用荧光实时定量PCR和ELISA方法测定人腹膜间皮细胞FN的表达;用ELISA方法观察AGEs对人腹膜间皮细胞PKC活性影响,应用PKC激活和抑制剂观察PKC在AGE-HSA促进人腹膜间皮细胞合成FN中的作用。结果:AGE-HSA呈时间剂量依赖性激活人腹膜间皮细胞中的PKC(P<0.05);AGE-HSA同时以时效和量效方式促进人腹膜间皮细胞中FN基因和蛋白的表达(P<0.05);PKC激活剂佛波酯(PMA)也能刺激人腹膜间皮细胞合成FN,先用PMA耗竭细胞内PKC或用PKC抑制剂calphostin C后,可抑制AGE-HSA诱导的FN分泌(P<0.05)。结论:AGEs可能部分通过活化PKC促进人腹膜间皮细胞合成和分泌FN,参与腹膜纤维化。     

A peptide-mediated and hydroxyl radical HO*-involved oxidative degradation of cellulose by brown-rot fungi

A special low-molecular-weight peptide named Gt factor, was isolated and purified from the extracellular culture of brown-rot fungi Gloeophyllum trabeum via gel filtration chromatography and HPLC. It has been shown to reduce Fe³⁺ to Fe²⁺. Electron paramagnetic resonance (EPR) spectroscopy revealed Gt factor was able to drive H₂O₂ generation via a superoxide anion O₂ ^.- intermediate and mediate the formation of hydroxyl radical HO^. in the presence of O₂. All the results indicated that Gt factor could oxidize the cellulose, disrupt the inter- and intrahydrogen bonds in cellulose chains by a HO^. -involved mechanism. This resulted in depolymerization of the cellulose, which made it accessible for further enzymatic hydrolysis.     

Organelle degradation during the lens and erythroid differentiation is independent of autophagy

Autophagy is a bulk degradation system within cells through which cytoplasmic components are degraded within lysosomes. Primary roles of autophagy are starvation adaptation and intracellular protein quality control. In contrast to the ubiquitin-proteasome system, autophagy can also degrade organelles. Here we examined a possible role of autophagy in organelle degradation during lens and erythroid differentiation. We observed that autophagy occurs in embryonic lens cells. However, organelle degradation in lens and erythroid cells occurred normally in autophagy-deficient Atg5(-/-) mice. Our data suggest that degradation system(s) other than autophagy play major roles in organelle degradation during these processes.     

Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family

Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.     

Wetland degradation leads to homogenization of the biota at local and landscape scales

1. We examined whether the anthropogenic degradation of wetlands leads to homogenization of the biota at local and/or landscape scales and, if so, what specific factors account for such an effect. We compared 16 isolated wetlands (Michigan, U.S.A.) that varied in surrounding land use: half had developed, and half undeveloped, riparian zones. Samples of macrophytes, epiphytic diatoms, zooplankton, macroinvertebrates and water chemistry were collected along three transects in each wetland. 2. Developed wetlands were more nutrient‐rich with higher Cl concentrations. The plant community at developed sites was dominated by Lemnaceae (duckweed), while undeveloped wetlands were dominated by rooted, floating‐leaved vegetation and sensitive plant species. Undeveloped wetlands contained heterogeneous and species‐rich plant communities, greater species richness of zooplankton and diatoms, and heterogeneous zooplankton distributions as compared to developed sites. 3. A comparison among wetlands showed that diatom and zooplankton assemblages in developed wetlands were nested subsets of richer biota found in less developed wetlands. Conversely, plant communities were more heterogeneously distributed among developed wetlands at the landscape level. This may be attributable to patchy invasions by exotic species, which were a feature of the degraded wetlands within developed landscapes. 4. Our results indicate that several taxonomic groups showed similar, probably inter‐dependent, responses to wetland degradation and habitat homogenization at both the local and landscape scales. This change in community structure from a species‐rich and heterogeneous community dominated by floating‐leaved plants in undeveloped wetlands, to nutrient‐rich wetlands dominated by duckweed may represent a shift to an alternate stable state.     

Species richness and species composition of fungal communities associated with cellulose decomposition at different altitudes on the Tibetan Plateau

Aims The aims of this study were to compare the fungal communities developing on cotton strips at three different altitudes on the Tibetan Plateau and to assess the environmental variables influencing them.Methods Cotton strips that had been buried in soil for a year were sampled at three sites at different altitudes (4500, 4950 and 5200 m) located on a southeast-facing slope on the Nyainqentanglha Mountains near Damxung. The fungi on the cotton strips were isolated using a modified washing method. The decomposition abilities and colony growth properties of the major species cultured in pure-culture conditions were investigated and compared. Canonical correspondence analysis (CCA) was used to evaluate the relationships between fungal community composition and environmental variables (altitude, soil depth, soil water content [SWC], plant root mass and gravel content).Important findings A total of 24 species were isolated from the cotton strips, and 12 species occurred frequently and were regarded as major species. The number of fungal species was lower at the 4950-m altitude site than at the other two sites, indicating that not only altitude but also other factors affected the number of species present. All of the major species were able to decompose the cotton strips. In the CCA ordination, automatic forward selection revealed that altitude, SWC and plant root mass significantly affected fungal species composition. Our results suggest that species number and the composition of cellulolytic fungal communities are highly correlated with environmental variables as well as altitude in the alpine meadow on the Tibetan Plateau.     

Distribution and diversity of members of the bacterial phylum Fibrobacteres in environments where cellulose degradation occurs

The Fibrobacteres phylum contains two described species, Fibrobacter succinogenes and Fibrobacter intestinalis, both of which are prolific degraders of cellulosic plant biomass in the herbivore gut. However, recent 16S rRNA gene sequencing studies have identified novel Fibrobacteres in landfill sites, freshwater lakes and the termite hindgut, suggesting that members of the Fibrobacteres occupy a broader ecological range than previously appreciated. In this study, the ecology and diversity of Fibrobacteres was evaluated in 64 samples from contrasting environments where cellulose degradation occurred. Fibrobacters were detected in 23 of the 64 samples using Fibrobacter genus-specific 16S rRNA gene PCR, which provided their first targeted detection in marine and estuarine sediments, cryoconite from Arctic glaciers, as well as a broader range of environmental samples. To determine the phylogenetic diversity of the Fibrobacteres phylum, Fibrobacter-specific 16S rRNA gene clone libraries derived from 17 samples were sequenced (384 clones) and compared with all available Fibrobacteres sequences in the Ribosomal Database Project repository. Phylogenetic analysis revealed 63 lineages of Fibrobacteres (95% OTUs), with many representing as yet unclassified species. Of these, 24 OTUs were exclusively comprised of fibrobacters derived from environmental (non-gut) samples, 17 were exclusive to the mammalian gut, 15 to the termite hindgut, and 7 comprised both environmental and mammalian strains, thus establishing Fibrobacter spp. as indigenous members of microbial communities beyond the gut ecosystem. The data highlighted significant taxonomic and ecological diversity within the Fibrobacteres, a phylum circumscribed by potent cellulolytic activity, suggesting considerable functional importance in the conversion of lignocellulosic biomass in the biosphere.     

黄河源区不同退化程度高寒草原群落生产力、物种多样性和土壤特性及其关系研究

近些年来,气候暖干化和过度放牧导致黄河源区高寒草原发生明显退化,严重影响了当地畜牧业和环境的可持续发展。退化后,植被群落生产力、物种多样性和土壤因子之间相互作用、相互影响,使生态系统持续恶化。以往的研究中研究人员对退化后群落生产力和物种多样性关系关注较多,但对退化过程中土壤要素变化的重视程度往往不够。因此,探究不同退化程度下高寒草原群落生产力、物种多样性和土壤特性及其关系对于认识高寒草地退化过程及退化草地恢复具有重要现实意义。在黄河源区采用空间分布代替时间演替的方法,根据植被和土壤特征选取了未退化到严重退化5个退化梯度,探讨不同退化程度下高寒草原群落生产力、物种多样性和土壤特性及其关系。结果表明：1）随着退化程度的加剧,群落地上和地下生物量均呈先稳定后降低的趋势,在轻度退化阶段达到最大值,重度和严重退化阶段显著降低;2）Shannon-Wiener多样性指数在轻度和中度退化阶段显著增加了20%和15%（P=0.025和P=0.039）,均匀度指数从未退化到重度退化变化不明显,严重退化阶段物种多样性指数均显著降低;3）土壤水分、各深度土壤有机碳、全氮、铵态氮和硝态氮均呈先稳定后降低的变化规律,土壤容重随着退化程度的加剧而显著增加;4）群落生物量、物种多样性与土壤养分呈正相关关系,与土壤容重呈负相关关系,冗余分析结果显示土壤容重、硝态氮、有机碳是退化过程中驱动植被因子变化的主要因素。因此,针对不同退化阶段采取不同的恢复治理措施,尤其是改善土壤养分和物理性质,同时对中度和重度退化两个关键阶段应该给予更多的关注。     

A light and electron microscope study of changes occurring at the cut ends following section of the dorsal roots of rat spinal nerves

Summary Rat dorsal spinal nerve roots were cut; 20 h later the axons in the vicinity of the cut were examined by light and electron microscopy. The changes in the cut tip distant from the ganglion were largely degenerative. On the ganglionic side of the cut a cap of free unmyelinated sprouts was formed. These sprouts contained clear and dense-core vesicles 40–150 nm in diameter, smooth endoplasmic reticulum and mitochondria. Some of the unmyelinated sprouts were extensions of myelinated axons, others arose from myelinated axons by lateral budding. In both myelinated and non-myelinated axons there was an accumulation of mitochondria, tubulo-vesicular smooth endoplasmic reticulum and large and small dense-core vesicles for a distance of approximately 500 m behind the tip. Dense-core vesicles were more common in nonmyelinated axons than in their myelinated counterparts. In areas of intense accumulation the non-myelinated fibres were grossly swollen and distorted. The myelinated axons and some of the sprouts contained an unusual type of mitochondrion. The similarity between these sprouts and pre-synaptic terminals is discussed.I.R.D. is supported by the Medical Research Council; P.K. thanks the Mental Health Trust for a project grant