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PubMed2000

Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis.

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.

Bacteria↗

PubMed2000

Microbial diversity during maturation and natural processing of coffee cherries of Coffea arabica in Brazil.

The magnitude and diversity of the microbial population associated with dry (natural) processing of coffee (Coffea arabica) has been assessed during a 2-year period on 15 different farms in the Sul de Minas region of Brazil. Peptone water-washed samples were taken of maturing cherries on trees (cherries, raisins and dried cherries) and from ground fermentations. The microbial load varied from 3 x 10(4) to 2.2 x 10(9) cfu/cherry with a median value of 1.6 x 10(7) cfu/cherry. The microbial load increased after heavy rainfall on cherries that were drying on the ground. At all stages, bacteria were usually the most abundant group, followed by filamentous fungi and finally yeasts. Counts of bacteria, yeasts and fungi varied considerably between farms and at different stages of maturation and processing and no consistent pattern could be seen. Yeasts showed an increase during the fermentation process. Median counts were not significantly different for fungi, yeasts and bacteria between the 2 years although Gram-negative bacteria dominated in the wet year and Gram-positive bacteria dominated in the dry year. Of a total of 754 isolates, 626 were identified to at least genus level comprising 44 genera and 64 different species. The 164 isolates of Gram-negative bacteria included 17 genera and 26 species, the most common of which were members of the genera Aeromonas, Pseudomonas, Enterobacter and Serratia. Of 191 isolates of Gram-positive bacteria, 23 were spore-forming and included six Bacillus species, and 118 were non-spore-formers of which over half were Cellulomonas with lesser numbers of Arthrobacter, Microbacterium, Brochothrix, Dermabacter and Lactobacillus. Of the 107 yeast isolates, 90 were identified into 12 genera and 24 different species and almost all were fermentative. The most common genera, in decreasing frequency, were Pichia, Candida, Arxula and Saccharomycopsis. There were many rarely described yeasts including Pichia lynferdii and Arxula adeninivorans. Almost all 292 fungal isolates were identified to genus level and 52 were identified to species level. Cladosporium, Fusarium and Penicillium each comprised about one third of the isolates and were found on all farms. Only 3% of the isolates were Aspergillus. Beauvaria, Monilia, Rhizoctonia and Arthrobotrys species were also occasionally found. The microbial flora is much more varied and complex than found in wet fermentations. The genera and species identified include members known to have all types of pectinase and cellulase activities.

Bacteria↗

PubMed2005

Survey of oral microbial diversity using PCR-based denaturing gradient gel electrophoresis.

Polymicrobial biofilms in the human oral cavity exhibit marked diversity. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) surveys microbial diversity by displaying PCR-generated 16S rDNA fragments that migrate at different distances, reflecting the differences in the base-pair (i.e., % G+C) composition of the fragment. This study examined DGGE-generated diversity profiles of cultivable bacteria from individuals with different caries status. Initially, we developed a set of PCR-DGGE running conditions appropriate to oral bacteria. Next, we assessed migration standards from known oral bacterial reference strains. To test the methods, we profiled 20 bacterial saliva samples cultivated from young adults. The study produced a battery of species-specific 16S rDNA amplicons that could be used as a migration distance standard necessary for computer-assisted profile analysis. From the clinical samples, we found a significantly greater diversity of oral microbes in caries-free individuals compared with caries-active individuals (P = 0.01). These findings suggest thtat a portion of oral microbiota of caries-active individuals may be absent, suppressed, or replaced.

Actinomyces↗

PubMed2005

Root exudation, phosphorus acquisition, and microbial diversity in the rhizosphere of white lupine as affected by phosphorus supply and atmospheric carbon dioxide concentration.

White lupine (Lupinus albus L.) was used as a phosphorus (P)-efficient model plant to study the effects of elevated atmospheric CO(2) concentrations on (i) P acquisition, (ii) the related alterations in root development and rhizosphere chemistry, and (iii) the functional and structural diversity of rhizosphere microbial communities, on a P-deficient calcareous subsoil with and without soluble P fertilization. In both +P (80 mg P kg(-1)) and -P treatments (no added P), elevated CO(2) (800 micromol mol(-1)) increased shoot biomass production by 20 to 35% and accelerated the development of cluster roots, which exhibit important functions in chemical mobilization of sparingly soluble soil P sources. Accordingly, cluster root formation was stimulated in plants without P application by 140 and 60% for ambient and elevated CO(2) treatments, respectively. Intense accumulation of citrate and increased activities of acid and alkaline phosphatases, but also of chitinase, in the rhizosphere were mainly confined to later stages of cluster root development in -P treatments. Regardless of atmospheric CO(2) concentrations, there was no significant effect on accumulation of citrate or on selected enzyme activities of C, N, and P cycles in the rhizosphere of individual root clusters. Discriminant analysis of selected enzyme activities revealed that mainly phosphatase and chitinase contributed to the experimental variance (81.3%) of the data. Phosphatase and chitinase activities in the rhizosphere might be dominated by the secretion from cluster roots rather than by microbial activity. Alterations in rhizosphere bacterial communities analyzed by denaturing gradient gel electrophoresis (DGGE) were related with the intense changes in root secretory activity observed during cluster root development but not with elevated CO(2) concentrations.

Atmosphere↗

PubMed1999

Evaluating the microbial diversity of soil samples: methodological innovations.

This manuscript is a review of the innovative methodologies that enable more precise evaluations of soil microbial diversity. Highlighting the molecular approach, which does not require the isolation of microorganisms and allows the inclusion of non-culturable genotypes in the analyses, the described methodologies revolutionised the environmental microbiology and opened gateways for an accurate understanding of the ecology and diversity of microorganisms. The application of techniques based on soil total DNA extraction, PCR amplification of genes or gene fragments, and sequence analysis revealed that the microbial universe is far more complex than ever imagined. Examples of applications of the molecular approach to study the diversity of soil diazotrophic bacteria are given.

Genetic Variation↗

PubMed2004

Microbial diversity and heterogeneity in sandy subsurface soils.

Microbial community diversity and heterogeneity in saturated and unsaturated subsurface soils from Abbott's Pit in Virginia (1.57, 3.25, and 4.05 m below surface) and Dover Air Force Base in Delaware (6.00 and 7.50 m below surface) were analyzed using a culture-independent small-subunit (SSU) rRNA gene (rDNA)-based cloning approach. Four to six dominant operational taxonomic units (OTUs) were identified in 33 to 100 unique SSU rDNA clones (constituting about 40 to 50% of the total number of SSU rDNA clones in the clone library) from the saturated subsurface samples, whereas no dominant OTUs were observed in the unsaturated subsurface sample. Less than 10% of the clones among samples from different depths at the same location were identical, and the proportion of overlapping OTUs was lower for the samples that were vertically far apart than for adjacent samples. In addition, no OTUs were shared between the Abbott's Pit and Dover samples. The majority of the clones (80%) had sequences that were less than 5% different from those in the current databases. Phylogenetic analysis indicated that most of the bacterial clones were affiliated with members of the Proteobacteria family (90%), gram-positive bacteria (3%), and members of the Acidobacteria family (3%). Principal component analysis revealed that samples from different geographic locations were well separated and that samples from the same location were closely grouped together. In addition, the nonsaturated subsurface samples from Abbott's Pit clustered together and were well separated from the saturated subsurface soil sample. Finally, the overall diversity of the subsurface samples was much lower than that of the corresponding surface soil samples.

Bacteria↗

PubMed2025

A deep metagenomic atlas of Qinghai-Xizang Plateau lakes reveals their microbial diversity and salinity adaptation mechanisms.

The Qinghai-Xizang Plateau (QXP), harboring the planet's highest density of plateau lakes, offers an exceptional biogeographic environment for studying extremophilic microbial communities and their adaptation to salinity. Through deep metagenomic sequencing, we construct the Qinghai-Xizang Lake Sediment Genome (QXLSG) catalog, a high-resolution genomic catalog comprising 5,866 metagenome-assembled genomes (MAGs), 58.16 million non-redundant protein encoding genes, and 19,008 biosynthetic gene clusters. Notably, 80.78% of the 2,742 species-level MAGs represent undescribed taxa, significantly expanding the known microbial diversity. Salinity emerges as the primary environmental factor influencing microbial community. Functional annotation highlights that the "salt-out" strategy, particularly the uptake of glycine betaine, is the main mechanism for salinity tolerance. This strategy is prevalent in both hypersaline lake communities and the dominant microbial phyla. Overall, this study provides a crucial genetic resource for future bioprospecting and deepens our understanding of the fundamental mechanisms of microbial adaptation to extreme saline environments.

Lakes↗

PubMed2002

Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis.

A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the groundwater in- and outflow.

Bacteria↗

PubMed2002

Microbial diversity and function in soil: from genes to ecosystems.

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.

Bacteria↗

PubMedDate not supplied

Extremely alkaline (pH > 12) ground water hosts diverse microbial community.

Chemically unusual ground water can provide an environment for novel communities of bacteria to develop. Here, we describe a diverse microbial community that inhabits extremely alkaline (pH > 12) ground water from the Lake Calumet area of Chicago, Illinois, where historic dumping of steel slag has filled in a wetland. Using microbial 16S ribosomal ribonucleic acid gene sequencing and microcosm experiments, we confirmed the presence and growth of a variety of alkaliphilic beta-Proteobacteria, Bacillus, and Clostridium species at pH up to 13.2. Many of the bacterial sequences most closely matched those of other alkaliphiles found in more moderately alkaline water around the world. Oxidation of dihydrogen produced by reaction of water with steel slag is likely a primary energy source to the community. The widespread occurrence of iron-oxidizing bacteria suggests that reduced iron serves as an additional energy source. These results extend upward the known range of pH tolerance for a microbial community by as much as 2 pH units. The community may provide a source of novel microbes and enzymes that can be exploited under alkaline conditions.

Alkalies↗

PubMed2024

Microbial diversity and metabolic pathways linked to benzene degradation in petrochemical-polluted groundwater.

The rapid advance in shotgun metagenome sequencing has enabled us to identify uncultivated functional microorganisms in polluted environments. While aerobic petrochemical-degrading pathways have been extensively studied, the anaerobic mechanisms remain less explored. Here, we conducted a study at a petrochemical-polluted groundwater site in Henan Province, Central China. A total of twelve groundwater monitoring wells were installed to collect groundwater samples. Benzene appeared to be the predominant pollutant, detected in 10 out of 12 samples, with concentrations ranging from 1.4 μg/L to 5,280 μg/L. Due to the low aquifer permeability, pollutant migration occurred slowly, resulting in relatively low benzene concentrations downstream within the heavily polluted area. Deep metagenome sequencing revealed Proteobacteria as the dominant phylum, accounting for over 63 % of total abundances. Microbial α-diversity was low in heavily polluted samples, with community compositions substantially differing from those in lightly polluted samples. dmpK encoding the phenol/toluene 2-monooxygenase was detected across all samples, while the dioxygenase bedC1 was not detected, suggesting that aerobic benzene degradation might occur through monooxygenation. Sequence assembly and binning yielded 350 high-quality metagenome-assembled genomes (MAGs), with 30 MAGs harboring functional genes associated with aerobic or anaerobic benzene degradation. About 80 % of MAGs harboring functional genes associated with anaerobic benzene degradation remained taxonomically unclassified at the genus level, suggesting that our current database coverage of anaerobic benzene-degrading microorganisms is very limited. Furthermore, two genes integral to anaerobic benzene metabolism, i.e, benzoyl-CoA reductase (bamB) and glutaryl-CoA dehydrogenase (acd), were not annotated by metagenome functional analyses but were identified within the MAGs, signifying the importance of integrating both contig-based and MAG-based approaches. Together, our efforts of functional annotation and metagenome binning generate a robust blueprint of microbial functional potentials in petrochemical-polluted groundwater, which is crucial for designing proficient bioremediation strategies.

Groundwater↗

PubMed2002

Characterizing man-made and natural modifications of microbial diversity and activity in coastal ecosystems.

The impacts of growing coastal pollution and habitat alteration accompanying human encroachment are of great concern at the microbial level, where much of the ocean's primary production and biogeochemical cycling takes place. Coastal ecosystems are also under the influence of natural perturbations such as major storwns and flooding. Distinguishing the impacts of natural and human stressors is essential for understanding environmentally-induced change in microbial diversity and function. The objective of this paper is to discuss the applications and merits of recently developed molecular, ecophysiological and analytical indicators and their utility in examining anthropogenic and climatic impacts on the structure and function of coastal microbial communities. The nitrogen-limited Neuse River Estuary and Pamlico Sound, North Carolina are used as examples of ecosystems experiencing both anthropogenic (i.e., accelerating eutrophication) and climatic stress (increasing frequencies of tropical storms and hurricanes). Additional examples are derived from a coastal monitoring site (LEO) on the Atlantic coast of New Jersey and Galveston Bay, on the Gulf of Mexico. In order to assess structure, function, and trophic state of these and other coastal ecosystems, molecular (DNA and RNA-based) characterizations of the microbial taxa involved in carbon, nitrogen and other nutrient transformations can be combined with diagnostic pigment-based indicators of primary producer groups. Application of these methods can reveal process-level microbial community responses to environmental variability over a range of scales. Experimental approaches combined with strategic monitoring utilizing these methods will facilitate: (a) understanding organismal and community responses to environmental change, and (b) synthesizing these responses in the context of ecosystem models that integrate physical, chemical and biotic variability with environmental controls.

Animals↗

PubMed2006

Evaluation of microbial diversity during the manufacture of Fior di Latte di Agerola, a traditional raw milk pasta-filata cheese of the Naples area.

Microbial diversity of the raw milk for the production of Fior di Latte di Agerola and its changes during cheesemaking were studied. Viable counts showed that at the end of curd ripening, loads of lactic acid bacteria, both mesophilic and thermophilic rods and cocci, higher than those commonly evidenced in similar cheeses produced by using natural or commercial starters, were detected. Identification of 272 isolates, supported by molecular diagnostic aids, evidenced representative cultures of a high number of bacterial taxa of interest as participating in the process, although most of the isolates belonged to Lactococcus lactis and Lactobacillus helveticus species. RAPD-PCR and REA-PFGE biotyping were performed for the isolates of the above species and it was shown that most of the strains isolated from the raw milk occurred during the whole cheesemaking process, and an active role of these strains in the fermentation was supposed. The results offer further proof of the importance of the raw milk as source of technologically interesting strains of lactic acid bacteria capable of driving the fermentation of traditional cheeses.

Animals↗

PubMed2006

Microbial diversity in water and sediment of Lake Chaka, an athalassohaline lake in northwestern China.

We employed culture-dependent and -independent techniques to study microbial diversity in Lake Chaka, a unique hypersaline lake (32.5% salinity) in northwest China. It is situated at 3,214 m above sea level in a dry climate. The average water depth is 2 to 3 cm. Halophilic isolates were obtained from the lake water, and halotolerant isolates were obtained from the shallow sediment. The isolates exhibited resistance to UV and gamma radiation. Microbial abundance in the sediments ranged from 10(8) cells/g at the water-sediment interface to 10(7) cells/g at a sediment depth of 42 cm. A major change in the bacterial community composition was observed across the interface. In the lake water, clone sequences affiliated with the Bacteroidetes were the most abundant, whereas in the sediments, sequences related to low G+C gram-positive bacteria were predominant. A similar change was also present in the archaeal community. While all archaeal clone sequences in the lake water belonged to the Halobacteriales, the majority of the sequences in the sediments were related to those previously obtained from methanogenic soils and sediments. The observed changes in the microbial community structure across the water-sediment interface were correlated with a decrease in salinity from the lake water (32.5%) to the sediments (approximately 4%). Across the interface, the redox state also changed from oxic to anoxic and may also have contributed to the observed shift in the microbial community.

Bacteroides↗

PubMed2004

Evaluation of a plasmid-based 16S-23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater.

A plasmid-based 16S-23S rDNA intergenic spacer region (ISR) array was developed and optimized for analysis of microbial diversity within complex environmental samples. Plasmid probes with 16S-23S rDNA ISR inserts (800-1500 bp) from industrial wastewater treatment plant (WWTP) microorganisms were arrayed onto glass slides. Hybridization of fluorescently labeled target sequences from two clones from the ISR WWTP library to arrayed probes showed that there was a good linear relationship between hybridization intensity and ISR similarity (r(2)=0.82). Hybridization was highly specific (average background from arrayed probes with less than 80% similarity in ISR sequence was less than 7%). Strong fluorescence intensity corresponded to near-perfect match clones (99% or greater similarity in ISR sequence). A majority of probes (79%) showed no background hybridization. However, weak background (less than 50% for arrayed probes with 90% and 95% similarity in the 16S rRNA genes) was observed from closely related microorganisms. Background fluorescence from the negative control (plasmid vector with no insert) was similar to water and dimethyl sulfoxide (DMSO)-negative controls. Hybridization using fluorescently labeled ISR sequences from a mixed community sample produced strong fluorescent signals with no background from negative controls. A Cy5-labeled reference standard, part of the vector and present in every spotted probe, was used to normalize hybridization values. These results indicate that arrayed plasmid containing ISR probe insert sequences provides specificity and sensitivity for microbial community analysis in a high-throughput array format.

Base Sequence↗

PubMed1998

Microbial Diversity and Community Structure in Two Different Agricultural Soil Communities.

Abstract In this study, two different agricultural soils were investigated: one organic soil and one sandy soil, from Stend (south of Bergen), Norway. The sandy soil was a field frequently tilled and subjected to crop rotations. The organic soil was permanent grazing land, infrequently tilled. Our objective was to compare the diversity of the cultivable bacteria with the diversity of the total bacterial population in soil. About 200 bacteria, randomly isolated by standard procedures, were investigated. The diversity of the cultivable bacteria was described at phenotypic, phylogenetic, and genetic levels by applying phenotypical testing (Biolog) and molecular methods, such as amplified rDNA restriction analysis (ARDRA); hybridization to oligonucleotide probes; and REP-PCR. The total bacterial diversity was determined by reassociation analysis of DNA isolated from the bacterial fraction of environmental samples, combined with ARDRA and DGGE analysis. The relationship between the diversity of cultivated bacteria and the total bacteria was elucidated. Organic soil exhibited a higher diversity for all analyses performed than the sandy soil. Analysis of cultivable bacteria resulted in different resolution levels and revealed a high biodiversity within the population of cultured isolates. The difference between the two agricultural soils was significantly higher when the total bacterial population was analyzed than when the cultivable population was. Thus, analysis of microbial diversity must ultimately embrace the entire microbial community DNA, rather than DNA from cultivable bacteria.

Journal Article↗

PubMed1999

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities.

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.

Bacteria↗

PubMed2005

Performance, feeding behaviour and microbial diversity in weaned piglets fed liquid diets based on water or wet wheat-distillers grain.

Performance, nutrient digestibility, feeding behaviour, diarrhoea frequency and faecal microbial diversity was studied in weaned piglets allotted to dry feed (diet A), liquid feed with water (diet B) or liquid feed with wet wheat-distillers grain (diet C). There were no differences in digestibility of organic matter and energy, and the performance was similar between piglets fed liquid diets. Piglets fed diet A spent more time on eating/drinking compared with piglets fed liquid diets. The frequency of diarrhoea decreased (p < 0.05) from from diet A (11 out of 16) to diet B (6 out of 16) and further to diet C (1 out 16). The type of diet did not affect the diversity of coliform bacteria or lactobacilli, but the composition of the Lactobacillus flora was changed. The lower diarrhoea frequency when feeding diet C may be due to a prophylactic effect induced by the inclusion of wet wheat-distillers grain in the diet.

Animal Feed↗