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PubMed2006

An assessment of the hypervariable domains of the 16S rRNA genes for their value in determining microbial community diversity: the paradox of traditional ecological indices.

Amplicon length heterogeneity PCR (LH-PCR) was investigated for its ability to distinguish between microbial community patterns from the same soil type under different land management practices. Natural sagebrush and irrigated mouldboard-ploughed soils from Idaho were queried as to which hypervariable domains, or combinations of 16S rRNA gene domains, were the best molecular markers. Using standard ecological indices to measure richness, diversity and evenness, the combination of three domains, V1, V3 and V1+V2, or the combined V1 and V3 domains were the markers that could best distinguish the undisturbed natural sagebrush communities from the mouldboard-ploughed microbial communities. Bray-Curtis similarity and multidimensional scaling were found to be better metrics to ordinate and cluster the LH-PCR community profiling data. The use/misuse of traditional ecological indices such as diversity and evenness to study microbial community profiles will remain a major point to consider when performing metagenomic studies.

Biodiversity↗
PubMed2002

Lipid analysis of the response of a sedimentary microbial community to polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants that can, under proper conditions, be degraded by microorganisms. The responses of a riverine sedimentary microbial community to PAH contamination were examined using an integrated biochemical assay that yielded data on PAH concentration, total microbial biomass, and microbial community structure and were interpreted using perturbation theory and the subsidy-stress gradient. Microbial mineralization of naphthalene, anthracene, fluorene, and phenanthrene was observed 24 h after their addition to all sediments sampled and ranged from 0.9 to 16.3% in ambient sediments and from 14.8 to 35.8% in contaminated sediments. Total microbial biomass, determined by phospholipid phosphate, increased in response to intermediate PAH concentration and decreased at sites with the highest PAH concentration (p < 0.05) during seven out of nine (78%) seasonal sampling periods. The two sampling periods that were not statistically different followed periods of high water and cold temperatures. Phospholipid fatty acid analysis of microbial community structure analysis indicated that increases in the relative abundance of gram-negative aerobes and heterotrophic eukaryotes were responsible, in part, for these observed increases in total microbial biomass. These findings (increased degradation rates, increased biomass at intermediate PAH concentrations, and altered community structure) indicate that a component of the microbial community responded to PAH as a usable input and are consistent with the predictions of perturbation theory and a subsidy-stress gradient.

Biomass↗
PubMed2005

Microbial community dynamics in manure composts based on 16S and 18S rDNA T-RFLP profiles.

Compost processing is assumed to be related to the microbial communities present. However, methods that will evaluate these relationships are not well understood. In this study, terminal restriction fragment length polymorphism (T-RFLP) analysis was used to evaluate the diversity of PCR-amplified bacterial 16S and fungal 18S rDNA communities from manure composts at different stages of composting (initial [day 0], thermophilic [day 24], and mature [day 104]). Results showed that the bacterial and fungal community profiles changed over the composting process, with bacterial communities showing a higher diversity compared with the fungal communities. During the thermophilic stage (day 24), the diversity of the bacterial communities increased, while the fungal communities decreased. As the compost reached maturity (day 104), a reverse pattern was observed between the diversity of bacterial and fungal communities. That is, the 18S rDNA T-RFLP-based diversity indices increased, while the 16S rDNA T-RFLP-based diversity decreased. Differences in temperature profiles at different stages of composting impacted the chemical properties and the diversity of the microbial communities. The day 104 compost (mature) had lower water, organic matter and C contents and higher C and OM loss compared with the day 0 (initial) and day 24 (thermophilic) composts, which affected the diversity of the microbial communities. The results presented here demonstrated that distinctive community patterns from manure composts could be rapidly generated using T-RFLP analysis. The succession of peaks in combination of increasing and decreasing peak heights at different stage of composting indicates the high potential of T-RFLP technique to monitor the dynamics of microbial communities, and their variation qualitatively and quantitatively.

Animals↗
PubMed2003

Differences in hyporheic-zone microbial community structure along a heavy-metal contamination gradient.

The hyporheic zone of a river is nonphotic, has steep chemical and redox gradients, and has a heterotrophic food web based on the consumption of organic carbon entrained from downwelling surface water or from upwelling groundwater. The microbial communities in the hyporheic zone are an important component of these heterotrophic food webs and perform essential functions in lotic ecosystems. Using a suite of methods (denaturing gradient gel electrophoresis, 16S rRNA phylogeny, phospholipid fatty acid analysis, direct microscopic enumeration, and quantitative PCR), we compared the microbial communities inhabiting the hyporheic zone of six different river sites that encompass a wide range of sediment metal loads resulting from large base-metal mining activity in the region. There was no correlation between sediment metal content and the total hyporheic microbial biomass present within each site. However, microbial community structure showed a significant linear relationship with the sediment metal loads. The abundances of four phylogenetic groups (groups I, II, III, and IV) most closely related to alpha-, beta-, and gamma-proteobacteria and the cyanobacteria, respectively, were determined. The sediment metal content gradient was positively correlated with group III abundance and negatively correlated with group II abundance. No correlation was apparent with regard to group I or IV abundance. This is the first documentation of a relationship between fluvially deposited heavy-metal contamination and hyporheic microbial community structure. The information presented here may be useful in predicting long-term effects of heavy-metal contamination in streams and provides a basis for further studies of metal effects on hyporheic microbial communities.

Alphaproteobacteria↗
PubMed2005

Effects of lead and cadmium nitrate on biomass and substrate utilization pattern of soil microbial communities.

A study was conducted to evaluate the effects of different concentrations of lead (Pb) and cadmium (Cd) applied as their nitrates on soil microbial biomass carbon (C(mic)) and nitrogen (N(mic)), and substrate utilization pattern of soil microbial communities. The C(mic) and N(mic) contents were determined at 0, 14, 28, 42 and 56 days after heavy metal application (DAA). The results showed a significant decline in the C(mic) for all Pb and Cd amended soils from the start to 28 DAA. From 28 to 56 DAA, C(mic) contents changed non-significantly for all other treatments except for 600 mgkg(-1) Pb and 100 mgkg(-1) Cd in which it declined significantly from 42 to 56 DAA. The N(mic) contents also decreased significantly from start to 28 DAA for all other Pb and Cd treatments except for 200 mgkg(-1) Pb which did not show significant difference from the control. Control and 200 mgkg(-1) Pb had significantly lower soil microbial biomass C:N ratio as compared with other Pb treatments from 14 to 42 DAA, however at 56 DAA, only 1000 mgkg(-1) Pb showed significantly higher C:N ratio compared with other treatments. No significant difference in C:N ratio for all Cd treated soils was seen from start to 28 DAA, however from 42 to 56 DAA, 100 mgkg(-1) Pb showed significantly higher C:N ratio compared with other treatments. On 56 DAA, substrate utilization pattern of soil microbial communities was determined by inoculating Biolog ECO plates. The results indicated that Pb and Cd addition inhibited the functional activity of soil microbial communities as indicated by the intensity of average well color development (AWCD) during 168 h of incubation. Multivariate analysis of sole carbon source utilization pattern demonstrated that higher levels of heavy metal application had significantly affected soil microbial community structure.

Biomass↗
PubMed2003

Microbial communities associated with geological horizons in coastal subseafloor sediments from the sea of okhotsk.

Microbial communities from a subseafloor sediment core from the southwestern Sea of Okhotsk were evaluated by performing both cultivation-dependent and cultivation-independent (molecular) analyses. The core, which extended 58.1 m below the seafloor, was composed of pelagic clays with several volcanic ash layers containing fine pumice grains. Direct cell counting and quantitative PCR analysis of archaeal and bacterial 16S rRNA gene fragments indicated that the bacterial populations in the ash layers were approximately 2 to 10 times larger than those in the clays. Partial sequences of 1,210 rRNA gene clones revealed that there were qualitative differences in the microbial communities from the two different types of layers. Two phylogenetically distinct archaeal assemblages in the Crenarchaeota, the miscellaneous crenarchaeotic group and the deep-sea archaeal group, were the most predominant archaeal 16S rRNA gene components in the ash layers and the pelagic clays, respectively. Clones of 16S rRNA gene sequences from members of the gamma subclass of the class Proteobacteria dominated the ash layers, whereas sequences from members of the candidate division OP9 and the green nonsulfur bacteria dominated the pelagic clay environments. Molecular (16S rRNA gene sequence) analysis of 181 isolated colonies revealed that there was regional proliferation of viable heterotrophic mesophiles in the volcanic ash layers, along with some gram-positive bacteria and actinobacteria. The porous ash layers, which ranged in age from tens of thousands of years to hundreds of thousands of years, thus appear to be discrete microbial habitats within the coastal subseafloor clay sediment, which are capable of harboring microbial communities that are very distinct from the communities in the more abundant pelagic clays.

Colony Count, Microbial↗
PubMed2000

Analysis of Microbial Communities in a Landfill Leachate Polluted Aquifer using a New Method for Anaerobic Physiological Profiling and 16S rDNA Based Fingerprinting.

Databases containing information regarding presence and activity of microbial communities will be very useful for determination of the potential for intrinsic bioremediation in landfill leachate polluted aquifers. Simple analyses such as community-level physiological profiling (CLPP) and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments yield large sets of data for inclusion into such databases. In this study we describe the development of a method for anaerobic CLPP, using commercially available Biolog plates. Incubation at the in situ temperature of the aquifer (10 degrees C) for 28 days was optimal for obtaining a specific, reproducible physiological profile. Anaerobic incubation was essential for profiling anaerobic communities. The anaerobic cultivation-dependent CLPP method and cultivation-independent DGGE were applied to groundwater and sediment samples from the aquifer near the Coupépolder landfill in The Netherlands. A combination of computer-assisted CLPP and DGGE analysis of both groundwater and sediment samples yielded the best separating power for characterizing microbial communities in the aquifer. Communities in groundwater were significantly different from those in the corresponding sediment. Microbial communities present in subsamples from sediment cores usually were similar for the various sampling locations. Variation was observed for the heterogeneous sediment beneath the landfill. Both anaerobic CLPP and DGGE analysis clearly separated microbial communities from the polluted aquifer underneath the landfill from those in the less or not polluted aquifer downstream and upstream of the landfill.

Journal Article↗
PubMed2002

Construction of a stable microbial community with high cellulose-degradation ability.

We bred a microbial community capable of degrading rice straw with high efficiency. The microbial community degraded more than 60% of rice straw within 4 days at 50 degrees C. The high stability of the community's degradation ability was demonstrated by its tolerance of being subcultured several times in medium with/without cellulosic material, being heated to 95 degrees C, and freezing at -80 degrees C. The community degraded both nonsterilized and sterilized substrate; and its degradation ability was not affected by pH changes in the medium (initial pH 5-9). PCR-denaturing gradient gel electrophoresis (DGGE) analyses based on 16S rDNA fragments showed that the community structure remained constant after multiple subcultures extending over 2 years. DNA sequence analyses of DGGE bands indicated the coexistence of both aerobic and anaerobic bacteria in the community.

Bacteria↗
PubMed2005

Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition.

Serial analysis of ribosomal sequence tags (SARST) is a novel technique for characterizing microbial community composition. The SARST method captures sequence information from concatemers of short 16S rDNA polymerase chain reaction (PCR) amplicons from complex populations of DNA. Here, we describe a similar method, serial analysis of V6 ribosomal sequence tags (SARST-V6), which targets the V6 hypervariable region of bacterial 16S rRNA genes. The SARST-V6 technique exploits internal primer sequences to generate compatible restriction digest overhangs, thereby improving upon the efficiency of SARST. Serial analysis of V6 ribosomal sequence tags of bacterial community composition in hydrothermal marine sediments from Guaymas Basin resembled results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identified the same major bacterial groups, but only SARST-V6 recovered thermodesulfobacteria and gamma-proteobacteria sequences, while only full-length PCR product cloning recovered candidate division OP11 se-quences. There were differences in the relative frequencies of some phylotypes. The disparities reflect differences in the amplicon pool obtained during initial amplification that may result from different primer affinities or DNA degradation. These results demonstrate the utility of SARST-V6 in collecting taxonomically informative data for high-throughput analysis of microbial communities.

Bacteria↗
PubMed1995

Impact of carbon and flooding on the metabolic diversity of microbial communities in soils.

The assumption that carbon and soil water content are major determinants of microbial community structure and function is rarely questioned because of substantial evidence of the impacts of these variables on specific populations and functions. The significance of carbon and water for metabolic diversity at the microbial community level was tested on the field scale in agricultural plots varying in carbon inputs and in whether they were flooded. Surface soils in which rice straw was incorporated or burned and which were flooded or unflooded were sampled at monthly intervals three times during the flooded winter period (January to March) and again 1 month postdraining. Biomass carbon and nitrogen were not affected by treatments, active bacterial counts showed slight increases, and respiration rates were increased by carbon inputs and flooding. Biolog microplates were inoculated with soil extracts to quantify the metabolic diversity of the soil microbial community. Canonical correspondence analysis and the Monte Carlo permutation testing showed that differences in substrate utilization patterns were significantly related (P < 0.001) to carbon and flooding treatments. Biolog substrates whose metabolism was altered by the treatments were consistent across dates and tended to be positively related (utilization enhancement) to carbon inputs and negatively related to winter flooding. The importance of carbon as an environmental variable increased over time after straw treatment, whereas the importance of water became evident after flooding and decreased after drainage. The effect of long-term rice straw incorporation on substrate utilization patterns at another field site was consistent with these results despite the dissimilarities of the two soils.

Journal Article↗
PubMed2004

Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient.

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R(2) = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R(2) = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the alpha, beta, and gamma-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.

Base Sequence↗
PubMed2004

Change of microbial community structure by respiratory quinone profile at intermittently aerated membrane bioreactor.

In the present study, the microbial community structure in an intermittently aerated submerged membrane bioreactor treating domestic wastewater was observed using the respiratory quinone profiles. The effects of different time interval for the aerobic and anoxic period on microbial community structure were examined with 60/90 min (Step 1) and 90/60 min (Step 2) as anoxic/aerobic periods. There was an observable slight difference in microbial community structure between Step 1 and Step 2 in the submerged membrane bioreactor. The dominant quinone types for Step 1 at both anoxic and aerobic conditions were UQ-8 followed by UQ-10 and MK-6, but those for Step 2 were UQ-8, MK-6 and MK-10(H4). The microbial diversity of Step 1 and Step 2 based on the composition of all quinones was 10.6-11.7 and 13.3-13.0 for anoxic and aerobic conditions, respectively. The present results suggest that the introduction of intermittent aeration into the submerged membrane bioreactor has little influence on the bacterial community structure.

Air Movements↗
PubMed2024

Environmental stress mediates groundwater microbial community assembly.

Community assembly describes how different ecological processes shape microbial community composition and structure. How environmental factors impact community assembly remains elusive. Here we sampled microbial communities and >200 biogeochemical variables in groundwater at the Oak Ridge Field Research Center, a former nuclear waste disposal site, and developed a theoretical framework to conceptualize the relationships between community assembly processes and environmental stresses. We found that stochastic assembly processes were critical (>60% on average) in shaping community structure, but their relative importance decreased as stress increased. Dispersal limitation and 'drift' related to random birth and death had negative correlations with stresses, whereas the selection processes leading to dissimilar communities increased with stresses, primarily related to pH, cobalt and molybdenum. Assembly mechanisms also varied greatly among different phylogenetic groups. Our findings highlight the importance of microbial dispersal limitation and environmental heterogeneity in ecosystem restoration and management.

Phylogeny↗
PubMed2005

Influence of soil zinc concentrations on zinc sensitivity and functional diversity of microbial communities.

Pollution induced community tolerance (PICT) is based on the phenomenon that toxic effects reduce survival of the most sensitive organisms, thus increasing community tolerance. Community tolerance for a contaminant is thus a strong indicator for the presence of that contaminant at the level of adverse concentrations. Here we assessed PICT in 11 soils contaminated with zinc runoff from galvanised electricity pylons and 11 reference soils sampled at 10 m distance from these pylons. Using PICT, the influence of background concentration and bioavailability of zinc on zinc sensitivity and functional diversity of microbial communities was assessed. Zinc sensitivity of microbial communities decreased significantly with increasing zinc concentrations in pore water and calcium chloride extracted fraction while no significant relationship was found with total zinc concentration in the soil. It was also found that functional diversity of microbial communities decreased with increasing zinc concentrations, indicating that increased tolerance is indeed an undesirable phenomenon when environmental quality is considered. The hypothesis that zinc sensitivity of microbial communities is related to background zinc concentration in pore water could not be confirmed.

Bacteria↗
PubMed2003

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities.

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.

Bacteria↗
PubMed2001

Comparative effects of a genetically engineered insect virus and a growth-regulating insecticide on microbial communities in aquatic microcosms.

The effects of a genetically engineered insect baculovirus on indigenous aquatic microbial communities were determined in closed, recirculating aquatic microcosms, and compared with the effects of a natural strain of the virus and of a growth-regulating insecticide, Dimilin. The recombinant virus was a nuclear polyhedrosis virus (NPV) of the spruce budworm (Choristoneura fumiferana (Cf)) with a lacZ marker gene inserted into the egt region of the CfNPV. The natural virus was Ireland strain CfNPV. Microbial measurement endpoints included decomposition activity (mass loss of organic material), respiration on two different substrates (O2 consumption), heterotrophic bacterial abundance (plate counts), and microbial community metabolic profiles (carbon source utilization patterns in Biolog GN microplates). Viral DNA of both the natural strain and the recombinant viruses, detected by polymerase chain reaction techniques, settled out of the microcosm water and accumulated on bottom substrates within 3 days of the microcosm inoculations. The viral DNA persisted in bottom substrates for the duration of the 21-day experimental period, although there was some evidence that the recombinant virus was less stable than the natural strain in particulate organic matter. No significant changes in microbial decomposition or respiration activity, bacterial abundance, or average metabolic responses were detected by a time trend analysis in microcosms inoculated with either the lacZ recombinant virus or the natural Ireland strain CfNPV. Significant effects on microbial decomposition and respiration activity were detected in microcosms treated with the growth-regulating insecticide at, and above, the expected environmental concentrations. Despite significant effects on microbial community functional attributes in Dimilin-treated microcosms, there were no detectable changes in community structure in terms of metabolic profiles or bacterial abundance.

DNA, Viral↗
PubMed2004

Biogeochemical features of lipids in endolithic microbial communities in the Ross Desert (McMurdo Dry Valleys), Antarctica.

Endolithic microbial communities inhabiting porous rocks in the cold, dry mountainous regions of Antarctica have been studied extensively as examples of life's adaptations to extreme environments. Here, we examine hydrocarbons and fatty acids occurring in these communities in order to clarify their biogeochemical features with respect to source organisms, microbial activity, fossilization processes and the influence of Gondwanaland sediments. Unusually, long-chain (>C19) n-alkanes and anteiso-alkanes were often the major hydrocarbons in the samples. A suite of n-alkanoic acids (n-C9-n-C32) and long-chain anteiso-alkanoic acids (a-C20-a-C30) were found, along with short-chain iso- and anteiso-alkanoic acids, and n-alkenoic acids. The relationship between long-chain n-alkanoic acids (n-C20-n-C32) and long-chain anteiso-alkanoic acids suggests that these compounds probably originated from the same group of microorganisms, such as bacteria or endolithic lichens, under moderate pH conditions (pH 3-5). Relatively high trans/cis-C16:1 alkenoic acid ratios suggest the presence of unfavorable environmental conditions in the endolithic microbial habitat. Normal-alkenoic/alkanoic acid ratios may be a useful marker for the fossilization of endolithic microbial communities. Thermally matured triterpanes and steranes from fossilized associations on Mount Fleming strongly suggest the presence of Gondwanaland sediments formed during Devonian and Jurassic (400-180 million years ago).

Alkanes↗
PubMed2003

A comparison of DNA profiling techniques for monitoring nutrient impact on microbial community composition during bioremediation of petroleum-contaminated soils.

Amplicon length heterogeneity PCR (LH-PCR) and terminal restriction fragment length polymorphisms (TRFLP) were used to monitor the impact that nutrient amendments had on microbial community dynamics and structural diversity during bioremediation of petroleum-contaminated soils. Slurried soils contaminated with petroleum hydrocarbons were treated in airlift bench-scale bioreactors and were either amended with optimal inorganic nutrients or left unamended. Direct DNA extraction and PCR amplification of whole eubacterial community DNA were performed with universal primers that bracketed the first two or three hypervariable regions of the 16S rDNA gene sequences. The LH-PCR method profiled a more diverse microbial community than did the TRFLP method. The LH-PCR method also tracked differences between the communities due to nutrient amendments. An in silico database search for bacterial genera with amplicon lengths represented in the community fingerprints was performed. It was possible to qualitatively identify different groups in the microbial community based on the amplicon length variations. A similar "virtual" search was performed for the TRFLP fragments using the web-based TAP-TRFLP program. Cloning and sequencing of the PCR products confirmed the in silico database matches. The application of the LH-PCR method as a monitoring tool for bioremediation could greatly enhance and extend the current understanding of the microbial community dynamics during the biodegradation of environmental contaminants.

Bacteria↗