城市污水细菌多样性及其生物安全性研究

为掌握镇江城市污水中微生物种群结构特征,笔者通过构建并分析城市污水中细菌16SrRNA基因文库,对其系统族群研究发现:污水中细菌16S rRNA基因主要来自变形细菌(proteobacte-ria)的各亚族,占总检序列的86.3％,还有脱铁杆菌门(Deferribacteres)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)等类群,表明城市污水中微生物丰富多样.通过生物安全评价发现,污水中存在多种病原微生物,包括Arcobacter; Sulfurospirillum;Acinetobacter; Aeromonas;Fusobacterium;Bacteroides; Esch-erichia coli;Enterobacter;Clostridium 等.研究表明,近似弓形杆菌属(Arcobacter)的细菌比例高达74.2％,为城市污水中的主要优势菌群,PCR扩增23S rDNA上的特异性序列后进一步证实污水中Arco-bacter为致病性弓形杆菌,该发现为制定治理城市污水生物性污染的措施提供了科学依据.     

纳污河流抗性基因和微生物群落相关性

通过Miseq高通量测序分析和荧光定量PCR技术,研究某纳污河流中四环素类与磺胺类抗性基因（ARGs）的分布特征、传播情况及微生物群落结构相关性.结果表明：在河流地表水与沉积物中均检测到四环素类抗性基因tetA和tetB与磺胺类抗性基因sul1和sul2,四环素类抗性基因和磺胺类抗性基因的绝对丰度分别为5.09×10³~1.26×10⁷,3.94×10⁵~1.32×10⁹copics/L,磺胺sul1抗生素抗性基因丰度显著高于其它基因;河流主要优势菌门为Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria和Cyanobacteria,其平均总相对丰度占总比例的95.62%,且总体差异较小.抗性基因与微生物群落冗余分析显示,Methylotenera菌属是影响tetA抗性基因分布丰度的主要因素,Dechloromonas和Clostridium sensu stricto 1菌属是影响tetB抗性基因分布丰度的主要因素,Dechloromonas、Clostridium sensu stricto 1和Methylotenera菌属是影响sul1、sul2抗性基因分布丰度的主要因素.     

产毒微囊藻mcyA基因荧光定量PCR方法的建立

针对微囊藻毒素合成酶基因mcyA探索一种适用于自然水样中微囊藻毒素产毒潜能检测的TaqMan实时荧光定量PCR方法.该方法从铜绿微囊藻FACHB-1279中的mcyA基因中扩增出目的基因片段,用TA克隆方法将基因片段插入Allel TA vector (ABP-CETAVEC020)中来制备质粒标准晶.通过不同连续稀释...     

DNA聚合酶对PCR方法检测基因点突变的干扰分析

以野生型斑马鱼p53基因外显子7为目的片段,运用变性高效液相色谱(DHPLC),比较分析三种常用的商品DNA聚合酶对聚合酶链式反应(PCR)技术检测基因点突变的影响.DHPLC检测结果显示,Promega Shanghai公司Taq DNA聚合酶,Promega公司Taq DNA聚合酶和BD Biosciences公司高保真酶Advantage-HF2的分子突变频率分别为14.3%,5.95%和0.76%.通过DNA直接测序法确认表明,Promega公司Taq DNA聚合酶和BD Biosciences公司高保真酶Advantage-HF2的碱基突变频率分别为3.61×10-4和7.69×10-5.研究表明,不同的DNA聚合酶对PCR技术的检测结果有明显影响,高保真酶Advantage-HF2所造成的碱基错配率明显低于其它两种商业化Taq酶.     

Application of an early warning indicator and CaO to maximize the time–space-yield of an completely mixed waste digester using rape seed oil as co-substrate

In order to increase the organic loading rate (OLR) and hereby the performance of biogas plants an early warning indicator (EWI-VFA/Ca) was applied in a laboratory-scale biogas digester to control process stability and to steer additive dosing. As soon as the EWI-VFA/Ca indicated the change from stable to instable process conditions, calcium oxide was charged as a countermeasure to raise the pH and to bind long-chain fatty acids (LCFAs) by formation of aggregates. An interval of eight days between two increases of the OLR, which corresponded to 38% of the hydraulic residence time (HRT), was sufficient for process adaptation. An OLR increase by a factor of three within six weeks was successfully used for biogas production. The OLR was increased to 9.5 kg volatile solids (VS) m^?3 d^?1 with up to 87% of fat. The high loading rates affected neither the microbial community negatively nor the biogas production process. Despite the increase of the organic load to high rates, methane production yielded almost its optimum, amounting to 0.9 m³ (kg VS)^?1. Beneath several uncharacterized members of the phylum Firmicutes mostly belonging to the family Clostridiaceae, a Syntrophomonas-like organism was identified that is known to live in a syntrophic relationship to methanogenic archaea. Within the methanogenic group, microorganisms affiliated to Methanosarcina, Methanoculleus and Methanobacterium dominated the community.     

新型填料A/O生物滤池处理低碳氮比农村污水脱氮

针对低碳氮比导致低污染农村污水生物处理时出水总氮(total nitrogen, TN)质量浓度高不能满足排放标准的问题,以普通砾石A/O生物滤池为对照组(1号),采用芦竹和活性炭分别作为缺氧段和好氧段填料的A/O生物滤池(2号)处理人工模拟农村污水并研究其脱氮效果.结果表明,当进水化学需氧量(chemical oxygen demand, COD)、氨氮(ammonia nitrogen, NH~+_4-N)和TN质量浓度分别为(79.47±14.21)、(34.49±2.08)和(34.73±3.87)mg·L~(-1)时,两套装置对COD、NH~+_4-N和TN的去除率分别为(88.00±7.00)%和(89.00±10.00)%、(90.00±2.00)%和(97.00±7.00)%、(37±15)%和(68±7)%,表明添加新型填料芦竹和活性炭能显著增强A/O生物滤池对NH~+_4-N和TN的去除.高通量测序结果显示, 1号装置中参与硝化过程的微生物主要为Proteobacteria(变形菌门), 2号则是变形菌门和Nitrospirae(硝化螺旋菌门)共同作用;1号装置缺氧段中发挥反硝化作用的主要细菌门类包括Chloroflexi(绿弯菌门)、变形菌门、Bacteroidetes(拟杆菌门)和Planctomycetes(浮霉菌门),而2号缺氧段中则主要是拟杆菌门、变形菌门、Firmicutes(厚壁菌门)和Patescibacteria.实时荧光定量聚合酶链式反应(quantitative real time polymerase chain reaction, qPCR)结果表明, 2号装置中生物膜的硝化功能基因(amoA和Nitrospira 16S rDNA)、反硝化功能基因(narG、nosZ、nirS和nirK)和厌氧氨氧化功能基因(ANAMMOX)丰度均高于1号装置,除narG和nosZ基因外,其余几种都有1～2个数量级的差别.     

Detection of Escherichia coli in a cattle manure composting process by selective cultivation and colony polymerase chain reaction

Livestock manure is suitable for use as a composting material. However, various intestinal microbes, such as Escherichia coli, are significant components of such manures. Thus, it is desirable that the level of intestinal microbes, and particularly opportunistic pathogens, in compost is inspected and counted regularly. The sensitivity and specificity of detection of E. coli in compost have been improved by selective cultivation followed by colony polymerase chain reaction (PCR) using the ECO primer. Indeed, the sensitivity of this method is higher than that of DNA extraction from compost and PCR. In this study, changes in numbers of E. coli present in a field-scale composting process over time was assessed using selective cultivation and colony PCR. Numbers of ECO-positive colonies after 24 h decreased, with a concomitant rise in compost temperature. ECO-positive colonies were not detected from 33 to 48 h. However, ECO-positive colony numbers increased beginning on day 4 and continuing until day 42. Thus, it seems likely that the high temperatures reached during the composting process did not affect E. coli numbers in the final compost. Additionally, selective cultivation followed by colony PCR using specific primers is an appropriate method of determining levels of cultivable pathogens in composted materials.     

活性污泥总DNA不同提取方法的比较

为了快速、简便和经济地获得活性污泥的总DNA,以用于分子生物学研究,采用5种不同的DNA提取方法提取活性污泥的总DNA,并通过提取的核酸总量、纯度、是否含有聚合酶链反应抑制剂等指标综合分析不同的提取方法对活性污泥总DNA提取效果的影响.结果表明:蛋白酶K-SDS-酚氯仿法和柱提取DNA试剂盒法提取活性污泥总DNA效果最好,提取DNA总量多,纯度高,可不经纯化直接进行PCR扩增反应。