Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil

Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats.     

Microbial diversity in a thermophilic aerobic biofilm process: analysis by length heterogeneity PCR (LH-PCR)

A two-stage pilot-scale thermophilic aerobic suspended carrier biofilm process (SCBP) was set up for the on-site treatment of pulp and paper mill whitewater lining. The microbial diversity in this process was analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA. The primer pair selected for PCR amplification was first evaluated by a computational analysis of fragment lengths in ten main phylogenetical eubacterial groups. The fragment contained the first third of the 16S rRNA gene, which was shown to vary naturally between 465 and 563 bp in length. The length heterogeneity analysis of polymerase chain reaction (LH-PCR) profile of the biomass attached to carrier elements was found to be diverse in both stages of the SCBP. During normal operating conditions, sequences belonging to beta-Proteobacteria, Cytophaga/Flexibacter/Bacteroides group and gamma-Proteobacteria were assigned to the most prominent LH-PCR peak. Samples from the suspended biomass consisted of completely different bacterial populations, which were, however, similar in the serial reactors. The pilot process experienced alkaline shocks, after which Bacillus-like sequences were detected in both the biofilm and suspended biomass. However, when the conditions were reversed, the normal microbial population in the biofilm recovered rapidly without further biomass inoculations. This study shows that LH-PCR is a valuable method for profiling microbial diversity and dynamics in industrial wastewater processes.     

Detection of infectious enteroviruses and adenoviruses in tap water in urban areas in Korea.

We investigated the viral contamination of tap water at 11 urban sites in Korea between 1997 and 1998 over a period of 11 months. A total of 23 tap water samples were examined for infectious enteroviruses and adenoviruses by a cell culture technique followed by polymerase chain reaction (PCR) amplification. To identify the recovered viruses, sequence analysis of PCR products was performed. Infectious enteroviruses and adenoviruses were detected in 11 (47.8%) and 9 (39.1%) of the samples, respectively. Both enteroviruses and adenoviruses were detected in five samples (21.7%). The level of viral contamination was quite high, ranging from 2 x 10(-3) to 2.9 x 10(-2) Most Probable Number of Infectious Unit L(-1), far above the recommended virus level in drinking water set by the US EPA. Poliovirus type I derived from vaccine was frequently detected and the remainder comprised coxsackievirus B type or echovirus type 6, which were causative agents of aseptic meningitis in Korea in 1997 and 1998, respectively. Several types of adenovirus were detected in tap water samples and some water samples were found to contain adenoviruses which were closely related to enteric adenovirus types 40 and 41.     

Improved coverage of fungal diversity in polluted groundwaters by semi-nested PCR

Traditional methods used for studying communities of aquatic hyphomycetes are based on the detection and identification of their asexual spores under a microscope. These techniques limit detection to aquatic fungi present in sufficient quantity and capable of sporulating under laboratory conditions. Our objective was to develop a molecular approach to detect and monitor all types of fungi (i.e. strictly or facultatively aquatic) in harsh habitats (i.e. groundwater wells and heavily polluted surface water) where fungal biomass may become limited. We developed a semi-nested PCR protocol for fungal 18S ribosomal RNA genes coupled to subsequent analysis of the PCR products by Temperature Gradient Gel Electrophoresis (TGGE) to monitor the fungal community structure in aquatic habitats characterized by a pollution gradient. Our TGGE-protocol was compared with the traditional morphological approach and revealed a higher diversity in groundwaters and in some polluted surface waters. Thus, PCR-TGGE is a promising alternative in particular in habitats with low fungal biomass. The dynamics of fungal biomass and sporulation rates during the first weeks of leaf colonization showed that habitats with adverse ecological conditions allow only reduced fungal growth, which might subsequently impact upper trophic levels and thus interfere with key ecological processes of leaf decomposition.     

Evaluation of molecular methods used for establishing the interactions and functions of microorganisms in anaerobic bioreactors

Molecular techniques have unveiled the complexity of the microbial consortium in anaerobic bioreactors and revealed the presence of several uncultivated species. This paper presents a review of the panoply of classical and recent molecular approaches and multivariate analyses that have been, or might be used to establish the interactions and functions of these anaerobic microorganisms. Most of the molecular approaches used so far are based on the analysis of small subunit ribosomal RNA but recent studies also use quantification of functional gene expressions. There are now several studies that have developed quantitative real-time PCR assays to investigate methanogens. With a view to improving the stability and performance of bioreactors, monitoring with molecular methods is also discussed. Advances in metagenomics and proteomics will lead to the development of promising lab-on chip technologies for cost-effective monitoring.     

Evaluation of DNA extraction methods for freshwater eukaryotic microalgae

The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy^® Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended.     

Molecular Detection of Phytophthora colocasiae of Taro Leaf Blight Based on PCR

The present PCR assay was conducted to develop rapid and sensitive detection of Phytophthora colocasiae,in order to provide a robust and reliable tool for healthy seedling production of taro and limiting the transmission and spread of the causal organism of taro leaf blight in taro planting regions.The samples were used to extract total DNA and to be detected by PCR with P.colocasiae specific primer pairs PCSP-RL F/PCSP-RL R and PCSP-T F/PCSP-T R,respectively.Distinct fragments of about 200 bp and 240 bp were amplified by PCR using primers PCSP-RL F/PCSP-RL R and PCSP-T F/PCSP-T R,respectively.The analysis of the nucleotide sequence of the PCR products were found to be 99% identical to sequence of RAS-related protein (Ypt1) and phospho-ribosylanthranilate isomerase (TRP1) in P.colocasiae,respectively.It is concluded that rapid and sensitive developed PCR assay for detection of P.colocasiae could be used in routine diagnosis and aid in management practices to mitigate taro leaf blight.     

重叠延伸聚合酶链反应克隆大鼠Foxo3a基因及序列测定

目的获得大鼠Foxo3a基因全长编码区基因。方法运用重叠延伸PCR方法从大鼠脾脏总RNA中扩增Foxo3a编码区基因全长,将其插入pMD19-T载体中进行酶切及测序鉴定。结果Foxo3a编码区基因全长2019bp,与GenBank中大鼠Foxo3a基因同源性为99．9％。结论运用重叠延伸PCR技术可成功克隆高GC含量大鼠Foxo3a的cDNA,为进一步研究该基因奠定了基础。     

上海软土地层咬合桩围护结构施工技术

介绍了咬合桩围护结构技术的施工工艺和施工顺序,并着重分析了导墙定位、垂直度控制和管涌控制等关键技术。通过上海某轨道交通深基坑工程实例,对咬合桩围护结构技术在上海软土地层中应用提出了几点建议。     

Using an intervening sequence of Faecalibacterium 16S rDNA to identify poultry feces

This study was designed to identify poultry feces-specific marker(s) within sequences of Faecalibacterium 16S rDNA for detecting poultry fecal pollution in water. Bioinformatics tools were used in the comparative analysis of 7,458 sequences of Faecalibacterium 16S rDNA, reportedly associated with various poultry (chicken and turkey) and animal species. One intervening sequence (IVS) within between the hypervariable region 1 and the conserved region 2, designated as IVS-p, was found to be unique to poultry feces. Based on this sequence, a PCR assay (PCR-p) was developed. The PCR-p produced an amplicon of 132 bp only in the test when fecal or wastewater samples from poultry were used, but not when using fecal or wastewater samples from other sources. The non-poultry sources included feces of beef or dairy cattle, dog, horse, human, domestic or wild geese, seagull, sheep, swine, and wild turkey. These data indicate that IVS-p may prove to be a useful genetic marker for the specific identification of poultry fecal pollution in environmental waterways. Furthermore, results of data mining and PCR assay indicate that the IVS-p may have a broad geographic distribution. This report represents initial evidence of the potential utility of ribosomal intervening sequences as genetic markers for tracking host sources of fecal pollution in waterways.     

Application of a rapid method for identifying fecal pollution sources in a multi-use estuary

We demonstrate the application of a new PCR assay to detect and differentiate human and ruminant sources of fecal pollution in natural water samples. We tested samples collected from Tillamook Bay, Oregon, which has a long history of fecal pollution levels that exceed acceptable standards. The most likely sources are from dairy operations and ineffective sewage treatment. Using a suite of three PCR primer pairs specific for human or ruminant bacterial 16S ribosomal DNA markers, we detected at least one marker in 17 of 22 samples. In general, host-specific fecal markers were detected in areas that are heavily impacted by anthropogenic activities. Nine out of 11 sites classified as either urban or near a sewage point source were positive for the human marker while only five of these same sites were positive for ruminant markers. Conversely, 12 out of 21 sites classified as rural or agricultural use were positive for ruminant markers, while only six of these sites were positive for human pollution. This suite of host-specific genetic markers holds promise for identifying non-point source fecal pollution in coastal waters.     

R·C·巨型框架结构抗震能力设计措施的探讨

以位于巨型梁上的次框架底层柱底截面组合弯矩设计值增强系数的不同取值为变量,进行多个典型巨型平面框架设计。采用平面结构弹塑性动力分析程序Drain-2d+,对每个典型巨型框架在地震作用下进行弹塑性时程分析,得出这些巨型框架在地震作用下的受力变形特点、塑性铰出现情况和整个结构的薄弱环节等,从中找出位于巨梁上的次框架底层柱底截面弯矩设计值增强系数取值的正确思路,并给出设计的合理取值,为钢筋混凝土巨型结构设计提出若干建议。     

Enhancement of biogas production by addition of hemicellulolytic bacteria immobilised on activated zeolite

Biogas from agricultural biomass and residues is a valuable source of renewable energy. However, recalcitrant plant cell structures represent a barrier in the fermentative biodegradation process in single- and two-stage reactors. Therefore, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required amongst others in order to optimise the fermentation efficiency and to increase methane yields. Here we show a new strategy for the enhancement of biogas production from hemicellulose-rich substrates. Hemicellulolytic populations from a common biogas fermenter consortium were successively enriched in batch-cultures using a synthetic medium containing xylan powder as single carbon source under anaerobic mesophilic conditions. Enriched hemicellulolytic bacteria were immobilised on trace metal activated zeolite to ensure a stable storage and easy application. Xylanase activity increased continuously during subsequent enrichment cycles by up to 162%. In batch-culture experiments we were able to observe an increase of methane by 53% compared to controls without additionally introduced microorganisms immobilised on zeolite. Specific enrichment of hemicellulolytic bacteria during the process was confirmed by using single strand conformation polymorphism (SSCP) analysis based on amplification of the eubacterial 16S rDNA fragments. Using sequence analysis conspicuous bands from SSCP patterns could be identified as belonging to the groups Bacteroides sp., Azospira oryzae (Dechlorosoma sp.) as well as to a wide spectrum of diverse species within the order of Clostridiales (Firmicutes).     

确定框架和排架柱计算长度的实用图表法

确定柱的计算长度是稳定验算的关键一环,对此,GB50017-2010《钢结构设计规范》中给出了一些相关的计算公式和表格,但这些公式和表格仍存在两方面的不足：一是这些公式和表格基于计算假定只适用一般情况,对于一些特殊情况,如框架各柱内的轴力或各排架柱的轴力和柱高相差较大时,这些公式和表格就不再适用了;二是使用这些公式和图表难以得到计算长度随各相关参数变化的直观表现。针对这些不足,本文作者利用：二阶位移法推导了相关的公式并制成了确定计算长度的实用图表,利用这些图表可快速地确定计算长度,并可得到计算长度随各参数变化的直观趋势。     

Discourse Analysis and its Utility for Urban Policy Research

In recent years, discourse analysis has been deployed by academics as a methodology to understand the urban policy implementation process, in particular, the ways in which key actors exercise power. Much of the discourse-based research in urban policy has drawn upon the writings of Norman Fairclough and Michel Foucault and seeks to provide a critical scrutiny of texts and utterances of policy makers and other key actors. The methodological assumption that informs discourse-based approaches is that politics is an arena in which different interest groups seek to establish a particular narrative or version of events as a means to pursue political objectives. This article begins by setting out the theoretical influences that have informed discourse analysis. There then follows a discussion of some of the studies that have deployed discourse-based research within urban policy, an evaluation of its strengths and weaknesses as a method and an assessment for new areas of enquiry. The article's conclusion is that discourse analysis provides the researcher with a set of tools to interpret urban policy in a theoretically informed and insightful way. However, there are some pitfalls associated with its techniques that require consideration before any analysis should commence.     

Experimental evaluation of a vibration-based leak detection technique for water pipelines

Conventional water pipeline leak detection surveys employ labour-intensive acoustic techniques, which are usually expensive and not amenable for continuous monitoring of distribution systems. Many previous studies attempted to address these limitations by proposing and evaluating a myriad of continuous, long-term monitoring techniques. However, these techniques have difficulty to identify leaks in the presence of pipeline system complexities (e.g. T-joints), offered limited compatibility with popular pipe materials (e.g. PVC), and were in some cases intrusive in nature. Recently, a non-intrusive pipeline surface vibration-based leak detection technique has been proposed to address some of the limitations of the previous studies. This new technique involves continuous monitoring of the change in the cross-spectral density of surface vibration measured at discrete locations along the pipeline. Previously, the capabilities of this technique have been demonstrated through an experimental campaign carried out on a simple pipeline set-up. This paper presents a follow-up evaluation of the new technique in a real-size experimental looped pipeline system located in a laboratory with complexities, such as junctions, bends and varying pipeline sizes. The results revealed the potential feasibility of the proposed technique to detect and assess the onset of single or multiple leaks in a complex system.     

Pedestrian-level wind conditions around buildings:Review of wind-tunnel and CFD techniques and their accuracy for wind comfort assessment

Information on pedestrian-level wind(PLW)speed for wind comfort assessment can be obtained by wind-tunnel measurements or Computational Fluid Dynamics(CFD)simulations.Wind-tunnel measurements for PLW are routinely performed with low-cost techniques such as hot-wire or hot-film anemometers,Irwin probes or sand erosion,while Laser-Doppler Anemometry(LDA)and Particle-Image Velocimetry(PIV)are less often used because they are more expensive.CFD simulations are routinely performed by the relatively low-cost steady Reynolds-AveragedNavier-Stokes(RANS)approach.Large-Eddy Simulation(LES)is less often used because of its larger complexity and cost.This paper reviews wind-tunnel and CFD techniques to determine PLW speeds expressed generally in terms of amplification factors defined as the ratio of local mean wind speed to mean wind speed at the same position without buildings present.Some comparative studies systematically indicate that the low-cost wind-tunnel techniques and steady RANS simulations can provide accurate results(~10%)at high amplification factors(1)while their accuracy can deteriorate at lower amplification factors(1).This does not necessarily compromise the accuracy of PLW comfort assessment,because the higher amplification factors provide the largest contribution to the discomfort exceedance probability in the comfort criterion.Although LDA,PIV and LES are inherently more accurate techniques,this paper supports the continued use of faster and less expensive techniques for PLW studies.Extrapolating a previous saying,we argue that pedestrian-level wind comfort is one of the few topics in wind engineering where nature is kind to us concerning turbulent flows.     

自来水厂中摇蚊rDNA系统学分析

在传统形态学分类的基础上,通过检索基因数据库 (GenBank)中摇蚊的rDNA同源序列,设计出特异性扩增引物,对南方某城市 4座自来水厂中摇蚊rDNA的内部转录间隔序列(ITS1)进行PCR扩增和限制性片段长度多态性分析(PCR-RFLP),同时对摇蚊ITS1区进行DNA序列测定,并根据ITS1区的序列差异构建了摇蚊的系统进化树。结果表明:摇蚊的ITS1可以作为摇蚊种分类阶元的理想分子标记。     

Seasonal bacterial community dynamics in a full-scale enhanced biological phosphorus removal plant

Activated sludge is one of the most abundant and effective wastewater treatment process used to treat wastewater, and has been used in developed countries for nearly a century. In all that time, several hundreds of studies have explored the bacterial communities responsible for treatment, but most studies were based on a handful of samples and did not consider temporal dynamics. In this study, we used the DNA fingerprinting technique called automated ribosomal intergenic spacer region analysis (ARISA) to study bacterial community dynamics over a two-year period in two different treatment trains. We also used quantitative PCR to measure the variation of five phylogenetically-defined clades within the Accumulibacter lineage, which is a model polyphosphate accumulating organism. The total bacterial community exhibited seasonal patterns of change reminiscent of those observed in lakes and oceans. Surprisingly, all five Accumulibacter clades were present throughout the study, and the total Accumulibacter community was relatively stable. However, the abundance of each clade did fluctuate through time. Clade IIA dynamics correlated positively with temperature (ρ = 0.65, p < 0.05) while Clade IA dynamics correlated negatively with temperature (ρ = −0.35, p < 0.05). This relationship with temperature hints at the mechanisms that may be driving the seasonal patterns in overall bacterial community dynamics and provides further evidence for ecological differentiation among clades within the Accumulibacter lineage. This work provides a valuable baseline for activated sludge bacterial community variation.