Purification and characterization of thermostable cellulase from Enterobacter cloacae IP8 isolated from decayed plant leaf litter

Cellulases are important in the hydrolysis of lignocellulosic materials and thereby contribute to biomass conversion into fuels and chemicals. A cellulase-producing bacterium was isolated from decayed plant leaf litter in soil of a botanical garden. Based on morphological, biochemical and 16S rRNA gene sequencing, it was identified as Enterobacter cloacae IP8, with gene bank accession number NR118568.1. The bacterial cellulase was purified in a three-step procedure using lyophilization, ion exchange chromatography (QAE Sephadex A-50) and gel filtration (Biogel P-100). Two isoforms of the enzyme were purified 1.21 and 1.23 folds, respectively, with yields of 30 and 29% for isoforms A and B, respectively. Apparent molecular weights of 36.61?±?1.40 and 14.1?±?0.10?kDa were obtained for isoforms A and B, respectively, using gel filtration chromatography. Kinetic parameters K_m and V_max were 0.13?±?0.04?mg/ml and 3.84?±?0.05?U/ml/min, respectively, for isoform A and 0.58?±?0.06?mg/ml and 13.8?±?0.10?U/ml/min, respectively, for isoform B. Optimum pH (7.0) and temperature (60?°C) of cellulase activity were determined for both isoforms A and B. Na⁺ and Ca²⁺ enhanced the activities of both isoforms. Mg²⁺ inhibited the enzyme activity at concentrations 4–15?mM but, while it stimulated the activity of isoform A at concentrations 15–200?mM, it inhibited that of isoform B at same concentration range. The strong inhibition of the enzyme by ethylenediaminetetraacetic acid (EDTA) confirmed the enzyme as a metalloenzyme. These results reveal the purified cellulase from E. cloacae IP8 as a thermostable, acidic to neutral metalloenzyme, suggesting that it has good potential for biotechnological applications.     

Measurement of saccharifying cellulase

This article sets forth a simple cellulase assay procedure. Cellulose is variable in nature, insoluble and resistant to enzymatic attack. As a result there have been a bevy of bewildering cellulase assays published that yielded irrational results. Certain protocols focused on the rapidity of the assay while ignoring that only the most readily susceptible cellulose regions were being hydrolyzed. Other assays simplified the system by using modified soluble substrates and yielded results that bore no relationship to the real world hydrolysis of insoluble cellulose. In this study Mandels, Andreotti and Roche utilized a common substrate, Whatman filter paper. Hydrolysis of a 50 mg sample of the paper was followed to roughly 4% degradation, which circumvented the problems of attack of only the most susceptible zones. This common hydrolysis target range also resulted in some balance with regard to the interaction of the several cellulase components. The method was subsequently widely adopted.     

Breeding systems in a temperate mediterranean-type climate montane sclerophyllous forest in central Chile

The frequency of dioecy in the predominantly biotically-pollinated native flora of a temperate montane sclerophyllous forest in central Chile, 33^oS, is determined. Experimental crosses and other tests were performed on a taxonomically diverse set of annual herbs, perennial herbs and woody species to detect genetic self-incompatibility, spontaneous selfing capacity and obligate agamospermy.
The overall frequency of dioecy in the community is 9%.Dioecy is unequally represented among life-forms, increasing in frequency with greater longevity: 0% in annual herbs; 2% in perennial herbs; 17% in shrubs; 57% in trees. Thirty-eight percent of 37 hermaphrodite species proved to be genetically self-incompatible. Self-incompatibility, like dioecy, increases in frequency with longevity: 0% in annual herbs; 50% in perennial herbs; 80% in shrubs. The self-compatible species showed a wide range of breeding habits from facultative outcrossing to strong autogamy. However, in most self-compatible species hand self-pollination increased fruit and seed set over spontaneous selfing. Obligate agamospermy was not detected among hermaphrodite specieS. Absolute and relative fecundity were strongly correlated with the degree of compatibility. However, reduced fecundity in obligate outbreeding species could not be unequivocally attributed to pollinator limitation.
For equivalent life-forms, the montane sclerophyllous forest exhibits proportionately more obligate outbreeding species than some tropical forests and other temperate communities. The strong tendency for obligate outcrossing in the Chilean community is seen as a result of interplay between life-history constraints and the intense abiotic and biotic pressures that are characteristic of the mediterranean-type climate community.     

亚热带森林树种多样性对凋落叶分解胞外酶活性的影响

森林生物多样性与生态系统功能关系是当前群落生态学的热点研究领域。然而, 以往研究更多聚焦在森林植物多样性丧失对群落生产力的影响, 而对森林凋落物分解的相关研究稍显不足。森林凋落叶分解的快慢直接受控于凋落物分解者分泌的胞外酶的活性, 后者更是指示森林生态系统养分循环的重要指标之一。本研究依托我国江西亚热带森林生物多样性与生态系统功能控制实验, 通过对不同植物多样性梯度样方内目标树种凋落叶胞外酶活性、理化性质以及腐生真菌的分析, 探索树种多样性丧失对胞外酶活性的影响及其调控机制, 以探讨森林树种多样性对地表、地下生态过程和功能的影响。结果表明, 样方水平树种多样性丧失显著影响胞外酶的活性, 除单种样方外, 随着样方水平树种丰富度的增加, 胞外酶活性呈现出增长趋势; 与碳周转相关的α-葡萄糖苷酶(AG)、β-葡萄糖苷酶(BG)、纤维二糖水解酶(CB)在树种多样性最大时活性达到最高; 而木糖苷酶(XS)以及与氮、磷和顽拗有机养分分解相关的N-乙酰-β-氨基葡萄糖苷酶(NAG)、酸性磷酸酶(AP)和多酚氧化酶(PPO)在树种多样性较低时活性较高。针对目标树种周围的邻居树种多样性进一步分析发现, 各胞外酶活性随着邻居树种多样性的变化呈“单峰”响应趋势, 酶活性大多在邻居树种丰富度为6时呈现峰值。研究发现真菌分解者在胞外酶活性对植物多样性的响应上可能存在重要的调控作用, 可以推测树种多样性通过改变腐生真菌分解者的群落结构和多度, 从而影响胞外酶活性。     

森林凋落物研究进展

对森林凋落物的概念、研究方法及主要研究内容作了阐述,特别就凋落物收集面积和分解袋孔径大小、凋落量时空动态和凋落物分解速率等问题进行了综合分析。目前森林凋落物研究的重要结论有：海拔和纬度因子是通过对光、温、水等生态因子的再分配来影响凋落量,其中主导气候因子是年均温。凋落物的分解与化学组成和环境因子有关,C／N和N含量在凋落物分解过程中起着重要作用。土壤水分是影响凋落物分解主要环境因子之一;土壤微生物对凋落物的影响,前期是通过真菌破碎凋落物表层使内居性动物得以侵入凋落物内部,后期则以细菌降解有机物为主。凋落量、凋落物分解的影响因子,以及凋落物的生态作用等内容应是凋落物研究的重要方向。     

Partial purification and characterization of extracellular cellulase from a strain ofTrichoderma viride isolated from forest soil

Partial purification of extracellular cellulase ofTrichoderma viride isolated from forest soil was done by ammonium sulfate precipitation of culture supernatant, centrifugation at higher speed, solubilization of protein in sodium acetate buffer and dialysis. The specific activity of cellulase in the culture supernatant, was 136 nkat/mg which was increased by 172% after the completion of final step (234 nkat/mg). The recovery of enzyme was 70%. The enzyme was characterized by demonstration of optimum activity at 55°C and pH 5.0 with 1% carboxymethyl cellulose as substrate.     

Measurement of seasonal variations in the oxygen uptake of various litter layers of an oak forest

Summary The oxygen uptake of decomposing oak leaves in three litter layers was determined with a Gilson respirometer at least once a month. Samples were taken from Meerdink forest over a period of two years. The determinations were made at the same temperatures as those prevailing in the soil in the field at the moment of sampling. Due probably to the supply of fresh litter, the respiration in the L layer is the same in the winter as in the summer, when high temperatures have a stimulatory effect on respiration. In the F and H layers the composition of the material is much more constant, and there is a distinct effect of soil temperature on respiration corresponding with a Q10 of 2. It must be taken into account here, however, that in the H layer root respiration accounts on average for 40 per cent of the total respiration. The moisture content of the decomposing material seems to have a marked effect only in the L layer during prolonged periods of drought. No relationship was found between the level of respiration and the degree of mycelial growth on nylon gauze, although the main contribution to respiration was made by fungi. The respiration of the various litter layers represents an energy consumption which is in good agreement with the annual decomposition of organic matter in the soil. R.I.N.-communication nr. 102. R.I.N.-communication nr. 102.     

Effects of heat on the germination of sclerophyllous forest species in the highlands of Madagascar

The effects of fire on germination have been extensively studied in many ecosystems. Several studies have shown that plant species in ecosystems frequently exposed to fire can survive through two main mechanisms: vegetative regeneration (re‐sprouts) and recruitment of new individuals from a fire‐resistant seed bank. In Africa, an increase in temperature can break seed dormancy and stimulate germination of some herbaceous and woody species. In Madagascar, the once widespread highland ecosystems dominated by woody species are now highly fragmented and dominated by anthropic grasslands and fields, with a significantly reduced area occupied by sclerophyllous forests referred to as ‘tapia woodlands’. Six species of this endemic vegetation type were studied: Abrahamia ibityensis (Anacardiaceae), Aphloia theiformis (Aphloiaceae), Carissa edulis (Apocynaceae), Pentachlaena latifolia (Sarcolaenaceae), Uapaca bojeri (Phyllanthaceae) and Vaccinium secundiflorum (Ericaceae). Germination tests were conducted (i) by soaking seeds in water for 24 h (imbibition) or (ii) by exposing the seeds to dry heat. Four different temperatures (40, 60, 80 and 120°C) were applied, and seeds were exposed for 10, 30, 60 and 90 min. To simulate hotter faster‐burning fires, two higher temperatures (100 and 120°C) were also used by exposing seeds to dry heat for 5 min. The results did not reveal any significant effect of 24‐h water imbibition on germination. For most species, germination decreased with increasing temperature of treatment using dry heat. Uapaca bojeri did not germinate under any treatment. Further studies on the biological and ecological characteristics of tapia woodland species in response to fire are needed to help guide conservation, management and restoration activities focusing on this endemic vegetation type.     

Estimation of fungal biomass in forest litter and soil

The contents of fungal biomass markers were analysed in the fruit bodies of dominant basidiomycetes from an ectomycorrhiza-dominated coniferous forest, and used to estimate the fungal biomass content in the litter and soil. The content of ergosterol (3.8 ± 2.0 mg g^?1 dry fungal biomass) and the phospholipid fatty acid 18:2ω6,9 (11.6 ± 4.3 mg g^?1) showed less variation than the internal transcribed spacer (ITS) copy numbers (375 ± 294 × 10⁹ copies g^?1). A high level of variation in the ITS copy numbers (per ng DNA) was also found among fungal taxa. The content of fungal biomass in the litter and soil, calculated using the mean contents, varied between 0.66 and 6.24 mg g^?1 fungal biomass in the litter, and 0.22 and 0.68 mg g^?1 in the soil. The ratio of fungal biomass in the litter to that in the soil varied greatly among the markers. The estimates of fungal biomass obtained with different biomarkers are not exactly comparable, and caution should be used when analysing taxon abundance using PCR amplification of fungal rDNA.     

Distribution and occurrence of myxomycetes on agricultural ground litter and forest floor litter in Thailand

The ecological distribution and seasonal patterns of occurrence of myxomycetes associated with the microhabitats represented by agricultural ground litter and forest floor litter were investigated in six study sites in northern Thailand during Oct 2004-Oct 2005. Both specimens that developed under natural conditions in the field and specimens obtained in moist chamber cultures were considered. Agricultural study sites were a banana plantation, a mango orchard and a sweet corn farm, whereas forest study sites consisted of a lowland forest, a midelevation forest and a highland forest. Seventy species of myxomycetes representing 20 genera were collected, with 34 species recorded during the dry season and 58 species recorded during the rainy season. Distinct differences were apparent for numbers of positive moist chambers, numbers of species recorded from moist chambers, numbers of species recorded as field collections and numbers of field collections when data obtained for the two seasons were compared. In the dry season only three species were collected in the field, but the percentage of positive moist chamber cultures (129/180 or 72%) and number of species recorded (33) from these cultures were high. In contrast, during the rainy season, 52 species of myxomycetes were collected in the field but only 15 species were recorded from the much lower percentage (75/180 or 42%) of positive moist chambers cultures.     

Review of mixed forest litter decomposition researches

The litter plays an important role in forest ecosystems. Decomposition of mixed leaf litters has recently become an active research area because it mimics the natural state of leaf litters in most of forests. Many studies reported effects of mixing litters on their decomposition, ranging from positive, negative to neutral. In this paper decomposition mechanisms of mixed litters concluded by researchers were summarized. Firstly, plant litter quality had been recognized as an important factor to affect decomposition rate. Some studies showed a positive significant correlation between initial N, P concentration and non-additive effect in litter mixture decomposition. Secondly, it has been suggested that litter mixture could increase abundance and diversity of fauna and microbial decomposers, especially fungi. Thirdly, compared with single litter decomposition, the nutrient exchange between different litter species is often considered as one of main non-additive effects observed in litter mixture. Some results showed that the active transport of nutrients by fungal hyphae derived positive effect on the decomposition of litter mixture. The multiple factors such as, leaf litter species, investigation method and plot, were also analyzed. In conclusion, it is necessary to enhance a further research on factors in mixed litter decomposition and an interaction between various factors due to the complex relationship. We are looking forward to using these theories of mixed litter decomposition to direct practical forest management.     

Review of mixed forest litter decomposition researches

The litter plays an important role in forest ecosystems. Decomposition of mixed leaf litters has recently become an active research area because it mimics the natural state of leaf litters in most of forests. Many studies reported effects of mixing litters on their decomposition, ranging from positive, negative to neutral. In this paper decomposition mechanisms of mixed litters concluded by researchers were summarized. Firstly, plant litter quality had been recognized as an important factor to affect decomposition rate. Some studies showed a positive significant correlation between initial N, P concentration and non-additive effect in litter mixture decomposition. Secondly, it has been suggested that litter mixture could increase abundance and diversity of fauna and microbial decomposers, especially fungi. Thirdly, compared with single litter decomposition, the nutrient exchange between different litter species is often considered as one of main non-additive effects observed in litter mixture. Some results showed that the active transport of nutrients by fungal hyphae derived positive effect on the decomposition of litter mixture. The multiple factors such as, leaf litter species, investigation method and plot, were also analyzed. In conclusion, it is necessary to enhance a further research on factors in mixed litter decomposition and an interaction between various factors due to the complex relationship. We are looking forward to using these theories of mixed litter decomposition to direct practical forest management.     

Streptosporangium isolated from forest litter in the Netherlands

Summary The organism described byCouch asStreptosporangium was isolated from forest litter in the Netherlands and Denmark. The development of this organism is followed in detail. Tevens verschenen als Mededeling Nr.36 I.T.B.O.N.     

Nutrients in the litter on jarrah forest soils

The amount of litter and its nutrient composition have been measured at seven sites on various lateritic soils within the jarrah (Eucalyptus marginata Donn ex Sm.) forest near Dwellingup, Western Australia. The weight of litter accumulated during 6 years ranges from 9 tonnes/ha for forest growing on yellow sand to 18 tonnes/ha for forest on reddish gravels. The litter on the reddish gravels contains more than twice the amounts of N, P, K and S in litter on yellow sand and grey and yellow gravels. The proportion of fine material in the forest floor litter increases with total litter weight. Phosphorus, which is less mobile than other nutrients tends to accumulate in this fine component. There are large differences between the foliar nutrient levels of jarrah and Banksia grandis Willd. (e.g. P: 0.041%, 0.025%; K: 0.57%, 0.34%; Mg: 0.43%, 0.21%; Mn: 177 μg/g. 730 μg/g). However, these differences are not reflected in the litter from sites with and without B. grandis understorey. Soil differences and the predominant contribution of the overstorey to the litter appear to be the main factors affecting the litter composition.     

森林凋落物与大型土壤动物相关关系的研究

以针阔混交林及落叶阔叶林为例,探讨了森林凋落物与大型土壤动物的相关关系。研究表明,森林凋落物中大型土壤动物的个体数依林型、凋落物厚度、凋落物量不同而异,凋落物的分解程度影响大型土壤动物的种类、数量分布,凋落物中矿质元素含量与大型土壤动物体内矿质元素含量相关较好,凋落物中微量元素含量与大型土壤动物体内微量元素含量明显不相关。本研究为揭示大型土壤动物在凋落物分解中的作用,进而为森林的经营和保护提供科学依据。     

森林凋落物分解重要影响因子及其研究进展

当前 ,森林凋落物分解被放在陆地生态系统碳平衡背景下进行研究 ,认识凋落物分解过程的影响因素和影响机理对理解地表碳平衡具有重要意义。凋落物在分解过程中 ,伴随有养分含量的变化 ,低品质凋落物在分解前期 (可达 2～ 3年 )会从环境中固定养分 ,特别是氮磷养分 ,而在后期则会释放出养分。凋落物本身的养分含量是影响分解速率的重要因素 ,高养分含量的凋落物分解快些 ,阔叶凋落物比针叶凋落物分解快些。有资料显示 ,在总分解率为2 9 4 %的构成中 ,理化因素、微生物因素与土壤动物因素对凋落物分解的贡献率分别为 7 2 %、8 0 %和 14 2 %。不同类型凋落物在分解过程中的土壤动物类群也不同 ,它也是造成凋落物分解速率不同的关键因素 ,通常阔叶树种凋落物分解过程中 ,会有更多的微节肢动物出现。CO2浓度升高将造成植物有机质含碳量与其它养分的比值升高 ,形成低品质的凋落物 ,从而间接影响凋落物分解速率 ,一般认为 ,全球CO2 浓度升高会加强土壤作为碳汇的功能。     

里氏木霉纤维素酶的分离纯化及酶学性质

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104。EGⅡ的最适反应pH是5.6,最适反应温度为65℃;EGⅠ的最适反应pH是4.4,最适反应温度为55℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km)分别为2.20 mg/mL、3.38 mg/mL。CBHⅠ的最适反应pH是5.8,最适反应温度为60℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km)为0.12 mg/mL。