纤维素降解真菌A25-2的分离、鉴定及其产纤维素酶的酶学特性

本研究以Avicel-刚果红选择培养基为初筛培养基,从云南哀牢山国家级自然保护区和广西猫儿山国家级自然保护区的土壤样品中分离筛选得到4200株真菌,从中筛选出透明圈与菌落直径比较大、透明程度较为清晰的12个菌株。通过液体培养发酵,测定其上清液中的羧甲基纤维素酶活力、滤纸酶活力和Avicel酶活力,最终筛选出一株产该三种酶且其活力均最高的真菌菌株A25-2。通过对菌株A25-2形态学观察和其内转录间隔区（internal transcribed spacer,ITS）序列同源性比对分析,将菌株A25-2鉴定为哈茨木霉（Hypocrea lixii）。酶活测定结果表明菌株A25-2产纤维素酶的酶活力较高,在最适作用pH4.5和最适作用温度55℃下,其羧甲基纤维素酶活力为2.26IU/mL,滤纸酶活力为0.58IU/mL,Avicel酶活力为0.39IU/mL。薄层层析实验表明A25-2具有完整的纤维素酶系统。因此,真菌A25-2可作为饲料加工等生产和纤维素酶相关研究的备选菌株。     

商陆根部多糖的分离、纯化及其体外抗氧化活性

本研究从商陆根部提取分离出商陆多糖,并对该多糖含量进行测定及体外抗氧化活性研究。商陆根部经石油醚回流脱脂、热水抽提、脱色、乙醇沉淀等处理可从中提取出粗多糖。多糖经三氯乙酸脱蛋白纯化,再分别以考马斯亮蓝法和蒽酮-硫酸法测定其蛋白质含量和糖含量,并通过采用清除羟自由基和超氧自由基能力测定法、β-胡萝卜素-亚油酸法,以抗坏血酸（Vc）为对照,来分析粗多糖和纯多糖的体外抗氧化能力。采用水提法提取的粗多糖,含有10.69%的蛋白质和60.91%的多糖;纯化后的多糖糖含量达到91.04%。商陆粗多糖、纯多糖及Vc对羟自由基（·OH）清除率的IC50分别是1.26mg/mL、4.78mg/mL和0.224mg/mL;对超氧自由基（O2-·）清除率的IC50分别为1.91mg/mL、2.28mg/mL和0.123mg/mL;对β-胡萝卜素-亚油酸体系的抑制作用的IC50分别为0.471mg/mL、0.692mg/mL和0.379mg/mL。实验结果表明商陆多糖是一种较好的抗氧化剂,可作为潜在天然抗氧化剂应用于食品和医药工业中。     

Effect of lithological substrate on microbial biomass and enzyme activity in brown soil profiles in the northern Apennines (Italy)

The aim of the present study was to investigate the microbial activity along forest brown soil profiles sequence developed on different lithological substrates (carbonate or non-carbonated cement in sandstone formations) at different altitudes. The main question posed was: does carbonate affect the biochemical activity of brown soil profiles at different altitudes? For the purpose of this study, four soil profiles with different amounts and compositions of SOM developed on different lithological substrates were selected: two with carbonate (MB and MZ) and the other two with non-carbonated cement in the sandstone formations (MF1 and MF2). Chemical and biochemical properties of soil were analysed along soil profiles in order to assess the SOM quantity and quality, namely total organic C (C_org), water extractable organic C (WEOC) and humification indices (HI, DH, HR). Microbial biomass (C_mic and N_mic) content, as well as the specific activities of acid phosphatase, β-glucosidase and chitinase enzymes were chosen as indicators of biochemical activity. The soil biochemical properties provided evidence of better conditions for microorganisms in MB than in MF1, MF2 and MZ soil profiles, since patterns of microbial biomass content and activity might be expected in response to the amount and quality of organic substances. The different lithological substrates did not show any clear effect on soil microbial biomass content, since similar values were obtained in MF1, MF2 (with non-carbonated cement) and MZ (with carbonate). However, the specific activities of acid phosphatase (per unit of C_org and per unit of C_mic) were higher in soils with no carbonate (MF1 and MF2) than in soils with carbonate (MB and MZ). In conclusion, the biochemical activity along brown soil profiles was mainly regulated by different soil organic matter content and quality, while the two different lithological substrates (with carbonate or non-carbonated cement in the sandstone formations) did not show any direct effect on microbial biomass and its activity. However, the activity of acid phosphatase per unit of C was particularly enhanced in soil with non-carbonate cement in the sandstone formations.     

Microbial respiration activities related to sequentially separated, particulate and water-soluble organic matter fractions from arable and forest topsoils

This study aimed to reveal differences in the relevance of particulate as well as water-soluble organic matter (OM) fractions from topsoils to the easily biodegradable soil organic matter (SOM). We selected eight paired sites with quite different soil types and soil properties. For each of these sites, we took samples from adjacent arable and forest topsoils. Physically uncomplexed, macro-, and micro-aggregate-occluded organic particle, as well as water-soluble OM fractions were sequentially separated by a combination of electrostatic attraction, ultrasonic treatment, density separation, sieving, and water extraction. The easily biodegradable SOM of the topsoil samples was determined by measuring microbial respiration during a short-term incubation experiment (OC_R). The organic carbon (OC) contents separated by i) the physically uncomplexed water-soluble OM, ii) the macro-, and iii) the micro-aggregate-occluded organic particle as well as water-soluble OM fractions were significantly correlated with OC_R. The correlation coefficients vary between 0.54 and 0.65 suggesting differences in the relevance of these OM fractions to the easily biodegradable SOM. The strongest correlation to OC_R was detected for the OC content separated by the physically uncomplexed water-soluble OM indicating the most distinct relation to the easily biodegradable SOM. This was found to be independent from land use or soil properties.     

Investigating the mechanisms for the opposing pH relationships of fungal and bacterial growth in soil

Soil pH is one of the most influential variables in soil, and is a powerful factor in influencing the size, activity and community structure of the soil microbial community. It was previously shown in a century old artificial pH gradient in an arable soil (pH 4.0-8.3) that bacterial growth is positively related to pH, while fungal growth increases with decreasing pH. In an attempt to elucidate some of the mechanisms for this, plant material that especially promotes fungal growth (straw) or bacterial growth (alfalfa) was added to soil samples of the pH gradient in 5-day laboratory incubation experiments. Also, bacterial growth was specifically inhibited by applying a selective bacterial growth inhibitor (bronopol) along the entire pH gradient to investigate if competitive interaction caused the shift in the decomposer community along the gradient. Straw benefited fungal growth relatively more than bacterial, and vice versa for alfalfa. The general pattern of a shift in fungal:bacterial growth with pH was, however, unaffected by substrate additions, indicating that lack of a suitable substrate was not the cause of the pH effect on the microbial community. In response to the bacterial growth inhibition by bronopol, there was stimulation of fungal growth up to pH 7, but not beyond, both for alfalfa and straw addition. However, the accumulation of ergosterol (an indicator of fungal biomass) during the incubation period after adding alfalfa increased at all pHs, indicating that fungal growth had been high at some time during the 5-day incubation following joint addition of alfalfa and bronopol. This was corroborated in a time-series experiment. In conclusion, the low fungal growth at high pH in an arable soil was caused to a large extent by bacterial competition, and not substrate limitation.     

Litter- and ecosystem-driven decomposition under elevated CO2 and enhanced N deposition in a Sphagnum peatland

Peatlands represent massive global C pools and sinks. Carbon accumulation depends on the ratio between net primary production and decomposition, both of which can change under projected increases of atmospheric CO₂ and N deposition. The decomposition of litter is influenced by 1) the quality of the litter, and 2) the microenvironmental conditions in which the litter decomposes. This study aims at experimentally testing the effects of these two drivers in the context of global change. We studied the in situ litter decomposition from three common peatland species (Eriophorum vaginatum, Polytrichum strictum and Sphagnum fallax) collected after one year of litter production under pre-treatment conditions (elevated CO₂: 560 ppm or enhanced N: 3 g m⁻² y⁻¹ NH₄NO₃) and decomposed the following year under treatment conditions (same as pre-treatment). By considering the cross-effects between pre-treatments and treatments, we distinguished between the effects on mass loss of 1) the pre-treatment-induced litter quality and 2) the treatment conditions under which the litters were decomposing. The combination between CO₂ pre-treatment and CO₂ treatment reduced Polytrichum decomposition by −24% and this can be explained by litter quality-driven decomposition changes brought by the pre-treatment. CO₂ pre-treatment reduced Eriophorum litter quality, although this was not sufficient to predict decomposition. The N addition pre-treatment reduced the decomposition of Eriophorum, due to enhanced lignin and soluble phenols concentrations in the initial litter, and reduced litter-driven losses of starch and enhanced litter-driven losses of soluble phenols. While decomposition indices based on initial litter quality provide a broad explanation of quantitative and qualitative decomposition, they can only be taken as first approximations. Indeed, the microbial ATP activity, the litter N loss and resulting litter quality, were strongly altered irrespective of the compounds' initial concentration and by means of processes that occurred independently of the initial litter-qualitative changes. The experimental design was valuable to assess litter- and ecosystem-driven decomposition pathways simultaneously or independently. The ability to separate these two drivers makes it possible to attest the presence of litter-qualitative changes even without any litter biochemical determinations, and shows the screening potential of this approach for future experiments dealing with multiple plant species.     

Priming effects: Interactions between living and dead organic matter

In this re-evaluation of our 10-year old paper on priming effects, I have considered the latest studies and tried to identify the most important needs for future research. Recent publications have shown that the increase or decrease in soil organic matter mineralization (measured as changes of CO₂ efflux and N mineralization) actually results from interactions between living (microbial biomass) and dead organic matter. The priming effect (PE) is not an artifact of incubation studies, as sometimes supposed, but is a natural process sequence in the rhizosphere and detritusphere that is induced by pulses or continuous inputs of fresh organics. The intensity of turnover processes in such hotspots is at least one order of magnitude higher than in the bulk soil. Various prerequisites for high-quality, informative PE studies are outlined: calculating the budget of labeled and total C; investigating the dynamics of released CO₂ and its sources; linking C and N dynamics with microbial biomass changes and enzyme activities; evaluating apparent and real PEs; and assessing PE sources as related to soil organic matter stabilization mechanisms. Different approaches for identifying priming, based on the assessment of more than two C sources in CO₂ and microbial biomass, are proposed and methodological and statistical uncertainties in PE estimation and approaches to eliminating them are discussed. Future studies should evaluate directions and magnitude of PEs according to expected climate and land-use changes and the increased rhizodeposition under elevated CO₂ as well as clarifying the ecological significance of PEs in natural and agricultural ecosystems. The conclusion is that PEs - the interactions between living and dead organic matter - should be incorporated in models of C and N dynamics, and that microbial biomass should regarded not only as a C pool but also as an active driver of C and N turnover.     

Hydrolysis of cellobiose by β-glucosidase in the presence of soil minerals - Interactions at solid-liquid interfaces and effects on enzyme activity levels

Extracellular enzymatic activities in soils are essential for the cycling of organic matter. These activities take place in multiphase environments where solid phases profoundly affect biocatalytic activities. Aspergillus niger is ubiquitous in soils; its β-glucosidase plays an important role in the degradation of cellulose, and therefore in the global carbon cycle and in the turnover of soil organic matter. However, the information on the interactions of this protein with soil minerals is very limited, and even less is known about their consequences for the hydrolysis of the natural substrate cellobiose. We therefore characterised the sorptive interactions of this enzyme with the soil minerals montmorillonite, kaolinite and goethite and quantified the resulting changes in the hydrolysis rate of cellobiose. Fractions of adsorbed protein, and the resulting catalytic activity loss, were lower for montmorillonite than for kaolinite and goethite at given experimental conditions; adsorption was 9.7 ± 7.3% for montmorillonite, 70.3 ± 3.1% for kaolinite and 71.4 ± 1.8% for goethite, respectively. Adsorption of the protein to the minerals caused a total decrease in the catalytic activity of 18.8 ± 3.4% for kaolinite and 17.9 ± 4.7% for goethite whereas it was not significant for montmorillonite. The average catalytic activity lost by the pool of adsorbed molecules was 26.8% for kaolinite and 25.0% for goethite. Both the amount of adsorbed protein and the resulting loss of catalytic activity were found to be independent of the specific surface areas yet were influenced by the electrical properties of the mineral surfaces. Under the experimental conditions, montmorillonite and kaolinite are negatively charged whereas goethite is positively charged. However, because of the adsorption of phosphate anions from the buffer, a charge reversal took place at the surface of goethite. This was confirmed by zeta (ζ)-potential measurements in phosphate buffer, revealing negative values for all the tested minerals. Indeed goethite interacted with the enzyme as a negatively charged surface: the amount of adsorbed protein and the resulting catalytic activity loss were very similar to those of kaolinite. Our results show that, even if an important fraction of β-glucosidase is adsorbed to the minerals, the catalytic activity is largely retained. We suggest that this strong activity retention in presence of soil minerals results from a selective pressure on A. niger, which benefits from the activity of the adsorbed, and thus stabilized, enzyme pool.     

Selection of Variovorax paradoxus-like bacteria in the mycosphere and the role of fungal-released compounds

At two occasions (2004 and 2006), a similar cluster of culturable bacteria was found to be selected in the mycosphere of the basidiomycetous fungus Laccaria proxima (Agaricales, Tricholomataceae) in the field. The bacteria, identified as related to Variovorax paradoxus, comprised 7.3-9% of the total culturable bacterial community in the L. proxima mycosphere, but were not found in corresponding bulk soil (<0.3%). One strain, denoted HB44, was selected in order to unravel the basis of the V. paradoxus mycosphere competence in in vitro experiments with the former Laccaria laccata, which was recently reclassified as Lyophyllum sp. strain Karsten (Agaricales, Tricholomataceae). In liquid culture experiments, L. strain Karsten was shown to be an avid producer of glycerol, next to acetate and formate, which constituted the most abundant carbonaceous compounds released. Strain HB44 was able to grow avidly at the expense of the glycerol liberated by the fungus, as evidenced by proton NMR analysis of the fungal exudates in the medium before and after bacterial growth. In sterilized field soil, strain HB44 survived significantly better in the presence than in the absence of L. strain Karsten. Addition of a glycerol series to the sterilized soil (without the fungus) resulted in the persistence or growth of strain HB44, but only if the pH of this soil was previously set at 5.5. Thus, we provide evidence for the contention that tricholomataceous fungi can create specific niches in soil for the V. paradoxus-related strain HB44 and that glycerol may be a main carbon source that drives the selection of this organism.     

供铁水平对不同苹果基因型生长及根系特性的影响

以小金海棠 (MalusxiaojinensisChengetJiang)和山定子 (MalusbaccataL .Borkh)为试材 ,研究供铁水平对其生长及根系特性的影响 ,结果表明 ,在幼苗生长量、新叶叶绿素含量、营养液pH日变化、根系活力、根系ATP酶活性诸方面 ,存在基因型差异。同一供铁水平 ,小金海棠有明显的铁胁迫反应 ,而山定子铁胁迫反应较弱。低铁胁迫处理 ,小金海棠叶片叶绿素含量、根系活力、根系ATP酶活性皆明显高于山定子。根系活力及根系ATP酶活性可用作铁高效基因型植物的筛选指标