FISH荧光原位杂交技术在污水生物脱氮研究中的应用*

简要介绍了荧光原位杂交(FISH)的基本原理,着重讨论近年来该技术在污水生物脱氮研究中的应用现状和特点。研究表明:FISH技术能够准确地表现污水处理反应器中脱氮菌群落的类型和结构形态。但在关于SRT、DO、C/N比等工艺参数的变化对脱氮反应器中微生物类型、数量和结构的影响等方面的研究还有待深入。FISH技术与PCR-DGGE和16SrRNA/rDNA序列分析等技术相结合是对污水处理构筑物中生物脱氮群落深入研究的发展方向。     

短程硝化活性污泥微生物群落结构及羟胺代谢对短程硝化的影响

活性污泥法随着技术的成熟,已应用在高氨氮污水/废水处理中,通过不断发展衍生出的很多新型工艺也成为研究热点,短程硝化反应作为代表已逐渐体现出优越性。短程硝化能达到高效净化污水的目的,其反应中的代谢产物羟胺也和微生物类群及反应产物之间有着至关重要的影响。反应器中活性污泥的微生物群落结构和动态密切相关,探究微生物群落结构能帮助生物强化、优化参数,提高脱氮效率。本文主要总结了近年来有关短程硝化/半短程硝化活性污泥微生物群落组成与结构及其与反应器处理效率之间的关系,以及羟胺代谢对短程硝化的影响等方面的研究进展,这些研究加深了对微生物群落结构和污水处理工艺之间的认识,但充分发掘生物信息、提高工艺效能之路仍然充满挑战,还需利用氮平衡方法、Real-time PCR法等多种生物技术手段对短程硝化进行全方位研究,为实践提供坚实的理论基础。     

厌氧氨氧化菌脱氮机理及其在污水处理中的应用

厌氧氨氧化细菌(anammox)可以将亚硝酸盐和氨氮转化为氮气从而缩短氨氮转化的过程,它已经成为新型生物污水脱氮技术研究的热点之一。当前,有关厌氧氨氧化菌特有的生理结构特点、种群分类及其功能酶等方面的研究取得了一定突破,为实现其工业应用奠定了良好的理论基础;同时分子生物学技术在厌氧氨氧化细菌种群分布、群落多样性及其共生关系等方面的应用也大大促进了污水生物脱氮技术的革新和进步。总结了厌氧氨氧化菌主要的生理生化特点、细胞结构特点、脱氮机理、污水处理体系中的应用以及分子生物学方法对污水处理体系中厌氧氨氧化菌种群分析的研究现状,并指出未来anammox细菌在生物特性及在污水脱氮处理实际应用的研究中的热点问题。生物特性方面的主要研究热点有：（1）anammox细菌除厌氧氨氧化作用外,其它新陈代谢途径有待探索;（2）anammox细菌在不同环境中分布的倾向性问题;（3）新型anammox细菌的确定。污水处理的实际应用方面的主要研究热点有：（1）anammox污泥的快速高效富集问题;（2）设计高特异性引物;（3）anammox细菌和其他微生物的共生关系。     

污水脱氮功能微生物的组学研究进展

生物脱氮是污水处理厂的核心,掌握生物脱氮过程相关微生物代谢特性,对于探索微生物资源和提高污水处理厂脱氮性能具有重要意义。近年来,分子生物学方法不断发展和改进,已被广泛应用于揭示脱氮微生物群落多样性、组成结构和潜在功能等方面,大幅提升了研究者们对污水生物脱氮系统中微生物,尤其是不可培养微生物的代谢机理、抑制调控原理及新型生物脱氮工艺途径的认识。本文对流行的分子生物学方法（16S rRNA基因测序、实时荧光定量PCR技术、宏基因组学、宏转录组学、宏蛋白质组学和代谢组学）进行了介绍,综述了其在硝化细菌、反硝化细菌、完全氨氧化细菌、厌氧氨氧化细菌、厌氧铁氨氧化细菌、硫酸盐型厌氧氨氧化细菌及亚硝酸盐/硝酸盐型厌氧甲烷氧化微生物等方面的研究进展,阐明了这些氮素转化微生物在氮循环过程的代谢途径和酶促反应,并从标准测定方法构建、不同方法的联用及跨学科结合和检测方法的简易化这3个方面展望了分子生物学方法的技术突破及其在污水生物处理系统中的应用前景。本综述从系统角度全面认识脱氮微生物群落及其结构,为未来污水处理生物脱氮微生物的研究提供了新方向。     

ZnO和CuO纳米颗粒对废水生物处理的影响及缓解毒性的研究进展

ZnO和CuO纳米颗粒(nanoparticles, NPs)在研究、医学和工业等领域的广泛使用,已引起人们对其生物安全性的忧虑。相关学者已在污水处理系统中检测到ZnO NPs和CuO NPs,由于其独特的理化性质,低含量NPs就对微生物群落结构和生长代谢产生毒性,进而影响污水脱氮的稳定运行。本文综述了ZnO NPs和CuO NPs对生物脱氮系统中相关功能细菌的毒性及机制,并总结了通过调节水环境因素(如pH值、离子强度、离子类型和天然有机物等)缓解ZnO NPs和CuO NPs的细胞毒性,以期为今后缓解和应急调控金属纳米颗粒(metal oxide nanoparticles, MONPs)对污水处理系统的冲击提供理论基础和支撑。     

Anammox反应器启动前后微生物群落结构差异性

接种A^2/O回流污泥启动Anammox-UASB反应器,研究了上升流速对系统脱氮性能影响,利用高通量测序对反应器中微生物群落结构变化进行了研究。结果表明,历时35 d成功启动Anammox反应器。上升流速升高可以明显促进脱氮效果,在最佳上升流速为1.14 m/h时TN去除率达84.74%,去除速率高达0.766 gTN/(L·d)。高通量分析表明,Anammox污泥群落Alpha多样性较接种污泥明显减少,Anammox污泥中的Anammox菌主要为Candidatus Jettenia和Candidatus Brocadia两个属,同时检测到大量的其他脱氮微生物菌属,系统中这些脱氮微生物的大量增值使系统脱氮能力逐步提高。     

改性生物填料载体强化厌氧氨氧化反应器脱氮研究

以上流式厌氧污泥床(UASB)为研究对象,主要探究2种改性生物填料载体对厌氧氨氧化反应器脱氮性能的影响,并以电子扫描显微镜(SEM)、脱氢酶活性检测及菌群结构分析等微观方面解析影响脱氮性能差异的原因.结果 表明:活性炭聚氨酯及改性聚乙烯塑料填料2种生物填料载体的投加均能在一定程度上提高厌氧氨氧化反应器的脱氮性能,当反应...     

厌氧氨氧化技术应用于主流城市污水处理的研究进展

长期以来,基于硝化和反硝化过程的传统生物脱氮工艺被广泛应用于废水中氮素污染物的去除,但其能耗物耗高,不能适应节能减排的时代发展要求。近年来新兴的厌氧氨氧化(Anammox)工艺,因具有低能耗、低剩余污泥量、无需外加碳源等优点而备受关注,在废水脱氮领域具有非常广阔的应用前景。综述了Anammox生物脱氮技术应用于城市污水处理的最新研究进展,探讨了主流污水处理应用Anammox所面临的挑战,并提出今后的研究重点。     

生物膜型污水脱氮系统中膜结构及微生物生态研究进展

生物膜法污水脱氮系统主要利用生物膜中脱氮功能微生物的代谢活动去除氮素,从而达到净化水质的目的,研究脱氮生物膜的微观结构和微生物生态是揭示生物膜脱氮机理从而提高脱氮效率的重要途径.本文综述了生物膜型污水脱氮系统类型、生物膜微观结构特征及其影响因素、生物膜型污水脱氮系统内氮素传质过程、脱氮机理和生物膜数学模型等方面的研究进展.另外,本文介绍了生物膜型污水脱氮系统内生物膜脱氮功能微生物分布特征,不同生物膜脱氮系统、底物、运行条件和时间对功能微生物群落影响,及新型脱氮功能微生物等方面的研究进展,为生物膜脱氮技术的深入研究提供参考.     

生物造粒流化床微生物落结构及其动态变化

为了研究生物造粒流化床污水处理反应器颗粒污泥中微生物群落结构及其动态变化,分别从生物造粒流化床10、60、110cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA。以细菌和古细菌16S rRNA基因通用引物530F/1490R,对活性污泥中提取的细菌基因组DNA进行PCR扩增,长约1kb的PCR扩增产物纯化后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱。结果显示,生物造粒流化床反应器颗粒污泥中的微生物群落非常丰富,在10cm处微生物的种属达到23种,60cm处为21种,110cm处为20种;生物造粒流化床不同高度都有一些各自的特有种属和共有种属,反应器不同高度的微生物群落演替不明显,微生物群落相似性为83.1%,群落结构较为稳定。     

活性污泥微生物菌群研究方法进展

活性污泥是活性污泥法处理污水系统的功能主体。人类对活性污泥微生物菌群的认识随着其研究方法的发展而逐步深入。传统培养方法只能检测到活性污泥中1％～15％的微生物。随着一系列基于免培养的分子生物学技术的出现,活性污泥中菌群的复杂性和多样性以惊人的速度被人们认识,大量依靠传统检测方法未能发现却在活性污泥中起关键作用的微生物逐渐被发现。许多模拟活性污泥菌群生存环境条件的现代培养技术开始发展,且已成功培养了一部分传统培养方法不能培养的细菌类群,这为研究基于免培养方法发现的大量新的微生物菌群的生理特性和作用机制提供了可能,也无疑将把人们对活性污泥菌群的认识推向一个新的层次．主要介绍活性污泥微生物菌群研究的一系列方法,从传统培养方法到基于免培养的现代分子生物学技术,再到现代培养技术,着重论述了现代分子生物学技术及其在活性污泥微生物菌群研究中的进展。     

Effects of substrate composition on the structure of microbial communities in wastewater using fluorescence in situ hybridisation

The microbial composition in a pulp and paper wastewater aerated lagoon system was analysed using fluorescence in situ hybridisation (FISH) to gain further understanding of the effect of substrate composition on microbial diversity for improved management of wastewater treatment systems. Few experiments have been conducted to tease apart the factors influencing the composition and abundance of certain groups within these wastewaters. Specific probes were used to investigate and enumerate the different bacterial groups present at particular stages through the treatment system over an extended period. Community composition and abundance of specific groups differed through the system however temporal stability was retained despite significant variability in the wastewater. Middle stream wastewater samples were enriched to explore the impact of different carbon/nitrogen/phosphorus (C:N:P) ratios on community composition and provide functionality to groups of micro-organisms within the microbial consortia. Nitrogen and phosphorus conditions did not impact community composition of methanol-fed cultures, which exhibited a dominance of Betaproteobacteria (>75%), namely Methylotrophic bacteria. This was confirmed through 16S rRNA gene sequencing and specific FISH probing, reflecting population observations at the beginning of the treatment system. We conclude that the nutrient and carbon combinations used in the enrichments created an interactive effect, altering the community composition and mimicking the main substrate load in the different stages of the treatment system. Finally, pulp and paper wastewater microbial composition was highly variable across the treatment system but was stable within the time sampled, with the enrichments emulating the substrate loads in the full scale system.     

Site-specific variation in Antarctic marine biofilms established on artificial surfaces

The community structure and composition of marine microbial biofilms established on glass surfaces was investigated across three differentially contaminated Antarctic sites within McMurdo Sound. Diverse microbial communities were revealed at all sites using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques. Sequencing of excised DGGE bands demonstrated close affiliation with known psychrophiles or undescribed bacteria also recovered from the Antarctic environment. The majority of bacterial sequences were affiliated to the Gammaproteobacteria, Cytophaga/Flavobacteria of Bacteroidetes (CFB), Verrucomicrobia and Planctomycetales. Principal components analysis of quantitative FISH data revealed distinct differences in community composition between sites. Each of the sites were dominated by different bacterial groups: Alphaproteobacteria, Gammaproteobacteria and CFB at the least impacted site, Cape Armitage; green sulfur and sulfate reducing bacteria near the semi-impacted Scott Base and Planctomycetales and sulfate reducing bacteria near the highly impacted McMurdo Station. The highest abundance of archaea was detected near Scott Base (2.5% of total bacteria). Multivariate analyses (non-metric multidimensional scaling and analysis of similarities) of DGGE patterns revealed greater variability in community composition between sites than within sites. This is the first investigation of Antarctic biofilm structure and FISH results suggest that anthropogenic impacts may influence the complex composition of microbial communities.     

Bacterial communities associated with benthic organic matter in headwater stream microhabitats

Bacterial communities associated with a variety of benthic detritus types were studied in three streams in the context of the chemical characteristics of the sediment material and the stream water. A cell purification assay was developed for a quantitative microscopic evaluation of bacterial community structure in detritus samples by fluorescence in situ hybridization (FISH). The efficiency of FISH with fluorescently monolabelled probes was compared with FISH with signal amplification by catalysed reporter deposition (CARD-FISH). In detritus types poor in organic carbon and nitrogen, the numbers of prokaryotes were related to the chemical characteristics of the stream water column, whereas no such relationship was found for detritus types rich in organic carbon and nitrogen. These results might help to provide criteria for the selection of detritus types for river ecosystem assessment and monitoring. The percentage of bacteria detected by FISH with monolabelled probes was correlated with the detritus total organic matter (OM). This is likely attributed to a higher ribosome content of microbial cells on substrates rich in OM. Cell detection by CARD-FISH did not show any correlation with OM content, indicating that this technique renders the results more independent from the activity state of cells. Fluorescence in situ hybridization with four group-specific probes suggested a relationship between substrate quality and the composition of the microbial assemblages on the various types of detritus. The improved protocol for cell purification and CARD-FISH may facilitate future investigations on the relationship between the riverine benthic detritus quality and microbial community composition.     

荧光原位杂交技术在医学微生物学中的应用

以16S rRNA 为靶序列的寡核苷酸探针荧光原位杂交技术已广泛应用于分析复杂环境中的微生物群落构成,包括监测和鉴定病原微生物以及未被培养微生物．通过对临床样品中微生物细胞的检测能提供微生物在人体中的种类、数量和空间分布等信息．其结果快速准确,较之传统的病原微生物诊断方法具有明显的优越性,在临床应用中有广泛的前景．     

Natural whey starter for Parmigiano Reggiano: culture‐independent approach

Aims: The aim of this work was to obtain a deeper insight into the knowledge of microbial composition of Parmigiano Reggiano natural whey starters through different culture‐independent methods. Methods and Results: Eighteen different Parmigiano Reggiano natural whey starters sampled from three different provinces of this cheese production area and the nonacidified wheys from which they arose have been studied by length heterogeneity polymerase chain reaction (LH‐PCR) and fluorescent in situ hybridization (FISH). A high microbial composition variability between different samples has been observed. Conclusions: Revealing different images of the same community, LH‐PCR and FISH have given a more accurate view of the not well‐known Parmigiano Reggiano whey starter ecosystem. Significance and Impact of the Study: New lights have been shed on Parmigiano Reggiano natural whey starters microbial composition, highlighting how culture‐independent approach could be used and improved to study this and other food ecosystems.     

Microbial community and physicochemical analysis of an industrial waste gas biofilter and design of 16S rRNA-targeting oligonucleotide probes

A study was conducted to investigate the microbial community structure, the physicochemical properties, and the relationships between these parameters of a full-scale industrial biofilter used for waste gas abatement in an animal-rendering plant. Fluorescence in situ hybridization (FISH) was successfully combined with digital image analysis to study the composition of the microbial community. Several new nucleic acid probes were designed and established based on published 16S rDNA sequences and on ones retrieved from the biomass of the biofilter under investigation. Bacterial detection rates varied greatly over time and filterbed depth between 27.2% and 88.1% relative to DAPI counts. Overall, members of the Betaproteobacteria followed by Actinobacteria, Alphaproteobacteria, Cytophaga-Flavobacteria, Firmicutes and Gammaproteobacteria were the most abundant groups. Among the groups below phylum level, members of the Alcaligenes/Bordetella lineage were on average the most abundant group accounting for up to 8.5% of DAPI-stained cells. Whereas the community composition generally showed no vertical gradient, the lower 50 cm of the biofilter proved to be the most active part for the degradation of aldehydes such as 2- and 3-methylbutanal, 2-methylpropanal, and hexanal. This zone of the filterbed being operated in up-flow direction degraded about 80% of these compounds. Dimethyldisulphide was the most common reduced sulphur compound. Statistical analysis of microbial versus waste gas parameters generally revealed only weak or non-significant correlations between the two. Possible explanations for this finding are discussed.     

现代分子生物学技术在冰川微生物生态研究中的应用

研究冰川中微生物的多样性对于揭示环境气候变迁,研究生物进化,开发微生物资源具有重要意义,现代分子生物学的发展为研究冰川微生物的多样性提供了行之有效的方法.简要综述了16S rDNA文库构建、变性梯度凝胶电泳、限制性片段长度多态性和荧光原位杂交等技术的原理及在冰川微生物生态研究中的应用现状.     

Effects of naphthalene on microbial community composition in the Delaware estuary

The effects of naphthalene on microbial communities in the bottom boundary layer of the Delaware Bay estuary were investigated in microcosms using denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization (FISH) with oligonucleotide probes. Three days after the addition of naphthalene, rates of bacterial production and naphthalene mineralization were higher than in no-addition controls and than in cases where glucose was added. Analyses using both DGGE and FISH indicated that the bacterial community changed in response to the addition of naphthalene. FISH data indicated that a few major phylogenetic groups increased in response to the glucose addition and especially to the naphthalene addition. DGGE also demonstrated differences in community composition among treatments, with four phylotypes being unique to naphthalene-amended treatments and three of these having 16S rRNA genes similar to known hydrocarbon degraders. The bacterial community in the naphthalene-amended treatment was distinct from the communities in the glucose-amended treatment and in the no-addition control. These data suggest that polycyclic aromatic hydrocarbons may have large effects on microbial community structure in estuaries and probably on microbially mediated biogeochemical processes.     

Deposition and postdeposition mechanisms as possible drivers of microbial population variability in glacier ice

Glaciers accumulate airborne microorganisms year by year and thus are good archives of microbial communities and their relationship to climatic and environmental changes. Hypotheses have focused on two possible drivers of microbial community composition in glacier systems. One is aeolian deposition, in which the microbial load by aerosol, dust, and precipitation events directly determines the amount and composition of microbial species in glacier ice. The other is postdepositional selection, in which the metabolic activity in surface snow causes microbial community shifts in glacier ice. An additional possibility is that both processes occur simultaneously. Aeolian deposition initially establishes a microbial community in the ice, whereas postdeposition selection strengthens the deposition patterns of microorganisms with the development of tolerant species in surface snow, resulting in varying structures of microbial communities with depth. In this minireview, we examine these postulations through an analysis of physical–chemical and biological parameters from the Malan and Vostok ice cores, and the Kuytun 51 Glacial surface and deep snow. We discuss these and other recent results in the context of the hypothesized mechanisms driving microbial community succession in glaciers. We explore our current gaps in knowledge and point out future directions for research on microorganisms in glacial ecosystems.