喀斯特土壤固氮微生物群落与植被、土壤的关系

固氮微生物作为土壤中重要的功能微生物群之一,其与地上植物群落、土壤环境之间的关系尚不清楚。在桂西北的环江县、都安县和大化县选取喀斯特典型植被类型(草丛、灌丛、次生林)建立样方,通过植被调查、测定土壤理化性质和构建克隆文库的方法,研究了土壤固氮微生物群落的结构与组成,分析了固氮微生物群落与植物群落、土壤理化性质之间的关系。结果表明:研究区典型植被类型土壤中的优势固氮微生物为慢生根瘤菌。Mantel相关性分析表明植物群落与固氮微生物群落显著相关(r=0.6116,P=0.011);结合PCo A分析和Venn图可看出,植物群落组成与结构越相似,土壤固氮微生物群落结构与组成也越相似。CCA分析前两轴的解释率之和仅为22.72%,其中总氮、有效态钾、有效态钙对固氮微生物群落的影响显著,这表明本研究涵盖的土壤理化性质指标并不能完全解释固氮微生物群落的变异,需要补充更多的土壤数据进行更深入的研究。由此可见,在喀斯特生态恢复过程中,不仅要关注地上植被群落的恢复与重建,同时也应重视地下功能微生物群落的恢复与重建。     

Differential microbial communities in hot spring mats from Western Thailand

The microbial communities of freshwater hot spring mats from Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Green-, red-, and yellow colored mat layers were analyzed. In order to detect the major components of the microbial communities constituting the mat as well as the microorganisms showing significant metabolic activity, samples were analyzed using DNA- and RNA-based molecular techniques, respectively. Microbial community fingerprints, performed by denaturing gradient gel electrophoresis (DGGE), revealed clear differences among mat layers. Thermophilic phototrophic microorganisms, Cyanobacteria and Chloroflexi, constituted the major groups in these communities (on average 65 and 51% from DNA and RNA analyses, respectively). Other bacteria detected in the mat were Bacteroidetes, members of the Candidate Division OP10, Actinobacteria, and Planctomycetes. Differently colored mat layers showed characteristic bacterial communities and the major components of the metabolically active fraction of these communities have been identified.     

未培养微生物的研究与微生物分子生态学的发展*

近年来现代分子技术和基因组学逐渐渗透到有关生命科学的整个领域,也为微生物生态学提供了新的研究方法和机遇。16S rRNA基因序列分析、DNA-DNA杂交、核酸指纹图谱以及宏基因组学等分子技术检查自然环境中的微生物,可以克服传统纯培养技术的不足,是一条探知未培养微生物、寻找新基因及其产物的新途径,开启了我们认识微生物多样性和获得新资源的大门。     

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.     

木质纤维素的微生物降解

木质纤维素广泛存在于自然界中,因结构复杂,其高效降解需要多种微生物的协同互作,由于参与木质纤维素降解的微生物种类繁多,其协同降解机理尚不完全明确。随着微生物分子生物学和组学技术的快速发展,将为微生物协同降解木质纤维素机制的研究提供新的方法和思路。笔者前期研究发现,细菌复合菌系在50℃下表现出强大的木质纤维素降解能力,菌系由可分离培养和暂时不可分离培养细菌组成,但是可分离培养细菌没有降解能力。通过宏基因组和宏转录组研究表明,与木质纤维素降解相关的某些基因表达量发生显著变化,通过组学方法有可能更加深入解释微生物协同降解木质纤维素的微生物学和酶学机理。文中从酶、纯培养菌株和复合菌群三个方面综述了木质纤维素微生物降解研究进展,着重介绍了组学技术在解析复合菌群作用机理方面的现状和应用前景,以期为探索微生物群落协同降解木质纤维素的机理提供借鉴。     

Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere

The rhizosphere is of central importance not only for plant nutrition, health and quality but also for microorganism-driven carbon sequestration, ecosystem functioning and nutrient cycling in terrestrial ecosystems. A multitude of biotic and abiotic factors are assumed to influence the structural and functional diversity of microbial communities in the rhizosphere. In this review, recent studies on the influence of the two factors, plant species and soil type, on rhizosphere-associated microbial communities are discussed. Root exudates and the response of microorganisms to the latter as well as to root morphology were shown to shape rhizosphere microbial communities. All studies revealed that soil is the main reservoir for rhizosphere microorganisms. Many secrets of microbial life in the rhizosphere were recently uncovered due to the enormous progress in molecular and microscopic tools. Physiological and molecular data on the factors that drive selection processes in the rhizosphere are presented here. Furthermore, implications for agriculture, nature conservation and biotechnology will also be discussed.     

红树林样品不经分离的微生物群体培养物生物活性研究

从海南、广西与广东三省的红树林区采集了181个样品,不进行微生物分离而直接作发酵剂接种到发酵培养基进行发酵,取发酵上清液进行抗细菌、抗真菌与肿瘤细胞毒活性的测定。同时对样品进行可培养微生物的分离与生物活性测定。结果显示:不同样品类型的生物活性差异较大。在15个具有强抗活性的样品中,有5个样品分离到的单株菌均无任何生物活性,说明这5个样品的生物活性可能是由微生物的群体作用产生的,也可能是某种没有培养出的微生物产生的。初步表明了探索微生物混合培养获得生物活性代谢产物的可能性。     

Do associated microbial abundances impact marine demosponge pumping rates and tissue densities?

The evolution of marine demosponges has led to two basic life strategies: one involving close associations with large and diverse communities of microorganisms, termed high microbial abundance (HMA) species, and one that is essentially devoid of associated microorganisms, termed low microbial abundance (LMA) species. This dichotomy has previously been suggested to correlate with morphological differences, with HMA species having a denser mesohyl and a more complex aquiferous systems composed of longer and narrower water canals that should necessitate slower seawater filtration rates. We measured mesohyl density for a variety of HMA and LMA sponges in the Florida Keys, and seawater pumping rates for a select group of these sponges using an in situ dye technique. HMA sponges were substantially denser than LMA species, and had per unit volume pumping rates 52–94% slower than the LMA sponges. These density and pumping rate differences suggest that evolutionary differences between HMA and LMA species may have resulted in profound morphological and physiological differences between the two groups. The LMA sponge body plan moves large quantities of water through their porous tissues allowing them to rapidly acquire the small particulate organic matter (POM) that supplies the majority of their nutritional needs. In contrast, the HMA sponge body plan is suited to host large and tightly packed communities of microorganisms and has an aquiferous system that increases contact time between seawater and the sponge/microbial consortium that feeds on POM, dissolved organic matter and the raw inorganic materials for chemolithotrophic sponge symbionts. The two evolutionary patterns represent different, but equally successful patterns and illustrate how associated microorganisms can potentially have substantial effects on host evolution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microbial communities adhering to the obverse and reverse sides of an oil painting on canvas: identification and evaluation of their biodegradative potential

In this study, we investigated and compared the microbial communities adhering to the obverse and the reverse sides of an oil painting on canvas exhibiting signs of biodeterioration. Samples showing no visible damage were investigated as controls. Air samples were also analysed, in order to investigate the presence of airborne microorganisms suspended in the indoor atmosphere. The diversity of the cultivable microorganisms adhering to the surface was analysed by molecular techniques, such as RAPD analysis and gene sequencing. DGGE fingerprints derived from DNA directly extracted from canvas material in combination with clone libraries and sequencing were used to evaluate the non-cultivable fraction of the microbial communities associated with the material. By using culture-dependent methods, most of the bacterial strains were found to be common airborne, spore-forming microorganisms and belonged to the phyla Actinobacteria and Firmicutes, whereas culture-independent techniques identified sequenced clones affiliated with members of the phyla Actinobacteria and Proteobacteria. The diversity of fungi was shown to be much lower than that observed for bacteria, and only species of Penicillium spp. could be detected by cultivation techniques. The selected strategy revealed a higher microbial diversity on the obverse than on the reverse side of the painting and the near absence of actively growing microorganisms on areas showing no visible damage. Furthermore, enzymatic activity tests revealed that the most widespread activities involved in biodeterioration were esterase and esterase lipase among the isolated bacterial strains, and esterase and N-acetyl-β-glucosaminidase among fungi strains.     

Biodegradation of atrazine in sand sediments and in a sand-filter

The potential of a microbial consortium for treating waters contaminated with atrazine was considered. In conventional liquid culture, atrazine and its two dealkylated by-products were equally metabolised by the microbial consortium. Transient production of hydroxyatrazine was observed during atrazine catabolism, indicating that the catabolic pathway was similar to the one reported for isolates capable of atrazine mineralisation. This consortium was then inoculated to sediments sampled from an artificial recharge site. These sediments were contaminated by atrazine and diuron and exhibited only a slow endogenous herbicide dissipation. Inoculated microorganisms led to extensive atrazine degradation and survived for more than 10 weeks in the sediments. A rudimentary bioreactor was then setup using a soil core originating from the same recharge site. Degrading microorganisms rapidly colonised the core and expressed their degrading activity. The efficiency of the bioreactor was improved in the presence of spiked environmental surface waters. Atrazine degraders thus possibly benefited from the other organic sources in developing and expressing their activity. The microbial consortium did not initially exhibit the capacity to degrade diuron, which was used as reference compound. No change in this characteristic was detected throughout the study. Received: 13 December 1999 / Received revision: 26 April 2000 / Accepted: 5 May 2000     

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。     

A survey of the methods for the characterization of microbial consortia and communities

A survey of the available literature on methods most frequently used for the identification and characterization of microbial strains, communities, or consortia is presented. The advantages and disadvantages of the various methodologies were examined from several perspectives including technical, economic (time and cost), and regulatory. The methods fall into 3 broad categories: molecular biological, biochemical, and microbiological. Molecular biological methods comprise a broad range of techniques that are based on the analysis and differentiation of microbial DNA. This class of methods possesses several distinct advantages. Unlike most other commonly used methods, which require the production of secondary materials via the manipulation of microbial growth, molecular biological methods recover and test their source materials (DNA) directly from the microbial cells themselves, without the requirement for culturing. This eliminates both the time required for growth and the biases associated with cultured growth, which is unavoidably and artificially selective. The recovered nucleic acid can be cloned and sequenced directly or subpopulations can be specifically amplified using polymerase chain reaction (PCR), and subsequently cloned and sequenced. PCR technology, used extensively in forensic science, provides researchers with the unique ability to detect nucleic acids (DNA and RNA) in minute amounts, by amplifying a single target molecule by more than a million-fold. Molecular methods are highly sensitive and allow for a high degree of specificity, which, coupled with the ability to separate similar but distinct DNA molecules, means that a great deal of information can be gleaned from even very complex microbial communities. Biochemical methods are composed of a more varied set of methodologies. These techniques share a reliance on gas chromatography and mass spectrometry to separate and precisely identify a range of biomolecules, or else investigate biochemical properties of key cellular biomolecules. Like the molecular biological methods, some biochemical methods such as lipid analyses are also independent of cultured growth. However, many of these techniques are only capable of producing a profile that is characteristic of the microbial community as a whole, providing no information about individual members of the community. A subset of these methodologies are used to derive taxonomic information from a community sample; these rely on the identification of key subspecies of biomolecules that differ slightly but characteristically between species, genera, and higher biological groupings. However, when the consortium is already growing in chemically defined media (as is often the case with commercial products), the rapidity and relatively low costs of these procedures can mitigate concerns related to culturing biases. Microbiological methods are the most varied and the least useful for characterizing microbial consortia. These methods rely on traditional tools (cell counting, selective growth, and microscopic examination) to provide more general characteristics of the community as a whole, or else to narrow down and identify only a small subset of the members of that community. As with many of the biochemical methods, some of the microbiological methods can fairly rapidly and inexpensively create a community profile, which can be used to compare 2 or more entire consortia. However, for taxonomic identification of individual members, microbiological methods are useful only to screen for the presence of a few key predetermined species, whose preferred growth conditions and morphological characteristics are well defined and reproducible.     

Surface Water Linkages Regulate Trophic Interactions in a Groundwater Food Web

Groundwaters are increasingly viewed as resource-limited ecosystems in which fluxes of dissolved organic carbon (DOC) from surface water are efficiently mineralized by a consortium of microorganisms which are grazed by invertebrates. We tested for the effect of groundwater recharge on resource supply and trophic interactions by measuring physico-chemistry, microbial activity and biomass, structure of bacterial communities and invertebrate density at three sites intensively recharged with surface water. Comparison of measurements made in recharge and control well clusters at each site showed that groundwater recharge significantly increased fluxes of DOC and phosphate, elevated groundwater temperature, and diminished dissolved oxygen (DO). Microbial biomass and activity were significantly higher in recharge well clusters but stimulation of autochthonous microorganisms was not associated with a major shift in bacterial community structure. Invertebrate assemblages were not significantly more abundant in recharge well clusters and did not show any relationship with microbial biomass and activity. Microbial communities were bottom-up regulated by DOC and nutrient fluxes but trophic interactions between microorganisms and invertebrates were apparently limited by environmental stresses, particularly DO depletion and groundwater warming. Hydrological connectivity is a key factor regulating the function of DOC-based groundwater food webs as it influences both resource availability for microorganisms and environmental stresses which affect energy transfer to invertebrates and top-down control on microorganisms.     

Dealing with water stress and microbial preservation

The relevance of preserving microorganisms has been well accepted for several decades. Interest is now shifting towards investigating adequate preservation methods to improve microbial survival rates and to preserve new taxa of previously considered unculturable microorganisms. In addition, a growing interest in preserving fragile microbial consortia or communities with biotechnological interest motivates the improvement of preservation methods. In the present study, we reviewed the effect of water availability in microbial diversity shift. We describe the effect of drought on microorganisms at the molecular level and their molecular responses to this life-threatening challenge focusing on the production of xeroprotectants. We also review the interspecies interactions of those drought-tolerant microorganisms with other sensitive organisms including neighbouring prokaryotes and eukaryotes such as plants, and the potential role of these microorganisms at determining the ecological composition of stressed environments. We emphasize the importance of applying the knowledge derived from the molecular mechanisms used by desiccation-tolerant microorganisms for the improvement of the preservation techniques. An overview of the current and newer techniques for preserving microorganisms and microbial communities is provided. The biotechnological interest in preserving pure cultures, microbial consortia and communities is also discussed.     

现代分子生物学技术在瘤胃微生态系统研究中的应用

瘤胃中栖息着大量的微生物,由于这些微生物组成复杂且有些细菌在体外无法培养,目前对这些微生物的了解仍然很少。现代分子生物学技术的发展为研究瘤胃微生物提供了有效的方法,利用核酸探针、基因序列分析、遗传指纹技术、全细胞杂交和实时定量PCR等技术可以对瘤胃微生物的分类及进化关系、区系结构图、重要酶的表达以及目的微生物的准确定量进行更为深入和透彻的研究。发展和利用这些技术不仅可以研究微生物之间的关系以及微生物与饲料颗粒之间时间与空间的关系,还能直接在细菌自然生长的环境中对其各种特征进行研究。     

稳定性同位素探测技术在微生物生态学研究中的应用

稳定性同位素标记技术同分子生物学技术相结合而发展起来的稳定性同位素探测技术（stableisotope probing,SIP）,在对各种环境中微生物群落组成进行遗传分类学鉴定的同时,可确定其在环境过程中的功能,提供复杂群落中微生物相互作用及其代谢功能的大量信息,具有广阔的应用前景.其基本原理是：将原位或微宇宙（microcosm）的环境样品暴露于稳定性同位素富集的基质中,这些样品中存在的某些微生物能够以基质中的稳定（性同位素为碳源或氮源进行物质代谢并满足其自身生长需要,基质中的稳定性同位素被吸收同化进入微生物体内,参与各类物质如核酸（DNA和RNA）及磷脂脂肪酸（PLFA）等的生物合成,通过提取、分离、纯化、分析这些微生物体内稳定性同位素标记的生物标志物,从而将微生物的组成与其功能联系起来.在介绍稳定性同位素培养基质的选择及标记方法、合适的生物标志物的选择及提取分离方法的基础上,举例阐述了此项技术在甲基营养菌、有机污染物降解菌、根际微生物生态、互营微生物、宏基因组学等方面的应用.     

昆虫体内微生物多样性的影响因素研究进展

昆虫体内的微生物种类繁多,在长期的协同进化中与其宿主昆虫形成了密切的共生关系。近年来,随着分子生物学技术的快速发展与应用,昆虫体内微生物多样性得到广泛的研究。本文综述了影响昆虫体内微生物多样性的各种因素。体内微生物多样性除与昆虫种类相关外,还与昆虫自身因素(性别、发育龄期和种群)、生态因子(食物、温度、CO_2浓度和辐照)以及技术本身的局限性等方面密切相关。     

Development and application of an enzymatic and cell flotation treatment for the recovery of viable microbial cells from environmental matrices such as anaerobic sludge

Efficient dissociation of microorganisms from their aggregate matrix is required to study the microorganisms without interaction with their native environment (e.g., biofilms, flocs, granules, etc.) and to assess their community composition through the application of molecular or microscopy techniques. To this end, we combined enzymatic treatments and a cell extraction by density gradient to efficiently recover anaerobic microorganisms from urban wastewater treatment plant sludge. The enzymes employed (amylase, cellulase, DNase, and pectinase) as a pretreatment softly disintegrated the extrapolymeric substances (EPS) interlocked with the microorganisms. The potential damaging effects of the applied procedure on bacterial and archaeal communities were assessed by studying the variations in density (using quantitative PCR), diversity (using capillary electrophoresis single-strand conformation polymorphism fingerprinting [CE-SSCP]), and activity (using a standard anaerobic activity test) of the extracted microorganisms. The protocol preserved the general capacity of the microbial community to produce methane under anaerobic conditions and its diversity; particularly the archaeal community was not affected in terms of either density or structure. This cell extraction procedure from the matrix materials offers interesting perspectives for metabolic, microscopic, and molecular assays of microbial communities present in complex matrices constituted by bioaggregates or biofilms.     

Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses,Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses.