人类特异性粪厌氧菌基因标记物实时荧光定量PCR检测方法的建立和应用

针对人类粪便污染,选择来源于拟杆菌和双歧杆菌16S rRNA的基因片段作为标记物,分别建立了相应的实时荧光定量PCR检测方法.选取不同来源的粪便样品进行检测,证实了引物的特异性.回收纯化从污水中扩增所得的目标PCR片段,经过连接转化,筛选阳性克隆提取其重组质粒作为实时荧光定量PCR的标准品.通过检测一系列梯度稀释的标准品,确定了拟杆菌基因标记物和双歧杆菌基因标记物定量PCR检测方法分别在1.98×102～1.98×107 copy/μL和2.12×101～2.12×107 copy/μL范围内具有良好的线性关系.     

生活污水中肠道病毒分布特性的定量PCR研究

为了明确污水中肠道病毒的分布及去除规律,采用定量PCR方法对西安市3个污水厂进行了为期一年的监测。结果表明,肠道病毒和人类肠道病毒在进水和二级出水中呈较高浓度,约分布在102~105 copies/mL和102~104 copies/mL范围内。肠道病毒和人类肠道病毒浓度呈现季节性变化,秋季浓度最高,冬季次之,春夏最低。经统计学分析发现,肠道病毒和人类肠道病毒浓度在3个水厂间无显著差异性(p<0.05)且肠道病毒和人类肠道病毒很好地服从对数正态分布。通过病毒的检测数据及分布特性,明确了函数参数并确定人类肠道病毒占肠道病毒的39%。经过常规二级处理,肠道病毒可去除1.12 log左右,与人类肠道病毒去除率相近,且不同水厂对病毒的去除率也相近。     

小型景观水体中病原微生物的分布特性

为了研究病原微生物在景观水体中的分布特性,采用定量PCR方法,监测了校园景观湖水中指示微生物(粪大肠菌群和大肠杆菌),病原性细菌(沙门氏菌和志贺氏菌)和病毒(肠道病毒、轮状病毒和诺如病毒)等典型肠道微生物的分布及变化情况.结果显示,粪大肠菌群未在景观水体中检出,而其他微生物均可检出,表明该水体未受到来自粪便源的污染,其他病原微生物来源于非粪便来源的面源污染.大肠杆菌、沙门氏菌和肠道病毒的检出率(>50%)比其他病原微生物的高;细菌和病毒的浓度分别分布在10-2~101 copies/mL和100~102 copies/mL.统计学分析结果表明,各病原微生物的浓度均服从对数正态分布,大肠杆菌与沙门氏菌和志贺氏菌无显著相关性.而肠道病毒与轮状病毒和诺如病毒在0.05置信水平上显著相关,相关系数分别为0.744和0.609.进一步分析病原微生物在景观水体中一年的变化,结果表明,随着降雨的汇入病原微生物的浓度明显增大,在高温和日光照射可致使病原微生物检出率和浓度明显降低.     

城市生活污水中志贺氏菌ipaH毒力基因的定量PCR检测

基于ipaH毒力基因的实时荧光定量PCR检测,建立适合城市污水中志贺氏菌的定量检测方法。使用从临床分离出的志贺氏菌构建重组质粒作为实时荧光定量PCR的标准品。在ipaH基因模板量2.58×100～2.58×106copy范围内具有良好的线性关系,每100 mL水样中含有2.58×101copy以上的ipaH基因即可被检出。从西安市生活污水分离得到2株野生型志贺氏菌,分析ipaH基因数量和菌体数量的关系,从而确定ipaH基因定量检测志贺氏菌的可行性。该方法灵敏、快速、特异性好,适用于城市生活污水中志贺氏菌的检测。     

Analysis of the effects diclofenac has on Japanese medaka (Oryzias latipes) using real-time PCR

The expression levels of cytochrome P450 1A, p53 and vitellogenin were investigated in three different tissues of male medaka fish after exposure to diclofenac that is one of the main concerns among pharmaceuticals frequently found in sewage treatment plant (STP) effluents. The results showed that cytochrome P450 1A, p53 and vitellogenin were highly expressed in tissue-specific gene expression patterns after exposure to 8 mg/l and 1 μg/l of diclofenac. These elevated expression levels of three biomarkers suggested that diclofenac has potential to cause cellular toxicity, p53-related genotoxicity and estrogenic effects. It is also noteworthy that diclofenac has the potential to cause these effects even at an environmentally relevant concentration of diclofenac, 1 μg/l.     

实时荧光定量PCR对A2/O短程硝化系统内氨氧化菌的定量分析

通过控制好氧区低DO浓度以及缩短好氧实际水力停留时间(actual hydraulic retention time,AHRT),在处理低C/N比实际生活污水的A2/O工艺中,成功启动并维持了短程硝化反硝化;系统亚硝酸盐积累率稳定维持在90%左右,氨氮去除率在95%以上。通过提取富集氨氧化菌(ammonia oxidizing bacteria,AOB)的基因组DNA,经两次常规PCR扩增和琼脂糖凝胶电泳,以纯化回收的DNA扩增片段作为实时荧光定量PCR检测AOB数量的DNA标准品,建立了检测AOB数量的实时荧光定量PCR标准曲线。利用实时荧光定量PCR技术比较了A2/O系统在不同运行条件及亚硝酸盐积累率情况下AOB菌群数量。结果表明,随着系统亚硝酸盐积累率的上升,系统内AOB菌群数量也大幅上升。全程硝化和短程硝化时,系统内的AOB菌群数量分别为5.28×109cells/g MLVSS和3.95×1010cells/g MLVSS。此外,亚硝酸盐积累率的下降相对于AOB菌群数量的下降有一定的滞后性。     

盐酸分级浸出赤泥中有价金属元素

采用盐酸2段分级浸出工艺回收氧化铝赤泥中的有价金属元素。通过考察液固比、反应温度、反应时间及盐酸使用量对浸出率的影响,确定了2段浸出的实验工艺。结果表明:在盐酸用量为理论用量的40%、90 ℃液固比为7∶1、反应时间1 h的条件下,Ca的浸出率为96.2%,Na的浸出率为82.47%,Al的浸出率为42.87%,其他元素几乎不浸出,这是第1段浸出;在盐酸用量为理论量的130%、90 ℃、盐酸浓度8.8 mol·L-1的条件下,浸出1段酸浸渣,Fe的浸出率99.65%,Sc的浸出率88.76%,V的浸出率93.58%,其他稀土元素的浸出率均达到了70%左右,这是第2段浸出。     

Binding and detoxification of chlorpyrifos by lactic acid bacteria on rice straw silage fermentation

This investigation examined the reduction of pesticide residues on straw inoculated with lactic acid bacteria (LAB) during ensiling. Lactobacillus casei WYS3 was isolated from rice straw that contained pesticide residues. Non-sterilized rice straw, which was inoculated with L. casei WYS3, showed increased removal of chlorpyrifos after ensiling, compared with rice straw that was not inoculated with L. casei WYS3 or sterilized rice straw. In pure culture, these strains can bind chlorpyrifos as indicated by high-performance liquid chromatography analysis. Viable L. casei WYS3 was shown to bind 33.3–42% of exogenously added chlorpyrifos. These results are similar to those of acid-treated cells but less than those of heat-treated cells, which were found to bind 32.0% and 77.2% of the added chlorpyrifos respectively. Furthermore, gas chromatography–mass spectrometry analysis determined that L. casei WYS3 detoxified chlorpyrifos via P-O-C cleavage. Real-time polymerized chain reaction analysis determined that organophosphorus hydrolase gene expression tripled after the addition of chlorpyrifos to LAB cultures, compared with the control group (without chlorpyrifos). This paper highlights the potential use of LAB starter cultures for the detoxification and removal of chlorpyrifos residues in the environment.     

Treatment of wastewater from red and tropical fruit wine production by zeolite anaerobic fluidized bed reactor

A study of the anaerobic treatment of wastewaters derived from red (RWWW) and tropical fruit wine (TFWWW) production was carried out in four laboratory-scale fluidized bed reactors with natural zeolite as bacterial support. These reactors operated at mesophilic temperature (35°C). Reactors R1 and R2 contained Chilean natural zeolite, while reactors R3 and R4 used Cuban natural zeolite as microorganism support. In addition, reactors R1 and R3 processed RWWW, while reactors R2 and R4 used TFWWW as substrate. The biomass concentration attached to zeolites in the four reactors studied was found to be in the range of 44–46 g volatile solids (VS)/L after 90 days of operation time. Both types of zeolites can be used indistinctly in the fluidized bed reactors achieving more than 80%–86% chemical oxygen demand (COD) removals for organic loading rates (OLR) of up to at least 20 g COD/L d. pH values remained within the optimal range for anaerobic microorganisms for OLR values of up to 20 and 22 g COD/L d for RWWW and TFWWW, respectively. Toxicity and inhibition levels were observed at an OLR of 20 g COD/L d in reactors R1 and R3 while processing RWWW, whereas the aforementioned inhibitory phenomena were not observed at an OLR of 24 g COD/L d in R2 and R4, treating TFWWW as a consequence of the lower phenolic compound content present in this substrate. The volatile fatty acid (VFA) levels were always lower in reactors processing TFWWW (R2 and R4) and these values (< 400 mg/L, as acetic acid) were lower than the suggested limits for digester failure. The specific methanogenic activity (SMA) was twice as high in reactors R2 and R4 than in R1 and R3 after 120 days of operation when all reactors operated at an OLR of 20 g COD/L d.     

Influence of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on ammonia-oxidizing bacteria and archaea in rhizosphere and bulk soil

In agricultural plant production nitrification inhibitors like 3,4-dimethylpyrazole phosphate (DMPP) are used to retard the microbial nitrification process of fertilized ammonium to enhance the nitrogen supply for cultivated crops and to reduce nitrogen losses from the production system. Besides the well-known ammonia-oxidizing bacteria (AOB) it is known for a few years that also ammonia-oxidizing archaea (AOA) are able to perform the first step in nitrification, hence being also a target for a nitrification inhibitor. However, so far no information are available concerning the effectiveness of DMPP and its extent towards AOB and AOA, neither in bulk soil nor in the root-rhizosphere complex. We investigated in a field experiment performed according to agricultural practice the effect of DMPP on the abundance of AOB and AOA two, four and eight weeks after fertilization. We observed impaired abundances of AOB but not of AOA in both soil compartments that were still visible eight weeks after application, possibly indicating a reduced effectiveness of the nitrification inhibitor in our study.     

UV/Fenton法处理酸性红B研究

采用UV/Fenton高级氧化技术对酸性红B模拟废水进行处理,当进水浓度为400mg/L时,确定了各影响因素的最佳投加量:H2O2投加量为2 mL/L,Fe2 投加量为0.08g/L,最佳pH值为4;并采用一级动力学公式对酸性红B降解速率进行拟合,研究了反应条件对速率常数的影响.最后通过对单独UV法、单独Fenton法和UV/Fenton法3种处理方法效果的比较,发现UV与Fenton试剂具有协同作用.     

UV／Fenton法处理酸性红B研究

采用UV／Fenton高级氧化技术对酸性红B模拟废水进行处理，当进水浓度为400mg／L时，确定了各影响因素的最佳投加量：H2O2投加量为2mL／L，Fe^2＋投加量为0．08g／L，最佳pH值为4；并采用一级动力学公式对酸性红B降解速率进行拟合，研究了反应条件对速率常数的影响。最后通过对单独UV法、单独Fenton法和UV／Fenton法3种处理方法效果的比较，发现UV与Fenton试剂具有协同作用。     

分子生态技术对厌氧脱氮除磷微生物优势菌群的鉴定

应用分子生态克隆技术,直接对A/O反应器所驯化培养的厌氧脱氮除磷微生物样品进行主要功能菌群的分子生态学鉴定。通过克隆文库的序列分析,发现驯化培养后厌氧污泥样品中的优势菌群主要是Azospirillum和Selenomonas两个属。这一结果表明,厌氧反应器中的某些红螺菌科和氨基酸球菌科的微生物,可能是在厌氧条件下对氮磷同时降解起关键作用的功能菌群。     

Altitudinal and thermal gradients of hepatic Cyp1A gene expression in natural populations of Salmo trutta from high mountain lakes and their correlation with organohalogen loads

The biomarker of xenobiotic exposure cytochrome p450A1 (Cyp1A) was used to analyze the biological response to chemical pollution in Salmo trutta (brown trout) from nine high mountain European lakes in Norway, Tatras, Tyrol, and central Pyrenees. Hepatic Cyp1A mRNA levels correlated both with the reciprocal of absolute annual average air temperatures of the sampled lakes and with muscle concentrations of several hydrophobic organohalogen compounds (OC), including chlorinated polychlorobiphenyls (PCB), DDE, and DDT. The correlation between Cyp1A expression and OC content was observed across the whole temperature range (between −0.7 °C and +6.2 °C), but also in the absence of any thermal gradient. We concluded that airborne pollutants accumulate in high mountain lake fish at concentrations high enough to increase Cyp1A expression, among other possible effects. As geographical distribution of semi-volatile OC is strongly influenced by air temperatures, future climate modifications will potentially enhance their physiological effects in lake ecosystems.     

耐热乳杆菌的分离及在食物垃圾乳酸发酵中的应用

食物垃圾在我国城市生活垃圾中占有较大比重.发酵食物垃圾生产乳酸是实现其资源化的有效方法.从厌氧发酵的食物垃圾中分离到一株耐热乳酸菌TY50,根据形态、生理生化特征和16S rDNA序列,确定该菌株属于乳杆菌属的干酪乳杆菌组群(Lactobacillus casei group),其最高生长温度为52℃.TY50发酵食物垃圾生产乳酸的最佳同液比为1:12,最适温度为45 ℃.在pH 5.5～6.0条件下,发酵食物垃圾产生36.29 g/L的乳酸,乳酸体积产牢和转化率(乳峻/垃圾干重)分别达到1.01 g/(L·h)和0.44.     

阳离子表面活性剂改性四氧化三铁-沸石复合材料对水中刚果红的去除作用

制备了四氧化三铁-沸石复合材料(磁性沸石)和阳离子表面活性剂改性磁性沸石(有机改性磁性沸石),采用X射线衍射分析对有机改性磁性沸石进行了表征,通过批量实验考察了有机改性磁性沸石对水中刚果红的吸附性能,并对相关的吸附机制进行了讨论。实验表明,有机改性磁性沸石对水中的刚果红具备良好的吸附能力,且有机改性磁性沸石对刚果红的吸附能力远远高于磁性沸石。有机改性磁性沸石对水中刚果红的吸附动力学符合准二级动力学模型,吸附平衡数据可以采用Langmuir、Freundlich和Dubinin-Radushkevich(D-R)等温吸附模型加以描述。根据Langmuir等温吸附模型计算得到的有机改性磁性沸石对刚果红的最大吸附容量为146 mg/g(pH 7和30℃)。有机改性磁性沸石对水中刚果红的吸附属于自发和放热的过程。有机改性磁性沸石吸附水中刚果红的作用机制包括静电吸引、有机相分配、氢键和表面配位。X射线衍射分析结果表明,有机改性磁性沸石含四氧化三铁,吸附刚果红后的有机改性磁性沸石可以很容易地通过外加磁场的作用快速地从水中分离出来。上述结果表明,有机改性磁性沸石适合作为一种吸附剂去除废水中的刚果红。     

蒌蒿(Artemisia selengensis)根际耐镉产酸细菌的筛选鉴定及其生物学特性

根际细菌的筛选研究有助于构建有效的植物-微生物联合修复体系.采用稀释平板法从蒌蒿根际土壤中分离耐Cd产酸的根际细菌,并对菌株Pb2+、Cu2+和Sb3+的耐受性,分泌吲哚乙酸(IAA)、铁载体、解磷特性及对土壤中Cd的活化效果进行了测定,基于形态特性、生理生化测定和16S rDNA 序列分析对目标菌株进行了分类鉴定.结果表明,从蒌蒿根际土壤中分离到能耐50 mg/L镉浓度且产酸能力较强的根际细菌1株T3,菌株T3对Pb2+、Cu2+和Sb3+均具一定的耐受性,能分泌一定量的IAA和铁载体,但不具解磷特性,能明显增加土壤中有效态镉的含量,经鉴定可初步确定为芽孢杆菌属(Bacillus sp.).为进一步研究其在蒌蒿修复镉污染土壤中的作用奠定了基础.     

盐酸浸出提取赤泥中铝和铁的工艺条件优化

系统研究盐酸浸出赤泥中铝和铁的过程,考察酸浸温度、盐酸浓度、酸浸时间、液固比以及赤泥粒度对铝、铁浸出率的影响。单因素实验和正交实验结果得出,在影响浸出率的酸浸温度、盐酸浓度、酸浸时间和液固比对铁、铝的浸出率的影响几个因素中,酸浸温度和盐酸浓度的影响最大,液固比和浸出时间其次。盐酸酸浸的最佳工艺条件为：赤泥粒度150 μm、酸浸温度80 ℃、盐酸浓度10 mol·L-1、液固比8∶1（V/m）、浸出时间150 min,此时铝的浸出率为96.7%,铁的浸出率为95.1%,铁铝总浸出率96.0%。     

Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.