活性污泥总DNA提取方法的比较

从处理某制药废水的MBR反应器中采集活性污泥,评价不同DNA提取方法对其总DNA提取效率的影响。DNA提取分细胞裂解和DNA纯化2步,对细胞裂解比较了珠磨匀浆法、反复冻融法、十二烷基磺酸钠（sodium dodecyl sulfate,SDS）裂解法等7种方法;对DNA纯化比较了酚/氯仿纯化法和胶回收纯化法。结果表明,SDS裂解法、酚氯仿纯化法最优。通过条件优化实验,确定SDS裂解酚/氯仿纯化法在污泥量1.1 g,10 000 r/min离心5 min的操作条件下,获得的DNA产量（10 774 μg/g泥重）和纯度（OD₂₆₀∶OD₂₈₀=1.84）等综合指标最好。     

废水处理系统中活性污泥总DNA的提取

对废水脱氮除磷厌氧-缺氧-好氧系统中的活性污泥样品经过或未经预处理,比较了超声波法、玻璃珠震荡法和冻融法3种细胞裂解方法对DNA提取效果的影响。吸光度和琼脂糖凝胶电泳检测结果表明:活性污泥经TENP和PBS预处理后采用冻融法加2%SDS裂解细胞的DNA提取操作,可以得到数量多、纯度高的活性污泥DNA样品,可不经纯化直接进行PCR扩增反应。     

河流沉积物中微生物DNA的提取方法比较研究

提取河流沉积物中DNA是研究河流沉积物微生物多样性的难点之一,实验开发了一种酚与SDS的DNA提取方法,与OMEGA和BioDee 2种DNA提取试剂盒方法进行了比较,并通过DNA纯度、PCR扩增结合16S rDNA克隆文库检验对几种方法进行了评价.结果表明,OMEGA DNA提取试剂盒法方法简单,提取的DNA纯度较高...     

间歇好氧硫酸盐废水处理系统微生物区系解析

应用PCR-DGGE(polymerase chain reaction-denaturing gradient gel electrophoresis)技术和16S rDNA序列测定对间歇好氧硫酸盐废水处理工艺的微生物群落结构进行了研究.采集味精厂好氧池原始污泥以及实验室内间歇好氧工艺驯化后不同条件下的活性污泥样品,通过基因组DNA的提取、PCR扩增和DGGE分离,初步分析了各污泥样品的微生物群落多样性,结果表明,PCR-DGGE方法可以在一定程度上反映工艺以及操作条件对微生物群落结构的影响.通过DGGE反复分离纯化及割胶回收,DGGE检验为单一条带后进行测序并提交到GenBank数据库比对,结果表明,间歇好氧硫酸盐系统中优势菌株大多数为未培养细菌,来源于不同的污染环境,具有重要污染物降解的生态功能,其中包括与硫酸盐还原菌(Desulfobulbus propionicus)在系统发育上非常接近的菌株.     

一种活性污泥总DNA提取方法的优化

对3种活性污泥DNA提取方法———传统蛋白酶K-SDS-氯仿异戊醇法(CPSCI法)、液氮研磨法和脱腐处理法进行了对比,并针对CPSCI法从污泥量、保温时间、裂解方式及沉淀时间等4个方面进行了优化。结果表明,优化的蛋白酶K-氯仿-异戊醇法(OPSCI法)采用污泥量0.10 g、37℃静置10 min,加SDS常温旋涡振荡及异丙醇直接离心等条件可获得长度在23.1 kb左右的DNA,OD260nm/OD280nm为1.86,稀释10倍后即可进行16S rDNA PCR。该方法重复性好,提取得率高,纯度好,操作简便,为常规实验室开展活性污泥微生物多样性研究提供了帮助。     

DGGE、T-RFLP 、LH-PCR对两种活性污泥的微生物种群多样性分析的比较

采用基于PCR扩增的分子生态技术DGGE、T-RFLP和LH-PCR,对2种典型污水处理系统中的活性污泥（生活污泥、焦化污泥）进行微生物种群多样性分析;并以此比较3种技术的优劣,提出不同应用条件下研究方法的选择依据。根据实验结果：DGGE得到的条带较多,但误差来源也最多;T-RFLP技术较为灵敏,但需要选择适当的内切酶,严格控制酶切条件,并且文库比对误差较大;而LH-PCR操作简单,结果稳定性较高。虽然目前尚无法判断3种方法的准确性,但LH-PCR在活性污泥的微生物种群多样性分析中已显示出潜在优势。     

全细胞脂肪酶(菌)对SBR系统中微生物群落演替的影响

将具有油脂污水处理功能的全细胞脂肪酶(菌)制剂,直接加入到SBR系统的活性污泥中,应用PCR-DGGE技术的应用,研究该制剂对活性污泥中微生物群落演替的影响。16S rDNA DGGE图谱表明投加全细胞脂肪酶(菌)制剂72h后,菌群丰富度Rs为57%、与空白组的48%相比变化不明显,不同样品相似性系数Cs在48.1%~74.4%之间,表明投加全细胞脂肪酶(菌)对活性污泥中原核微生物群落的演替没有明显影响。18S rDNA DGGE图谱表明,投加全细胞脂肪酶(菌)制剂72 h后,菌群丰富度Rs为72%、与空白组的22%相比存在显著性的差异,不同样品相似性系数Cs在14.5%~73.7%之间,而且优势菌体也发生动态变化,表明投加全细胞脂肪酶(菌)后,对活性污泥中的真核微生物群落演替影响明显。     

有机碳源条件下厌氧氨氧化SBBR中的微生物多样性

研究有机碳源对SBBR厌氧氨氧化菌群等微生物的影响。采用16S rDNA序列与PCR-DGGE分析技术相结合的方法,对稳定运行的反应器内的活性污泥和生物膜样品,进行细菌多样性图谱分析,同时采用巢式PCR-DGGE技术对浮霉状菌属(Planctomycetes)细菌进行分析。结果表明,在有机碳源反应系统细菌条带数和多样性指数均高于无机系统,与活性污泥相比,生物膜表尤为明显。当进水不含有机碳源时,氨氧化细菌(ammonia oxidizing bacteria,AOB),厌氧氨氧化菌(anaerobic ammonia oxidizing bacteria,ANAMMOX)为优势功能菌;当进水含有机碳源时,系统中存在的AOB以亚硝化单胞菌(Nitrosomonas sp.)为优势菌群,同时存在反硝化菌,如索氏菌(Thauera sp.)以及厌氧氨氧化菌,它们共同作用完成N的去除。此外,与无机碳源系统相比,有机碳源的存在,有利于浮霉状菌的积累,但压缩了ANAMMOX的生存空间。本研究可为厌氧氨氧化工艺处理低C/N比有机废水提供了理论依据。     

城市污水处理系统进水水质对曝气池微生物种群结构的影响

以芜湖市天门山污水处理厂为例,研究城市污水处理系统进水水质对曝气池活性污泥微生物种群结构的影响。采集多个曝气池活性污泥样品进行DNA提取和聚合酶链式反应(PCR)扩增,对采样期间进水主要水质指标进行测定,运用变性梯度凝胶电泳(DGGE)技术分析各活性污泥样品DNA的PCR扩增产物,采用非加权组平均(UPGMA)聚类法分析活性污泥样品的DGGE图谱,选择基于线性模型的冗余分析(RDA)对影响微生物种群结构的水质理化因子进行排序。分析结果表明,污水处理系统处理效果整体比较稳定,COD的去除率在62.3%～97.0%,NH3-N的去除率在81.6%～96.4%,TP的去除率在95.3%～99.7%,系统出水均能达到设计要求。微生物种群结构不稳定,随着进水水质的变化呈现出动态变化过程。其中,BOD5与微生物种群结构的关联度最高。由此得出,系统功能稳定的污水处理厂污泥中微生物的种群结构仍会存在一定变化。     

以游泳馆污水为处理对象的SBR中不同污泥负荷下氨氧化菌群落的演变

为了研究污泥负荷对SBR系统内活性污泥微生物中氨氧化菌群落结构的影响,应用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,对不同污泥负荷条件下SBR处理经投加葡萄糖调节的游泳馆污水的活性污泥中氨氧化菌进行了分析。研究结果表明,氨氧化菌的群落结构在不同污泥负荷条件下变化明显,在有机碳源较低的情况下生长旺盛,随着污泥负荷的提高其DGGE图谱条带数量逐渐减少,亮度逐渐减弱;在高污泥负荷环境下,氨氧化菌受到严重抑制,多样性指数大幅下降,并从系统中消失。SBR系统内氨氧化菌大部分为不可培养的变形菌,最常见的氨氧化菌是β变形菌中的亚硝化螺菌和亚硝化单细胞菌。     

Effects of different methods of DNA extraction for activated sludge on the subsequent analysis of bacterial community profiles

The effect of different DNA extraction protocols on activated sludge DNA yield and bacterial community composition was evaluated by temperature gradient gel electrophoresis (TGGE). Nine different procedures to extract DNA were compared-sonication (30s), sonication (40s), sonication (50s), freezing-thawing, bead milling, sodium dodecyl sulfate (SDS)-lysozyme, SDS-proteinase K, SDS-lysozyme-proteinase, and a commercial extraction kit. It was found that the TGGE profiles and the DNA band numbers made significant differences via various extraction methods. The yield and purity of DNA extracted by sonication and other physical methods were not satisfactory, while the DNA purity extracted by SDS and other chemical-biological methods were better. Crude DNA extracts isolated by sonication and other physical methods passed the polymerase chain reaction, despite the absence of purification and acquired affluent DNA bands in TGGE. The affluence of bands in TGGE was not consistent with the yield and purification of DNA, but was correlative with extraction protocols. To analyze the activated sludge bacterial community by TGGE fingerprint, it is necessary to make a synthesis of the TGGE fingerprint profiles of chemical and physical DNA extraction methods to overcome the representative bias.     

A+OSA污泥减量工艺的微生态特性

采用16S rDNA序列与PCR-DGGE(polymerase chain reaction-denaturing gradient gel electrophoresis)分析技术相结合的方法,研究了A+OSA(the anoxic+oxic-settling-anaerobic)污泥减量工艺在不同工况下的减量效果及其微生态特性。结果显示,在自然条件下,A+OSA工艺可有效减少剩余污泥27%左右。分子生物研究表明,解耦联池的插入可以明显改变系统微生物的群落结构,且随着解耦联池水力停留时间的延长,系统中部分微生物被"淘洗",微生物丰富度和多样性指数均有所降低。相似性分析表明,参照系统和A+OSA工艺分属于2个不同的集群,但在A+OSA工艺内部各反应池样品间具有较高的相似性,且各反应池在HRT为5.16 h和7.14 h时,表现为显著相似。通过上述研究可为该工艺优化及调控提供理论指导。     

污泥好氧颗粒化过程中微生物群落结构的演变与分析

为了揭示颗粒污泥形成过程中微生物群落结构多样性的演变过程,以人工配水为进水,在SBR中采用厌氧/好氧循环的手段成功培育出具有聚磷特性的颗粒污泥,利用基于16S rDNA的PCR-DGGE技术获得了微生物群落的DNA特征指纹图谱,对条带进行了统计分析和切胶测序,并建立了系统发育树。结果表明,污泥沉降性能的改善要先于颗粒污...     

Atrazine,chlorpyrifos, and iprodione effect on the biodiversity of bacteria,actinomycetes, and fungi in a pilot biopurification system with a green cover

The use of biopurification systems can mitigate the effects of pesticide contamination on farms. The primary aim of this study was to evaluate the effect of pesticide dissipation on microbial communities in a pilot biopurification system. The pesticide dissipation of atrazine, chlorpyrifos and iprodione (35 mg kg^?1 active ingredient [a.i.]) and biological activity were determined for 40 days. The microbial communities (bacteria, actinomycetes and fungi) were analyzed using denaturing gradient gel electrophoresis (DGGE). In general, pesticide dissipation was the highest by day 5 and reached 95%. The pesticides did not affect biological activity during the experiment. The structure of the actinomycete and bacterial communities in the rhizosphere was more stable during the evaluation than that in the communities in the control without pesticides. The rhizosphere fungal communities, detected using DGGE, showed small and transitory shifts with time. To conclude, rhizosphere microbial communities were not affected during pesticide dissipation in a pilot biopurification system.     

Applicability of a modified MCE filter method with Button Inhalable Sampler for monitoring personal bioaerosol inhalation exposure

In this study, a “modified” mixed cellulose ester (MCE) filter culturing method (directly placing filter on agar plate for culturing without extraction) was investigated in enumerating airborne culturable bacterial and fungal aerosol concentration and diversity both in different environments. A Button Inhalable Sampler loaded with a MCE filter was operated at a flow rate of 5 L/min to collect indoor and outdoor air samples using different sampling times: 10, 20, and 30 min in three different time periods of the day. As a comparison, a BioStage impactor, regarded as the gold standard, was operated in parallel at a flow rate of 28.3 L/min for all tests. The air samples collected by the Button Inhalable Sampler were directly placed on agar plates for culturing, and those collected by the BioStage impactor were incubated directly at 26 °C. The colony forming units (CFUs) were manually counted and the culturable concentrations were calculated both for bacterial and fungal aerosols. The bacterial CFUs developed were further washed off and subjected to polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) for diversity analysis. For fungal CFUs, microscopy method was applied to studying the culturable fungal diversity obtained using different methods. Experimental results showed that the performance of two investigated methods varied with sampling environments and microbial types (culturable bacterial and fungal aerosols). For bacterial aerosol sampling, both methods were shown to perform equally well, and in contrast the “modified” MCE filter method was demonstrated to enumerate more culturable fungal aerosols than the BioStage impactor. In general, the microbial species richness (number of gel bands) was observed to increase with increasing collection time. For both methods, the DGGE gel patterns were observed to vary with sampling time and environment despite of similar number of gel bands. In addition, an increase in sampling time from 20 to 30 min was found not to substantially alter the species richness. Regardless of the sampling methods, more species richness was observed in the outdoor environment than the indoor environment. This study described a new personal bioaerosol exposure assessment protocol, and it was demonstrated applicable in monitoring the personal bioaerosol exposure in replace of an Andersen-type impactor.     

Assessing microbial communities for a metabolic profile similar to activated sludge.

To search for reliable testing inocula alternatives to activated sludge cultures, several model microbial consortia were compared with activated sludge populations for their functional diversity. The evaluation of the metabolic potential of these mixed inocula was performed using the Biolog EcoPlates and GN and GP MicroPlates (Biolog, Inc., Hayward, California). The community-level physiological profiles (CLPPs) obtained for model communities and activated sludge samples were analyzed by principal component analysis and hierarchic clustering methods, to evaluate the ability of Biolog plates to distinguish among the different microbial communities. The effect of different inocula preparation methodologies on the community structure was also studied. The CLPPs obtained with EcoPlates and GN MicroPlates showed that EcoPlates are suitable to screen communities with a metabolic profile similar to activated sludge. New, well-defined, standardized, and safe inocula presenting the same metabolic community profile as activated sludge were selected and can be tested as surrogate cultures in activated-sludge-based bioassays.