Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR.

In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.     

Evaluating real-time PCR for the quantification of distinct pathogens and indicator organisms in environmental samples.

We evaluated quantitative real-time PCR (qPCR) and RTqPCR (for RNA species) for their ability to quantify microorganisms and viruses in problematic environmental samples such as cattle manure, digester material, wastewater and soil. Important developments included a standard spiking approach which compensated for methodological bias and allowed sample-to-sample comparison and reliable quantification. Programme CeTe was developed to calculate endogenous concentrations of target organisms (nucleic acid copies) for each sample separately from the generated standard curves. The approach also permitted assessment of the detection limit of the complete method, including extraction. It varied from sample to sample, due to different extraction efficiencies and variable co-extraction of PCR inhibitors. False negative results were thereby avoided. By using this approach we were able to optimise a DNA extraction protocol from the different tested sample types. Protocols for the extraction of RNA species from environmental samples were also optimised. DNA was (almost) not degraded after lethal shock (autoclaving) in the sterile environment. In contrast, the parallel selective cultivation and qPCR results for various microbial parameters from an anaerobic digester chain suggested that DNA from decaying organisms was readily recycled in metabolically active environments. It may, therefore, be used to determine viable organisms in samples exhibiting substantial metabolic turnover. It is proposed that our standard spiking approach, including data evaluation by the program CeTe, should be considered in future standardisation and norms for the quantification of nucleic acid containing organisms in environmental and product samples.     

Evaluation of methods for isolation of DNA for polymerase chain reaction (PCR)-based identification of pathogenic bacteria from pure cultures and water samples

Polymerase chain reaction (PCR) provides a reliable detection of pathogenic bacteria in water samples. However, this method can be adversely influenced by the purity of the DNA template. This is a particularly important obstacle when the bacterial DNA is directly extracted from water samples. In this study we compared the suitability of 8 different methods for isolation of bacterial DNA from pure cultures and 10 different methods for isolation of DNA from water samples. The quality of extracted DNA was assessed by PCR amplification of target sequences derived from uid (E. coli and Shigella sp.), tuf (Enterococcus sp.) and hns (Salmonella sp.). Results indicated that there are differences among the methods tested and only a few of them gave satisfactory results. The method based on alkaline lysis of bacterial suspension, which was developed in our laboratory, seemed to be efficient enough for the detection of bacteria from pure cultures. Detection of bacteria directly from water samples was more difficult. The modified method developed by Slusarenko was found as the best of the tested methods.     

Enumeration of viable Escherichia coli by real-time PCR with propidium monoazide

A photo-inducible DNA-binding dye, propidium monoazide (PMA), was used to distinguish viable and dead Escherichia coli cells. Microscopic observations using a combination of the dyes 4',6-diamidino-2-phenylindole and PMA indicated that PMA stained only dead cells, with membrane damage, red. Mixtures of viable and heat-treated E. coli cells were subjected to real-time polymerase chain reaction (PCR) with PMA treatment. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells in the mixtures of viable and heat-treated cells, as long as the ratio of dead cells to viable cells was no greater than 10. In the wastewater treatment plants, total, viable and culturable E. coli were enumerated by real-time PCR, real-time PCR coupled with PMA treatment and the most probable number method using EC-MUG medium, respectively. The concentrations of viable E. coli in the wastewater treatment plants were much higher than those of culturable cells. In addition, viable cells were even more chlorine resistant than culturable ones.     

Biofilms, thermophilic amoebae and Legionella pneumophila--a quantitative risk assessment for distributed water.

A simplistic quantitative microbial risk assessment (QMRA) based on the maximum risk curve (r = 1) was developed for Legionella within a water distribution system. Both biofilms and a thermophilic isolate of acanthamoebae were shown to increase the resistance of Legionella to conventional thermal disinfection by between one and two logs respectively. The level of risk presented to consumers was shown to exceed the USEPA 10(-4) benchmark in many cases tested. This was caused, in part, by the sensitivity of the risk model but also through a lack of reliable dose-response data for Legionella. Not withstanding this, the current study provided comparative information on the efficacy of conventional disinfection against Legionella. Combined chlorine was shown to reduce the risk of infection by as much as 1-log when compared to free chlorine, although thermal disinfection provided the most effective means of risk reduction. Biofilm detachment and the interaction of Legionella with acanthamoebae were two important ecological factors that significantly increased the risk of legionellosis, and thus should be further considered in the refinement of QMRA models.     

Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption

A quantitative real-time polymerase chain reaction (qPCR) is a robust means by which to monitor toxin-producing cyanobacteria. However, qPCR usually requires DNA extraction, which is a time-consuming, labor-intensive pretreatment. To be able to quickly determine the potential of cyanotoxin contamination in the field, a rapid pretreatment method for DNA extraction and a portable qPCR device are needed. In this study, we applied a microwave-based method for the qPCR pretreatment and a multicolor portable qPCR with UPL and TaqMan probes to quantify toxigenic and total Microcystis. The method was tested using laboratory cultures of toxigenic Microcystis aeruginosa PCC7820. The qPCR results showed the cycle thresholds value (Ct value) correlated well with cell numbers, with detection limit at about 1,000 cells/ml. This scheme was applied in 22 environmental samples from six drinking water reservoirs (DWRs) in Taiwan. Although the results for qPCR were about four times higher than those of microscopic observation, good correlation between qPCR and microscope methods were obtained (r-square: 0.79, P < 0.01). The ratios of toxigenic Microcystis to total Microcystis in two reservoirs, Sin-Shan Reservoir and Shih-men Reservoir, were less than 10%. In three other reservoirs, Ren-Yi-Tan Reservoir, Nan-Hua Reservoir and Bao-Shan Reservoir, much higher (>46.1%) ratios were obtained. The scheme may assist quick assessment of the risk associated with toxic cyanobacteria in DWRs.     

让政策评估为流域水生态环境保护保驾护航

我国有七大流域,河流众多,1km^2以上的湖泊有3100多个。过去30年来,在经济社会高速发展的过程中,由于不合理的开发利用方式等原因,许多河湖受到严重污染,甚至处于生态极度退化的境地,引发了各种利益冲突和社会矛盾。     

探地雷达与管线探测仪联合探测技术分析

结合东莞市东江与水库联网供水水源工程输水线路选址中地下综合管线探测中的工程实例,对管线探测仪与探地雷达探测的原理以及联合探测应用进行分析,得出了一些经验性规律,可供类似工程参考。     

A fate and transport model for Asian carp environmental DNA in the Chicago area waterways system

Detection of environmental DNA (eDNA) is widely employed to infer the presence of endangered or invasive species in the aquatic environment. Detection of eDNA, however, does not guarantee the presence of the species in question. The location, time, and nature of the eDNA source are unknown. An eDNA fate and transport model can help to address these unknowns. Construction of such a model requires resolution of multiple issues including: 1) Quantification of eDNA concentration in the environment; 2) Quantification of the eDNA source; 3) Quantification of decay rate; and, 4) Model application and validation. We address these issues and present the results of a fate and transport model for eDNA originating from an invasive species, silver carp (Hypophthalmicthys molitrix), in the Chicago Area Waterways System, USA. Results indicate the presence of roughly 4600?kg of silver carp, distributed along the major axes of the system, is required to produce the eDNA detected in routine monitoring. Positive detection of eDNA in a sample suggests a source within days and km of the sample time and location.     

Microbial challenge-testing of treatment processes for quantifying stormwater recycling risks and management.

Pathogenic microorganisms have been identified as the main human health risks associated with the reuse of treated urban stormwater (runoff from paved and unpaved urban areas). As part of the Smart Water initiative (Victorian Government, Australia), a collaborative evaluation of three existing integrated stormwater recycling systems, and the risks involved in non-potable reuse of treated urban stormwater is being undertaken. Three stormwater recycling systems were selected at urban locations to provide a range of barriers including biofiltration, storage tanks, UV disinfection, a constructed wetland, and retention ponds. Recycled water from each of the systems is used for open space irrigation. In order to adequately undertake exposure assessments, it was necessary to quantify the efficacy of key barriers in each exposure pathway. Given that none of the selected treatment systems had previously been evaluated for their treatment efficiency, experimental work was carried out comprising dry and wet weather monitoring of each system (for a period of 12 months), as well as challenging the barriers with model microbes (for viruses, bacteria and parasitic protozoa) to provide input data for use in Quantitative Microbial Risk Assessment.     

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16-18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.     

Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment

Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25 microg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.     

利用ASP进行实时雨水情信息查询检索探讨

实时雨水情信息是防汛、抗旱指挥决策和水资源管理及国民经济建设不可或缺的基础性数据,目前使用的实时雨水情信息数据库是典型的关系型数据库,采用SQL SERVER 2000实现.本文对利用ASP编程技术结合ADO数据接口、SQL语言实现实时雨水情信息查询、检索进行了相关探索.     

里墩水库大坝渗漏的雷达探测与分析

里墩水库建于软土地基上,施工过程中曾出现多组裂缝,蓄水1年半左右发生渗漏问题.在综合分析坝址和坝体的地质地貌、地球物理特征及周围环境的基础上,采用探地雷达技术探测了疏松薄弱带、裂缝及洞体的主要分布范围和深度变化情况.该结果与已回填的原河道位置、地层变化及实际观测到的裂缝有较好的一致性,并为开挖揭示所证实.     

堤防管涌渗漏实时监测技术研究与应用

汛期堤防管涌渗漏的实时监测对堤防的紧急抢险具有非常重要的意义．本在分析了已知各种用于堤防管涌渗漏监测工作方法的优缺点的基础上,提出了用流场拟合法进行堤防管涌渗漏入水口的监测．通过物理模拟实验以及实际应用表明流场拟合法可以有效地用于堤防管涌渗漏入水口的实时监测．     

Use of real-time PCR to examine the relationship between ammonia oxidizing bacterial populations and nitrogen removal efficiency in a small decentralized treatment system 'Johkasou'.

The aim of this study was to examine the relationship between ammonia oxidizing bacterial populations and biological nitrogen removal in a small on-site domestic wastewater treatment system "Johkasou". The population dynamics of ammonia oxidizing bacteria (AOB) in six full-scale advanced Johkasous was surveyed using real-time PCR assay over a period of one year. These Johkasous were selected to compare the AOB populations in different treatment performance. When the effluent NH4-N concentration was higher than 2 mg L(-1), it was difficult to meet the effluent standard of advanced Johkasous (T-N 10 mg L(-1)). In contrast, the nitrogen removal efficiency was hardly affected by nitrite oxidation and denitrification in these systems. In other words, ammonia oxidation was a rate-limiting step. Furthermore, we focused on the relationship between NH4-N loading per AOB cell and nitrogen removal. Real time PCR monitoring results demonstrated that it is important to regulate NH4-N loading per AOB cell below 210 pg cell(-1) day(-1) to meet the effluent standard of advanced Johkasou. It is considered that NH4-N loading per AOB cell is a useful parameter for determining suitable nitrogen loading and small decentralized system design.     

重要实时水情短信息发布查询系统设计

重要实时水情短信息发布查询系统,是利用现代移动通信、数据库应用和计算机网络等技术手段,结合黄河实际,不仅实现了水情信息实时发布和查询,使得各级防汛决策部门可以在任意地点及时获取黄河重要实时水情信息,争取防汛指挥的主动性,而且能够推动"数字防汛"和黄河水文测报现代化的建设。本文对系统的设计和实现方法作了介绍。     

确保粮食安全和环境可持续发展的水土资源利用

主要讨论灌溉农业所面临的挑战.随着人均可用水量的不断下降,灌溉农业必须提高水资源利用效率,同时进一步采用有利于环境的经营和管理方式.灌溉界面临的压力是如何通过提高作物产量和用水效率来实现生产率的最大化.今后的发展方向是以更加高效率、有成效的方式利用水资源,而实现从"提高每滴水的粮食产量"向"少用水,多产粮"的转变,则有助于该目标的实现.为了尽可能减小外在因素的影响,将来的努力方向是在全面性和整体性上下工夫.