重庆市河水中粪源微生物污染特征及源解析

以重庆主要河流水体作为研究对象,使用传统培养技术评估了细菌总数、粪链球菌、肠球菌、脆弱拟杆菌、总大肠菌群及粪大肠菌群的污染水平;同时以拟杆菌作为特异性指示菌,选取人源性粪便专一指示菌引物（HF183）和猪源性粪便专一指示菌引物（Pig-2-Bac）进行源追踪.微生物培养结果表明：重庆市主要河流在春季有15.4%的研究断面未达到Ⅲ类水质,秋季有61.5%的研究断面未达到Ⅲ类水质.在春季,主城区河流主要受人类粪便污染,区县河流主要受动物粪便污染;在秋季,主城区和区县河流都主要受人类粪便污染.指示微生物指标间Pearson相关性分析结果表明粪链球菌、粪大肠菌群、肠球菌两两之间有显著相关性,肠球菌与脆弱拟杆菌有显著相关性.猪源性拟杆菌特异性生物标记Pig-2-Bac和人源拟杆菌特异性生物标记HF183对人和动物粪便污染区分成功率达100%;用这两种特异性引物对春季水样DNA进行扩增,发现13个采样点均未受到猪源粪便污染,唐家沱、朝天门、鸡冠石、合川受到人源粪便污染.     

基于拟杆菌16S rRNA基因进行微生物溯源的研究进展

微生物溯源方法可利用粪便中的微生物区分来自人或动物的粪便污染.其中,拟杆菌以其丰度高、不能体外繁殖和宿主特异性强等优势被广泛应用于微生物溯源研究中.本文以拟杆菌16S rRNA基因为标记物,总结了拟杆菌及其标记物在环境中的衰减、拟杆菌引物的敏感性和特异性以及分子生物学技术在微生物溯源中的运用,可为粪便污染源解析提供一定的科学参考依据.     

肠道菌群的环境毒理学研究:现状与展望

肠道菌群对人类和动物的健康发挥着重要作用,已经成为近年来国内外环境毒理学研究的又一新兴课题.由于肠道和肠道菌群系统是大部分污染物进入生物体后第一个暴露接触点,污染物必然要通过这层屏障进一步诱导毒性效应.因此,研究环境污染物对肠道菌群的影响及其与宿主健康互作关系具有重要意义.结果表明:①污染物对肠道菌群丰度和多样性的干扰作用是目前研究的焦点,个人护理品、医用药物、重金属、农药、新污染物、空气污染物、持久性有机污染物等七大类常见污染物的毒理学研究中均发现肠道菌群的改变.②有益菌群可以彼此影响并与肠道系统相互作用,参与调控宿主营养物质消化、免疫系统反应、内分泌调节、神经元活动等多方面的生理过程;相关分析表明,对拟杆菌、厚壁菌、乳酸杆菌和双歧杆菌等有益菌群的改变是污染物毒性效应的关键途径.③环境污染物暴露可以影响肠道菌群结构和代谢活动,通过肠道菌群的介导作用,进而破坏肠道屏障功能和完整性,影响免疫反应、代谢调控、内分泌信号通路、神经发育等一系列生理生化过程最终影响宿主健康.研究显示,肠道菌群可以作为环境毒理学研究中的新型终点,对深入研究污染物的毒性效应和作用机制具有重要意义.      

珠三角河网地区粪便污染源解析

准确识别水体中微生物污染物的宿主来源是进行针对性污染治理的基础.应用实时荧光定量PCR(q PCR)技术筛选出适用于珠江三角洲河网地区的宿主特异性拟杆菌引物,并结合总细菌引物(BACT1369F/PROK1541R)、大肠埃希氏菌引物(EC23S857F/EC23S857R)、拟杆菌通用引物(Gen Bac3F/Gen Bac3R)及粪大肠菌群(Fecal coliform)和大肠埃希氏菌(Escherichia coli)等常规水质指标对研究区域的水体进行分析,结果表明:人源(q HS601F/q Bac725R)、牛源(Bac B2-590F/Bac708Rm)、猪源(Bac41F/Bac163R)及鸡源(q C160F-HU/q Bac265R-HU)特异性拟杆菌引物在珠江三角洲地区同时具有较高的灵敏度和较强的特异性,适用于目标研究区域.选取的14处城市地表水均受到粪便污染,污染源分别为人、反刍动物和禽类粪便.水体中各污染源强度为人源反刍动物鸡源,其中人源特异性拟杆菌浓度与粪大肠菌群(Faecal coliform)、大肠埃希氏菌(Escherichia coli)及大肠埃希氏菌引物(EC23S857F/EC23S857R)浓度具有较好的相关性(P0.05),说明人粪是各地表水中微生物污染的主要贡献源.     

特异性拟杆菌引物在珠江三角洲的适应性研究

在珠江三角洲地区采集人、猪、牛、狗以及家禽等33个粪便样品,高效提取基因组DNA,选取14种特异性拟杆菌引物进行检验分析,并进一步采用已知污染源类型的水样对其进行验证.结果表明:采用粪便样品DNA专属提取试剂盒和水体样品DNA专属提取试剂盒提取的基因组模版纯度和提取率均符合后续实验要求.拟杆菌通用引物2#(Bac32F/Bac708R)检出率为85%(28/33),特别是对哺乳类动物和鸡的粪便具有更高的检出率.人的拟杆菌特异性引物3#(HF134F/ Bac708R)和4#(HF183F/Bac708R)、反刍动物的拟杆菌特异性引物8#(CF128F/Bac708R)以及猪的拟杆菌特异性引物10#(PF163F/Bac708R)在珠江三角洲地区同时具有较高的灵敏度和较强的特异性.水体样品验证实验与实际污染类型相符,说明拟杆菌通用引物2#、人的特异性引物3# 和4# 以及猪的特异性引物10# 在珠江三角洲地区具有很好的适用性.     

植物内生菌16S rRNA基因扩增子高通量测序中降低宿主污染的方法

植物内生菌能够帮助植物生长,增强其抗逆性与抗病性,研究植物内生菌对了解植物入侵机制、保护生态环境与农业的健康发展具有重要意义.通过阅读相关文献,总结归纳了降低植物内生菌16SrRNA基因扩增子高通量测序(16S-seq)中宿主基因污染的4种方法,即:①寻找特异性错配引物用来扩增细菌16S rRNA基因;②添加特异性阻断...     

基于现代分子生物学法追溯水体中粪便污染源

研究表明不同生物体粪便中含有已知或未知的病原微生物,所以动物体粪便的排放会携带这些病原微生物到自然水体中,严重污染了水体环境甚至危害人体健康。如何快速、准确鉴定出水体受到的粪便污染来源对进一步研究水体中可能存在的病原微生物至关重要,这也成为目前科学研究的热点。传统的通过监测粪便指示菌的方法指示污染不仅耗时费力,最重要的是它并不能区别出粪便污染的来源。微生物溯源技术(microbial source tracking,MST)的逐渐发展弥补了传统方法的不足,尤其是利用宿主特异性DNA标记物来指示污染源的方法已经取得了突破性进展,并具有高效、快速的特点。该文主要是针对微生物溯源技术的介绍以及如何利用现代分子生物学技术快速追溯水体中粪便污染源的概述。     

西江流域地表水微生物污染定量源解析

水体微生物污染标记物的源强特征是定量解析微生物污染来源的关键.应用实时荧光定量PCR（qPCR）技术分别对人源（qHS601F/qBac725R）、牛源（BacB2-590F/Bac708Rm）、猪源（Bac41F/Bac163R）及禽类（qC160F-HU/qBac265R-HU）特异性拟杆菌引物的源强特征进行了探索,并进一步对西江流域广东段进行微生物污染来源定量解析,结果表明：针对不同目标宿主,不同特异性拟杆菌引物的源强特征为人源（qHB） > 猪源（qPB） > 牛源（qRB） > 禽类（qCB）,DNA模板浓度对引物的源强特征影响较小.选取的目标研究区域水体普遍受到人、猪、牛及禽类粪便污染.干流中qHB、qCB及qPB自上游至下游缓慢升高.各支流中qHB、qCB及qPB的平均浓度较干流均上升了1个数量级,qRB在不同河段变化较小.说明下游河段的生活污水、猪源及禽类粪便污染较为严重,牛源污染的影响较小.统计分析显示在污染水平较高的各支流中,人源特异性拟杆菌引物与传统指示菌具有一定的相关性（P<0.05）,说明生活污水是传统指示菌浓度较高的主要原因,且为持续排放源.     

基于FIB的北京温榆河流域粪便污染物分布特征和源解析

目前,北京市温榆河流域存在着较为严重的粪源微生物污染问题,对流域周边暴露人群的健康带来了潜在的威胁.为了明确温榆河粪便污染的分布特征及宿主来源,本研究采用传统培养法与定量聚合酶链式反应(qPCR)分别对常规粪便指示细菌(Fecal indicator bacteria, FIB),包括粪大肠菌群(Fecal Coliform, FC)、大肠杆菌(Escherichia coli, EC)与肠球菌(Enterococcus,ENT)和溯源FIB,包括不同宿主来源的特异性生物标记进行检测,并探究其相关性.研究结果表明,流域内粪源微生物污染较为严重,常规FIB浓度介于0～4.38×10~7 CFU·L~(-1)之间,溯源FIB处于1.37×10~6～4.29×10~(10) copies·L~(-1)之间.从时空分布来看,由于来水中各大支流及污水处理厂的贡献,从温榆河起点沙河闸至终点北关闸,所有的指示菌浓度呈现出大幅度的升高趋势.其中,龙道河为干流来水中粪便污染的首要地域,污水处理厂的尾水可能是清河河口及坝河处微生物污染的主要来源;降雨径流对粪源微生物含量的时空变化存在着非常显著的影响,使得FIB最高可达2个数量级的增长.微生物溯源结果显示温榆河流域内的粪源微生物来源主要为人类粪便污染,而非畜禽粪便污染,这同北京市近年来有关畜禽养殖管控密切相关.相关性分析表明,虽然FC与EC、ENT之间皆存在着统计学意义上的显著正相关性,但传统的FIB指标与拟杆菌及人源拟杆菌指标之间不存在显著相关性.本研究通过传统FIB培养和微生物溯源方法结合,明确了北京温榆河粪源污染程度及其主要来源,为北京温榆河污染防控提供了新的思路.     

菲反硝化降解菌群的富集及其群落结构解析

从潜在多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)污染的油田区域采集土壤样品,以菲为唯一碳源且添加硝酸根的培养基来富集土壤中的菲反硝化降解菌群.随后,通过定量PCR(Polymerase Chain Reaction)测定了获取的富集菌群中反硝化相关功能基因(硝酸还原酶基因nar G、亚硝酸还原酶基因nir S)的丰度,并通过Illumina Mi Seq测序对其中的细菌群落结构进行解析.结果表明,获取到的3个菌群(PDN-1、PDN-2和PDN-3)12 d内对菲的降解率分别为45.18%、34.04%和25.92%.各富集培养菌群中nar G的丰度均高于nir S,且菲降解率最高的PDN-1中的反硝化相关基因丰度较低.Illumina Mi Seq测序结果表明,菲降解率最高的富集菌群PDN-1同时也具有较高的细菌多样性指数,变形菌门(Proteobacteria)、疣微菌门(Verrucomicrobia)和拟杆菌门(Bacteroidetes)是各富集菌群中的优势菌门,且Proteobacteria在3个富集菌群PDN-1(97.78%)、PDN-2(96.57%)、PDN-3(93.90%)中的比例均最高.变形菌门的Pseudomonas(γ-Proteobacteria)和Methylophilus(β-Proteobacteria)则是各富集菌群中最大的优势菌属,前者为公认的PAHs降解菌,而后者则为能够利用还原型"一碳化合物"的特殊菌属.细菌多样性与菲的降解率呈正相关,表明菲的反硝化降解可能是多种细菌参与的共同结果.上述结果可为揭示典型PAHs反硝化降解的微生物机制提供理论依据,同时为深入研究反硝化与菲代谢的偶联机理打下基础.     

Toxic effects of acetochlor, methamidophos and their combination on nifH gene in soil

Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis （DGGE） and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity （both in gene richness and diversity index values） and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patterns showed that nifH gene composition which had been affected by low concentration of acetochlor （50 mg/kg） recovered firstly. Methamidophos reduced nifH gene richness that except at 4 weeks. The medium concentration of methamidophos （150 mg/kg） caused the most apparent changes in nifH gene diversity at the first week while the high concentration of methamidophos （250 mg/kg） produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes （bands） responded differently to the impact of agrochemicals： four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha- and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.     

EDTA-enhanced phytoremediation of lead contaminated soil by Bidens maximowicziana

Phytoremediation is a potential cleanup technology for the removal of heavy metals from contaminated soils.Bidens maximowicziana is a new Pb hyperaccumulator,which not only has remarkable tolerance to Pb but also extraordinary accumulation capacity for Pb.The maximum Pb concentration was 1509.3 mg/kg in roots and 2164.7 mg/kg in overground tissues.The Pb distribution order in the B. maximowicziana was:leaf>stem>root.The effect of amendments on phytoremediation was also studied.The mobility of soil Pb and the Pb concentrations in plants were both increased by EDTA application.Compared with CK(control check),EDTA application promoted translocation of Pb to overground parts of the plant.The Pb concentrations in overground parts of plants was increased from 24.23-680.56 mg/kg to 29.07-1905.57 mg/kg.This research demonstrated that B.maximowicziana appeared to be suitable for phytoremediation of Pb contaminated soil,especially,combination with EDTA.     

Distribution and ecological effect of mercury in Laogang landfill, Shanghai, China

Laogang landfill near Shanghai is the largest landfill in China, and receives about 10000 t of daily garbage per day, Samples of topsoil and plants were analyzed to evaluate mercury pollution from the landfill. For topsoil samples, there were significant correlations among total mercury （HgT）, combinative mercury （Hgc） and gaseous mercury （HgG）, and content of total organic carbon （TOC）, but, no significantly relationship was found between Hg content and filling time. Hg content changes in vertical profiles with time showed that the average Hgv of profiles 1992, 1996, and 2000 was similar, but their average HgG was quite different. HgT was significantly correlated with Hgc in profile 1992 and 2000, and Hgv was significantly correlated with Hg6 in profile 1996. HgG/Hgv ratio in profile samples decreased in the order of （HgG,/HgT）1992〉（HgG/HgT）1996〉〉（HgG/HgT）2000. A simple outline of Hg release in landfill could be drawn： with increasing of filling time, degradation undergoes different biodegradation, accordingly, gaseous mercury goes through small, more, and small proportion to total mercury. Distribution of Hg in plants was inhomogeneous, following the order of leaf〉root〉stem. The highest value of leaf may be associated with higher atmospheric Hg from landfill. Ligneous plants （e.g. Phyllostachys glanca, Prunus salicina and Ligustrum lucidum） are capable of enriching more Hg than herbaceous plants.     

Arsenic uptake by arbuscular mycorrhizal maize （Zea mays L.） grown in an arsenic-contaminated soil with added phosphorus

The effects of arbuscular mycorrhizal (AM) fungus (Glomus mosseae) and phosphorus (P) addition (100 mg/kg soil) on arsenic (As) uptake by maize plants (Zea mays L.) from an As-contaminated soil were examined in a glasshouse experiment.Non-mycorrhizal and zero-P addition controls were included.Plant biomass and concentrations and uptake of As,P,and other nutrients,AM colonization,root lengths,and hyphal length densities were determined.The results indicated that addition of P significantly inhibited root colonization and development of extraradical mycelium.Root length and dry weight both increased markedly with mycorrhizal colonization under the zero-P treatments,but shoot and root biomass of AM plants was depressed by P application.AM fungal inoculation decreased shoot As concentrations when no P was added,and shoot and root As concentrations of AM plants increased 2.6 and 1.4 times with P addition,respectively.Shoot and root uptake of P,Mn,Cu,and Zn increased,but shoot Fe uptake decreased by 44.6%,with inoculation, when P was added.P addition reduced shoot P,Fe,Mn,Cu,and Zn uptake of AM plants,but increased root Fe and Mn uptake of the nonmycorrhizal ones.AM colonization therefore appeared to enhance plant tolerance to As in low P soil,and have some potential for the phytostabilization of As-contaminated soil,however,P application may introduce additional environmental risk by increasing soil As mobility.     

Enzymatic oxidation of aqueous pentachlorophenol

The influence of the nonionic surfactant Tween 80 on pentachlorophenol （PCP） oxidation catalyzed by horseradish peroxidase was studied. The surfactant was tested at concentrations below and above its critical micelle concentration （CMC）. Enhancement of PCP removal was observed at sub-CMCs. The presence of Tween 80 in the reaction mixture reduced enzyme inactivation which occurred through a combination of free radical attack and sorption by precipitated products. A simple first-order model was able to simulate time profiles for enzyme inactivation in the presence or absence of Tween 80. At supra-CMCs, the surfactant caused noticeable reductions in PCP removal, presumably through micelle partitioning of PCP which precluded the hydrophobic PCP molecule from interacting with the enzyme.     

Decomposition of alachlor by ozonation and its mechanism

Decomposition and corresponding mechanism of alachlor, an endocrine disruptor in water by ozonation were investigated. Results showed that alachlor could not be completely mineralized by ozone alone. Many intermediates and final products were formed during the process, including aromatic compounds, aliphatic carboxylic acids, and inorganic ions. In evoluting these products, some of them with weak polarity were qualitatively identified by GC-MS. The information of inorganic ions suggested that the dechlorination was the first and the fastest step in the ozonation of alachlor.     

Degradation of metabolites accumulated of benzo[a]pyrene by coupling Penicillium Chrysogenum with KMnO4

Several main metabolites of benzo[a]pyrene （BaP） formed by Penicillium chrysogenum, Benzo[a]pyrene-1,6-quinone （BP 1,6- quinone）, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene （BP 7,8-diol）, 3-hydroxybenzo[a]pyrene （3-OHBP）, were identified by high-performance liquid chromatography （HPLC）. The three metabolites were liable to be accumulated and were hardly further metabolized because of their toxicity to microorganisms. However, their further degradation was essential for the complete degradation of BaP. To enhance their degradation, two methods, degradation by coupling Penicillium chrysogenum with KMnO4 and degradation only by Penicillium chrysogenum, were compared; Meanwhile, the parameters of degradation in the superior method were optimized. The results showed that （1） the method of coupling Penicillium chrysogenum with KMnO4 was better and was the first method to be used in the degradation of BaP and its metabolites; （2） the metabolite, BP 1,6-quinone was the most liable to be accumulated in pure cultures; （3） the effect of degradation was the best when the concentration of KMnO4 in the cultures was 0.01% （w/v）, concentration of the three compounds was 5 mg/L and pH was 6.2. Based on the experimental results, a novel concept with regard to the bioremediation of BaP-contaminated environment was discussed, considering the influence on environmental toxicity of the accumulated metabolites.     

Impact of atrazine and nitrogen fertilizers on the sorption of chlorotoluron in soil and model sorbents

Sorption of chlorotoluron in ammonium sulfate, urea and atrazine multi-solutes system was investigated by batch experiments. The results showed application of nitrogen fertilizers to the soil could affect the behavior of chlorotoluron. At the same concentration of N, sorption of chlorotoluron decreased as the concentration of atrazine increased on the day 0 and 6 in soil, respectively. The sorption of chlorotoluron increased from 0 to 6 d when soils were preincubated with deionized water, ammonium sulfate and urea solution for 6 d. That indicated incubation time was one of the most important factors for the sorption of chlorotoluron in nitrogen fertilizers treatments. The individual sorption isotherms of chlorotoluron in rubbery polymer and silica were strictly linear in single solute system, but there were competition sorption between pesticides or between pesticides and nitrogen fertilizers. That indicated the sorption taken place by concurrent solid-phase dissolution mechanism and sorption on the interface of water-organic matter or water-mineral matter.     

基于综合水质标识指数和对应分析法的校园河道水质评价

以三峡大学的校园河道求索溪为研究对象,利用综合水质标识指数法确定求索溪水质类别,分析其水质时空变化规律,并利用对应分析法得出求索溪中不同监测点的主要污染因子.研究结果表明:求索溪整体的综合水质标识指数为7.423,整体水质为劣V类(地表水环境质量标准GB 3838-2002)且黑臭.从时间变化来看,求索溪4月份的水质最差,5月份次之,4、5月份所有监测点的水质都劣于V类且黑臭;8月份水质最好,水质为Ⅳ类;从空间分布来看,8个监测点综合水质标识指数均超过6.0,水质为劣V类,其中6号监测点的水质相对最好,监测点3号的水质相对最差;对应分析法得出求索溪的整体水体污染程度受总氮因子的影响最大,其次为总磷.该研究拟为求索溪及类似校园河道的水环境治理研究提供基础依据和参考.     

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.