Microbial diversity of benthic mats along a tidal desiccation gradient

We investigated the influence of desiccation frequency, indicated by tidal position, on microbial community structure, diversity and richness of microbial mats. We independently characterized cyanobacterial, bacterial and archaeal communities, and their spatial variability for two distinct microbial mat systems: subtidal hypersaline mats and intertidal sand flat mats. Community fingerprints based on 16S rDNA were obtained via denaturing gradient gel electrophoresis using polymerase chain reaction primers specific for each group. Fingerprints for all three groups were consistently similar [> or =85% according to Weighted Pair Group with Arithmetic Mean (WPGMA) analysis] along a 1-km-long transect in subtidal mats. Here, pair-wise comparison analysis yielded minimal variation in diversity and richness for all groups. Fingerprints of three sites along an intertidal transect were heterogenous (> or =32% similarity according to WPGMA analysis) with clear shifts in community structure in all three microbial groups. Here, all groups exhibited statistically significant decreases in richness and diversity with tidal height (as desiccation frequency increases). Regression analysis yielded a strong correlation between diversity or richness estimates and position along the tidal gradient, for both Archaea and Bacteria, with Cyanobacteria exhibiting a weaker correlation. These results suggest that desiccation frequency can shape the structure of microbial mat communities, with Archea being least tolerant and Cyanobacteria most tolerant.     

Temporal and spatial patterns of eukaryotic and bacterial communities found in vernal pools

In this study, we examined the effects of physicochemical variability on the microbial communities of vernal pools. Denaturing gradient gel electrophoresis revealed temporal changes to be more pronounced than spatial changes in eukaryotic and bacterial communities. Sequencing revealed high degrees of richness in decomposers, which supports the notion that vernal pools are heterotrophic habitats.     

Differences in vegetation composition and plant species identity lead to only minor changes in soil-borne microbial communities in a former arable field

To examine the relationship between plant species composition and microbial community diversity and structure, we carried out a molecular analysis of microbial community structure and diversity in two field experiments. In the first experiment, we examined bacterial community structure in bulk and rhizosphere soils in fields exposed to different plant diversity treatments, via a 16S rRNA gene clone library approach. Clear differences were observed between bacterial communities of the bulk soil and the rhizosphere, with the latter containing lower bacterial diversity. The second experiment focused on the influence of 12 different native grassland plant species on bacterial community size and structure in the rhizosphere, as well as the structure of Acidobacteria and Verrucomicrobia community structures. In general, bacterial and phylum-specific quantitative PCR and PCR-denaturing gradient gel electrophoresis revealed only weak influences of plant species on rhizosphere communities. Thus, although plants did exert an influence on microbial species composition and diversity, these interactions were not specific and selective enough to lead to major impacts of vegetation composition and plant species on below-ground microbial communities.     

黑河下游额济纳绿洲植物群落物种多样性的空间异质性

采用地统计学方法研究了黑河下游额济纳绿洲物种多样性的空间异质性.结果表明:Margalef丰富度指数(Ma)、Shannon多样性指数(H')、Simpson多样性指数(Ds)和Pielou均匀度指数(J')均服从正态分布,变异系数(CV)为55.8%～67.8%,均属中等变异;Ma和H'符合指数模型,Ds和J'符合球体模型;H'的空间变异程度最高,其次是Ma和J',Ds最小;在变程范围内,各物种多样性指数空间变异的结构性因子占主导地位,所占比例为81.1%～93.0%.各多样性指数沿河流方向的格局变化明显,绿洲核心区达来呼布镇(42°N,101°E)附近为显著高值区,在100°—101°E和102°—102°30'E的带状范围内呈明显的低值区,主要包括东、西戈壁及巴丹吉林沙漠腹地.     

Spatial and temporal changes in microbial community structure associated with recharge-influenced chemical gradients in a contaminated aquifer

In a contaminated water-table aquifer, we related microbial community structure on aquifer sediments to gradients in 24 geochemical and contaminant variables at five depths, under three recharge conditions. Community amplified ribsosomal DNA restriction analysis (ARDRA) using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) using bacterial 16S rDNA primers indicated: (i). communities in the anoxic, contaminated central zone were similar regardless of recharge; (ii). after recharge, communities at greatest depth were similar to those in uncontaminated zones; and (iii). after extended lack of recharge, communities at upper and lower aquifer margins differed from communities at the same depths on other dates. General aquifer geochemistry was as important as contaminant or terminal electron accepting process (TEAP) chemistry in discriminant analysis of community groups. The Shannon index of diversity (H) and the evenness index (E), based on DGGE operational taxonomic units (OTUs), were statistically different across community groups and aquifer depths. Archaea or sulphate-reducing bacteria 16S rRNA abundance was not clearly correlated with TEAP chemistry indicative of methanogenesis or sulphate reduction. Eukarya rRNA abundance varied by depth and date from 0 to 13% of the microbial community. This contaminated aquifer is a dynamic ecosystem, with complex interactions between physical, chemical and biotic components, which should be considered in the interpretation of aquifer geochemistry and in the development of conceptual or predictive models for natural attenuation or remediation.     

Dynamics of an oligotrophic bacterial aquifer community during contact with a groundwater plume contaminated with benzene, toluene, ethylbenzene, and xylenes: an in situ mesocosm study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

应用DGGE研究微生物群落时的常见问题分析

变性梯度凝胶电泳(DGGE)是通过核酸片段对微生物群落进行研究,可以监测未培养细菌及其功能基因,被广泛地应用于微生物群落多样性和动态分析,并成为微生物分子生态学研究中的重要手段之一。文中论述了DGGE操作过程中遇到的常见问题,并提出了相应的解决方法。全面分析了样品预处理过程和PCR扩增效果对DGGE分析的影响,探讨了DGGE图谱的优化过程和图谱分析方法,并对DGGE的应用前景进行了综述。     

Empirical Relationships between Species Richness, Evenness, and Proportional Diversity

Diversity (or biodiversity) is typically measured by a species count (richness) and sometimes with an evenness index; it may also be measured by a proportional statistic that combines both measures (e.g., Shannon-Weiner index or H'). These diversity measures are hypothesized to be positively and strongly correlated, but this null hypothesis has not been tested empirically. We used the results of Caswell's neutral model to generate null relationships between richness (S), evenness (J'), and proportional diversity (H'). We tested predictions of the null model against empirical relationships describing data in a literature survey and in four individual studies conducted across various scales. Empirical relationships between log S or J' and H' differed from the null model when <10 species were tested and in plants, vertebrates, and fungi. The empirical relationships were similar to the null model when >10 and <100 species were tested and in invertebrates. If >100 species were used to estimate diversity, the relation between log S and H' was negative. The strongest predictive models included log S and J'. A path analysis indicated that log S and J' were always negatively related, that empirical observations could not be explained without including indirect effects, and that differences between the partials may indicate ecological effects, which suggests that S and J' act like diversity components or that diversity should be measured using a compound statistic.     

Spatio-temporal patterns of microbial communities in a hydrologically dynamic pristine aquifer

Seasonal patterns of groundwater and sediment microbial communities were explored in a hydrologically dynamic alpine oligotrophic porous aquifer, characterized by pronounced groundwater table fluctuations. Rising of the groundwater level in consequence of snow melting water recharge was accompanied by a dramatic drop of bacterial Shannon diversity in groundwater from H' = 3.22 ± 0.28 in autumn and winter to H' = 1.31 ± 0.35 in spring and summer, evaluated based on T-RFLP community fingerprinting. Elevated numbers of bacteria in groundwater in autumn followed nutrient inputs via recharge from summer rains and correlated well with highest concentrations of assimilable organic carbon. Sterile sediments incubated to groundwater in monitoring wells were readily colonized reaching maximum cell densities within 2 months, followed by a consecutive but delayed increase and leveling-off of bacterial diversity. After 1 year of incubation, the initially sterile sediments exhibited a similar number of bacteria and Shannon diversity when compared to vital sediment from a nearby river incubated in parallel. The river bed sediment microbial communities hardly changed in composition, diversity, and cell numbers during 1 year of exposure to groundwater. Summing up, the seasonal hydrological dynamics were found to induce considerable dynamics of microbial communities suspended in groundwater, while sediment communities seem unaffected and stable in terms of biomass and diversity.     

Determining Microeukaryotic Plankton Community around Xiamen Island,Southeast China,Using Illumina MiSeq and PCR-DGGE Techniques

Microeukaryotic plankton are important components of aquatic environments and play key roles in marine microbial food webs; however, little is known about their genetic diversity in subtropical offshore areas. Here we examined the community composition and genetic diversity of the microeukaryotic plankton in Xiamen offshore water by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), clone-based sequencing and Illumina based sequencing. The Illumina MiSeq sequencing revealed a much (approximately two orders of magnitude) higher species richness of the microeukaryotic community than DGGE, but there were no significant difference in species richness and diversity among the northern, eastern, southern or western stations based on both methods. In this study, Copepoda, Ciliophora, Chlorophyta, Dinophyceae, Cryptophyta and Bacillariophyta (diatoms) were the dominant groups even though diatoms were not detected by DGGE. Our Illumina based results indicated that two northern communities (sites N2 and N3) were significantly different from others in having more protozoa and fewer diatoms. Redundancy analysis (RDA) showed that both temperature and salinity were the significant environmental factors influencing dominant species communities, whereas the full microeukaryotic community appeared to be affected by a complex of environmental factors. Our results suggested that extensive sampling combined with more deep sequencing are needed to obtain the complete diversity of the microeukaryotic community, and different diversity patterns for both abundant and rare taxa may be important in evaluating the marine ecosystem health.     

Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

Sediment Enzyme Activities and Microbial Community Diversity in an Oligotrophic Drinking Water Reservoir,Eastern China

Drinking water reservoir plays a vital role in the security of urban water supply, yet little is known about microbial community diversity harbored in the sediment of this oligotrophic freshwater environmental ecosystem. In the present study, integrating community level physiological profiles (CLPPs), nested polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and clone sequence technologies, we examined the sediment urease and protease activities, bacterial community functional diversity, genetic diversity of bacterial and fungal communities in sediments from six sampling sites of Zhou cun drinking water reservoir, eastern China. The results showed that sediment urease activity was markedly distinct along the sites, ranged from 2.48 to 11.81 mg NH₃-N/(g·24h). The highest average well color development (AWCD) was found in site C, indicating the highest metabolic activity of heterotrophic bacterial community. Principal component analysis (PCA) revealed tremendous differences in the functional (metabolic) diversity patterns of the sediment bacterial communities from different sites. Meanwhile, DGGE fingerprints also indicated spatial changes of genetic diversity of sediment bacterial and fungal communities. The sequence BLAST analysis of all the sediment samples found that Comamonas sp. was the dominant bacterial species harbored in site A. Alternaria alternate, Allomyces macrogynus and Rhizophydium sp. were most commonly detected fungal species in sediments of the Zhou cun drinking water reservoir. The results from this work provide new insights about the heterogeneity of sediment microbial community metabolic activity and genetic diversity in the oligotrophic drinking water reservoir.     

Microbial community fingerprinting by differential display-denaturing gradient gel electrophoresis

Complex microbial communities exhibit a large diversity, hampering differentiation by DNA fingerprinting. Herein, differential display-denaturing gradient gel electrophoresis is proposed. By adding a nucleotide to the 3' ends of PCR primers, 16 primer pairs and fingerprints were generated per community. Complexity reduction in each partial fingerprint facilitates sample comparison.     

Effects of fertilization on bacterial community structure and function in a black soil of Dehui region estimated by Biolog and PCR-DGGE methods

Soil microbial community structure and function are commonly used as indicators for soil quality and fertility. In this paper, the bacterial community structure and function in a black soil of Dehui region influenced by fertilization were investigated by Biolog and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) methods. Biolog examination showed that substrate richness and catabolic diversities of bacterial communities were the highest in the treatment of farm yard manure, and the lowest in the chemical fertilizer treatment. DGGE fingerprint showed that the majority of bands were similar among all treatments, suggesting that microbial communities with those bands were stable, and not influenced by fertilization. In general, chemical fertilizer decreased the diversity of soil bacterial communities. The PCA (principal component analysis) plots of Biolog and DGGE revealed that the structure and function of bacterial communities were similar in the non-fertilized control and the treatment of farm yard manure alone, which inferred that the application of farm yard manure increased the quantity of soil microbes but had less effect on the changes of community structure. The catabolic function was similar, but the composition structure differed between the treatments of chemical fertilizer alone and combined application of farm yard manure with chemical fertilizer. These results suggest that the use of chemical fertilizer mainly decreased the catabolic activity of the fast growth bacteria or eutrophic bacteria.     

Bacterial community structure associated with the Antarctic soft coral, Alcyonium antarcticum

The structure and composition of microbial communities inhabiting the soft coral Alcyonium antarcticum were investigated across three differentially contaminated sites within McMurdo Sound, Antarctica. Diverse microbial communities were revealed at all sites using culture-based analysis, denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone-library analysis, and FISH. Phylogenetic analysis of isolates and retrieved sequences demonstrated close affiliation with known psychrophiles from the Antarctic environment and high similarity to Gammaproteobacteria clades of sponge-associated microorganisms. The majority of bacteria detected with all techniques reside within the Gammaproteobacteria, although other phylogenetic groups including Alpha- and Betaproteobacteria, Bacteroidetes, Firmicutes, Actinomycetales, Planctomycetes, and Chlorobi and bacteria from the functional group of sulfate-reducing bacteria were also present. Multivariate (nMDS) analysis of DGGE banding patterns and principal component analysis of quantitative FISH data revealed no distinct differences in community composition between differentially contaminated sites. Rather, conserved coral-associated bacterial groups were observed within and between sites, providing evidence to support specific coral-microbial interactions. This is the first investigation of microbial communities associated with Antarctic soft corals, and the results suggest that spatially stable microbial associations exist across an environmental impact gradient.     

Effect of (a)synchronous light fluctuation on diversity,functional and structural stability of a marine phytoplankton metacommunity

Disentangling the mechanisms that maintain the stability of communities and ecosystem properties has become a major research focus in ecology in the face of anthropogenic environmental change. Dispersal plays a pivotal role in maintaining diversity in spatially subdivided communities, but only a few experiments have simultaneously investigated how dispersal and environmental fluctuation affect community dynamics and ecosystem stability. We performed an experimental study using marine phytoplankton species as model organisms to test these mechanisms in a metacommunity context. We established three levels of dispersal and exposed the phytoplankton to fluctuating light levels, where fluctuations were either spatially asynchronous or synchronous across patches of the metacommunity. Dispersal had no effect on diversity and ecosystem function (biomass), while light fluctuations affected both evenness and community biomass. The temporal variability of community biomass was reduced by fluctuating light and temporal beta diversity was influenced interactively by dispersal and fluctuation, whereas spatial variability in community biomass and beta diversity were barely affected by treatments. Along the establishing gradient of species richness and dominance, community biomass increased but temporal variability of biomass decreased, thus highest stability was associated with species-rich but highly uneven communities and less influenced by compensatory dynamics. In conclusion, both specific traits (dominance) and diversity (richness) affected the stability of metacommunities under fluctuating conditions.     

DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria

A denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of dsrB (dissimilatory sulfite reductase beta-subunit)-genes in sulfate-reducing communities. For this purpose a PCR primer pair was optimized for the amplification of a approximately 350 bp dsrB gene fragment that after DGGE gel electrophoresis enabled us to discriminate between dsrB genes of different SRB-subgroups,-genera and -species. The dsrB-DGGE method revealed considerable genetic diversity when applied to DNA extracts obtained from aquifer samples that were derived from monitoring wells of an in situ metal precipitation (ISMP) pilot project conducted at the site of a non-ferrous industry or from environmental heavy metal contaminated samples. The sequences of the excised and sequenced DGGE bands represented dsrB genes of different SRB-subgroups,-genera and -species, thus confirming the broad applicability of the PCR primer pair. Linking the results of the physico-chemical follow-up of the field and lab experiments to the dsrB-DGGE data will provide a better understanding of the contribution of the SRB populations to the ongoing ISMP processes.     

Temporal distribution of the archaeal community in the Changjiang Estuary hypoxia area and the adjacent East China Sea as determined by denaturing gradient gel electrophoresis and multivariate analysis

The archaeal community and the effects of environmental factors on microbial community distribution were investigated at five sampling sites in the Changjiang Estuary hypoxia area and the adjacent East China Sea in June, August, and October 2006. Profiles of the archaeal communities were generated by denaturing gradient gel electrophoresis of 16S rRNA genes followed by DNA sequence analysis, and the results were analyzed by multivariate statistical analysis. Denaturing gradient gel electrophoresis band patterns were analyzed by cluster analysis to assess temporal changes in the genetic diversity of the archaeal communities. Most of the October samples grouped together separately from those of June and August. Analysis of DNA sequences revealed that the dominant archaeal groups in the Changjiang Estuary hypoxia area and the adjacent East China Sea were affiliated with Euryarchaeota (mainly marine group II) and Crenarchaeota. The effects of environmental factors on the archaeal community distribution were analyzed by the ordination technique of canonical correspondence analysis. Salinity had a significant effect on the archaeal community composition.     

Molecular method to assess the diversity of Burkholderia species in environmental samples

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.