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Bacterial diversity in subbituminous coal and soil from coal mine of South Sumatra, Indonesia
Abstract
Coal and soil around coal seam environment provide potential factors that support bacterial life. Study of environmental metagenome to determine relationship among the bacteria that make up the community in coal and soil layers is reported. DNA extractions were performed in three different methods, namely direct extraction, filtration, and filtration with blending. The DNA of coal and soil samples were subject to PCRamplification to get V5-V6 16S rRNA gene fragments, then separated by denaturing gradient gel electrophoresis (DGGE). Results showed that the indirect methods are advantageous and can detect more bands compared to the direct method. Among the samples tested, a high number of bacterial ribotypes (Shannon diversity index), but low evenness of the bacterial community were observed in the coal samples. Cluster analysis of DGGE bands showed that the coal mixed soil clustered separate from its parent, the coal seam and the soil samples. Phylogenetic trees of their sequences showed that the coal boomed more Firmicutes and Actinobacteria compared to the soil samples. It confirmed that the physico-chemical properties of soil strongly influence evolutionary distance of coal and soil bacteria though they are separated by little physical distance. This is the first report in which the disturbed coal which is represented by coal mixed soil was not artificially reconstructed as a microcosm, and is in a natural situation in which the virgin coal seam is compared to the adjacent virgin soil layers and soil mixed coal as the disturbed soil.
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In soil microbial ecology, the effects of environmental factors and their gradients, temporal changes or the response to specific experimental treatments of microbial communities can only be effectively analyzed using methods that address the structural differences among whole communities. Fingerprinting methods are the most appropriate technique for this task when multiple samples must be analyzed. Among the methods currently used to compare microbial communities based on nucleic acid sequences, the techniques based on differences in the melting properties of double-stranded molecules, denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE), are the most widely used. Their main advantage is that they provide the possibility to further analyze whole sequences contained in fingerprints using molecular methods. In addition to the analysis of microbial communities based on DNA extracted from soils, DGGE/TGGE can also be used for the assessment of the active part of the community based on the analysis of RNA-derived sequences or for the analysis of sequences of functional genes encoding for proteins involved in important soil processes.
Two hydrocarbon degraders isolated from an abandoned coal power plant soil in Lagos, Nigeria, were tested. The isolates were identified as belonging to the genus Pseudomonas. P. putida G1 exhibited strong degradative ability on crude oil, kerosene, fair ability on engine oil, brake oil and petrol and a weak ability on pyrene, while P. aeruginosa K1 had corresponding values of 0.21days-1 and 3.31 days. The amount of crude oil degraded by strain G1 and K1 after a 21 day incubation period were 92.05% and 90.89% respectively. Data obtained from gas chromatographic analysis of oil recovered from culture fluids of G1 and K1 confirmed a near-disappearance of aliphatic fractions and a significant reduction of aromatic fractions in the hydrocarbon mixture.
This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.
Separation of polymerase chain reaction (PCR)-amplified 16S rDNA products using denaturing gradient gel electrophoresis (DGGE) was tested as a means to study microbial community composition in bulk soil samples. DNA was extracted from six soils from agroecosystems in Norway and the USA under different agronomic treatments (crop, rotation, and tillage); one soil is contaminated with polyaromatic hydrocarbons (PAH, 700 mg kg-1). Two sets of primers specific for Bacteria (V3 and the V6/V9 regions of 16S rRNA) and another for Archaea (V3 region of 16S rRNA) were used to determine the contribution of each domain to the microbial community. Reproducible, characteristic profiles of the communities were obtained by DGGE separation of the PCR amplification products. The number of fragments resolved by DGGE indicated bacterial diversity was far greater than that of the Archaea in the agricultural soils examined. Only the soil contaminated with PAHs had reduced bacterial diversity, evidenced by a distinct DGGE profile. The results showed that the method is useful as an initial step to discriminate among communities because it is rapid and multiple samples can be easily screened. There are some limitations, but under highly selective conditions it is possible to distinguish communities from different soils and to indicate the presence of numerically dominant populations.
Although recent work has shown that both deterministic and stochastic processes are important in structuring microbial communities, the factors that affect the relative contributions of niche and neutral processes are poorly understood. The macrobiological literature indicates that ecological disturbances can influence assembly processes. Thus, we sampled bacterial communities at 4 and 16 weeks following a wildfire and used null deviation analysis to examine the role that time since disturbance has in community assembly. Fire dramatically altered bacterial community structure and diversity as well as soil chemistry for both time-points. Community structure shifted between 4 and 16 weeks for both burned and unburned communities. Community assembly in burned sites 4 weeks after fire was significantly more stochastic than in unburned sites. After 16 weeks, however, burned communities were significantly less stochastic than unburned communities. Thus, we propose a three-phase model featuring shifts in the relative importance of niche and neutral processes as a function of time since disturbance. Because neutral processes are characterized by a decoupling between environmental parameters and community structure, we hypothesize that a better understanding of community assembly may be important in determining where and when detailed studies of community composition are valuable for predicting ecosystem function.
There is a growing interest in the links between humus forms and soil biota, and little is known about these links in Mediterranean ecosystems. Culture-independent techniques, such as DNA extraction followed by DGGE and enzyme activities, allowed us to compare microbial communities in two horizons of a forest soil in different seasonal conditions. Direct in situ lysis was applied for extraction of DNA from soil; intracellular DNA was separated from extracellular and used to represent the composition of microflora. The aims were to describe how biochemical and microbiological parameters correlate with topsoil properties in typical Mediterranean Moder humus. Changes in bacterial and fungal community composition were evident from DGGE profiles. Degrees of similarity and clustering correlation coefficients showed that the seasonal conditions may affect the composition and activity of bacterial and fungal communities in the OH horizon, while in the E horizon the two communities were hardly modified. In the same season, OH and E horizons showed a different composition of bacterial and fungal communities and different enzyme activities, suggesting similar behaviour of eubacteria and fungi relatively to all the variables analysed. Evidently, different organic carbon content in soil horizons influenced microflora composition and microbial activities involved in the P and N cycles.
This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.
Despite the important ecosystem role played by arbuscular mycorrhizal fungi (AMF), little is known about spatial and temporal variation in soil AMF communities. We used pyrosequencing to characterise AMF communities in soil samples (n = 44) from a natural forest ecosystem. Fungal taxa were identified by BLAST matching of reads against the MaarjAM database of AMF SSU rRNA gene diversity. Sub-sampling within our dataset and experimental shortening of a set of long reads indicated that our approaches to taxonomic identification and diversity analysis were robust to variations in pyrosequencing read length and numbers of reads per sample. Different forest plots (each 10 × 10 m and separated from one another by 30 m) contained significantly different soil AMF communities, and the pairwise similarity of communities decreased with distance up to 50 m. However, there were no significant changes in community composition between different time points in the growing season (May-September). Spatial structure in soil AMF communities may be related to the heterogeneous vegetation of the natural forest study system, while the temporal stability of communities suggests that AMF in soil represent a fairly constant local species pool from which mycorrhizae form and disband during the season.
Extensive use of chromium (Cr) and arsenic (As) based preservatives from the leather tanning industry in Pakistan has had a deleterious effect on the soils surrounding production facilities. Bacteria have been shown to be an active component in the geochemical cycling of both Cr and As, but it is unknown how these compounds affect microbial community composition or the prevalence and form of metal resistance. Therefore, we sought to understand the effects that long-term exposure to As and Cr had on the diversity and structure of soil microbial communities. Soils from three spatially isolated tanning facilities in the Punjab province of Pakistan were analyzed. The structure, diversity and abundance of microbial 16S rRNA genes were highly influenced by the concentration and presence of hexavalent chromium (Cr (VI)) and arsenic. When compared to control soils, contaminated soils were dominated by Proteobacteria while Actinobacteria and Acidobacteria (which are generally abundant in pristine soils) were minor components of the bacterial community. Shifts in community composition were significant and revealed that Cr (VI)-containing soils were more similar to each other than to As contaminated soils lacking Cr (VI). Diversity of the arsenic resistance genes, arsB and ACR3 were also determined. Results showed that ACR3 becomes less diverse as arsenic concentrations increase with a single OTU dominating at the highest concentration. Chronic exposure to either Cr or As not only alters the composition of the soil bacterial community in general, but affects the arsenic resistant individuals in different ways.
DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.
In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.
Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.
Bacterial communities play a central role in ecosystems, by regulating biogeochemical fluxes. Therefore, understanding how multiple functional interactions between species face environmental perturbations is a major concern in conservation biology. Because bacteria can use several strategies, including horizontal gene transfers (HGT), to cope with rapidly changing environmental conditions, potential decoupling between function and taxonomy makes the use of a given species as a general bioindicator problematic. The present work is a first step to characterize the impact of a recent polymetallic gradient over the taxonomical networks of five lacustrine bacterial communities. Given that evolutionary convergence represents one of the best illustration of natural selection, we focused on a system composed of two pairs of impacted and clean lakes in order to test whether similar perturbation exerts a comparable impact on the taxonomical networks of independent bacterial communities. First, we showed that similar environmental stress drove parallel structural changes at the taxonomic level on two independent bacterial communities. Second, we showed that a long-term exposure to contaminant gradients drove significant taxonomic structure changes within three interconnected bacterial communities. Thus, this model lake system is relevant to characterize the strategies, namely acclimation and/or adaptation, of bacterial communities facing environmental perturbations, such as metal contamination.
The early years of soil genesis during mine spoil reclamation are critical for vegetative establishment and may help predict reclamation success. Mine spoils in the Halle-Leipzig region of Germany were analyzed for microbial changes following a hay mulch-seeding treat- ment without topsoil or fertilizer application. Microbial biomass car- bon (Cmic and dehydrogenase activity(DHA)of spoils were measured each year in the first 3 yr after treatment. In the third year, bacterial communityDNAfingerprints were compared with those from a refer- ence soil. Microbial indicators were measured at three depths in the upper 10 cm of spoils at three sites with contrasting parent materials: glacial till (sandy loam), limnic tertiary sediments (high-lignite sandy clay loam), and quaternary sand and gravel (loamy sand). Before reclamation, Cmic (01 cm) were 9 6, 39 11, and 38 16 mg kg1 means and standard deviations of surface spoils for the loamy sand, high-lignite sandy clay loam, and sandy loam spoils, respectively. Within one year, mean Cmic 229 64, and 497 167 mg kg1 at the surface increased to 148 70, , respectively, and was significantly higher at 0 to 1 cm than at lower depths. Highest DHA and DNA yields were obtained in the 0- to 1-cm depth of the sandy loam spoils. Microbial biomass C values exhibited significant correlations with DHA, DNA yield, and extractable C for all three mine spoils. Soil microbial indices were more responsive than plant measurements to differences in parent materials
The spatial scale of disturbance is a factor potentially influencing the relationship between disturbance and diversity. There has been discussion on whether disturbances that affect local communities and create a mosaic of patches in different successional stages have the same effect on diversity as regional disturbances that affect the whole landscape. In a microcosm experiment with metacommunities of aquatic protists, we compared the effect of local and regional disturbances on the disturbance–diversity relationship. Local disturbances destroyed entire local communities of the metacommunity and required reimmigration from neighboring communities, while regional disturbances affected the whole metacommunity but left part of each local community intact. Both disturbance types led to a negative relationship between disturbance intensity and Shannon diversity. With strong local disturbance, this decrease in diversity was due to species loss, while strong regional disturbance had no effect on species richness but reduced the evenness of the community. Growth rate appeared to be the most important trait for survival after strong local disturbance and dominance after strong regional disturbance. The pattern of the disturbance–diversity relationship was similar for both local and regional diversity. Although local disturbances at least temporally increased beta diversity by creating a mosaic of differently disturbed patches, this high dissimilarity did not result in regional diversity being increased relative to local diversity. The disturbance–diversity relationship was negative for both scales of diversity. The flat competitive hierarchy and absence of a trade-off between competition and colonization ability are a likely explanation for this pattern.
Electronic supplementary material
The online version of this article (doi:10.1007/s00442-011-2140-8) contains supplementary material, which is available to authorized users.
Advances in DNA sequencing have allowed us to characterize microbial communities--including those associated with the human body--at a broader range of spatial and temporal scales than ever before. We can now answer fundamental questions that were previously inaccessible and use well-tested ecological theories to gain insight into changes in the microbiome that are associated with normal development and human disease. Perhaps unsurprisingly, the ecosystems associated with our body follow trends identified in communities at other sites and scales, and thus studies of the microbiome benefit from ecological insight. Here, we assess human microbiome research in the context of ecological principles and models, focusing on diversity, biological drivers of community structure, spatial patterning and temporal dynamics, and suggest key directions for future research that will bring us closer to the goal of building predictive models for personalized medicine.
Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present
study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved
in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [13C]acetate and H2-13CO2, were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates,
samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic
(DGGE) analyses were taken over 6 months. Surprisingly, the formation of [13C]methane was linked to acetoclastic methanogenesis in both the [13C]acetate- and the H2-13CO2-amended cultures of coal and timber. H2-13CO2 was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic
microbial community appears to be highly adapted to the low-H2 conditions found in coal mines.
The global atmospheric carbon dioxide (CO2) concentration is expected to increase continuously over the next century. However, little is known about the responses of
soil bacterial communities to elevated CO2 in terrestrial ecosystems. This study aimed to partition the relative influences of CO2, nitrogen (N), and the spatial factor (different sampling plots) on soil bacterial communities at the free-air CO2 enrichment research site in Duke Forest, North Carolina, by two independent techniques: an entirely sequencing-based approach
and denaturing gradient gel electrophoresis. Multivariate regression tree analysis demonstrated that the spatial factor could
explain more than 70% of the variation in soil bacterial diversity and 20% of the variation in community structure, while
CO2 or N treatment explains less than 3% of the variation. For the effects of soil environmental heterogeneity, the diversity
estimates were distinguished mainly by the total soil N and C/N ratio. Bacterial diversity estimates were positively correlated
with total soil N and negatively correlated with C/N ratio. There was no correlation between the overall bacterial community
structures and the soil properties investigated. This study contributes to the information about the effects of elevated CO2 and soil fertility on soil bacterial communities and the environmental factors shaping the distribution patterns of bacterial
community diversity and structure in temperate forest soils.
Four extraction methods, including a novel one, were compared for their efficiencies in producing DNA from three contrasting
agricultural soils. Molecular analyses (PCR-denaturing gradient gel electrophoresis [DGGE] and clone libraries) focusing on
different microbial groups were used as assessment criteria. Per soil, the DNA yields differed between extraction methods.
Clear effects of method on apparent richness and community structure were found. Actinobacterial diversity based on soil DNA
produced by two divergent methods revealed that a hitherto-undescribed group was obtained by the novel method.
DNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental
microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and
conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal
gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction
fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for
bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups
such as Acidobacteria, Gemmatimonades, and Verrucomicrobia were higher in the first extraction than in the sixth but that the reverse was true for Proteobacteria and Actinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction
bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional
measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.
Attachment of Mycobacterium avium subsp. paratuberculosis to soil particles could increase their availability to farm animals, as well as influence the transportation of M. avium subsp. paratuberculosis to water sources. To investigate the possibility of such attachment, we passed a known quantity of M. avium subsp. paratuberculosis through chromatography columns packed with clay soil, sandy soil, pure silica, clay-silica mixture, or clay-silica complexes
and measured the organisms recovered in the eluent using culture or quantitative PCR. Experiments were repeated using buffer
at a range of pH levels with pure silica to investigate the effect of pH on M. avium subsp. paratuberculosis attachment. Linear mixed-model analyses were conducted to compare the proportional recovery of M. avium subsp. paratuberculosis in the eluent between different substrates and pH levels. Of the organisms added to the columns, 83 to 100% were estimated
to be retained in the columns after adjustment for those retained in empty control columns. The proportions recovered were
significantly different across different substrates, with the retention being significantly greater (P < 0.05) in pure substrates (silica and clay-silica complexes) than in soil substrates (clay soil and sandy soil). However,
there were no significant differences in the retention of M. avium subsp. paratuberculosis between silica and clay-silica complexes or between clay soil and sandy soil. The proportion retained decreased with increasing
pH in one of the experiments, indicating greater adsorption of M. avium subsp. paratuberculosis to soil particles at an acidic pH (P < 0.05). The results suggest that under experimental conditions M. avium subsp. paratuberculosis adsorbs to a range of soil particles, and this attachment is influenced by soil pH.
Phylogenetic and "fingerprinting" analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing approximately 5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.
Denaturing gradient gel electrophoresis was used to identify bacterial communities from Gedongsongo hot spring, Indonesia, by culture-independent and -dependent procedures. Predominant species were found to be closely related to beta-proteobacteria, particularly from genus Ralstonia and Delftia. Other species present include alpha- and gamma-proteobacteria and Thermus groups. Some of the strains showed unique 16S rDNA sequences.
A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.
The presence of bacteria in a deep clay sediment was analyzed in a 20-m-long core horizontally drilled from a mine gallery at a depth of 224 m in the Boom clay formation (Mol, Belgium). This clay deposit is the result of a marine sedimentary process that occurred 35 million years ago. Bacterial activities were estimated by measuring respiration on [14C]glucose. Using the same samples, universal primers for the genes coding for eubacterial 16S rRNA were used to amplify extracted DNA. PCR products were then cloned, sequenced, and analyzed by molecular phylogeny. Our data showed a decrease in bacterial densities as a function of distance from the gallery, with few bacteria detectable by culture at more than 80 cm from the gallery wall. PCR experiments showed the presence of bacteria in all samples, and phylogenetic analyses were then used to tentatively identify these organisms. Because of low bacterial densities in deep clay samples, direct counts and enumeration of viable bacteria on diverse culture media remained negative. All experiments, both cultures and PCR, demonstrated the difficulty of analyzing samples that contain only a few poorly active bacteria as it is difficult to avoid a small contamination by active bacteria during sampling. Since the porosity of the Boom clay formation is less than the expected size of bacteria, it is possible that some of the bacteria present in this 35-million-year-old deep clay deposit derive from cells initially trapped during the sedimentation process.
A simple method that combines an adapted ribosome isolation method and a common RNA extraction step has been developed for selective recovery of intact rRNA from natural microbial communities in soil. After mechanical cell lysis, ribosomes are separated by centrifugation steps, avoiding massive humic acid contamination and RNA degradation. The protocol accommodates the complex composition of soils by blocking adsorbing surfaces and humic acids with polyvinylpyrrolidone and bovine serum albumin. A usual RNA extraction step yields rRNA accessible for hybridization or reverse transcription-PCR. Hybridization with specific oligonucleotide probes was used for group-specific yield comparison. By using a probe hybridizing to the 16S rRNA of the bacterial kingdom, total bacterial rRNA yield was estimated to be in the range of 0.2 microgram per g for different soils. Group-specific probes did not indicate a selectivity of the isolation procedure and differentiated the compositions of different soil microbial communities. The sequence diversity of the isolated RNA population was also revealed by temperature gradient gel electrophoresis of reverse transcription-PCR amplification products by using a region of the 16S rRNA as a target. The pattern obtained by this analysis differed from a similar one resulting from the separation of amplification products of community DNA preparations. This different view of the community composition is attributable to the correlation of ribosome numbers to the metabolic activity of bacteria in the habitat under observation.
Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5' end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR-single-stranded DNA approach for microbial community analysis.
Culture-independent molecular phylogenetic methods were used to explore the breadth of diversity and environmental distribution
of members of the division-level “candidate” phylogenetic group WS6, recently discovered in a contaminated aquifer and with
no cultivated representatives. A broad diversity of WS6-affiliated sequences were cloned from 7 of 12 environments investigated:
mainly from anaerobic sediment environments. The number of sequences representing the WS6 candidate division was increased
from 3 to 60 in this study. The extent of phylogenetic divergence (sequence difference) in this candidate division was found
to be among the largest of any known bacterial division. This indicates that organisms representing the WS6 phylogenetic division
offer a broad diversity of undiscovered biochemical and metabolic novelty. These results provide a framework for the further
study of these evidently important kinds of organisms and tools, the sequences, with which to do so.
For aligning DNA sequences that differ only by sequencing errors, or by equivalent errors from other sources, a greedy algorithm can be much faster than traditional dynamic programming approaches and yet produce an alignment that is guaranteed to be theoretically optimal. We introduce a new greedy alignment algorithm with particularly good performance and show that it computes the same alignment as does a certain dynamic programming algorithm, while executing over 10 times faster on appropriate data. An implementation of this algorithm is currently used in a program that assembles the UniGene database at the National Center for Biotechnology Information.
In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain Pseudomonas fluorescens DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous Pseudomonas organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (Pseudomonas) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting Streptomyces and closely related genera. DR54 constituted more than 75% of the Pseudomonas population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous Pseudomonas organisms negatively, whereas imazalil affected Pseudomonas numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for Pseudomonas during plant growth, but after plant harvest Pseudomonas-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence, Pseudomonas was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.
Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (alpha, beta, and gamma subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.
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.
Organisms belonging to the OP11 candidate phylogenetic division of Bacteria have been detected only in rRNA-based sequence surveys of environmental samples. Preliminary studies indicated that such organisms represented by the sequences are abundant and widespread in nature and highly diverse phylogenetically. In order to document more thoroughly the phylogenetic breadth and environmental distribution of this diverse group of organisms, we conducted further molecular analyses on environmental DNAs. Using PCR techniques and primers directed toward each of the five described subdivisions of OP11, we surveyed 17 environmental DNAs and analyzed rRNA gene sequences in 27 clonal libraries from 14 environments. Ninety-nine new and unique sequences were determined completely, and approximately 200 additional clones were subjected to partial sequencing. Extensive phylogenetic comparisons of the new sequences to those representing other bacterial divisions further resolved the phylogeny of the bacterial candidate division OP11 and identified two new candidate bacterial divisions, OP11-derived 1 (OD1) and Sulphur River 1 (SR1). The widespread environmental distribution of representatives of the bacterial divisions OD1, OP11, and SR1 suggests potentially conspicuous biogeochemical roles for these organisms in their respective environments. The information on environmental distribution offers clues for attempts to culture landmark representatives of these novel bacterial divisions, and the sequences are specific molecular signatures that provide for their identification in other contexts.
Changes in soil microbial community structure and diversity may reflect environmental impact. We examined 16S rRNA gene fingerprints of bacterial communities in six agroecosystems by PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) separation. These soils were treated with manure for over a century or different fertilizers for over 70 years. Bacterial community structure and diversity were affected by soil management practices, as evidenced by changes in the PCR-DGGE banding patterns. Bacterial community structure in the manure-treated soil was more closely related to the structure in the untreated soil than that in soils treated with inorganic fertilizers. Lime treatment had little effect on bacterial community structure. Soils treated with P and N-P had bacterial community structures more closely related to each other than to those of soils given other treatments. Among the soils tested, a significantly higher number of bacterial ribotypes and a more even distribution of the bacterial community existed in the manure-treated soil. Of the 99 clones obtained from the soil treated with manure for over a century, two (both Pseudomonas spp.) exhibited 100% similarity to sequences in the GenBank database. Two of the clones were possible chimeras. Based on similarity matching, the remaining 97 clones formed six major clusters. Fifty-six out of 97 were assigned taxonomic units which grouped into five major taxa: alpha-, beta-, and gamma-Proteobacteria (36 clones), Acidobacteria (16 clones), Bacteroidetes (2 clones), Nitrospirae (1 clone), and Firmicutes (1 clone). Forty-one clones remained unclassified. Results from this study suggested that bacterial community structure was closely related to agroecosystem management practices conducted for over 70 years.
The effect of fermentable carbohydrates (sugar beet pulp and fructooligosaccharides) on the faecal bacterial communities of weaning piglets was analysed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in piglet faecal bacterial community structure were determined based on the Dice coefficients for pairwise comparison of the DGGE fingerprints and revealed significant changes in the faecal microbiota immediately after weaning. Piglets fed with fermentable carbohydrates showed a higher bacterial diversity and a more rapid stabilisation of the bacterial community compared with that of the animals fed with the control diet. Thirteen dominant DGGE bands were matched with sequences that showed 91-97% similarity to those derived from the Clostridium coccoides group and the Clostridium leptum subgroup. Amplicons related to Ruminococcus-like species were found in all DGGE fingerprints derived from pigs on the diet containing sugar beet pulp and fructooligosaccharides, but not in pigs on the control diet. These results indicate that these bacteria may play a role in the utilisation of dietary fibres.
A laboratory-scale model system was developed to investigate the transport mechanisms involved in the horizontal movement of bacteria in overland flow across saturated soils. A suspension of Escherichia coli and bromide tracer was added to the model system, and the bromide concentration and number of attached and unattached E. coli cells in the overland How were measured over time. Analysis of the breakthrough curves indicated that the E. coli and bromide were transported together, presumably by the same mechanism. This implied that the E. coli was transported by advection with the flowing water. Overland-flow transport of E. coli could be significantly reduced if the cells were preattached to large soil particles (> 45 mu m). However, when unattached cells were inoculated into the system, the E. coli appeared to attach predominantly to small particles (< 2 mu m) and hence remained unattenuated during transport. These results imply that in runoff generated by saturation-excess conditions, bacteria are rapidly transported across the surface and have little opportunity to interact with the soil matrix.
Analysis Considerations Accuracy and Precision Bias Reporting Coal Analysis Interrelationships of the Data Coal Classification The Future References
The Handbook deals with the various aspects of coal analysis and provides a detailed explanation of the necessary standard tests and procedures that are applicable to coal in order to help define usage and behavior relative to environmental issues. It provides details of the meaning of various test results and how they might be applied to predict coal behavior during use. Emphasis is on ASTM standards and test methods but ISO and BSI standards methods are included. Chapter headings are: Coal analysis; Sampling and sample preparation; Proximate analysis; Ultimate analysis; Mineral matter; Physical and electrical properties; Thermal properties; Mechanical properties; Spectroscopic properties; Solvent properties; and Glossary.
Surface coal mining and reclamation methods in the Appalachians have changed dramatically since the passage of the Surface Mining Control and Reclamation Act (SMCRA) of 1977 and subsequent improvements in mining and reclamation technology. In this study, 30 pre‐SMCRA mine soil profiles (4–20 yr old) were examined and sampled in 1980 and compared with 20 mine soil profiles (8–13 yr old) described in the same area in 2002 after it had been completely remined by modern deep cut methods. Mine soils in both sampling years had high rock fragment content (42–81%), relatively well‐developed A horizons, and generally exhibited A‐C or A‐AC‐C horizonation. Although six Bw horizons were described in 1980, only two met all requirements for cambic horizons. The 1980 mine soils developed in overburden dominated by oxidized, preweathered material due to relatively shallow mining cuts. The 1980 mine soils had lower rock fragment content, finer textures, lower pH, and tended to be more heterogeneous in horizonation, morphology, and texture than soils observed in 2002, which had formed primarily in unweathered overburden from deeper cuts. Half the pedons sampled in both years had densic materials within 70 cm of the surface. Four poorly to very poorly drained soil profiles were described in each sampling year containing distinct hydric soil indicators in surface horizons. While older pre‐SMCRA mine soils do have many properties in common with newer mine soils, their properties are highly influenced by the fact that they generally have formed in more weathered overburden from higher in the geologic column. Overall, Appalachian mine soils are much more complex in subsoil morphology than commonly assumed, and differential compaction greatly complicates their internal drainage and limits their overall productivity potential.
The initial adhesion of micro-organisms on solid surfaces strongly affects their transport and fate in soil and aquatic environments. Experiments on Bacillus subtilis with various soil minerals (including kaolinite, montmorillonite, goethite, birnessite, quartz and mica) were conducted to determine the role of surface properties in adhesion and to test the validity of the extended Derjaguin, Landau, Verwey and Overbeek (DLVO) theory for bacterial adhesion. Adhesion of B. subtilis on all six minerals conformed to the Langmuir equation. Adhesion capacity and affinity showed a significant correlation with the specific external surface area (SESA) of the minerals and the calculated electrostatic energy barrier, respectively, but no significant correlation was observed between hydrophobicity and the adhesion parameters. These results demonstrate that adhesion capacity and affinity are primarily controlled by the SESA of the minerals and the surface electrical properties, respectively. The dependence of the adhesion capacity on SESA may be explained by the cell–mineral interaction model. Adhesion could be well predicted by the extended DLVO theory. The initial adhesion of bacteria with soil minerals can be evaluated by using independently measured surface properties of these components based on the extended DLVO theory combined with the Langmuir equation.
In the process of opencast mining, several changes occur in physical, chemical, and microbiological properties of soil as a result of mining and storage. Inability to preserve topsoil is one of the basic hindrances to restoration of mined land. The acute problem in preserving mine soil is discussed. Every year large areas are continually becoming unfertile in spite of efforts to grow vegetation on the degraded mined land One large opencast coal project of Eastern Coalfields Ltd. (ECL) is investigated to assess the deterioration of soil properties due to stripping and stockpiling. Different age classes of mine soil dumps are identified for the study. Mine soil characteristics of the dumps are compared with those of unmined soil and analyzed critically to evaluate deterioration of soil properties with respect to time of stockpiling. The changes in soil quality were found to be drastic in the first year and continually deteriorating every year, and ultimately the soil became unfertile.
Our goal was to determine the effects of surface mining on forest land productivity in the eastern coalfields of the USA before the passage of the Surface Mining Control and Reclamation Act of 1977 (SMCRA), and to determine the extent to which selected mine soil properties influenced forest productivity. The site productivity of 14 mined and eight nonmined sites in the eastern and midwestern coalfields were compared. Results show that site productivity of nonmined sites and 12 of the 14 mined sites was similar. Sites with low productivity were shallow, had high coarse fragment contents, and had lower fertility. Regression analysis identified five influential soil properties affecting site quality, which included soil profile base saturation (BS), total coarse fragments, total available water, C horizon total porosity, and soil profile electrical conductivity (EC). These five properties explained 52% of the variation in tree growth. Forests on most prelaw mined sites were just as productive as the forests on unmined adjacent sites and can be used as a benchmark to assess the impacts of current reclamation on mine soil quality and forest productivity.
Recently lignite represents mainly and alternatively exploited fuel of lower heating value. Similarly to other naturally occurring raw materials the economical efficiency of lignite mining is strongly influenced by plenty of different economical, technological as well as political factors, all of which are subjects of permanent changes. Fossil materials, including lignite, in general possess some specific properties (as a consequences of their com-position), which enable their miscellaneous and often economically very valuable non-energetic utilization. As a rule quantitatively as well as qualitatively rich humic substance and low toxic and heavy metal content, specific physical structure, not negligible sorption properties, suitable sensorial features allow lignite usage as a fertiliser, soil conditioner, bioregulator, regulator of humidity, metal sorbent or agrochemical agent. The presented paper intends to contribute to summarization of recent knowledge concerning lignite -a natural raw material of huge amount and of permanently rising importance.
Rhizoplane bacteria in white clover roots (0–5 cm), perennial ryegrass roots (0–5 and 5–10 cm) and surrounding rhizosphere soil from a long-term pasture were studied for the rate of colony development over a 10 day period. Serial dilutions were inoculated onto 0.1 strength tryptose soy agar (TSA) and 0.1 strength modified King's medium B (KB). Bacterial counts were made eight times over 10 days. Bacterial colony development (CD) and eco-physiological (EP) indices were calculated from these data. There were no significant differences in CD and EP indices of fluorescent pseudomonads growing on KB medium. After 5 days, the number of colonies developing on KB as a proportion of total colonies appearing over 10 days was lower for ryegrass roots than for soil (P < 0.05). The community structure derived from the colonies growing on 0.1 strength TSA showed that the rhizoplanes of white clover and ryegrass had a greater proportion of K-strategists and a smaller proportion of r-strategists than did the rhizosphere soil (P < 0.001). CD values for roots were significantly lower than those for soil (P < 0.001), while EP values were not significantly different. Randomly selected bacterial isolates were identified using the Microlog™ system. The fastest growing (24 h) strains belonged to the genera Pseudomonas and Bacillus spp. Other genera identified included Acinetobacter, Sphingomonas, Streptococcus, Cellulomonas, Comamonas, Xanthomonas and Erwinia. The slowest growing bacteria belonged to the genera Cellulomonas, Rhizobium, Xanthomonas and Erwinia.
In the Lusatian mining district, rehabilitated mine soils contain substantial amounts of lignite in addition to recent carbon derived from plant litter. The aim of this study was to examine the importance of the two organic matter types as substrates for soil microbial biomass in mine soils containing organic matter with a contrasting degree of humification. Samples were taken from the lignite-containing overburden material, from a mine soil under 14-year-old black pine (Pinus nigra) and from a mine soil under 37-year-old red oak (Quercus rubra). Overburden material was ameliorated with alkaline ash and incubated in an identical manner as the 14-year-old and 37-year-old mine soils for 16 months. Carbon mineralisation was monitored throughout. After 0, 3, 6, 12 and 16 months, samples were removed and analysed for chemical parameters and for microbial biomass. In addition, ¹⁴C activity measurements in bulk soil and microbial biomass were used to estimate their lignite content.
River systems are exposed to anthropogenic disturbances, including chemical pollution and eutrophication. This may affect the phylogenetic diversity as well as the abundance of various functional groups within sediment-associated microbial communities. To address such potential effects, mesocosms filled with Ebro delta sediment covered with river water were exposed to chlorinated organic compounds or to a high nutrient concentration as used for fertilization. Changes in the abundance of selected functional microbial groups, i.e. total aerobes, nitrate, sulfate and iron reducers, organohalide-respiring microorganisms as well as methanogens, were examined using culture-dependent most probable number and culture-independent PCR methods targeting phylogenetic as well as functional gene markers. It was concluded that the abundance of functional groups was neither affected by pollution with 1,2-dichloroethane and tetrachloroethene nor by elevated nutrient loads, although changes in the bacterial community composition were observed using 16S rRNA gene-targeted fingerprint techniques. This study reinforced the notion that complementary culture-dependent and molecular methods, focusing on different fractions of the microbial community (cultivable, active or total), should be used in combination for a comprehensive description of phylogenetic diversity and functional potential.
The effect of fermentable carbohydrates (sugar beet pulp and fructooligosaccharides) on the faecal bacterial communities of weaning piglets was analysed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in piglet faecal bacterial community structure were determined based on the Dice coefficients for pairwise comparison of the DGGE fingerprints and revealed significant changes in the faecal microbiota immediately after weaning. Piglets fed with fermentable carbohydrates showed a higher bacterial diversity and a more rapid stabilisation of the bacterial community compared with that of the animals fed with the control diet. Thirteen dominant DGGE bands were matched with sequences that showed 91-97% similarity to those derived from the Clostridium coccoides group and the Clostridium leptum subgroup. Amplicons related to Ruminococcus-like species were found in all DGGE fingerprints derived from pigs on the diet containing sugar beet pulp and fructooligosaccharides, but not in pigs on the control diet. These results indicate that these bacteria may play a role in the utilisation of dietary fibres.
Soils sustain an immense diversity of microbes, which, to a large extent, remains unexplored. A range of novel methods, most of which are based on rRNA and rDNA analyses, have uncovered part of the soil microbial diversity. The next step in the era of microbial ecology is to extract genomic, evolutionary and functional information from bacterial artificial chromosome libraries of the soil community genomes (the metagenome). Sophisticated analyses that apply molecular phylogenetics, DNA microarrays, functional genomics and in situ activity measurements will provide huge amounts of new data, potentially increasing our understanding of the structure and function of soil microbial ecosystems, and the interactions that occur within them. This review summarizes the recent progress in studies of soil microbial communities with focus on novel methods and approaches that provide new insight into the relationship between phylogenetic and functional diversity.
Since the delineation of 12 bacterial phyla by comparative phylogenetic analyses of 16S ribosomal RNA in 1987 knowledge of microbial diversity has expanded dramatically owing to the sequencing of ribosomal RNA genes cloned from environmental DNA. Currently, only 26 of the approximately 52 identifiable major lineages, or phyla, within the domain Bacteria have cultivated representatives. Evidence from field studies indicates that many of the uncultivated phyla are found in diverse habitats, and some are extraordinarily abundant. In some important environments, including seawater, freshwater, and soil, many biologically and geochemically important organisms are at best only remotely related to any strain that has been characterized by phenotype or by genome sequencing. Genome sequence information that would allow ribosomal RNA gene trees to be related to broader patterns in microbial genome evolution is scant, and therefore microbial diversity remains largely unexplored territory.