荧光原位杂交技术在环境微生物生态学解析中的应用研究

近年来，诸多国家在环境微生物领域先后开展了分子生物学研究方法的建立和生物学评价工作。一些不依靠纯培养的微生物群落的分析方法已得到广泛应用和发展。荧光原位杂交(FISH)技术，具有细胞在测定过程中不被破坏、形状不改变、特异性强、能够真实反映在自然环境下微生物的情况及分布等特点，在环境微生物群落探测分析中已逐渐被广泛应用。该技术利用带有荧光标记的特异性寡核苷酸探针，与细胞内相应的靶核糖体结合，能将微生物探测、鉴定到属和种的水平。运用于硝化细菌、除磷细菌和丝状微生物等废水处理中常见的特征性微生物种群和群落生态学研究中，颇为高效。该技术的应用避免了传统培养方法进行鉴定和计数的局限性，在环境微生物生态学解析中具有较高应用价值。     

绿色荧光蛋白分子标记在环境微生物学研究中的应用

绿色荧光蛋白(green fluorescent protein,GFP)分子标记是现有遗传标记中最简单方便的一种方法，GFP及GFP突变体在微生物降解污染物、生物膜菌群构架、环境生态学和环境检测生物传感器等研究领域取得了很好的应用效果。     

分子生物技术在环境工程微生物领域中的应用

目前分子生物技术发展日新月异,已渗透到各相关学科。本文综述了分子生物技术的基本原理与方法,及其在环境工程微生物领域的应用。讨论了分子生物技术在环境工程微生物的检测应用及在污泥、生物膜、底泥和土壤等微生物种群的多样性分析方面,以及环境工程菌的挑选和培养等方面的研究成果及其巨大研究前景,对该技术在环境工程领域的应用与发展提出了一些见解。     

微生物在水处理技术研究中的新进展

微生物水处理法具有投资少、操作简单、处理时间短等优点,广泛应用于水处理工程之中.2000年3月,在东京大学召开了名为"运用复杂微生物群落机能的高级水处理技术体系的建立与评估"国际研讨会.本文主要就该国际会议从以下五个方面--微生物群落分析技术、水净化微生物技术、生物营养物去除、污泥减量与资源化技术和亚洲热带与亚热带地区的水处理技术等进行综述,并指出微生物在水处理技术中进一步发展的方向和趋势.     

PCR-DGGE技术解析A2/O工艺好氧单元中微生物群落结构

应用PCR-DGGE方法,追踪了汉沽工业废水处理中好氧工艺的活性污泥系统中微生物群落结构动态变化过程及其微生物群落结构组成。研究结果表明:系统中的微生物群落结构随水质变化而变化,随着培养时间的延长,微生物群落结构趋于稳定,分别属于5大类群,与γ、δ、α、ε变形杆菌(Proteobacterias)、芽孢杆菌(Bacilli)的亲缘关系较近。其中γ变形杆菌是该废水处理过程中的主要菌群,包括Pseudomonas sp.、Rheinheimera sp.、Citrobacter sp.、Klebsiella sp.、Enterbacte-riaceae、Stenotrophomonas maltophilia、Acinetobacter。在整个系统中uncultured Pseudomonas sp.、Halobacillus sp.、Pseudomonassp.、Pseudomonas stutzeri、Acinetobacter sp.可稳定存在于系统中,为该污水处理系统中的优势微生物。因此,提高Halobacillussp.、Pseudomonas sp.、Pseudomonas stutzeri、Acinetobacter sp.菌属在系统中的数量和质量,有利于提高废水生化处理的效果。     

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.     

PCR-DGGE技术解析A~2/O工艺好氧单元中微生物群落结构

应用PCR-DGGE方法,追踪了汉沽工业废水处理中好氧工艺的活性污泥系统中微生物群落结构动态变化过程及其微生物群落结构组成。研究结果表明：系统中的微生物群落结构随水质变化而变化,随着培养时间的延长,微生物群落结构趋于稳定,分别属于5大类群,与γ、δ、α、ε变形杆菌（Proteobacterias）、芽孢杆菌（Bacilli）的亲缘关系较近。其中γ变形杆菌是该废水处理过程中的主要菌群,包括Pseudomonas sp.、Rheinheimera sp.、Citrobacter sp.、Klebsiella sp.、Enterbacte-riaceae、Stenotrophomonas maltophilia、Acinetobacter。在整个系统中uncultured Pseudomonas sp.、Halobacillus sp.、Pseudomonassp.、Pseudomonas stutzeri、Acinetobacter sp.可稳定存在于系统中,为该污水处理系统中的优势微生物。因此,提高Halobacillussp.、Pseudomonas sp.、Pseudomonas stutzeri、Acinetobacter sp.菌属在系统中的数量和质量,有利于提高废水生化处理的效果。     

分子生态技术对厌氧脱氮除磷微生物优势菌群的鉴定

应用分子生态克隆技术,直接对A/O反应器所驯化培养的厌氧脱氮除磷微生物样品进行主要功能菌群的分子生态学鉴定。通过克隆文库的序列分析,发现驯化培养后厌氧污泥样品中的优势菌群主要是Azospirillum和Selenomonas两个属。这一结果表明,厌氧反应器中的某些红螺菌科和氨基酸球菌科的微生物,可能是在厌氧条件下对氮磷同时降解起关键作用的功能菌群。     

膜生物反应器中不同阶段微生物群落结构演变的研究

为了研究膜生物反应器（membrane bioreactor, 简称MBR）中微生物群落结构的演变情况,对不同阶段运行状况下(包括培养驯化阶段、初期增长阶段、污泥流失阶段和恢复成熟阶段4个阶段)污泥中的微生物进行了考察。从MBR污泥中提取细菌总基因组DNA,进行聚合酶链式反应变性梯度凝胶电泳(PCR-DGGE)图谱的直观分析,以及总细菌的Shannon多样性指数、各污泥样品的相似性分析和聚类分析。研究表明,反应器中的微生物群落比较丰富。不同的阶段状况下,微生物的群落结构变化比较明显,能够比较好地反映MBR的运行状况和系统处理效能的关系,并且发现微生物的群落结构能够随着反应器的运行状况的变化做出调整恢复。     

不同污泥负荷下SBR处理游泳馆污水的微生物群落的演变

污泥负荷直接影响微生物的生长模式,当污泥负荷发生变化时,短时间内微生物群落结构将发生明显变化。为了研究污泥负荷冲击对SBR系统内活性污泥微生物群落结构的影响,应用聚合酶链式反应-变性梯度凝胶电泳(PCRDGGE)技术,对不同污泥负荷冲击时,SBR处理游泳馆污水中的活性污泥微生物进行了考查。研究表明,在不同污泥负荷冲击的条件下,以MBR污泥为接种污泥,SBR工艺处理游泳馆污水系统内活性污泥微生物群落结构变化明显,多样性指数随着污泥负荷升高而逐渐增加并趋于稳定,但污泥冲击负荷过高多样性指数反而下降,SBR系统内微生物菌种大部分为未经培养菌种,肠杆菌属、甲苯单胞菌属以及γ-变形菌纲细菌等。微生物通过对不同负荷阶段环境条件的适应及演变,逐渐形成了适应相应污泥负荷的微生物种群。     

Whole cell hybridisation for monitoring harmful marine microalgae

Fluorescence in situ hybridisation (FISH) is a powerful molecular biological tool to detect and enumerate harmful microorganism in the marine environment. Different FISH methods are available, and especially in combination with automated counting techniques, the potential for a routine monitoring of harmful marine microalgae is attainable. Various oligonucleotide probes are developed for detecting harmful microalgae. However, FISH-based methods are not yet regularly included in monitoring programmes tracking the presence of harmful marine microalgae. A limitation factor of the FISH technique is the currently available number of suited fluorochromes attached to the FISH probes to detect various harmful species in one environmental sample at a time. However, coupled automated techniques, like flow cytometry or solid-phase cytometry, can facilitate the analysis of numerous field samples and help to overcome this drawback. A great benefit of FISH in contrast to other molecular biological detection methods for harmful marine microalgae is the direct visualisation of the hybridised target cells, which are not permitted in cell free formats, like DNA depending analysis methods. Therefore, an additional validation of the FISH-generated results is simultaneously given.     

Molecular tools for bathing water assessment in Europe: Balancing social science research with a rapidly developing environmental science evidence-base

The use of molecular tools, principally qPCR, versus traditional culture-based methods for quantifying microbial parameters (e.g., Fecal Indicator Organisms) in bathing waters generates considerable ongoing debate at the science–policy interface. Advances in science have allowed the development and application of molecular biological methods for rapid (~2 h) quantification of microbial pollution in bathing and recreational waters. In contrast, culture-based methods can take between 18 and 96 h for sample processing. Thus, molecular tools offer an opportunity to provide a more meaningful statement of microbial risk to water-users by providing near-real-time information enabling potentially more informed decision-making with regard to water-based activities. However, complementary studies concerning the potential costs and benefits of adopting rapid methods as a regulatory tool are in short supply. We report on findings from an international Working Group that examined the breadth of social impacts, challenges, and research opportunities associated with the application of molecular tools to bathing water regulations.     

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C−MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C−MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.     

An updated review on advancement in fermentative production strategies for biobutanol using Clostridium spp.

A significant concern of our fuel-dependent era is the unceasing exhaustion of petroleum fuel supplies. In parallel to this, environmental issues such as the greenhouse effect, change in global climate, and increasing global temperature must be addressed on a priority basis. Biobutanol, which has fuel characteristics comparable to gasoline, has attracted global attention as a viable green fuel alternative among the many biofuel alternatives. Renewable biomass could be used for the sustainable production of biobutanol by the acetone-butanol-ethanol (ABE) pathway. Non-extinguishable resources, such as algal and lignocellulosic biomass, and starch are some of the most commonly used feedstock for fermentative production of biobutanol, and each has its particular set of advantages. Clostridium, a gram-positive endospore-forming bacterium that can produce a range of compounds, along with n-butanol is traditionally known for its biobutanol production capabilities. Clostridium fermentation produces biobased n-butanol through ABE fermentation. However, low butanol titer, a lack of suitable feedstock, and product inhibition are the primary difficulties in biobutanol synthesis. Critical issues that are essential for sustainable production of biobutanol include (i) developing high butanol titer producing strains utilizing genetic and metabolic engineering approaches, (ii) renewable biomass that could be used for biobutanol production at a larger scale, and (iii) addressing the limits of traditional batch fermentation by integrated bioprocessing technologies with effective product recovery procedures that have increased the efficiency of biobutanol synthesis. Our paper reviews the current progress in all three aspects of butanol production and presents recent data on current practices in fermentative biobutanol production technology.

     

混汞法采金地区的汞污染研究进展

混汞法是一种设备简单、操作简便且应用历史悠久的提金方法 ,在世界范围内得到了普遍使用 ,同时也导致了严重的汞污染。本文介绍了混汞法的流程以及汞的释放过程和释放因子 ;综述了应用混汞法提金的不同国家和地区 ,对由此引发的大气、水体、生物 (包括人体 )和土壤汞污染的研究现状 ,并讨论了采金地区的环境管理政策     

环境污染物的分子毒理机制研究进展

以分子生物学技术为基础研究污染物的毒理机制．从分子水平揭示了污染物毒作用的本质。综述了国内外分子毒理机制的研究进展。详细阐述了毒理芯片、单细胞凝胶电泳等分子生物学技术应用与毒理学研究中的特点、应用前景以及存在的问题。提出了分子毒理机制今后的研究方向。     

Filamentous scum bacteria in activated sludge plants: detection and identification quality by conventional activated sludge microscopy versus fluorescence in situ hybridization.

Detection of filamentous bacteria morphotypes involved in scum formation in activated sludge wastewater treatment plants by conventional sludge microscopy is often doomed to fail because of morphological and taxonomical variations. The aim of this study is to compare detection, identification, and quantification quality of filamentous "scum bacteria" found by conventional activated sludge microscopy and fluorescence in situ hybridization (FISH). In the case of filamentous Microthrix parvicella and Eikelboom morphotypes 1863 and 1851, conventional activated sludge microscopy and FISH results correspond well. In contrast, conventional activated sludge microscopy overlooks nocardioform actinomycete and type 1863 single cells. On the other hand, FISH underestimates filamentous nocardioform actinomycetes and morphotypes 0041/0675 or 0092 because of insufficient cell wall permeability or because of their taxonomic variability, with a resulting inadequacy of previously published probes. Nostocoida limicola morphotype results are still inconclusive because of low bacteria numbers being available in situ and the enormous taxonomic variability within this group.     

低温等离子体杀菌的实验研究

由于传统的化学水处理方式有化学成分残留和环境污染问题，因而迫切需要开发新型的水处理技术。本研究针对现有杀菌消毒方法的不足之处，利用低温等离子体进行杀菌处理的实验研究，对电压、水层厚度、气体种类及电极形式的影响进行考察。结果表明，低温等离子体用于杀菌不仅效果显著，而且使用方便、快捷、安全并无残留，因而适于饮用水或者生活污水的杀菌处理。     

Nitrifying community analysis in a single submerged attached-growth bioreactor for treatment of high-ammonia waste stream.

This study investigated the nitrifying community structure in a single-stage submerged attached-growth bioreactor (SAGB) that successfully achieved stable nitrogen removal over nitrite of a high-strength ammonia wastewater. The reactor was operated with intermittent aeration and external carbon addition (methanol). With influent ammonia and total Kjeldahl nitrogen ranging from 537 to 968 mg/L and 643 to 1510 mg/L, respectively, 85% nitrogen removal was obtained, and effluent was dominated by nitrite (NO2-/NOx > 0.95). Nitrifying community analysis using fluorescence in situ hybridization (FISH), with a hierarchical set of probes targeting known ammonia-oxidizing bacteria (AOB) within beta-proteobacteria, showed that the AOB community of the biofilter consists almost entirely of members of the Nitrosomonas europaea/eutropha and the Nitrosococcus mobilis lineages. Image analysis of FISH pictures was used to quantify the identified AOB, and it was estimated that Nitrosomonas europaea/eutropha-like AOB accounted for 4.3% of the total volume of the biofilm, while Nitrosococcus mobilis-like AOB made up 1.2%; these numbers summed up to a total AOB fraction of 5.5% of the total volume on the biofilm. Nitrite-oxidizing bacteria (NOB) were not detectable in the biofilm samples with probes for either Nitrospira sp. or Nitrobacter sp., which indicated that NOB were either absent from the biofilters or present in numbers below the detection limit of FISH (< 0.1% of the total biofilm). Nitrite oxidizers were likely outcompeted from the system because of the free ammonia inhibition and the possibility that the aeration period (from intermittent aeration) was not sufficiently long for the NOB to be released from the competition for oxygen with heterotrophs and AOB. The nitrogen removal via nitrite in a SAGB reactor described in this study is applicable for high-ammonia-strength wastewater treatment, such as centrate or industrial wastes.     

Effects of long exposure to low temperatures on nitrifying biofilm and biomass in wastewater treatment

Attached growth biological treatment systems are a promising solution to ammonia removal in cold-temperature climates. Environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy in combination with fluorescent in situ hybridization (FISH) was used to investigate the effects of 4 months of exposure to 4 degrees C on nitrifying biofilm and biomass. These molecular and microscopic methods were modified to minimize loss of mass and distortion of in situ perspectives. Environmental scanning electron microscopy revealed that nitrifying biofilm did not exhibit significant changes in volume with exposure to 4 degrees C. Confocal laser scanning microscopy in combination with FISH showed that the number of ammonia-oxidizing bacteria (AOB) cells present in the biofilm was statistically consistent during exposure to 4 degrees C. The RNA content of AOB cells remained sufficient for FISH enumeration. The number of nitrite-oxidizing bacteria cells remained steady during exposure to 4 degrees C; however, the RNA content of the cells appeared to decrease with exposure to 4 degrees C, thereby preventing their enumeration using FISH.