Quantitative Isolation of Microbial DNA from Different Types of Soils of Natural and Agricultural Ecosystems

A novel procedure was developed for direct quantitative isolation of microbial DNA from soil. This technique was used to evaluate microbial DNA pools in soils of contrasting types (chernozems and brown forest soils) under different anthropogenic loads. A strong correlation was found between microbial biomass and DNA contents in soils of different types (R ²= 0.799). The ratio of soil CO₂ emission rate to the amount of extractable DNA in the soil was shown to reflect the physiological state of the soil microbial community; this ratio can be used as an ecophysiological parameter similarly to the metabolic quotient qCO₂.     

Reassessment of the Microbial Role in Mn-Fe Nodule Genesis in Andean Paleosols

The presence of Mn-Fe nodules in the epipedons (surface horizons) of paleosols of presumed Upper Neogene age in the northwestern Venezuelan Andes have been interpreted as products of inorganic oxidation and reduction processes operating over the full range of glacial and interglacial cycles that affected paleosol morphogenesis. New microscopic/chemical data from combined SEM-EDS-FIB analyses of representative Mn-Fe nodules indicate microbes play an important role in Mn/Fe precipitation leading to their genesis in alpine Mollisols (Argiustolls). Although the prevailing new data are based mainly on fossil forms of filamentous bacteria and fungi and other biogenic pseudomorphs that may represent the former resident bacteria, the presence of extant microbes must await field experiments/collection, followed by a molecular microbiology approach to determine the biological drivers of metal precipitation. As in other terrestrial niche environments, microbes are seen here to play a role, perhaps a key one, in the morphogenesis of paleosols of importance in upper Neogene paleoenvironmental reconstruction.     

Microcalorimetric assessment of microbial activity in long-term fertilization experimental soils of Southern China

Microcalorimetry, plate count and PCR–denaturing gradient gel electrophoresis (DGGE) were employed to investigate microbial diversity and activity in soils from the Red Soil Experimental Station of the Chinese Academy of Agricultural Sciences, Hunan Province, China, where a wheat–corn rotation with 12 fertilization treatments was established in 1990. Fertilization greatly increased microbial biomass carbon (C) and nitrogen (N) (C_mic and N_mic) as well as the activities of phosphatase, urease, invertase, protease, catalase and dehydrogenase. Manure alone (M) enhanced the number of denitrifying and aerobic bacteria by 54.4% and 20.5%, respectively, whereas fallow (H) increased the number of aerobic cellulose decomposing bacteria by 31.4%. Fallow and soils amended with mineral fertilizers plus pig manure or straw increased both the DGGE band patterns and the Shannon index compared with mineral fertilizers or the control. Mineral treatments with lower bacterial numbers enhanced the values of the peak time ( t _max) more than did organic treatments. The peak height ( P _max) was positively correlated ( P <0.01), with soil enzymes, C_mic and N_mic, and the number of microorganisms, whereas the peak time ( t _max) was negatively connected ( P <0.01) with these parameters. The microbial growth rate constant ( k ) was linked to bacteria ( P <0.01), actinomycetes ( P <0.05) and catalase ( P <0.05). The total heat evolution ( Q ) had no relationships with any soil microbial properties (except for catalase). We propose that P _max and t _max could be used as indices of soil microbial activity, while the values of k and Q are poor indicators.     

复合微生态菌剂对黄瓜根际土壤微生物数量及酶活的影响

采用盆栽试验的方法,研究黄瓜移苗时施入复合微生态菌剂对黄瓜不同生育时期根际土壤中微生物数量和土壤酶活动态变化的影响,结果显示复合微生态菌剂施入前期可提高土壤中细菌和放线菌的数量,随生育时期的延长效果变弱,于生育后期可有效降低真菌和尖孢镰刀菌的数量;复合微生态菌剂处理可提高土壤脲酶、过氧化氢酶、蔗糖酶和中性磷酸酶的活性,但随生育时期的延长,对中性磷酸酶和脲酶的效果减弱。可见复合土壤微生态菌剂的施入应该于适宜时期补施,才能保证其积极的功效。     

黄土高原石油污染土壤微生物群落结构及其代谢特征

针对污染胁迫下土壤微生物群落变化和代谢变异等问题,基于平板稀释法和Biolog微平板分析方法,研究了陕北黄土高原石油污染土壤微生物群落结构、代谢特征及其功能多样性。结果表明,不同类群的土壤微生物对石油污染胁迫的响应不同,污染土壤细菌和真菌数量高出清洁土壤1个数量级,而污染土壤的放线菌数量极显著减少(P0.01);污染土壤和清洁土壤微生物对糖类和多聚物类碳源较易利用,污染土壤微生物总体上代谢碳源的种类和活性均低于清洁土壤。微生物群落主成分分析(PCA)表明,石油污染土壤和清洁土壤的微生物群落存在显著差异(P0.01),起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类;随着土壤石油含量增加,典型变量值变异(离散)增大,土壤微生物群落结构稳定性降低。微生物群落多样性分析表明,Shannon丰富度指数(H)、McIntosh均一度指数(U)和Simpson优势度指数(1/D)均达到极显著差异(P0.01),污染土壤微生物群落H和U低于清洁土壤,但是一定浓度的石油污染可以刺激土壤微生物群落中优势种群的生长,1/D增高。研究结果为陕北黄土高原石油污染区土壤微生物修复提供理论基础。     

Microbial Community Structures in Terrestrial Subsurface Sediments from the Southern Kanto Plain,Japan

Abstract

Microbial community structure reflects the surrounding natural environment and changes to that environment. Although the subsurface at 5–100?m depth is important for human activities and there are potential risks of environmental pollution in this region, there have been only a few reports of subsurface microbial community structures in terrestrial areas. We investigated the diversity and community compositions of Bacteria and Archaea in boring cores collected from various depths at three different sites in the southern Kanto Plain, Japan. The results of 16S rRNA gene amplicon sequencing using MiSeq showed that the microbial community composition varied with the geological unit. Proteobacteria (Alphaproteobacteria and Gammaproteobacteria) were dominant members within sediments accumulated during the Pleistocene in the Musashino Upland. In contrast, Acidobacteria and Chloroflexi characteristically appeared in the Holocene layers of the Arakawa Lowland. These data suggest that the subsurface microbial composition is controlled by the geological features of the sediments.     

Microbial community dynamics and evaluation of bioremediation strategies in oil-impacted salt marsh sediment microcosms

Microbial community dynamics in wetlands microcosms emended with commercial products (surfactant, a biological agent, and nutrients) designed to enhance bioremediation was followed for 3 months. The effectiveness of enhanced degradation was assessed by determining residual concentrations of individual petroleum hydrocarbons by GC/MS. The size and composition of the sediment microbial community was assessed using a variety of indices, including bacterial plate counts, MPNs, and DNA hybridizations with domain- and group-specific oligonucleotide probes. The addition of inorganic nutrients was the most effective treatment for the enhancement of oil degradation, resulting in marked degradation of petroleum alkanes and a lesser extent of degradation of aromatic oil constituents. The enhanced degradation was associated with increases in the amount of extractable microbial DNA and Streptomyces in the sediment, although not with increased viable counts (plate counts, MPN). Bacteria introduced with one of the proprietary products were still detected in the microcosms after 3 months, but were not a major quantitative constituent of the community. The biological product enhanced oil degradation relative to the control, but to a lesser extent than the nutrient additions alone. In contrast, application of the surfactant to the oil-impacted sediment decreased oil degradation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 72–79. Received 18 March 2001/ Accepted in revised form 09 June 2001     

新疆绿洲农田不同连作年限棉花根际土壤微生物群落多样性

以南北疆不同连作年限棉花根际土壤为研究对象,采用Biolog技术,并结合传统平板培养法和土壤酶的测定,研究连作对棉花根际土壤微生物群落多样性的影响。Biolog分析结果表明,不同连作年限棉花根际土壤微生物碳源利用和功能多样性差异显著。荒地土壤微生物活性较低;在连作年限较短时(5—10a),根际土壤微生物群落的平均颜色变化率(AWCD)和Shannon指数较高;长期连作(15—20a),则呈明显下降趋势。主成分分析表明,不同连作年限的棉花根际土壤微生物碳源利用特征有明显不同。第一、二组开垦与未开垦土壤分别在PC1和PC2上出现差异,未开垦土壤得分均为负值,开垦土壤均为正值;而正茬与连作多年的棉花土壤在PC1上差异显著。其中在PC1上起分异作用的碳源主要是羧酸类和聚合物类,这两类碳源可能是影响连作棉花根际土壤微生物的主要碳源。可培养微生物数量的测定结果表明,荒地细菌数量最少;在连作年限较低时(5—10a左右),细菌数量呈上升趋势;而长期连作(>15a)后,细菌数量呈现下降趋势。真菌数量在连作多年后(10—15a)也开始增加。放线菌变化趋势不明显。四种土壤酶活性在连作的初中期(5—15a),连作障碍表现明显,土壤酶活性呈下降(过氧化氢酶和磷酸酶)或先升高后下降(脲酶和蔗糖酶)趋势,但随着连作年限的延长(15—20a),这4种土壤酶活性均表现出增高趋势。综上所述,棉花长期连作使棉花根际土壤微生物群落多样性降低,发生连作障碍,进而导致棉花产量降低。     

Microbial Activity in High-Sulfur Reclaimed Coal Mine Overburden Sites

The high-sulfur containing coalmine overburden (OB) dumping ground of the Tirap colliery under the northeastern coalfields in Assam, India, was reclaimed by phytoremediation techniques. Native plant species were planted with line and field methods and by amending with cow dung and unmined soil. In this study, the changes in microbial activity of the reclaimed mine OB wasteland of the Tirap colliery are examined. The enzyme activities of urease, dehydrogenase, and phosphatase were compared over time. Improvements in soil pH, bulk density, and water-holding capacity in the mine OB were found. After four years of reclamation, 1.01% of particles were soil sized (=0.255 mm grain size) with the maximum degradation in the mine OB grain sizes of ?20 mm to +12 mm and of +25 mm. A 1,000-fold increase in microbial colony forming units (CFU) in the four years at the remediated site along with an increase in total DNA were evident for the mine OB reclaimed sites. The increase of microbial CFUs in the mine OB was related to a 100- to 2,000-fold increase in enzyme activity for the reclaimed areas. From this investigation, it can be concluded that the improvement of enzyme activities and the increase in microbial populations of reclaimed mine OB will yield information pertaining to the reclamation index of mine OB.     

热带次生林利用方式与土壤微生物活性

热带次生林利用方式与土壤微生物活性许炼烽,朱伍坤（国家环保局华南环境科学研究所,广州５１０６５５）ＷａｙｓｏｆＵｔｉｌｉｚｉｎｇＴｒｏｐｉｃａｌＳｅｃｏｎｄａｒｙＦｏｒｅｓｔａｎｄＳｏｉｌＭｉｃｒｏｂｉａｌＡｃｔｉｖｉｔｙ．￥ＸｕＬｉａｎｆｅｎｇ;Ｚ...     

污染土壤微生物群落结构分子鉴定技术研究进展

土壤微生物群落结构多样性检测是土壤修复、监测、评估时的一个重要参数。由于绝大多数微生物在实验室条件下是不可培养,因而早期依赖于微生物培养的检测结果代表性不强。20世纪90年代以来,不依赖于微生物培养的分子生物学方法是研究微生物群落结构的重要手段。该文对近年来土壤污染微生物群落结构研究所采用的主要分子生物学方法按照其原理进行了比较、分析、总结。根据不同技术的灵敏度、优缺点分析了其适用范围。指出了目前技术中存在的一些共性问题和缺陷并展望了土壤修复领域分子生物学技术的发展趋势。     

狮子头热泉菌席样品环境总DNA提取方法的比较研究

通过对狮子头热泉7个环境菌席样品所提取的总DNA进行纯度检测、提取得率计算和DGGE分析,比较了3种直接和1种间接DNA提取方法。结果表明：综合利用多种裂解方式比单一裂解方式更能充分释放环境DNA;其中3种方法获得的DNA片段能够进行后续16S rDNA扩增;针对同一样品,不同方法提取的环境DNA,可获得不同DGGE群落指纹图谱;间接提取法提取的总DNA,能更好地反映狮子头热泉菌席的微生物多样性。     

Microbial characteristics of soils on a latitudinal transect in Siberia

Soil microbial properties were studied from localities on a transect along the Yenisei River, Central Siberia. The 1000 km‐long transect, from 56°N to 68°N, passed through tundra, taiga and pine forest characteristic of Northern Russia. Soil microbial properties were characterized by dehydrogenase activity, microbial biomass, composition of microbial community (PLFAs), respiration rates, denitrification and N mineralization rates. Relationships between vegetation, latitude, soil quality (pH, texture), soil organic carbon (SOC) and the microbial properties were examined using multivariate analysis. In addition, the temperature responses of microbial growth (net growth rate) and activity (soil respiration rate) were tested by laboratory experiments. The major conclusions of the study are as follows: 1. Multivariate analysis of the data revealed significant differences in microbial activity. SOC clay content was positively related to clay content. Soil texture and SOC exhibited the dominant effect on soil microbial parameters, while the vegetation and climatic effects (expressed as a function of latitude) were weaker but still significant. The effect of vegetation cover is linked to SOC quality, which can control soil microbial activity. 2. When compared to fine‐textured soils, coarse‐textured soils have (i) proportionally more SOC bound in microbial biomass, which might result in higher susceptibility of SOC transformation to fluctuation of environmental factors, and (ii) low mineralization potential, but with a substantial part of the consumed C being transformed to microbial products. 3. The soil microbial community from the northernmost study region located within the permafrost zone appears to be adapted to cold conditions. As a result, microbial net growth rate became negative when temperature rose above 5 °C and C mineralization then exceeded C accumulation.     

不同生态条件下油松(Pinus tabulaeformis)菌根根际土壤微生物群落

利用BIOLOG代谢指纹方法分析了陕南商南和陕北安塞不同生态条件下油松菌根根际土壤微生物群落。结果表明,安塞油松和商南油松菌根根际微生物对糖类和氨基酸类碳源较易利用,商南油松菌根根际微生物总体上代谢碳源的种类和活性远大于安塞油松,而且对同类碳源的代谢商南油松的AWCD比安塞油松均高出2倍多。安塞油松菌根根际微生物以氨基酸类代谢群为优势类群,商南油松以糖类代谢群为优势类群。微生物群落多样性指数和微生物群落主成分分析（PCA）指标均表明商南油松和安塞油松菌根根际土壤微生物群落有明显不同,起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类。商南油松菌根根际土壤微生物群落AWCD极显著高于安塞油松（P〈0．01）,细菌数量显著高于安塞油松（P〈0．05）,Shannon指数和丰富度指数达极显著性差异（P〈0．01）,商南油松和安塞油松菌根侵染率差异不显著,但菌根生物量差异达极显著水平（P〈0．01）。相关性分析表明,菌根生物量与丰富度指数、AWCD呈极显著正相关,与Shannon指数呈显著正相关,但是与菌根侵染率相关性不显著。在商南温暖潮湿丘陵区油松菌根根际微生物活性、群落大小和多样性高于安塞油松,在安塞黄土高原干旱区微生物群落稳定性强于商南油松。     

重离子诱变微生物育种的研究进展

近年来,重离子束作为一种新的辐射诱变源在微生物育种领域已多有应用.与传统的辐照源相比,重离子束具有更高的传能线密度,可以产生更强的辐射损伤生物效应,因此诱变效率高.本文综述了近年来重离子诱变在微生物育种中取得的进展、诱变后突变体菌株的筛选策略、重离子束引起微生物遗传物质改变的直接和间接机制以及突变后的修复机理,并对其在...     

不同生态条件下油松(Pinus tabulaeformis)菌根根际土壤微生物群落

利用BIOLOG代谢指纹方法分析了陕南商南和陕北安塞不同生态条件下油松菌根根际土壤微生物群落。结果表明,安塞油松和商南油松菌根根际微生物对糖类和氨基酸类碳源较易利用,商南油松菌根根际微生物总体上代谢碳源的种类和活性远大于安塞油松,而且对同类碳源的代谢商南油松的AWCD比安塞油松均高出2倍多。安塞油松菌根根际微生物以氨基酸类代谢群为优势类群,商南油松以糖类代谢群为优势类群。微生物群落多样性指数和微生物群落主成分分析（PCA）指标均表明商南油松和安塞油松菌根根际土壤微生物群落有明显不同,起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类。商南油松菌根根际土壤微生物群落AWCD极显著高于安塞油松（P<0.01）,细菌数量显著高于安塞油松（P<0.05）,Shannon指数和丰富度指数达极显著性差异（P<0.01）,商南油松和安塞油松菌根侵染率差异不显著,但菌根生物量差异达极显著水平（P<001）。相关性分析表明,菌根生物量与丰富度指数、AWCD呈极显著正相关,与Shannon指数呈显著正相关,但是与菌根侵染率相关性不显著。在商南温暖潮湿丘陵区油松菌根根际微生物活性、群落大小和多样性高于安塞油松,在安塞黄土高原干旱区微生物群落稳定性强于商南油松。     

单细胞、显微计数和高通量测序典型水稻土微生物组的技术比较

【目的】比较传统显微计数、单细胞分选和现代分子方法研究典型水稻土微生物组的细胞数量、物种组成及好氧甲烷氧化菌生理生态过程的技术特点。【方法】针对水稻土中可提取微生物细胞(土壤细胞)及其DNA(细胞DNA)、单细胞DNA、土壤微生物组总DNA(土壤DNA),利用传统显微计数和实时荧光定量PCR(qPCR)方法,研究水稻土好氧甲烷氧化过程中微生物数量的变化规律;通过高通量测序16S rRNA基因技术,研究微生物物种组成的变化规律。【结果】水稻土微生物组的传统显微计数结果显著低于现代分子方法 qPCR,最高可达3个数量级。基于DAPI染色、CARD-FISH、细胞DNA及土壤DNA的qPCR定量结果分别为:(5.8–7.4)×10~7、(1.7–1.9)×10~7、(2.8–6.3)×10~8、(1.5–2.7)×10~(10) cells/g。基于qPCR的水稻土好氧甲烷氧化菌数量为1.1×10~7 cells/g,比传统显微计数方法高3个数量级。然而,当水稻土氧化高浓度甲烷后,所有方法均发现甲烷氧化菌显著增加,增幅分别为54倍(CARD-FISH)、388倍(细胞DNA)和45倍(土壤DNA)。在微生物分类学门的水平,细胞DNA(25个门)与土壤DNA(30个门)结果基本一致,均能较好地反映水稻土微生物组的群落结构,而单细胞DNA尽管检测到20个门,但偏好性较大,95%以上均为Proteobacteria。在微生物分类学属的水平,土壤DNA、细胞DNA和单细胞DNA分析均表明背景土壤含有7个好氧甲烷氧化菌的pmo A基因型,但氧化高浓度甲烷后,γ-Proteobacteria的2个属Methylobacter/Methylosarcina则成为优势类群。【结论】土壤微生物的传统显微计数(DAPI和CARD-FISH)结果显著低于qPCR技术,相差1–3个数量级。qPCR定量土壤DNA和细胞DNA表明:水稻土可提取微生物细胞约占土壤微生物总量的2%左右,而好氧甲烷氧化菌的提取效率最高可达6%。细胞DNA在门水平能较好地反映水稻土微生物组成,但在属水平和土壤DNA有着较大差异。Planctomycetes微生物门的细胞易被提取,Acidobacteria门的微生物则较难被提取,而单细胞分选技术则偏好Proteobacteria。尽管传统方法和分子技术的分辨率明显不同,但均能较好地表征水稻土甲烷氧化的微生物生理生态过程。未来土壤微生物组研究应更加重视科学问题本身对技术手段的内在需求,最大限度发挥各种先进技术的优势。     

白及根腐病植株根际土壤微生物群落组成特征分析

分析白及根腐病植株根际土壤微生物群落组成特征,为理解白及根腐病发病机理并针对性开展防治工作提供科学依据。利用Illumina HiSeq高通量测序技术,比较分析白及根腐病与健康植株根际土壤微生物群落结构特征,并对土壤理化性质与酶活性进行定量分析。白及根际土壤中优势真菌为子囊菌门(Ascomycota)和Mortierellomycota,优势细菌为变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。与健康植株相比,发病白及根际土壤Ascomycota相对丰度显著升高,而Mortierellomycota相对丰度显著降低;镰刀菌属(Fusarium)和白粉菌属(Erysiphe)等致病菌群相对丰度显著升高(P<0.05)。多元统计分析表明,健康与发病白及根际土壤细菌与真菌群落存在明显分化。此外,健康植株根际土壤脲酶、蛋白酶与蔗糖酶活性和有效钾含量较高,pH、有机质、铵态氮与硝态氮含量较低。白及根腐病的发生可能与土壤致病菌相对丰度升高、微生物群落结构变化、酶活性降低、养分失衡等因素有关。     

秦岭石生苔藓结皮的微生物群落组成和多样性特征北大核心CSCD

苔藓结皮是地表重要的活性覆盖物,具有改善土壤性状、固土持水、固碳固氮等多种生态功能,微生物群落是苔藓结皮发挥生态功能的主要成分。为了探究湿润区石生苔藓结皮的微生物群落组成及其多样性特征,揭示其发育形成机制以及功能机理,该研究以秦岭北麓的泥峪、车峪、骆峪5个样地的石生苔藓结皮为研究对象,利用高通量测序技术,分析了石生苔藓结皮层的微生物群落组成、丰度、多样性,并测定分析养分与微生物群落的关系,为裸露岩石创面的生态修复提供理论依据。结果表明:(1)秦岭石生苔藓结皮层的全氮和有机质含量在4.16~8.26 g/kg、89.44~131.05 g/kg之间,且样点间均差异显著;全磷和全钾含量较低,分别为0.88~1.21 g/kg和13.50~18.10 g/kg。(2)秦岭石生苔藓结皮层中细菌占微生物群落组成的80%以上,且细菌的多样性也远高于真菌;其中细菌优势菌群为变形菌门(Proteobateria)、拟杆菌门(Bateroidetes)、放线菌门(Actinobacteria)和酸杆菌门(Acidobacteria),真菌优势菌群为子囊菌门(Ascomycota)和被孢霉菌门(Mortierellomycota)。(3)冗余分析(RDA)显示,全钾和有机质对优势细菌群落结构影响显著,变形菌和拟杆菌适宜生活在湿度大、营养状况良好的基质中;全磷和有机质对优势真菌群落结构影响显著,被孢霉菌门在低磷基质中更具生长优势。研究发现,细菌是秦岭石生苔藓结皮层的优势菌群,与旱区土生状况相比,秦岭石生苔藓结皮层的放线菌和酸杆菌的含量有所下降,而拟杆菌含量显著增加;对于真菌而言,子囊菌门和担子菌门的相对丰度明显减少,被孢菌门丰度显著增加;有机质是影响结皮层微生物群落组成最主要的养分因子,丰富的养分使湿润区石生苔藓结皮层的微生物群落结构更加复杂多样、物种组成更加丰富。     

Extractable microbial DNA pool and microbial activity in paleosols of Southern Ural

An evaluation of microbial DNA pools was performed using direct quantitative isolation of DNA from contemporary soils of Southern Urals and paleosols sealed under burial mounds of early Bronze Age more than 5000 years B.P. Significant regression dependence was found between the biomass and DNA contents in these soils (R2 = 0.97). Activity and dominant ecological strategies of microbial communities of paleosols and contemporary southern black soil were compared from growth parameters obtained by analysis of respiratory curves. The ratio of maximum specific growth rates of soil microorganisms on glucose and on yeast extract was shown to provide an auxotrophy index for soil microbial communities.