Detection of dioxygenase genes present in various activated sludge

GOAL, SCOPE AND BACKGROUND: Activated sludge from refineries contains various microorganisms that could utilize aromatics under aerobic conditions due to the oxygenase enzymes. Dioxygenase enzymes are oxygenases, which are involved in the ring cleavage step of aromatic hydrocarbons. In this study, the selected catabolic loci involved in ring cleavage have been monitored in the activated sludge samples at different time intervals. The investigation of the dioxygenase genes in the Effluent Treatment Plants (ETPs) and evaluation of their presence at different time points provides a clue for the aromatic utilizing potential of the inherent microbial flora. METHODS: The catabolic gene loci pheB, xylE, tod-isp, bed and nahG responsible for the enzymes catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, toluene dioxygenase-iron-sulphur protein component, benzene dioxygenase and naphthalene dioxygenase were used respectively. The time dependent change in eubacterial population was demonstrated by the amplification of 16S rDNA product, followed by restriction digestion. The template DNA was obtained from the activated sludge collected from ETPs. The supporting physiological data for the overall performance of sludge was developed using respirometric analysis. The on-site COD and MLSS analysis for ETP was used in final evaluation. The study was carried out with samples collected from three different ETPs and also from a selected ETP at different time intervals. RESULTS AND DISCUSSION: The respirometric studies were carried out with phenol, catechol, toluene, and naphthalene to arrive at the target genotypes for further study by PCR protocol. The respirometric analysis coupled with the COD and MLSS analysis represented the physiological capacity of the various sludges. Initially, the tracking protocol was optimized by using different sludge samples, which were collected from refineries. The selected genotypes were amplified and their presence has been confirmed using Southern analysis. The gene loci tod-isp, bed and xylE were commonly observed at various time intervals of the sludge from the same source. The gene loci pheB and nahG were found to be relatively rare. CONCLUSION: The 16S rDNA PCR products after restriction digestion produced different DNA fingerprint patterns, suggesting that the microbial community composition was diverse in the three sources. Similarly, the presence of the catechol 2,3-dioxygenase, benzene dioxygenase and toluene dioxygenase genes confirmed the aromatic degrading potential in the various sludges. The probes could not pick the nahG and pheB genes. However, the respirometeric assay suggested that the oxidative capacity to use naphthalene as a substrate exists. RECOMMENDATION AND PERSPECTIVE: Our study of the diversity at various time points from the ETP provided an overview of the shifts of the catabolic composition of the sludge. This also depends on the influential parameters like the incoming pollutant level and the environmental conditions that are prevailing and often changing from time to time. The results of direct DNA extraction and PCR amplification do reflect the relative abundance of a particular catabolic genotype, which could be used to monitor the efficiency of treatment.     

基于浮动车数据的机动车排放实时测算模型

根据浮动车检测技术可针对单车采集,所收集数据中的平均速度按照采样间隔连续的特点,利用道路实测数据建立了面向其应用的轻型车尾气排放实时测算模型.模型中引入平均速度增量(ASI)指标对平均速度进行细分,并以其来反映相同平均速度下的排放变化.结果表明,与实测数据相比,模型对CO2的计算误差在10%以内;NOx、HC和CO的计算误差在15%以内.同时提出了模型使用方法,可以实现对路网中交通尾气污染的实时测算和动态评估.     

生活垃圾堆肥过程中细菌群落演替规律

应用PCR-DGGE技术研究生活垃圾堆肥过程中的细菌群落演替规律,对堆肥不同时期的宏基因组DNA进行提取,扩增16S rDNA的V3区,分析生活垃圾堆肥过程中细菌群落的变化. DGGE图谱表明,随着堆体温度的升高,DNA条带表现出了明显的动态变化,降温期出现了新的优势条带并趋于稳定,说明堆肥不同时期的细菌群落发生了更替. 对条带分布进行聚类分析,结果表明:以55 ℃为界,将14个堆肥样品划分为2个族,族间的相似性仅为13%,说明堆肥过程中常温期(<55 ℃)和高温期(>55 ℃)微生物群落结构差别较大. 对优势条带回收测序的结果表明:在升温期,堆肥堆体中检测到H. obtusa和人类排泄物中的细菌;但随着温度的升高,具有纤维素降解功能的嗜热微生物Clostridium thermocellum成为堆肥高温期的优势细菌;当堆体温度小于55 ℃时出现了大量的未培养微生物.      

蚯蚓金属硫蛋白定量PCR检测方法及其分子诊断

根据GenBank中蚯蚓(Eisenia fetida)金属硫蛋白(MT)基因序列设计合成引物,采用RT-PCR扩增出目的基因片段,将目的基因片段插入pEASY-Blunt中制备质粒标准品.通过不同梯度稀释倍数的质粒标准品,建立了目的基因MT与内参基因β-actin的SYBR Green I荧光定量RT-PCR检测方法.通过土壤染毒培养实验,将蚯蚓分别暴露于50,500,1000mg/kg 重金属Cd染毒的自然土壤中培养14,28d,研究蚯蚓体内MT mRNA转录水平的表达变化.结果表明,重组质粒测序后显示目的片段序列同源性好,证明所设计的MT、β-actin引物能成功用于蚯蚓MT mRNA的检测.两基因构建的荧光定量标准曲线线性关系分别为0.994,0.999,且PCR扩增效率也皆接近于100%.染毒实验发现,Cd可诱导蚯蚓体内MT mRNA的表达,其表达水平与Cd污染暴露呈现出剂量-效应和时间-效应关系,表明蚯蚓MT基因可作为潜在分子标志物,诊断环境中Cd污染及其暴露水平.     

猪场土壤中5种四环素抗性基因的检测和定量（Quantification of Five Tetracycline Resistance Genes in Soil from a Swine Feedlot）

近年来由于四环素在畜禽养殖业中的大量使用,诱导了环境中四环素抗性微生物的产生,然而有关四环素抗性基因在土壤中存在、迁移和扩散的研究目前还很少.论文提取了北京一规模化养猪场周边土壤的微生物DNA,利用普通PCR检测到5种四环素抗性基因(tet(B/P)、tet(M)、tet(O)、tet(T)、tet(W)),这5种基因都属于编码核糖体保护蛋白的一类.进一步建立了定量PCR程序对这5种基因进行了定量.结果显示,除tet(T)和tet(M)含量接近外(p=0.367),其余几种基因含量之间均差异显著(p<0.05),其中tet(W)的含量最高((2.16±0.20)×108copies·g-1(干土)),比含量最低的tet(B/P)((2.89±0.54)×106copies·g-(1干土))高出约两个数量级.抗性基因tet(W)、tet(T)、tet(M)、tet(O)含量均较高,为猪场土壤中优势抗性基因.这些基因曾被报道广泛存在于猪和牛的肠道中,显示抗性基因很可能是通过基因横向转移(HGT)等机制从养殖动物体内传播到周围土壤中土著微生物体内的.论文所建立的实验方法为进一步系统研究抗生素抗性基因在土壤中的环境行为及其生态风险提供了基础.     

一种羟胺氧化酶基因序列的PCR扩增及其生物信息学分析

采用PCR法获得不动杆菌Acinetobactor sp.YY-5的一种羟胺氧化酶(Hydroxylamine oxidoreductase,HAO)编码基因序列并进行生物信息学分析.根据自养菌的HAO基因序列保守区设计4对引物,通过RT-PCR获得预期大小的部分序列,再采用Genome walking PCR法向所得的部分序列两侧延伸;对所得序列在NCBI中进行blast分析,并用FGeneSB进行开放滨码框(Open read frame,ORF)预测.结果表明,RT-PCR获得了一段462 bp的序列,通过Genome walking PCR向两侧延伸后,获得了一段3 152 bp长的序列;ORF预测结果显示所得序列可能有4个ORF,RT-PCR获得的462 bp序列所在ORF长555 bp,对应氨基酸序列相对分子质量(M)大小为20.2×103;在Genebank中进行的Blast比对分析显示,除保守区的引物序列外,未发现有与此ORF存在明显相似性的HAO基因,表明这可能是一种新型羟胺氧化酶基因.图5表3参14     

计算机图像处理技术在污水处理絮凝效果检测中的应用

采用图像处理技术,分析污水处理过程中投药量与絮凝图像的等效直径、分形维数和浓度特征参数之间的关系。考虑到投药自动控制的实际运行环境和实时性要求,采用Retinex算法解决光照不均现象,在图像处理主要环节上均采用小计算量算法。分析结果表明:在实际的自动控制中具有较好的实用性。     

B/S构架下基于JavaEE与Comet技术的油气泄漏实时监控系统

针对传统WEB监控技术实时性较差、服务器负担重、监控人员任务繁琐等问题,提出了一种基于JavaEE(Java 2 Platform Enterprise Edition)与Comet技术(服务器推技术)的油气泄漏实时在线监控技术。该技术采用多层B/S(Browser/Server)模式软件体系结构,充分利用JavaEE分布式计算平台的优越性,建立了基于Comet目标参数实时在线监控技术的系统模型。通过实时监控实例,对其工作性能进行了验证,实现了实时、远程应急管理与控制。     

褐菖鲉肝HSC70基因的环介导等温扩增技术的建立及对石油污染的剂量效应研究

从褐菖鲉肝脏中克隆了热休克蛋白HSC70基因,利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)建立HSC70基因的定量检测方法。为检测该方法的可行性,将褐菖鲉分别暴露于石油水溶性成分(water-soluble fraction,WSF) 20、60、180 μg·L^-1 1 d后,利用real-time PCR及LAMP技术同时测定褐菖鲉肝HSC70 mRNA表达量,两种方法测定结果基本一致,证实LAMP技术可用于褐菖鲉肝HSC70基因的定量。为更细致了解石油WSF影响褐菖鲉肝脏HSC70基因表达的剂量－效应关系,将褐菖鲉分别暴露于25、50、75、100、125、150、175 μg·L^-1 WSF中,5天后采样,用LAMP技术定量检测HSC70 mRNA。结果表明,HSC70 mRNA表达量在50 μg·L^-1浓度组即被显著诱导,在75 μg·L^-1浓度下达到最大值,这说明褐菖鲉肝HSC70基因对石油污染较敏感,有潜力作为海洋石油污染的生物标志物。