基因芯片技术在环境微生物群落研究中的应用

基因芯片技术作为一种快速、敏感、高通量的检测技术,近几年来在环境微生物群落研究中的应用越来越广泛并且得到充分的发展.它不仅可以研究环境微生物群落的微生物分布、种类、功能、动力学变化,还能分析环境污染等环境因素改变对其微生物生态的影响.本文按照基因芯片探针的设计方法,将环境样品群落研究基因芯片分为系统寡核苷酸芯片、功能基因芯片、群落基因组芯片、宏基因组芯片,并简要综述了该技术在活性污泥、土壤、水等环境样品微生物群落研究上的应用,最后,本文展望了该技术的研究方向和在寻找不同环境微生物群落之间差异微生物、差异基因或差异表达基因研究中的应用前景.     

Simultaneous Rates of Ribonucleic Acid and Deoxyribonucleic Acid Syntheses for Estimating Growth and Cell Division of Aquatic Microbial Communities

A method for measuring rates of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) syntheses using a single radioactive precursor has been devised and tested using bacterial cultures and natural assemblages of marine and freshwater microorganisms. The procedure is based upon the uptake and incorporation of exogenous [³H]adenine into cellular adenosine triphosphate and deoxyadenosine triphosphate pools which serve as the immediate precursors for the adenine incorporated into RNA and DNA, respectively. It is proposed that the DNA/RNA rate ratio is correlated with the specific growth rate of microorganisms and can be used as an index for estimating and comparing the productivities of microbial assemblages in nature. This technique can also be used to detect discontinuous growth and cell division processes which frequently occur in surface plankton populations. The DNA/RNA rate ratios measured in a variety of aquatic ecosystems ranged from 3.3 to 31.8% without significant correlation to total microbial biomass.     

微生物的交流信号

多年来微生物一直被认为是相对孤立的个体,在环境中独立地生存,但近些年的研究使人们认识到微生物也使用复杂多样的方式进行种内、种间,甚至与其他生物间的跨界信息交流。这些交流由特定的信号分子来完成,称之为微生物语言。借助这些交流语言使微生物在特定的生态位中与其相邻个体或种群建立了多样的互动关系,包括合作、竞争与资源共享等,通过协调群体行为,共同应对多变的环境。随着现代分子科学对自然微生物群落的不断深入研究,人们对微生物交流也逐渐有了更为清晰的认知。本综述总结了原核和真核微生物所使用的主要信号物质(如群体感应、群体猝灭、抗生素等)和交流方式,讨论了这些通讯语言在种内(同种微生物)、种间(异种微生物),以及跨界(微生物与宿主)交流上的表现。旨在更为深入地解读这一有趣的多学科交叉研究领域,更好地理解微生物交流语言的形式、机制和目的,为微生物行为的解读和生态事件的解析获取基于化学生态学的新思路。     

An in situ method for cultivating microorganisms using a double encapsulation technique

The lack of cultured microorganisms represents a bottleneck for advancement in microbiology. The development of novel culturing techniques is, therefore, a crucial step in our understanding of microbial diversity in general, and the role of such diversity in the environment, in particular. This study presents an innovative method for cultivating microorganisms by encapsulating them within agar spheres, which are then encased in a polysulfonic polymeric membrane and incubated in a simulated or natural environment. This method stimulates growth of the entrapped microorganisms by allowing them access to essential nutrients and cues from the environment. It allows for the discovery of microorganisms from dilutions that are 10–100-fold greater than possible with conventional plating techniques. Analysis of microorganisms grown in such spheres incubated in and on a number of different substrates yielded numerous novel ribotypes. For example, spheres incubated on the mucus surface of a Fungiid coral yielded numerous ribotypes, with only 50% sharing similarity (85–96%) to previously identified microorganisms. This suggests that many of the species represent novel ribotypes. Hence, the technique reported here advances our ability to retrieve and successfully culture microorganisms and provides an innovative tool to access unknown microbial diversity.     

Phenotypic biomonitoring using multivariate flow cytometric analysis of multi-stained microorganisms

A new method for monitoring phenotypic profiles of pure cultures and complex microbial communities was evaluated. The approach was to stain microorganisms with a battery of fluorescent dyes prior to flow cytometry analysis (FCM) and to analyse the data using multivariate methods, including principal component analysis and partial least squares. The FCM method was quantitatively evaluated using different mixtures of pure cultures as well as microbial communities. The results showed that the method could quantitatively and reproducibly resolve both populations and communities of microorganisms with 5% abundance in a diverse microbial background. The feasibility of monitoring complex microbial communities over time during the biodegradation of naphthalene using the FCM method was demonstrated. The biodegradation of naphthalene occurred to differing extents in microcosms representing three different types of aromatic-contaminated groundwater and a sample of bio-basin water. The FCM method distinguished each of these four microbial communities. The phenotypic profiles were compared with genotypic profiles generated by random-amplified polymorphic DNA analysis. The genotypic profiles of the microbial communities described only the microbial composition, and not their functional change, whereas the phenotypic profiles seemed to contain information on both the composition and the functional change of the microorganisms. Furthermore, event analysis of the FCM data showed that microbial communities with initially differing compositions could converge towards a similar composition if they had a capacity for high levels of degradation, whereas microbial communities with similar initial compositions could diverge if they differed in biodegrading ability.     

Microbial populations in wetwood of European white fir (Abies alba Mill.)

Abstract A method for extraction of microbial populations from wood samples was worked out which gave good recovery of both aerobic and anaerobic microorganisms in agar shake dilution and plating enumerations. This method was applied to the quantification of microbial populations in three European white firs ( Abies alba Mill.) which were afflicted with the European fir disease. Low numbers of aerobic microorganisms (10²−10⁴ colony- forming units (cfu) per g fresh tissue) were detected in sapwood irrespective of the degree of affliction. Anaerobic bacteria were usually 1–2 orders of magnitude less frequent. Wetwood of highly diseased firs contained significantly higher numbers of aerobic microorganisms (10⁵−10⁷), whereas the number of anaerobes was not enhanced significantly. Among the prevalent aerobic microorganisms in wetwood were Protaminobacter, Pseudomonas strains, and a yeast. In anaerobic counts from wetwood, Klebsiella and Vibrio strains predominated. The sapwood contained Bacillus, Beijerinckia, Staphylococcus , and Clostridium spp. High numbers of aerobic microorganisms were also detected in the roots and lower stem of a diseased vine plant ( Vitis vinifera L.). The importance of microbial populations in wetwood formation and disease expression is discussed.     

Microbial associations of soil types

Microorganisms are characterized by wide ranges of distribution. Some groups, however, are known to have zones of active proliferation, and the development of specific populations in discrete zones results in rather specific microbial associations in some soil types. The soils formed in warm climates are richer in microorganisms and contain more bacilli and actinomycetes. The spectrum of dominant microbial groups varies in different soil types.     

A decomposition method for the stability analysis of large microbial systems

The primary objective of this paper is to present a method for the stability analysis of microbial systems consisting of a large number of different populations of microorganisms. The overall system is decomposed into easily analyzable subsystems and its stability characteristics are deduced from those of a properly constructed linear system of lower dimension. Several examples are provided which demonstrate the use of the method in studying the dynamics of some interacting microbial populations which grow under continuous flow conditions and, after a parametric analysis, the regions of the parameters which guarantee coexistence are found.     

Screening for lectin-like protein-producing microorganisms based on cell surface proteins

A method for screening lectin-producing microorganisms was developed. The presence of lectin on microbial cell surfaces was used as an index for their selective isolation. The lectin-producing microorganisms adhered to sugar-modified agarose beads and were selectively eluted with specific saccharide solutions. Spin columns were an effective tool for excluding non-lectin producers. Eighty-seven percent of the microorganisms that were eluted from the beads showed hemagglutination. The results of sequence analysis indicated that some of the eluted microorganisms have not been previously identified as lectin-producing microorganisms.     

Marker and reporter genes: illuminating tools for environmental microbiologists

Fluorescent and luminescent marker and reporter genes provide easily detectable phenotypes to microbial cells and are therefore valuable tools for the study of microorganisms in the environment. Although these tools are becoming widely adopted, there are still issues that remain to be solved, such as the dependence of the reporter output on the physiological status of the cell. Eventually it might be the use of marker and reporter genes themselves that will contribute towards better understanding of the physiological status of specific microbial populations in nature.     

Stable carbon isotopes of lipid biomarkers: analysis of metabolites and metabolic fates of environmental microorganisms

Lipid biomarkers are specific compounds that are characteristic of certain groups or species of microorganisms. The use of natural or labeled carbon isotopes of lipid biomarkers has enabled a better understanding of carbon flow pathways at the molecular level. Recent advances include, but are not limited to, the elucidation of mechanisms of anaerobic methane oxidation mediated by syntrophic sulfate-reducing bacteria and Archaea, linking microbial populations with specific microbial processes or bacterial transport mechanisms in natural or contaminated environments, and elucidation of the biosynthetic pathways of cellular material.     

Dynamics of water-soluble carbon substances and microbial populations during the composting of de-inking paper sludge

Composting is an alternative method to dispose of de-inking paper sludge (DPS). Today, few studies have investigated the water-soluble carbon (WSC) substances as indicators of the decomposition process and the microbial changes taking place during the composting of DPS. Accordingly, the goal is to study their dynamics during the composting of DPS at three nitrogen levels, 0.6%, 0.7% or 0.9% total N, using mechanical turning. The changes in WSC substances, microbial biomass carbon (MBC) and, total and DPS microbial populations were monitored during 24 weeks. Also, microorganisms were identified and tested for the production of selected enzymes. Regardless of N treatments, the dynamic of WSC substances indicated that cellulose and hemicellulose fractions of DPS fibers were mainly biodegraded during the first 8 weeks while the more resistant carbon (C) fractions were biodegraded thereafter. MBC also evolved regardless of N treatments but was correlated to WSC substances. Its high values decline mostly after 12 weeks indicating the exhaustion of this source of C energy for microbial growth and the stabilisation of DPS organic matter. The dynamic and identified microorganisms were comparable to those observed in other composting processes. However, the results pointed out that those mostly implicated in the hydrolysis of DPS fibers were the thermophilic actinomycetes and fungi and, by comparison to the 0.6% or 0.7% N treatment, they decreased in presence of the 0.9% N treatment. Most microorganisms were hemicellulolytic bacteria, while actinomycetes and fungi were capable of degrading a wide variety of substrates. Overall, dynamics of WSC substances and microbial populations indicated that during composting, DPS decomposition obey a two phase decay while, contrary to the lowest N treatment, the 0.9% N treatment has slowed down this process by harming the important microbial populations implicated in the degradation of DPS fibers.     

Assessing populations of a disease suppressive microorganism and a plant pathogen using DNA arrays

Understanding the relationships between disease suppressive microbial populations and plant pathogens is essential to develop procedures for effective and consistent disease control. Currently, DNA array technology is the most suitable technique to simultaneously detect multiple microorganisms. Although this technology has been successfully applied for diagnostic purposes, its utility to assess different microbial populations, as a basis for further study of population dynamics and their potential interactions, has not yet been investigated. In this study, a DNA macroarray with multiple levels of phylogenetic specificity was developed to measure population densities of a specific disease suppressive microorganism, Trichoderma hamatum isolate 382, and the plant pathogen Rhizoctonia solani. Amongst others, the DNA array contained genus-, species- and isolate-specific detector oligonucleotides and was optimized for sensitive detection and reliable quantification of the target organisms in potting mix samples. Furthermore, this DNA array was used to quantify disease severity as well as incidence of severe disease based on pathogen population densities in the growing medium. Taking into account the unlimited expanding possibilities of DNA arrays to include detector oligonucleotides for other and more microorganisms, this technique has the potential for studying the population dynamics and ecology of several target populations in a single assay.     

Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high-resolution flow cytometry

The two primary kinetic constants for describing the concentration dependency of nutrient uptake by microorganisms are shown to be maximal rate of substrate uptake and, rather than the Michaelis constant for transport, specific affinity. Of the two, the specific affinity is more important for describing natural aquatic microbial processes because it can be used independently at small substrate concentrations. Flow cytometry was used to evaluate specific affinities in natural populations of aquatic bacteria because it gives a convenient measure of biomass, which is an essential measurement in the specific-affinity approach to microbial kinetics. Total biomass, biomass in various filter fractions, and the specific affinity of the bacteria in each fraction were determined in samples from a near-arctic lake. The partial growth rate of the pelagic bacteria from the 25 micrograms/liter of dissolved amino acids present (growth rate from the amino acid fraction alone) was determined to be 0.78 per day. By measuring activity in screened and whole-system populations, the biomass of the bacteria associated with particles was computed to be 427 micrograms/liter.     

近自然纯培养法对细菌培养的初步研究

依据微生物在自然生境中协作生长的基本特性,提出一种对传统纯培养技术的改进思路及方法：设计一种有孔培养皿,皿内覆盖有不允许细菌通过、但营养物质可以自由流动的微孔滤膜。培养时将该培养皿放入被分离微生物所需生境中,可以克服传统纯培养难以提供外源活性物质的缺陷,一定程度上弥补了混合培养法和传统纯培养法的弱点,从而达到增强部分微生物可培养性、甚至培养出未培养微生物的目的。     

<Emphasis Type="Italic">Eisenia fetida</Emphasis> (Oligochaeta: Lumbricidae) Modifies the Structure and Physiological Capabilities of Microbial Communities Improving Carbon Mineralization During Vermicomposting of Pig Manure

Although microorganisms are largely responsible for organic matter decomposition, earthworms may also affect the rates of decomposition directly by feeding on and digesting organic matter and microorganisms, or indirectly affect them through their interactions with the microorganisms, basically involving stimulation or depression of the microbial populations. We tested the general hypothesis that microbial populations, and especially fungi, are enhanced by earthworm activity, and also whether earthworms are able to modify the biodiversity of microbial populations, and its relation to the function of the system. In addition, we examined the metabolic quotient and the effect of labile organic C to assess the relationships between earthworm and microbes. We found that decomposition of pig manure has two stages characterized by the presence or absence of earthworms. Thus, the presence of earthworms was related with increases in overall microbial biomass and activity, which decreased when earthworms left the substrate; the same pattern was observed for fungi. Furthermore, earthworms modified the physiological profiles of microbial communities of pig manure, increasing the diversity of substrates utilized. In addition, earthworms promoted a more efficient use of energy of microbial communities, as the metabolic quotient showed. The rate of carbon loss was almost twice where earthworms were present, revealing faster decomposition. Our data match with the recent findings that to maintain essential processes the functional properties of present species are at least as important as the number of species per se. This is in accordance with the “insurance hypothesis,” which states that a large number of species is probably essential for maintaining stable processes in changing environments, as the presence of earthworms would have promoted in pig manure.     

Quantification of the presence and activity of specific microorganisms in nature

Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.     

Electrode system for determination of microbial cell populations in polluted water.

Microbial cell populations in polluted water were determined by using a fuel cell-type electrode. The electrode was composed of a Pt anode, a Pt-K3Fe(CN)6-K4Fe(CN)6 cathode, and a cation-exchange membrane for separating two electrode compartments. The principle of microbial cell number determination is based on sensing a redox dye reduced by microorganisms with the electrode. Sample solutions containing microorganisms, a redox dye (thionine), and peptone were purged with oxygen-free nitrogen during the determination. A linear relationship was obtained between the increasing rate of current and the number of microbial cells measured by the colony count method above 10(4) cells per ml. The determination time varied with the number of microbial cells determined from 20 to 60 min for 3.6 x 10(6) and 3.6 x 10(4) cells per ml, respectively.     

Dissecting the Rhizosphere complexity: The truffle-ground study case

The word Rhizosphere describes the part of the soil which is immediately adjacent to and affected by plant roots. This is a very dynamic environment where plants, soil and microorganisms interact. The plant releases, in addition to biologically active substances, nutritive substances (exudates), which create a privileged habitat for many microbial populations. The same microbes that live in the rhizosphere may be useful for the plant. Interest in this fascinating environment has increased over the years. However, our knowledge of the biology and diversity of microbial populations in the rhizosphere is still limited, because it has always been linked to traditional culture-based techniques. These methods, which only allow the study of cultured microorganisms, do not allow the majority of the organisms existing in nature to be characterized. Over the last few years, this limitation has been overcome through the introduction of methodologies that are independent of culture techniques. This different approach, which has revolutionized scientific research, is known as metagenomics. In this review, the rhizosphere environment is considered with particular attention to the fungal and symbiotic organisms, which populate it. The new environmental genomic techniques and how these have been applied to the study of the various environments and the rhizosphere are described. Finally, a specific rhizosphere, a truffle-ground, is described as our study case.      

Electrode system for determination of microbial cell populations in polluted water

Microbial cell populations in polluted water were determined by using a fuel cell-type electrode. The electrode was composed of a Pt anode, a Pt-K3Fe(CN)6-K4Fe(CN)6 cathode, and a cation-exchange membrane for separating two electrode compartments. The principle of microbial cell number determination is based on sensing a redox dye reduced by microorganisms with the electrode. Sample solutions containing microorganisms, a redox dye (thionine), and peptone were purged with oxygen-free nitrogen during the determination. A linear relationship was obtained between the increasing rate of current and the number of microbial cells measured by the colony count method above 10(4) cells per ml. The determination time varied with the number of microbial cells determined from 20 to 60 min for 3.6 x 10(6) and 3.6 x 10(4) cells per ml, respectively.