城市河流异养菌耐药状况及微生物群落结构特征

为深入了解城市河流中细菌的耐药状况和微生物群落结构特征,在西安市浐河和灞河河段8个采样点以及某污水处理厂二级出水排放口采集水样,利用细菌培养技术、PCR检测和DGGE(变性梯度凝胶电泳)分析技术,对河流中异养菌耐药率、四环素抗性基因以及微生物群落结构进行研究. 结果表明:浐河和灞河水样中异养菌对SMZ(磺胺甲恶唑)、TET(四环素)、CIP(环丙沙星)和CTX(头孢噻肟)的平均耐药率分别为18.3%、6.2%、2.7%和1.3%,耐药率高低顺序与污水厂排放水中的异养菌相同. 城市河流中异养菌浓度与其耐药率并不存在显著的相关性. 与上游河段相比,污水厂排放口下游河段的异养菌浓度没有明显增加,但对多种抗生素的耐药率却有所升高. 浐河和灞河中四环素抗性基因主要是tetA和tetB,检出率分别为100%和75%,这意味着城市河流中异养菌对TET的耐药性可能主要通过编码外输泵蛋白的抗性机制来实现. 污水厂排放水进入城市河流,并未显著影响河流中的微生物群落结构,但却使某些原有的敏感物种消失,而病原菌或条件致病菌在邻近污水厂排放口的下游河段则有所增加.      

连续施用发酵猪粪对土壤中四环素抗性基因数量的影响

为了研究连续施用发酵猪粪之后,土壤中抗性基因的数量以及发酵猪粪的施用量对土壤抗性基因的影响,采用实时荧光定量PCR方法,对稻麦轮作模式下连续6年施用4.5t/hm2和9.0t/hm2发酵猪粪的土壤进行了四环素抗性基因(TRGs)的检测和定量分析.在施用发酵猪粪的土壤中能够检测到9种TRGs.施用发酵猪粪显著增加了土壤中tet G、tet L、tet B(P)、tet O、tet W的绝对数量,其中tet B(P)、tet W、tet O的绝对数量受到了施肥用量的影响;而tet Z、tet C和tet S的绝对数量则没有受到施肥的影响.上述8种TRGs在同一施肥处理的0~5cm、5~10cm以及10~20cm土壤中的相对丰度分布均无显著差异.发酵猪粪的施用也显著增加了tet G、tet L、tet B(P)和tet O的相对丰度,但是仅有tet O的相对丰度受到了施肥用量的显著影响.本研究表明,在稻麦轮作模式下,农田土壤中的TRGs数量会受到发酵猪粪中残留TRGs的影响,连续施用该粪肥会显著增加土壤中tet G、tet L、tet B(P)和tet O的绝对数量和相对丰度.因此,需要优化畜禽粪堆肥发酵工艺,以减少抗性基因在粪肥中的残留.     

厌氧和好氧处理过程中四环素抗药基因的丰度

为了了解抗生素生产废水不同处理过程(厌氧生物处理和好氧生物处理)中抗药基因的行为,本文以两种四环素(土霉素、金霉素)生产废水处理系统为调查对象,采用PCR和实时定量PCR(qPCR)方法考察厌氧和好氧处理过程中常见的6种四环素抗药基因(tet(A), tet(C), tet(G), tet(Q), tet(W), tet(X))及2种转移因子(I型整合子intI1, 异常插入序列ISCR3)的丰度特征.结果表明,tet(C)在所有样品中均未检出,其它基因在所有样品中检出.四环素生产废水处理系统中,厌氧污泥中tet(A)、tet(G)、tet(X)的相对丰度(与16S rRNA基因的比值)范围为(1.25±0.16)×10^-4~(4.52±0.002)×10^-2,显著低于好氧污泥[(9.88±0.67)×10^-5~(2.70±0.29)×10^-1],而tet(Q)、tet(W)在厌氧污泥中的相对丰度为(1.66±0.03)×10^-2~(7.48±1.22)×10^-2,比好氧污泥中[(1.94±0.12)×10^-3~(2.85±0.16)×10^-2]高1个数量级;转移因子intI1和ISCR3在厌氧污泥中相对丰度范围为(1.48±0.01)×10^-3~(2.61±0.31)×10^-2,显著低于好氧污泥[(1.18±0.15)×10^-1~(8.99±0.75)×10^-1],表明厌氧处理过程中由这两种转移因子介导的水平转移潜力较小.研究表明,好氧处理促进了tet(A)、tet(G)、tet(X)的传播,但对tet(Q)和tet(W)有控制效果,而厌氧处理过程与之相反.抗药基因的分布与水平转移因子、抗药机制、群落结构有关.     

污水处理厂尾水排放对受纳河流细菌和真菌微生物群落的影响

污水处理厂尾水的持续排放会重塑受纳河流的理化和生物条件,对河流水质和水生态产生潜在影响.选取惠州市东江沿线2座污水处理厂的受纳河流进行水样采集及分析,利用高通量测序进行样品DNA分析,系统研究细菌和真菌群落的组成结构及其分布,结合水体的理化指标更全面地反映尾水排放对受纳河流的影响.结果表明,污水处理厂尾水排放口处TP和NH₄⁺-N等营养盐指标均高于受纳河流上游,尾水排放对受纳河流微生物的影响显著,细菌群落的丰富度明显降低.对受纳河流微生物群落组成和水体理化指标进行统计分析,发现DOM、TP和NH₄⁺-N的变化对浮游微生物群落结构可能起关键作用.通过对浮游细菌和真菌群落结构及水体的理化性质综合分析研究,能够更加全面地揭示污水处理厂尾水排放对受纳河流引起的复合生态效应,对污水处理厂提标改造和城市河流的保护具有重大意义.     

四环素对污泥蚯蚓粪中微生物种群和抗性基因的影响

城市污泥中高含量的四环素可能会致使污泥蚯蚓堆肥产物中携带高丰度的抗性基因,降低其利用价值.本实验采用赤子爱胜蚓处理添加不同含量四环素(100、500和1 000 mg·kg-1)的新鲜污泥,以不添加为对照组,分析微生物种群和四环素抗性基因(tet C、tet G、tet M、tet W和tet X)及整合子基因(int I1)在不同四环素选择性压力下的变化,揭示四环素含量对污泥蚯蚓堆肥中微生物种群和抗性基因的影响.结果表明,添加四环素降低了污泥蚯蚓堆肥产物中变形菌门的丰度但增加了拟杆菌门的丰度,且四环素的含量与细菌的Shannon和Pielou指数有明显的负相关关系.同时,添加四环素致使int I1增加了4. 25倍;四环素类抗性基因增加了4. 7～186. 9倍,其中tet M基因丰度与四环素含量呈现出显著的正相关关系.因此初始污泥中较高含量的四环素会改变污泥蚯蚓粪的微生物种群结构,增加抗生素抗性基因的丰度和传播风险.     

人类活动影响下的高原湿地四环素类抗生素抗性基因赋存与微生物群落共现性

抗生素的广泛使用导致抗生素大量进入环境中,进而使微生物产生耐药性.近年来,随着高原区域人类活动的加剧,高原湿地中抗生素耐药基因(ARGs)的赋存和迁移研究备受关注.以地处云贵高原的鹤庆草海国家湿地公园为研究区,分析河流上游(泉眼附近原始生境)和河流下游(居民生活污水排放口)沉积物中的四环素类、磺胺类、喹诺酮类和大环内酯类共4类抗生素的含量分布,其中四环素类抗生素检出含量为103.65～2 185μg·kg^-1,是含量最高的抗生素种类.为进一步探究四环素类抗性基因的赋存特征和影响因素,通过相关性分析和网络分析揭示了人类活动影响下环境因子、细菌群落结构和病原菌对四环素类ARGs的影响.结果发现,上、下游沉积物中共检出15种四环素类抗性基因,其中,上游检出tetPA、tetD和tetPB等7种抗性基因,下游检出tetPA、tetE、tetM和tetX等13种抗性基因,下游新增的8种抗性基因占下游基因丰度的43.44%;四环素类抗生素含量及有效磷、总有机碳、硝态氮和总磷等理化指标是影响四环素类ARGs分布的主要环境因子;上、下游沉积物中检出细菌分属于64个细菌门,其中变形...     

四环素抗生素对污泥中四环素抗性基因丰度和表达水平的作用影响

为研究四环素(TC)抗生素对其抗性基因(TC-ARGs)转录表达的作用影响,以从活性污泥中筛选获得的四环素抗性细菌(TRB)作为研究对象,采用荧光定量PCR和逆转录PCR方法检测了7种TC-ARGs,包括tet A、tet C、tet G、tet M、tet O、tet W和tet X基因的丰度和表达水平,并探讨了TC与TC-ARGs丰度及其表达水平之间的相关关系.结果表明,在整个培养周期内,tet A、tet G和tet W基因丰度随TC暴露浓度的增大总体呈现上升趋势,但其余TC-ARGs基因丰度整体波动较大. TC胁迫对不同TC-ARGs的转录表达水平作用影响差别较大,其中,tet A基因表达水平相对稳定,且随TC浓度的升高而上调,在TC浓度为100 mg·L~(-1)时,其上调倍数高达5. 3倍.在短期(1 d) TC胁迫下,TC-ARGs转录表达水平随TC浓度的升高整体呈现上调趋势.由相关性分析可知,tet A和tet W基因丰度与其转录表达水平之间存在显著的线性相关性,表明其基因丰度可在一定程度上衡量和评价其抗性表达水平,进而反映其功能活性和环境风险.     

北运河沉积物中氨氧化微生物的群落特征

采用T-RFLP、RT-qPCR和克隆测序等分子生物学技术,以氨单加氧酶基因(amoA)为分子标记,研究了北运河表层沉积物中氨氧化古菌(AOA)和氨氧化细菌(AOB)的群落多样性、丰度、系统发育及其与环境因子的响应关系.结果表明,沉积物中AOB的群落多样性和丰度均高于AOA,是北运河沉积物中氨氧化过程的主要功能微生物.沉积物中氨氧化微生物群落结构沿干流和支流存在明显的空间分异,而AOA的种类组成空间差异较小;沉积物的氨氮(NH4+)和硝态氮(NO3﹣+NO2﹣)是影响氨氧化微生物群落特征的主要因子,AOB对环境变化的敏感性更高;AOA和AOB的amoA基因拷贝数分别为1.32×105~1.91×106copies/g、5.39×105~8.3×106copies/g.闸坝下游沉积物的氨氧化微生物丰度最高.系统发育分析表明,amoA基因序列多属于土壤/沉积物分支,较多AOB的克隆序列与土壤亚硝化螺菌属(Nitrosospira)的类群相似性可达98%.受污水处理厂退水的影响,部分amoA基因序列与污水处理厂废水和活性污泥中发现的类群同源性高.污染物质来源、支流汇入和闸坝拦截对河流沉积物氨氧化微生物的群落特征影响显著.     

布吉河丰水期总细菌和氨氧化细菌的定性和定量研究

河流中微生物的数量和群落结构能在一定程度上反映水环境状况.氨氧化细菌驱动的硝化作用是氮素转化的主要机制,为了解氮素污染河流中氨氧化细菌的群落组成及数量,采用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和Real-time PCR技术分析了布吉河丰水期不同断面水样中总细菌和氨氧化细菌的群落结构以及数量变化.结果表明,水样中总细菌(16S rRNA)和氨氧化细菌(16S rRNA)数量变化范围分别为4.73×1010～3.90×1011copies.L-1和5.44×106～5.96×108copies.L-1.冗余度分析表明影响微生物数量和群落结构的水环境因子不同:对于总细菌,与其数量显著相关的环境因子是硝氮(P<0.05),与其群落结构显著相关的环境因子是氮素(三氮)和金属(Mn和Zn)(P<0.05);对于氨氧化细菌(ammonia-oxidizing bacteria,AOB),与其数量显著相关的是氨氮和Zn(P<0.05),与其群落组成显著相关的是氨氮、Mn和Zn(P<0.05).测序结果表明在布吉河水样中微生物属于变形菌门(Proteobacterium)的Epsilon-Proteobacteria、Gamma-Proteobacteria、Beta-Proteobacteria和Delta-Proteobacteria这4个纲,氨氧化细菌与Nitrosomonas sp.和Nitrosospira sp.属的细菌相似度较高,且Nitrosospira sp.为优势菌属.由于污染影响,布吉河上游和下游微生物群落结构明显不同.     

畜禽粪便中多重耐药细菌及耐药基因的分布特征

为了解畜禽粪便中多重抗生素耐药细菌及耐药基因的污染特征,采用微生物培养的方法调查了鸡粪、猪粪中多重耐药细菌的数量,并挑取部分菌株进行16S rDNA鉴定和抗生素敏感性试验;进一步通过高通量测序技术解析多重耐药细菌的群落结构,利用高通量定量PCR对粪便中176种耐药基因的分布情况进行研究.结果表明,不同鸡粪、猪粪中对四环素、环丙沙星和庆大霉素同时耐药的多重耐药细菌比例在7.96%~12.40%;单菌株鉴定和群落结构分析均显示,可培养的多重耐药细菌主要集中在Escherichia(埃希氏杆菌属)、Acinetobacter(不动杆菌属)和Proteus(变形杆菌属)中.与未饲用抗生素的猪粪相比,猪粪样品中耐药基因的总富集倍数达到1.96×10~4~1.54×10~5倍,各类耐药基因的富集情况为:四环素类β-内酰胺类MLSB(大环内酯、林可酰胺和链阳性菌素B类)氨基糖苷类FCA(氟喹诺酮、喹诺酮、氟苯尼考、氯霉素和酰胺醇类)磺胺类万古霉素类.     

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.     

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.     

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.     

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.     

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.     

Effects of chitosan on growth of an aquatic plant (Hydrilla verticillata) in polluted waters with different chemical oxygen demands

Effects of chitosan on a submersed plant, Hydrilla verticillata, were investigated. Results indicated that H. venicillata could prevent ultrastructure phytotoxicities and oxidativereaction from polluted water with high chemical oxygen demand (COD). Superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents in H. verticillata treated with 0.1% chitosan in wastewater increased with high COD (980 mg/L) and decreased with low COD (63 mg/L), respectively. Ultrastructural analysis showed that the stroma and grana of chloroplast basically remained normal. However, plant cells from the control experiment (untreated with chitosan) were vacuolated and the cell interval increased. The relict of protoplast moved to the center, with cells tending to disjoint. Our findings indicate that wastewater with high COD concentration can cause a substantial damage to submersed plant, nevertheless, chitosan probably could alleviate the membrane lipid peroxidization and ultrastructure phytotoxicities, and protect plant cells from stress of high COD concentration polluted water.