Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR (PMA-qPCR)

The detection of viable bacteria in wastewater treatment plants(WWTPs) is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide(PMA) combined with the quantitative polymerase chain reaction(PMA-qPCR) to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 μmol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.     

基于大肠杆菌的CellSense生物传感器毒性分析性能研究

采用聚碳酸酯膜直接固定法制备并优化了大肠杆菌（E.coli Top10）CellSense生物传感器微生物电极,探讨了其在重金属和有机污染物生物急性毒性分析中的应用性能.结果表明,基于对数生长后期和稳定期E.coli微生物电极的CellSense生物传感器具有良好的毒性分析性能,基于衰减期E.coli菌株的CellSense生物传感器毒性分析的稳定性和灵敏性降低;CellSense生物传感器测试得Hg²⁺、Cu²⁺、Zn²⁺、邻氯苯酚和对硝基苯酚对E.coli的EC50分别为0.6、3.1、5.8、180和94 μg/mL,制备的E.coli微生物电极在冰箱中4℃保存2个月,仍能很好地满足毒性分析的需要.     

电化学消毒法对水中大肠杆菌的灭活特性

本研究通过电化学消毒小试装置,系统考察了电极板材料、极板间距、p H、水温、Na Cl浓度等因素对水中大肠杆菌CGMCC 1.3373灭活效果的影响,并进一步考察了电化学消毒对抗生素抗性大肠杆菌CGMCC 1.1595的灭活效果及其抗性变化.结果发现,在相同的电化学消毒条件下,阳极采用钌铱涂层的钛电极板的灭活效果显著优于铅涂层和铱钽涂层电极板,并且极板间距为50 mm时灭活率最高.进一步研究表明,电化学消毒法对大肠杆菌的灭活率随水的p H升高而降低,随水温和Na Cl浓度升高而升高.在相同消毒条件下,电化学法对四环素抗性大肠杆菌的灭活率显著低于普通大肠杆菌.消毒后存活的抗性菌对四环素、青霉素、氯霉素的抗性,随消毒时间延长呈现先下降后上升的规律.     

微生物杀灭效果试验中的游离氯转化研究

在实验室研究游离氯灭活微生物的试验中发现,由于试验体系中有机氮的存在,投加游离氯消毒后,游离氯会迅速与之反应转化为无消毒作用的有机氯胺.在对大肠杆菌、铁细菌、铜绿假单胞菌的研究中均发现同样的问题,投加2 mg·L^-1的游离氯消毒剂对初始菌浓度为10⁸ CFU·mL^-1的大肠杆菌进行灭活试验,反应5　min后游离氯就已经为0,而一氯胺和二氯胺的量分别为0.92　mg·L^-1和0.4 mg·L^-1.为了降低有机氮的干扰,采用多次离心纯化菌液、膜过滤纯化菌液和增加受试菌液的稀释倍数的方法进行测试,发现当菌液浓度降至10⁵CFU·mL^-1以下时,游离氯转氯胺的比例可以明显降低;而2种纯化过程并不能分离菌液中的有机氮,进而改善试验中出现的游离氯转氯胺的现象.该结论也说明参加反应的有机氮应来源于细菌自身的细胞物质,有机氮的干扰在消毒的相关研究中需要引起关注.     

氯及氯胺灭活大肠杆菌的消毒动力学模型

分别研究了游离氯和一氯胺在pH7.0、20℃时对大肠杆菌的灭活作用,利用6种不同消毒动力学模型:Chick’slaw,Chick-Watson,Hom,Rational,Hom-Powerlaw,Selleckmodel对试验数据进行了拟合,通过对拟合结果的比较分析得出:在消毒剂符合一级衰减动力学情况下,游离氯和一氯胺灭活大肠杆菌的最佳拟合模型分别是3参数的Rational模型和2参数的Selleck模型.     

连续流光催化消毒器灭活E.coli及病毒的性能

紫外消毒是污水处理常用技术.紫外线仅能破坏微生物的遗传物质,阻断其繁殖,并不能彻底杀死微生物,一旦停止紫外光照射,微生物可在修复酶的作用下恢复活性.光催化过程能产生强氧化性·OH,破坏细胞壁和细胞膜,彻底杀死微生物.为考察光催化技术消毒效率、抑制复活性能及应用潜力,在市售紫外消毒器中安装了光催化组件,搭建了连续流动式光催化消毒器.以大肠杆菌(E.coli)和噬菌体为目标物评价其消毒能力.紫外剂量50 m J·cm-2条件下,初始浓度3.40×106CFU·m L-1的E.coli原水流经紫外消毒器后被去除4.12 log10的E.coli,而流经光催化消毒器后去除4.79 log10.同时光催化处理后E.coli的光复活率仅为紫外消毒后的17%.连续运行40 h的灭菌能力稳定.光催化设备对噬菌体F2和噬菌体MS2的去除率分别为6.37 log10和6.00 log10,表明该设备也具有病毒去除能力.     

贵州万山汞矿区苔藓植物对汞的吸附和富集特征

在所设定的贵州万山汞矿区调查范围内,有6种苔藓植物,其中常见种为东亚褶叶藓和大羽藓.结果表明,苔藓能高度富集汞,东亚褶叶藓和大羽藓的汞含量随污染源距离和相对高度的增加而降低;不同苔藓对汞的吸附等温线具共同性,吸附量随平衡液浓度的增加而增大,东亚褶叶藓对Hg的吸附量比大羽藓大,吸附特征都能用Liner、Freundlic...     

A flow cytometer based protocol for quantitative analysis of bloom-forming cyanobacteria (Microcystis) in lake sediments

A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W × 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3×10⁴ cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 μupm in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium.     

污水河道污泥中大肠杆菌抗生素抗性与金属耐受性的耦合分析

城市污水河道受人为活动的影响,可能作为抗性细菌与抗性基因的储库,给人类生产生活带来巨大的威胁.本研究以中国寒区城市污水河道污泥为取样点,研究大肠杆菌(Escherichia coli)的抗生素与金属的耐药现状,以评估中国寒区城市水环境受抗性细菌污染的情况与传播抗性的风险.利用选择性培养基从河道淤泥中分离出455株肠杆菌,并从中筛选出110株E.coli.抗生素表型结果显示:69.1%的E.coli分离株对至少1种抗生素具有抗性,其中,抗性最高的是氧四环素(42.7%);所有的E.coli对亚胺培南和呋喃妥因没有抗性;分离株E.coli DL27对检测的17种抗生素中的13种具有抗性,分离株E.coli DL18、分离株E.coli DL46对12种抗生素具有抗性.金属抗性表型检测结果显示:40.9%的E.coli对银(Ag)具有耐受性,3.6%的分离株对汞(Hg)、锌(Zn)具有耐受性,所有菌株对铜(Cu)无耐受性;且发现分离株E.coli DL35对9种抗生素和2种金属具有抗性.对E.coli群体15种抗生素抗性基因(Antibiotic Resistance Genes,ARGs)...     

UV/H2O2降解美罗培南的影响因素及毒性研究

利用波长为254 nm的紫外灯活化过氧化氢(H_2O_2)氧化降解美罗培南(MPN),考察了H_2O_2投加量、初始pH值、水中常见共存阴离子(Cl~-、HCO~-_3、NO~-_3)和天然有机化合物(NOM)等重要影响因素对MPN降解的影响.结果表明,紫外光功率为4 W,初始pH为7.0,n(H_2O_2)/n(MPN)=20∶1时,反应20 min后,MPN的降解率达到97.8%.H_2O_2投加量的增加会加快MPN的降解速率,Cl~-和HCO~-_3对UV/H_2O_2体系中MPN去除效果的影响较小,极少量的NO~-_3会促进MPN的降解,当NO~-_3的浓度≥10 mg·L~(-1)时,MPN的降解受到抑制,且离子浓度越高,抑制程度表现越明显.NOM的存在对MPN的降解有抑制作用.与纯水相比,MPN在实际水体中的去除受到抑制,归因于水体基质的影响.大肠杆菌的急性毒性实验研究表明,MPN的中间转化产物保留了一些抗菌性能,但光解的最终产物无抗菌性能.     

紫外线和次氯酸钠对Escherichia coli和Poliovirus的消毒作用

本研究选用大肠杆菌(Escherichia coli)和脊髓灰质炎病毒(poliovirus)分别作为典型的细菌和病毒,利用培养和定量PCR的检测技术,对比研究紫外线消毒和次氯酸钠消毒对细菌和病毒的作用特点.结果表明:脊髓灰质炎病毒比大肠杆菌更难被灭活,达到1-log所需的氯剂量分别为19.2 mg·L~(-1)·min和10.14 mg·L~(-1)·min;所需的紫外线剂量分别为6.37 m J·cm~(-2)和1.81 m J·cm~(-2).定量PCR方法检测大肠杆菌和脊髓灰质炎病毒达到1-log的核酸损伤所需的紫外线剂量和氯剂量要比培养法高出1~2数量级,紫外线消毒对脊髓灰质炎病毒的RNA损伤量明显大于对大肠杆菌的DNA损伤,病毒的单链RNA对紫外线的敏感性更强,该结果与培养法正好相反.达到1-log核酸损伤脊髓灰质炎病毒所需的紫外线剂量为135 m J·cm~(-2),大肠杆菌所需的剂量为270.3 m J·cm~(-2),核酸损伤需要更多的消毒剂量,可能由于消毒过程微生物进入活性但处于非可培养状态(VBNC),以及灭活对微生物其他分子的损伤和微生物死后核酸的持续性.     

共培养体系中石莼和江蓠对赤潮异弯藻生长的影响

研究共培养体系中2种大型海藻石莼(Ulva pertusa)和江蓠(Gracilaria lemaneiformis)的鲜组织和培养液滤液对赤潮异弯藻(Heterosigma akashiwo)生长的影响.结果发现:①石莼和江蓠均能明显影响赤潮异弯藻生长,鲜组织的作用更明显,能在短时间内完全灭杀共培养的赤潮异弯藻;石莼对微藻的影响强于江蓠的作用.②除菌作用和补充充足的碳酸盐不能消除或缓解石莼和江蓠对赤潮异弯藻生长的影响,说明细菌和碳限制不是导致大藻对微藻作用的原因.③对共培养体系中硝酸盐和磷酸盐的跟踪测定结果显示,营养盐限制不是造成石莼对赤潮异弯藻影响的原因,但却在江蓠的作用中起重要作用.当向江蓠体系中补充f/2营养盐后发现,充足的营养盐能够减轻但不能完全消除江蓠对赤潮异弯藻生长的影响.另一方面,石莼的起始浓度与其对赤潮异弯藻的生长影响之间存在明显的相关性,石莼的起始浓度越高,对赤潮异弯藻的影响越明显.④实验初步说明,石莼可能通过相生相克作用影响共培养体系中赤潮异弯藻的生长,而相生相克和营养竞争的共同作用是导致江蓠作用的根本原因.     

农地土壤重金属Pb和Cd有效性测定方法的筛选与评价

作物重金属累积主要受土壤中重金属有效性的制约,由于土壤种类和污染特征的差异,目前尚无公认的有效态测定方法.为筛选建立适宜土壤铅(Pb)和镉(Cd)有效性评价的方法,本文选择重庆市4种性质差异较大的典型农地土壤:酸性紫色土、中性紫色土、石灰性黄壤和钙质紫色土,系统比较了氯化钙(CaCl2)、醋酸铵(NH4OAc)、盐酸(...     

Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay (NPA-SH)

Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China. In this study, sandwich hybridization integrated with nuclease protection assay (NPA-SH) was used to qualitatively and quantitatively detect P. globosa. Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields. The linear regression equation for P. globosa was obtained, and the lowest detection number of cells was 1.8 × 10⁴ cells. Statistics showed that there was no distinct difference between the results of detecting the microalgae by NPA-SH and traditional microscopy. This technique has good reliability, accuracy, and can give a remarkably high sample processing rate. Sandwich hybridization integrated with nuclease protection assay will provide an efficient alternative to microscopic method for monitoring and investigating the bloom of P. globosa.     

人工纳米材料富勒烯（C60）低剂量长期暴露对鲫鱼的氧化伤害

为更好地判断人工纳米材料(富勒烯,C60)对水生生物的潜在健康危害,以鲫鱼(Carassius auratus)幼鱼为受试生物,研究了低剂量C60(0.04～1.0 mg·L-1)长期(32 d)暴露对鲫鱼的氧化伤害.结果表明,各实验组中鲫鱼幼鱼脑、肝脏、鳃组织中的还原型谷胱甘肽(GSH)含量都发生显著降低(p＜0.05),其中1 mg·L-1 nC60,aq的暴露对鳃组织GSH含量的抑制率为14.3%,高于对鱼脑、肝脏组织中的抑制率;肝脏组织中过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性,以及鳃组织中Na -K -ATP酶活性均被显著激活,其最大活性分别是对照的121.34%(0.04 mg·L-1暴露组)、114.80%(0.04 mg·L-1暴露组)和348.59%(0.20mg·L-1暴露组).实验结果揭示,长期暴露引起机体组织的氧化应激可能是水环境中C60的致毒机制之一.     

Comparative cytotoxicity of nanoparticles and ions to Escherichia coli in binary mixtures

The objective of this study was to understand toxicity of mixture of nanoparticles (NPs) (ZnO and TiO₂) and their ions to Escherichia coli. Results indicated the decrease in percentage growth of E. coli with the increase in concentration of NPs both in single and mixture setups. Even a small concentration of 1 mg/L was observed to be significantly toxic to E. coli in binary mixture setup (exposure concentration: 1 mg/L ZnO and 1 mg/L TiO₂; 21.15% decrease in plate count concentration with respect to control). Exposure of E. coli to mixture of NPs at 1000 mg/L (i.e., 1000 mg/L ZnO and 1000 mg/L TiO₂) resulted in 99.63% decrease in plate count concentration with respect to control. Toxic effects of ions to E. coli were found to be lesser than their corresponding NPs. The percentage growth reduction was found to be 36% for binary mixture of zinc and titanium ions at the highest concentration (i.e., 803.0 mg/L Zn and 593.3 mg/L Ti where ion concentrations are equal to the Zn ions present in 1000 mg/L ZnO NP solution and Ti^+ 4 ions present in 1000 mg/L TiO₂ NP solution). Nature of mixture toxicity of the two NPs to E. coli was found to be antagonistic. The alkaline phosphatase (Alp) assay indicated that the maximum damage was observed when E. coli was exposed to 1000 mg/L of mixture of NPs. This study tries to fill the knowledge gap on information of toxicity of mixture of NPs to bacteria which has not been reported earlier.     

微囊藻毒素和温度对萼花臂尾轮虫热激蛋白Hsp70水平的影响

微囊藻毒素(MCs)是由有毒蓝藻(尤其是铜绿微囊藻)产生的一类藻毒素,可对水生生物和人类健康造成严重危害,然而目前有关浮游动物对MC-LR胁迫的应激响应研究较为少见.因此,本文探究了微囊藻毒素MC-LR和温度对采自青海西宁的萼花臂尾轮虫(Brachionus calyciflorus)热激蛋白Hsp70水平的影响.结果表明,温度、MC-LR及其交互作用对萼花臂尾轮虫Hsp70含量具有显著影响.在各温度条件下,MC-LR均诱导了轮虫Hsp70水平的上调;虽然Hsp70的表达水平在低剂量MC-LR处理下上调不显著,但在高剂量MC-LR(6 mg·L-1)处理下上调却极为显著;另外,以24℃处理为对照组,在20和28℃温度胁迫下各MC-LR处理组轮虫的Hsp70含量均显著增加,但16℃实验组与24℃对照组间未见显著差异,这可能与诱导轮虫Hsp70表达的环境温度阈值或机体对低温环境的长期适应性进化和调节机制有关.     

甲烷催化还原NOx中Co基/分子筛催化剂的助剂和载体优化

采用共浸渍法制备了系列Co基/分子筛催化剂,在固定床催化反应器中考察了不同催化剂在甲烷催化还原NO_x中的催化行为,优化了Co基/分子筛催化剂的助剂和载体,并通过催化表征手段阐明了其构效关系.结果表明,Fe和SAPO-34分别作为优化助剂和载体制备得到的Co-Fe/SAPO-34催化剂具有最高的催化活性,在450℃时,NO_x的最大转化率可以达到52.7%.CO_2对Co-Fe/分子筛催化剂活性无明显抑制作用,H_2O对催化剂的活性抑制是可逆的,SO_2对催化剂活性抑制作用明显.Co-Fe/SAPO-34催化剂表面钴物种以CoO和Co(OH)_2为主,Co-Fe/ZSM-5催化剂表面钴物种则以Co_3O_4和Co(OH)_2为主,Co-Fe/Beta催化剂则可能以CoO、Co Al2O_4和Co_3O_4为主.各Co-Fe/分子筛催化剂表面Fe~(2+)/Fe~(3+)含量比依次为Co-Fe/ZSM-5(3.98)Co-Fe/SAPO-34(0.52)Co-Fe/Beta(0.43).活性组分钴物种的形态和合适的Fe~(2+)/Fe~(3+)含量比对Co-Fe/分子筛催化剂上甲烷催化还原NO_x至关重要.Co-Fe/分子筛催化剂上CH_4-SCR反应机制为:NO在Br?nsted酸位上吸附氧化成NO~+,同时CH_4在Br?nsted酸位上吸附活化成—C=O和—COO等活性物种,生成的NO~+在Co2+和Fe~(3+)等催化活性位上转化成硝酸盐等中间产物,中间产物硝酸盐与活化物种(—C=O和—COO)反应生成N_2和CO_2.     

Histopathological studies and oxidative stress of synthesized silver nanoparticles in Mozambique tilapia (Oreochromis mossambicus)

To evaluate the potential environmental effects of engineered nano metals, it is important to determine the adverse effects of various nanomaterials on aquatic species. Adult tilapia (Oreochromis mossambicus) were maintained in 10 L glass aquaria, and exposed to a graded series of synthesized silver nanoparticles (Ag-NPs) at 25, 50 and 75 mg/L for eight days. The LC₅₀ value was 12.6 mg/L. Reduced activities of antioxidant enzymes and the contents of antioxidants were lowered in the gills and liver of fishes treated with Ag-NPs, which resulted in heavy accumulation of free radicals. Histopathological results imply that the balance between the oxidative and antioxidant system in the fish was broken down during Ag-NPs exposure. The principal concern related with the release of nanomaterials and their smaller particle may change the materials transport and potential toxicity to aquatic organisms compared to larger particles.     

Application of internal standard method in recombinant luminescent bacteria test

Mercury and its organic compounds have been of severe concern worldwide due to their damage to the ecosystem and human health. The development of effective and affordable technology to monitor and signal the presence of bioavailable mercury is an urgent need. The Mer gene is a mercury-responsive resistant gene, and a mercury-sensing recombinant luminescent bacterium using the Mer gene was constructed in this study. The mer operon from marine Pseudomonas putida strain SP1 was amplified and fused with prompterless luxCDABE in the pUCD615 plasmid within Escherichia coli cells, resulting in pTHE30–E. coli. The recombinant strain showed high sensitivity and specificity. The detection limit of Hg^2 + was 5 nmol/L, and distinct luminescence could be detected in 30 min. Cd^2 +, Cu^2 +, Zn^2 +, Ca^2 +, Pb^2 +, Mg^2 +, Mn^2 +, and Al^3 + did not interfere with the detection over a range of 10^− 5–1 mM. Application of recombinant luminescent bacteria testing in environmental samples has been a controversial issue: especially for metal-sensing recombinant strains, false negatives caused by high cytotoxicity are one of the most important issues when applying recombinant luminescent bacteria in biomonitoring of heavy metals. In this study, by establishing an internal standard approach, the false negative problem was overcome; furthermore, the method can also help to estimate the suspected mercury concentration, which ensures high detection sensitivity of bioavailable Hg^2 +.