Molecular community analysis of microbial diversity

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.     

Changes in soil microbial community structure and function in an alpine dry meadow following spring snow melt

Previous work in an alpine dry meadow in the Front Range of the Rocky Mountains has shown that microbial biomass is high during winter and declines rapidly as snow melts in the spring, and that this decline is associated with changes in temperature regime and substrate availability. In this study we tested the hypothesis that the summer and winter microbial communities differ in function and composition. Shifts in species composition between pre- and post-snowmelt communities were detected using reciprocal hybridization of community DNA; DNA extracted from soils sampled at different times was significantly less homologous relative to spatial replicates sampled at the same time. Fungal/bacterial ratios, as measured by direct microscopic counts and by substrate-induced respiration experiments with specific inhibitors, were higher in winter soils. Specific activity of cellulase (absolute cellulase activity per unit microbial biomass C) was higher in the winter soils than in summer soils, while specific amylase activity was not different between winter and summer. Based on most-probable number measurements, the use of the phenolic compound vanillic acid was highest in the winter, while the use of the amino acid glycine was lowest in the winter. Winter and summer soil respiration responded differently to temperature; at 0 degrees C, winter soils respired at a higher proportion of the 22 degrees C rate than did summer soils.     

A thiotrophic microbial community in an acidic brine lake in Northern Chile

The endorheic basins of the Northern Chilean Altiplano contain saline lakes and salt flats. Two of the salt flats, Gorbea and Ignorado, have high acidic brines. The causes of the local acidity have been attributed to the occurrence of volcanic native sulfur, the release of sulfuric acid by oxidation, and the low buffering capacity of the rocks in the area. Understanding the microbial community composition and available energy in this pristine ecosystem is relevant in determining the origin of the acidity and in supporting the rationale of conservation policies. Besides, a comparison between similar systems in Australia highlights key microbial components and specific ones associated with geological settings and environmental conditions. Sediment and water samples from the Salar de Gorbea were collected, physicochemical parameters measured and geochemical and molecular biological analyses performed. A low diversity microbial community was observed in brines and sediments dominated by Actinobacteria, Algae, Firmicutes and Proteobacteria. Most of the constituent genera have been reported to be either sulfur oxidizing microorganisms or ones having the potential for sulfur oxidation given available genomic data and information drawn from the literature on cultured relatives. In addition, a link between sulfur oxidation and carbon fixation was observed. In contrast, to acid mine drainage communities, Gorbea microbial diversity is mainly supported by chemolithoheterotrophic, facultative chemolithoautotrophic and oligotrophic sulfur oxidizing populations indicating that microbial activity should also be considered as a causative agent of local acidity.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide spring in the north Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide spring (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Sphaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism’s population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or “threads” of natural sulfide springs.     

Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem

Rumen microbial community comprising of bacteria, archaea, fungi, and protozoa is characterized not only by the high population density but also by the remarkable diversity and the most complex microecological interactions existing in the biological world. This unprecedented biodiversity is quite far from full elucidation as only about 15-20?% of the rumen microbes are identified and characterized till date using conventional culturing and microscopy. However, the last two decades have witnessed a paradigm shift from cumbersome and time-consuming classical methods to nucleic acid-based molecular approaches for deciphering the rumen microbial community. These techniques are rapid, reproducible and allow both the qualitative and quantitative assessment of microbial diversity. This review describes the different molecular methods and their applications in elucidating the rumen microbial community.     

Cyanobacterial composition of microbial mats from an Australian thermal spring: a polyphasic evaluation

Cyanobacterial composition of microbial mats from an alkaline thermal spring issuing at 43-71 degrees C from tropical north-eastern Australia are described using a polyphasic approach. Eight genera and 10 species from three cyanobacterial orders were identified based on morphological characters. These represented taxa previously known as thermophilic from other continents. Ultrastructural analysis of the tower mats revealed two filamentous morphotypes contributed the majority of the biomass. Both types had ultrastructural characteristics of the family Pseudanabaenaceae. DNA extracts were made from sections of the tentaculiform towers and the microbial community analysed by 16S cyanobacteria-specific PCR and denaturing-gradient gel electrophoresis. Five significant bands were identified and sequenced. Two bands clustered closely with Oscillatoria amphigranulata isolated from New Zealand hot springs; one unique phylotype had only moderate similarity to a range of Leptolyngbya species; and one phylotype was closely related to a number of Geitlerinema species. Generally the approaches yielded complementary information, however the results suggest that species designation based on morphological and ultrastructural criteria alone often fails to recognize their true phylogenetic position. Conversely some molecular techniques may fail to detect rare taxa suggesting that the widest possible suite of techniques be applied when conducting analyses of cyanobacterial diversity of natural populations. This is the first polyphasic evaluation of thermophilic cyanobacterial communities from the Australian continent.     

Population structure and physiological changes within a hot spring microbial mat community following disturbance.

The influence of disturbance on a hot spring cyanobacterial mat community was investigated by physically removing the top 3.0 mm, which included the entire cyanobacterial layer. Changes in 16S rRNA-defined populations were monitored by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments. Some previously absent cyanobacterial populations colonized the disturbed areas, while some populations which were present before the disturbance remained absent for up to 40 days. Changes in physiological activity were measured by oxygen microelectrode analyses and by 14CO2 incorporation into cyanobacterial molecular components. These investigations indicated substantial differences between the disturbed and undisturbed mats, including an unexplained light-induced oxygen consumption in the freshly exposed mat, increased carbon partitioning by phototrophs into growth-related macromolecules, bimodal vertical photosynthesis profiles, and delayed recovery of respiration relative to photosynthesis.     

Soil microbial community structure across a thermal gradient following a geothermal heating event

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65 degrees C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50 degrees C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50 degrees C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.     

Influence of sulfide and temperature on species composition and community structure of hot spring microbial mats

In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80 degrees C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90 degrees C, and a Chloroflexus mat is formed at 65 to 70 degrees C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one Chloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the Chloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter(-1)) below 70 degrees C, whereas Aquificales were dominant in the high-sulfide spring (12 mg liter(-1)) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.     

Sheep-urine-induced changes in soil microbial community structure

Soil microbial communities play an important role in nutrient cycling and nutrient availability, especially in unimproved soils. In grazed pastures, sheep urine causes local changes in nutrient concentration which may be a source of heterogeneity in microbial community structure. In the present study, we investigated the effects of synthetic urine on soil microbial community structure, using physiological (community level physiological profiling, CLPP), biochemical (phospholipid fatty acid analysis, PLFA) and molecular (denaturing gradient gel electrophoresis, DGGE) fingerprinting methods. PLFA data suggested that synthetic urine treatment had no significant effect on total microbial (total PLFA), total bacterial or fungal biomass; however, significant changes in microbial community structure were observed with both PLFA and DGGE data. PLFA data suggested that synthetic urine induced a shift towards communities with higher concentrations of branched fatty acids. DGGE banding patterns derived from control and treated soils differed, due to a higher proportion of DNA sequences migrating only to the upper regions of the gel in synthetic urine-treated samples. The shifts in community structure measured by PLFA and DGGE were significantly correlated with one another, suggesting that both datasets reflected the same changes in microbial communities. Synthetic urine treatment preferentially stimulated the use of rhizosphere-C in sole-carbon-source utilisation profiles. The changes caused by synthetic urine addition accounted for only 10-15% of the total variability in community structure, suggesting that overall microbial community structure was reasonably stable and that changes were confined to a small proportion of the communities.     

Microenvironments and microbial community structure in sediments

The aim of this study was to explore the potential of a combined chemical and microbiological approach as part of a study of organic carbon oxidation processes in sediments. An assessment of microbiological diversity using molecular techniques was carried out in combination with high resolution chemical measurements at the sediment-water interface of a coastal lagoon affected by eutrophication in autumn 2000. There was a 0.2 mm overlap between the O2 and H2S profiles. pH showed a maximum just above the sediment-water interface coinciding with an oxygen maximum, suggesting photosynthetic activity, and a minimum coinciding with the O2-H2S interface. The redox potential was high in bottom water and surface sediment, reflecting the presence of oxygen and oxides, and reached low values after a step-wise decrease at -18 mm. Reduction of Fe occurred within the biofilm at the O2-H2S interface and was mostly due to reduction by H2S. The elevated concentrations of dissolved Mn in the oxic water may have been caused either by in situ production within organic aggregates or lateral water flow from sites nearby at which Mn2+ diffuses out of the sediment. Sequences related to sulphur chemolitotrophs were retrieved from the biofilm samples, which is consistent with the small overlap between O2 and H2S observed in this biofilm. Although the resolution of techniques used was different, sequencing results were consistent with chemical data in delineating the same horizons according to redox, pH or ecological properties.     

Deep sequencing analysis of bacterial community structure of Soldhar hot spring,India

We investigated the bacterial community structure of Soldhar hot spring with extreme high temperature 95°C located in Uttarakhand, India using high throughput sequencing. Bacterial phyla Proteobacteria (88.8%), Deinococcus-Thermus (7.5%), Actinobacteria (2.3%), and Firmicutes (1.07%) were predominated in the sequencing survey, whereas Bacteroidetes, Verrucomicrobia, Aquificae and Acidobacteria were detected in relatively lower abundance in Soldhar hot spring. At the family level, Comamonadaceae (52.5%), Alteromonadaceae (15.9%), and Thermaceae (7.5%) were mostly dominated in the ecosystem followed by Chromatiaceae, Microbacteriaceae, and Cyclobacteriaceae. Besides, members of Rhodobacteraceae, Moraxellaceae, Xanthomonadaceae, Aquificaceae, Enterobacteriaceae, Bacillaceae, Methylophilaceae, etc. were detected as a relatively lower abundance. In the present study we discuss the overall microbial community structure and their relevance to the ecology of the Soldhar hot spring environment.     

Drivers of microbial community structure in forest soils

Applied Microbiology and Biotechnology - Forests are essential biomes for global biogeochemical cycles, and belowground microorganisms have a key role in providing relevant ecosystem services. To...     

Spatial and temporal analysis of the microbial community in the tailings of a Pb-Zn mine generating acidic drainage

Analysis of spatial and temporal variations in the microbial community in the abandoned tailings impoundment of a Pb-Zn mine revealed distinct microbial populations associated with the different oxidation stages of the tailings. Although Acidithiobacillus ferrooxidans and Leptospirillum spp. were consistently present in the acidic tailings, acidophilic archaea, mostly Ferroplasma acidiphilum, were predominant in the oxidized zones and the oxidation front, indicating their importance to generation of acid mine drainage.     

Illumina-based analysis of microbial community diversity

Microbes commonly exist in milieus of varying complexity and diversity. Although cultivation-based techniques have been unable to accurately capture the true diversity within microbial communities, these deficiencies have been overcome by applying molecular approaches that target the universally conserved 16S ribosomal RNA gene. The recent application of 454 pyrosequencing to simultaneously sequence thousands of 16S rDNA sequences (pyrotags) has revolutionized the characterization of complex microbial communities. To date, studies based on 454 pyrotags have dominated the field, but sequencing platforms that generate many more sequence reads at much lower costs have been developed. Here, we use the Illumina sequencing platform to design a strategy for 16S amplicon analysis (iTags), and assess its generality, practicality and potential complications. We fabricated and sequenced paired-end libraries of amplified hyper-variable 16S rDNA fragments from sets of samples that varied in their contents, ranging from a single bacterium to highly complex communities. We adopted an approach that allowed us to evaluate several potential sources of errors, including sequencing artifacts, amplification biases, non-corresponding paired-end reads and mistakes in taxonomic classification. By considering each source of error, we delineate ways to make biologically relevant and robust conclusions from the millions of sequencing reads that can be readily generated by this technology.     

浓香型白酒窖池微生物群落结构特征

窖池是中国白酒,尤其是浓香型大曲酒生产颇具特色的固态生物反应器,窖龄与微生物群落结构关系密切且复杂,对产品质量影响非常显著.本研究以微生物细胞膜的特征组分磷酸脂肪酸(PLFA)为指标,研究了不同窖龄(5年、100年和300 年)窖池窖泥、糟醅和黄水的微生物群落结构特征.结果表明:窖泥中总PLFA含量最高,糟醅次之,黄水最低.PLFA的组成因窖龄而异,黄水中总PLFA含量随窖池窖龄的增长而减小.窖泥中直链饱和脂肪酸含量最高,为PLFA总量的50.7%～73.9%,其中300年窖池窖泥最高.窖泥中表征革兰氏阳性(G+)厌氧细菌的PLFA含量较高,而糟醅和黄水中均以表征革兰氏阴性(G-)厌氧菌的PLFA含量较高.100年窖泥中表征G+菌、G-菌和厌氧菌的PLFA含量高于其他窖龄相应样品.5年窖窖泥、糟醅和黄水中真菌PLFA含量均高于其他窖龄相应样品.经主成分分析,5年窖和100年窖中主要变异菌群是G+菌和真菌,300年窖中主要变异菌群是细菌.描述窖池微生物群落特征的多样性指数可以选用PLFA的频次、Simpson优势度指数和Shannon多样性指数.     

Response of the microbial community to copper oxychloride in acidic sandy loam soil

AIMS: Determining the response of different microbial parameters to copper oxychloride in acidic sandy loam soil samples using cultivation-dependent and direct microscopic techniques. METHODS AND RESULTS: Culturable microbial populations were monitored for 245 days in a series of soil microcosms spiked with different copper oxychloride concentrations. Microbial populations responded differently to additional Cu. Protistan numbers and soil metabolic potential decreased. Experiments with more soil samples revealed that metabolic potential was not significantly affected by < or =100 mg kg(-1) additional Cu. However, a negative impact on protista was noted in soil containing only 15 mg kg(-1) EDTA-extractable Cu. The negative impact on protistan numbers was less severe in soils with a higher phosphorous and zinc content. CONCLUSIONS: Bacterial populations responded differently, and protista were most sensitive to elevated Cu levels. Protistan numbers in soil from uncultivated land were higher and seemed to be more sensitive to additional Cu than the numbers of these organisms in soil originating from cultivated land. SIGNIFICANCE AND IMPACT OF THE STUDY: Protistan sensitivity to small increases in Cu levels demonstrates the vulnerability of the soil ecosystem to Cu perturbations, especially when the importance of protista as link in the flow of energy between trophic levels is considered.     

Molecular analysis of a bacterial chitinolytic community in an upland pasture

The effects of agricultural-improvement treatments on the chitinolytic activity and diversity of a microbial community were investigated within an upland pasture. The treatments of interest were lime and treated sewage sludge, both commonly applied to pasture land to improve fertility. Burial of chitin-containing litter bags at the field site resulted in enrichment of bacteria according to 16S rRNA fingerprinting. Chitinolytic-activity measurements showed that the highest activity occurred in those bags recovered from sludge-amended plots, which correlated well with increased counts of actinobacteria in samples from these chitin bags. Our findings suggest that sewage sludge increases the fertility of the soil in terms of chitinase activity. Ten clone libraries were constructed from family 18 subgroup A chitinases, PCR amplified from litter bags buried in soil in July 2000 or in September 2000, in a separate study. Analysis of these libraries by restriction fragment length polymorphism and sequencing showed that they were dominated by actinobacterium-like chitinase sequences. This suggests that actinobacteria have an important chitinolytic function in this soil ecosystem. Our findings showed that sludge application increased chitinolytic activity but decreased the diversity of chitinases present.