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PubMed2005

Molecular diversity and ecology of microbial plankton.

The history of microbial evolution in the oceans is probably as old as the history of life itself. In contrast to terrestrial ecosystems, microorganisms are the main form of biomass in the oceans, and form some of the largest populations on the planet. Theory predicts that selection should act more efficiently in large populations. But whether microbial plankton populations harbour organisms that are models of adaptive sophistication remains to be seen. Genome sequence data are piling up, but most of the key microbial plankton clades have no cultivated representatives, and information about their ecological activities is sparse.

Adaptation, Physiological↗
PubMed2003

Diversity and dynamics of microbial communities in soils from agro-ecosystems.

Soil microbial communities are integrally involved in biogeochemical cycles and their activities are crucial to the productivity of terrestrial ecosystems. Despite the importance of soil microorganisms, little is known about the distribution of microorganisms in the soil or the manner in which microbial community structure responds to changes in land management. We investigated the structure of microbial communities in the soil over two years in a series of replicated plots, that included, cultivated fields, fields abandoned from cultivation and fields with no history of cultivation. Microbial community structure was examined by monitoring the relative abundance of ribosomal RNA (rRNA) from seven of the most common bacterial groups in soil (the Alpha and Beta Proteobacteria, Actinobacteria, Cytophagales, Planctomycetes, Verrucomicrobia and the Acidobacteria) and the Eukarya. These data reveal that soil microbial communities are dynamic, capable of significant change at temporal scales relative to seasonal events. However, despite temporal change in microbial community structure, the rRNA relative abundance of particular microbial groups is affected by the local environment such that recognizable patterns of community structure exist in relation to field management.

Agriculture↗
PubMed2006

Prokaryotic diversity and metabolically active microbial populations in sediments from an active mud volcano in the Gulf of Mexico.

In this study, ribosomes and genomic DNA were extracted from three sediment depths (0-2, 6-8 and 10-12 cm) to determine the vertical changes in the microbial community composition and identify metabolically active microbial populations in sediments obtained from an active seafloor mud volcano site in the northern Gulf of Mexico. Domain-specific Bacteria and Archaea 16S polymerase chain reaction primers were used to amplify 16S rDNA gene sequences from extracted DNA. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from each sediment depth that had been subjected to reverse transcription polymerase chain reaction amplification. Twelve different 16S clone libraries, representing the three sediment depths, were constructed and a total of 154 rDNA (DNA-derived) and 142 crDNA (RNA-derived) Bacteria clones and 134 rDNA and 146 crDNA Archaea clones obtained. Analyses of the 576 clones revealed distinct differences in the composition and patterns of metabolically active microbial phylotypes relative to sediment depth. For example, epsilon-Proteobacteria rDNA clones dominated the 0-2 cm clone library whereas gamma-Proteobacteria dominated the 0-2 cm crDNA library suggesting gamma to be among the most active in situ populations detected at 0-2 cm. Some microbial lineages, although detected at a frequency as high as 9% or greater in the total DNA library (i.e. Actinobacteria, alpha-Proteobacteria), were markedly absent from the RNA-derived libraries suggesting a lack of in situ activity at any depth in the mud volcano sediments. This study is one of the first to report the composition of the microbial assemblages and physiologically active members of archaeal and bacterial populations extant in a Gulf of Mexico submarine mud volcano.

Archaea↗
PubMed2006

[16S rDNA-RFLP analysis of structure and diversity of an aerobic microbial community degrading hexachlorobenzene].

Hexachlorobenzene is a chlorinated aromatic hydrocarbon that was widely used as a seed dressing for prevention of fungal growth on crops, and is also a component of fireworks, ammunition, and synthetic rubbers. Because of the bioaccumulation and persistence of hexachlorobenzene as well as its potential toxicity, hexachlorobenzene must be removed from environment. The potential for aerobic dechlorination of hexachlorobenzene by a hexachlorobenzene-adapted mixed culture was investigated. An aerobic microbial community which was able to grow at the presence of hexachlorobenzene was enriched from sediment from contaminated site after incubating about 2 months. During the growth of the mixed microorganisms on hexachlorobenzene, the accumulating consumption of oxygen, the microbial population curve and the release of Cl- were investigated. The data suggest the rapid degradation of hexachlorobenzene to support microbial growth and the aerobic decholrination of hexachlorobenzene was observed. The result showed that the mixed microorganisms were able to utilize hexachlorobenzene as sole carbon and energy source. It was shown that up to 55% of HCB could be degraded during 18 days incubation at 30 degrees C in mineral salts medium (pH 7.0) with 4.5mg/L HCB. The calculated rate of hexachlorobenzene biodegradation was 137.5 microg/ (L x d). The 16S rDNA genes were amplified from community DNA by using primers specific to bacteria and were subsequently cloned. The cloned 16S rDNA fragments were reamplified, and restriction analysis was performed following separate digestion with enzymes Hae III and Rsa I. Application of restriction fragment length polymorphism screening approach revealed 9 clusters, and 3 major clusters were sequenced. Nearly complete 16S rDNA sequence analysis show that the microbial community was dominated by Alcaligenes and Azospirillum groups. This is the first report describing aerobic dechlorination of hexachlorobenzene via dehalorespiration by a microbial community which was enriched from contaminated site. The microbial community can be used to degrade highly recalcitrant chlorinated pollutants.

Bacteria, Aerobic↗
PubMed2006

The influence of tropical plant diversity and composition on soil microbial communities.

There is growing interest in understanding the linkages between above- and belowground communities, and very little is known about these linkages in tropical systems. Using an experimental site at La Selva Biological Station, Costa Rica, we examined whether plant diversity, plant community composition, and season influenced microbial communities. We also determined whether soil characteristics were related to differences in microbial communities. Phospholipid fatty acid (PLFA) composition revealed that microbial community composition differed across a plant diversity gradient (plots contained 1, 3, 5, or over 25 species). Plant species identity also was a factor influencing microbial community composition; PLFA composition significantly varied among monocultures, and among three-species combinations that differed in plant species composition. Differences among treatments within each of these comparisons were apparent in all four sampling dates of the study. There was no consistent shift in microbial community composition between wet and dry seasons, although we did see significant changes over time. Of all measured soil characteristics, soil C/N was most often associated with changes in microbial community composition across treatment groups. Our findings provide evidence for human alteration of soil microbial communities via the alteration of plant community composition and diversity and that such changes are mediated in part by changes in soil carbon quality.

Bacteria↗
PubMedDate not supplied

Protistan diversity estimates based on 18S rDNA from seawater incubations in the Western North Atlantic.

Cloning/sequencing and fragment analysis of ribosomal RNA genes (rDNA) are becoming increasingly common methods for the identification of microbial taxa. Sequences of these genes provide many additional taxonomic characters for species that otherwise have few distinctive morphological features, or that require involved microscopy or laboratory culture and testing. These same approaches are now being applied with great success in ecological studies of natural communities of microorganisms. Extensive information on the composition of natural microbial assemblages is being amassed at a rapid pace through genetic analyses of environmental samples and comparison of the resulting genetic information with well-established (and rapidly growing) public databases. We examined microbial eukaryote diversity in a natural seawater sample from the coastal western North Atlantic Ocean using two molecular biological approaches: the cloning and sequencing of rRNA genes and by fragment analysis of these genes using the terminal restriction fragment length polymorphism (T-RFLP) method. A simple experiment was carried out to examine changes in the overall eukaryote (largely protistan) diversity and species composition (phylotype diversity) of a natural microbial assemblage when a seawater sample is placed in a container and incubated at ambient light and temperature for 72 h. Containment of the natural seawater sample resulted in relatively minor changes in the overall eukaryote diversity (species richness) obtained by either molecular method at three time points (time-zero, time-24 h, time-72 h). However, substantial changes in the dominance of particular eukaryote phylotypes took place between the three sampling times. Only 18% of the total number of phylotypes observed in the study were observed at all three time points, while 65% (108 of 165) phylotypes were observed only at a single time point (54 unique phylotypes initially, 37 more unique phylotypes at 24 h, and 17 more at 72 h). The results of this study indicate that a high diversity of protistan taxa existed in the original seawater sample at very low abundance, and thus were not observed in the initial characterization of community structure. Containment resulted in significant shifts in the dominance of these taxa, enabling the presence of previously unobserved phylotypes to be documented after 24 or 72 h of incubation.

Animals↗
PubMed2002

The diversity and function of soil microbial communities exposed to different disturbances.

To improve understanding of the relationship between the diversity and function of the soil ecosystem, we investigated the effect of two different disturbances on soil bacterial communities -- long-term exposure to the heavy metal mercury and transient exposure to the antibiotic tylosin. In the mercury-contaminated soil the diversity (Shannon index) was reduced as assessed from denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil community DNA and from colony morphology typing of the culturable bacterial population. However, analysis of the substrate utilization profiles did not reveal any differences in diversity. In the tylosin-treated soil, DGGE revealed a small difference in the diversity of 16S rDNA compared to the control soil, whereas analysis of the colony morphology typing or substrate utilization results did not reveal any differences in diversity. Soil function was also affected by mercury contamination. The lag time before soil respiration increased following addition of glucose or alfalfa substrate was longer in the mercury-contaminated soil than in the control soil. Moreover, it was markedly prolonged in mercury-contaminated soil subjected to heat treatment prior to substrate addition, thus indicating reduced resistance to a new disturbance in the mercury-contaminated soil as compared to the control soil. Tylosin treatment did not have any significant effect on any of the respiration parameters measured, either with or without prior heat treatment of the soil.

Bacteria↗
PubMed2025

Functional metaproteomics for enzyme discovery.

Discovery of microbial biocatalysts traditionally relied on activity screening of isolated bacterial strains. However, since most microorganisms cannot be cultivated in the lab, such an approach leaves the majority of the microbial enzyme diversity untapped. Metagenomic approaches, in which the DNA from a microbial community is directly isolated and then used either for the creation of an expression library or for sequencing and metagenome annotation have alleviated this shortcoming to an extent, but have their own limitations: the generation of large expression libraries is time-consuming and their screening is costly, while metagenome annotation can infer biocatalytic function only from prior knowledge. We have thus developed a functional metaproteomic approach, which combines the immediacy of traditional activity screening with the comprehensiveness of a meta-omics approach. Briefly, the whole metaproteome of an environmental sample is separated on a 2-D gel, biocatalytically active proteins are visualized in-gel through zymography, and those candidate biocatalysts are then identified through mass spectrometry, searching against a metagenome-derived database obtained from the very same environmental sample. Here we explain the process in detail, with a focus on esterases, and give guidelines on how to develop a functional metaproteomic workflow for enzyme discovery.

Proteomics↗
PubMed2006

Structural and functional responses of a sewage microbial community to dilution-induced reductions in diversity.

The relationship between functional redundancy and microbial community structure-diversity was examined using laboratory incubations to ensure constant environmental conditions. Serial dilutions of a sewage microbial community were prepared, used to inoculate sterile sewage, and maintained in batch culture. Probability suggests that dilution of the initial community should remove rare organism types, creating mixtures of cells differing in diversity. Regrowth of the diluted mixtures generated communities similar in abundance but differing in community structure and relative diversity (as determined using two DNA fingerprinting techniques and dilution-to-extinction analysis of community-level physiological profiles). The in situ function of each regrown community was examined by monitoring the short-term uptake of five different (14)C-labeled compounds (glucose, acetate, citrate, palmitic acid, and an amino acid mixture). No significant differences were detected between treatments in either the rate of uptake of a substrate or the efficiency with which each community assimilated each compound. The fact that the activity of the original community was the same as that of a community regrown from an inoculum containing fewer that 100 cells (10(-6) dilution) indicates that functional redundancy was quite high in this system. For each organism type eliminated during the dilution process, at least one of the remaining types was able to provide the same function at the same level as the lost one. Further research is necessary to determine what impact this functional redundancy may have on overall ecosystem function and stability.

Biodiversity↗
PubMed2003

New approaches to analyzing microbial biodiversity data.

Modern molecular techniques have revealed an extraordinary diversity of microorganisms, most of which are as yet uncharacterized. This poses a major challenge to microbial ecologists: how can one compare the microbial diversity of different environments when the vast majority of microbial taxa are usually unknown? Three statistical approaches developed by ecologists and evolutionary biologists--parametric estimation, nonparametric estimation and community phylogenetics--are proving to be promising tools to meet this challenge. The combination of these tools with molecular biology techniques allow the rigorous estimation and comparison of microbial diversity in different environments.

Bacteria↗
PubMed2006

Diversity and biotechnological potential of the sponge-associated microbial consortia.

Sponges are well known to harbor diverse microbes and represent a significant source of bioactive natural compounds derived from the marine environment. Recent studies of the microbial communities of marine sponges have uncovered previously undescribed species and an array of new chemical compounds. In contrast to natural compounds, studies on enzymes with biotechnological potential from microbes associated with sponges are rare although enzymes with novel activities that have potential medical and biotechnological applications have been identified from sponges and microbes associated with sponges. Both bacteria and fungi have been isolated from a wide range of marine sponge, but the diversity and symbiotic relationship of bacteria has been studied to a greater extent than that of fungi isolated from sponges. Molecular methods (e.g., rDNA, DGGE, and FISH) have revealed a great diversity of the unculturable bacteria and archaea. Metagenomic approaches have identified interesting metabolic pathways responsible for the production of natural compounds and may provide a new avenue to explore the microbial diversity and biotechnological potential of marine sponges. In addition, other eukaryotic organisms such as diatoms and unicellular algae from marine sponges are also being described using these molecular techniques. Many natural compounds derived from sponges are suspected to be of bacterial origin, but only a few studies have provided convincing evidence for symbiotic producers in sponges. Microbes in sponges exist in different associations with sponges including the true symbiosis. Fungi derived from marine sponges represent the single most prolific source of diverse bioactive marine fungal compounds found to date. There is a developing interest in determining the true diversity of fungi present in marine sponges and the nature of the association. Molecular methods will allow scientists to more accurately identify fungal species and determine actual diversity of sponge-associated fungi. This is especially important as greater cooperation between bacteriologists, mycologists, natural product chemists, and bioengineers is needed to provide a well-coordinated effort in studying the diversity, ecology, physiology, and association between bacteria, fungi, and other organisms present in marine sponges.

Animals↗
PubMed2026

Probiotic supplementation increases fecal TLR4 agonists without improving disease activity in juvenile idiopathic arthritis: a randomized placebo-controlled trial.

Gut dysbiosis has been implicated in the pathogenesis of juvenile idiopathic arthritis (JIA), suggesting that microbiota-targeted interventions may influence immune signalling during early immune development. We conducted the PERMAJI multicentre randomized, double-blind, placebo-controlled trial to evaluate the effects of probiotic supplementation (VSL#3) on host-microbiota immune interactions and disease activity in children with oligoarticular or RF-negative polyarticular JIA. Participants were randomly assigned (1:1) to receive VSL#3 or placebo for 3 months in addition to standard therapy. Stool and serum samples collected at baseline and month 3 were used to assess gut microbiota composition, fecal innate immune agonists, intestinal permeability, and systemic cytokines. The primary clinical endpoint was the proportion achieving an ACR Pedi 30 response at 3 months. Forty-four children were enrolled between September 2017 and July 2022. Clinical responses did not differ between groups (ACR Pedi 30: 47% with VSL#3 vs 63% with placebo; p = 0.33), and conservative worst-case assumptions for missing data suggested lower response rates with VSL#3 (36% vs 68%; p = 0.03). Probiotic supplementation significantly increased fecal Toll-like receptor 4 (TLR4) agonist activity, whereas gut microbiota diversity, intestinal permeability, and systemic cytokine levels remained unchanged. These findings indicate that probiotic supplementation can modify microbial innate immune signalling without detectable changes in microbial community diversity and may increase exposure to pro-inflammatory microbial stimuli in early-life autoimmune disease. The results highlight the complexity of host-microbiota immune interactions and underscore the need for careful evaluation of microbiome-targeted therapies in paediatric autoimmune disorders.

Humans↗
PubMed2002

Composition and physiological profiling of sprout-associated microbial communities.

The native microfloras of various types of sprouts (alfalfa, clover, sunflower, mung bean, and broccoli sprouts) were examined to assess the relative effects of sprout type and inoculum factors (i.e., sprout-growing facility, seed lot, and inoculation with sprout-derived inocula) on the microbial community structure of sprouts. Sprouts were sonicated for 7 min or hand shaken with glass beads for 2 min to recover native microfloras from the surface, and the resulting suspensions were diluted and plated. The culturable fraction was characterized by the density (log CFU/g), richness (e.g., number of types of bacteria), and diversity (e.g., microbial richness and evenness) of colonies on tryptic soy agar plates incubated for 48 h at 30 degrees C. The relative similarity between sprout-associated microbial communities was assessed with the use of community-level physiological profiles (CLPPs) based on patterns of utilization of 95 separate carbon sources. Aerobic plate counts of 7.96 +/- 0.91 log CFU/g of sprout tissue (fresh weight) were observed, with no statistically significant differences in microbial cell density, richness, or diversity due to sprout type, sprout-growing facility, or seed lot. CLPP analyses revealed that the microbial communities associated with alfalfa and clover sprouts are more similar than those associated with the other sprout types tested. Variability among sprout types was more extensive than any differences between microbial communities associated with alfalfa and clover sprouts from different sprout-growing facilities and seed lots. These results indicate that the subsequent testing of biocontrol agents should focus on similar organisms for alfalfa and clover, but alternative types may be most suitable for the other sprout types tested. The inoculation of alfalfa sprouts with communities derived from various sprout types had a significant, source-independent effect on microbial community structure, indicating that the process of inoculation alters the dynamics of community development regardless of the types of organisms involved.

Brassica↗
PubMed2004

Effect of gas velocity and influent concentration on biofiltration of gasoline off-gas from soil vapor extraction.

This study was conducted to evaluate the effects of gas inlet concentration and velocity on the biofiltration of gasoline vapor. Gasoline vapor was treated using a compost biofilter operated in an upflow mode for about 3 months. The inlet concentration of gasoline total petroleum hydrocarbon (TPH) ranged from about 300 to 7000 mgm(-3) and gas was injected at velocities of 6 and 15 mh(-1) (empty bed residence time (EBRT)=10 and 4 min, respectively). The maximum elimination capacities of TPH at 6 and 15 mh(-1) found in this research were over 24 and 19 gm(-3) of filling material h(-1), respectively. TPH removal data was fit using a first-order kinetic relationship. In the low concentration range of 300-3000 mg m(-3), the first-order kinetic constants varied between about 0.10 and 0.29 min(-1) regardless of gas velocities. At TPH concentrations greater than 3000 mgm(-3), the first-order kinetic constants were about 0.09 and 0.07 min(-1) at gas velocities of 6 mh(-1) and 15 mh(-1), respectively. To evaluate microbial dynamics, dehydrogenase activity, CO2 generation and microbial species diversity were analyzed. Dehydrogenase activity could be used as an indicator of microbial activity. TPH removal corresponded well with CO2 evolution. The average CO2 recovery efficiency for the entire biofilter ranged between 60% and 70%. When the gas velocity was 6 mh(-1), most of the microbial activity and TPH removal occurred in the first quarter of the biofilter. However, when the gas velocity was 15 mh(-1), the entire column contributed to removal. Spatial and temporal variations in the biofilter microbial population were also observed. Nearly 60% of the colonies isolated from the compost media prior to biofiltration were Bacillus. After 90 days of biofiltration, the predominant species in the lower portion (0-50 cm) of the filter were Rhodococcus, while Pseudomonas and Acinetobacter dominated the upper portion (75-100 cm).

Bacteria↗
PubMed2001

Microbiological analysis of multi-level borehole samples from a contaminated groundwater system.

A range of bacteriological, geochemical process-related and molecular techniques have been used to assess the microbial biodegradative potential in groundwater contaminated with phenol and other tar acids. The contaminant plume has travelled 500 m from the pollutant source over several decades. Samples were obtained from the plume using a multi-level sampler (MLS) positioned in two boreholes (boreholes 59 and 60) which vertically transected two areas of the plume. Activity of the microbial community, as represented by phenol degradation potential and ability to utilise a range of substrates, was found to be influenced by the plume. Phenol degradation potential appeared to be influenced more by the concentration of the contaminants than the total bacterial cell numbers. However, in the areas of highest phenol concentration, the depression of cell numbers clearly had an effect. The types of bacteria present were assessed by culture and DNA amplification by polymerase chain reaction (PCR). Bacterial groups or processes associated with major geochemical processes, such as methanogenesis, sulphate reduction and denitrification, that have the potential to drive contaminant degradation, were detected at various borehole levels. A comparative molecular analysis of the microbial community between samples obtained from the MLS revealed the microbial community was diverse. The examination of microbial activity complemented those results obtained through chemical analysis, and when combined with hydrological data, showed that MLS samples provided a realistic profile of plume effects and could be related to the potential for natural attenuation of the site.

Bacteria, Aerobic↗
PubMed2003

Cyanobacterial diversity in natural and artificial microbial mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a morphological and molecular approach.

Currently, there is no consensus concerning the geographic distribution and extent of endemism in Antarctic cyanobacteria. In this paper we describe the phenotypic and genotypic diversity of cyanobacteria in a field microbial mat sample from Lake Fryxell and in an artificial cold-adapted sample cultured in a benthic gradient chamber (BGC) by using an inoculum from the same mat. Light microscopy and molecular tools, including 16S rRNA gene clone libraries, denaturing gradient gel electrophoresis, and sequencing, were used. For the first time in the study of cyanobacterial diversity of environmental samples, internal transcribed spacer (ITS) sequences were retrieved and analyzed to complement the information obtained from the 16S rRNA gene. Microscopy allowed eight morphotypes to be identified, only one of which is likely to be an Antarctic endemic morphotype. Molecular analysis, however, revealed an entirely different pattern. A much higher number of phylotypes (15 phylotypes) was found, but no sequences from Nodularia and Hydrocoryne, as observed by microscopy, were retrieved. The 16S rRNA gene sequences determined in this study were distributed in 11 phylogenetic lineages, 3 of which were exclusively Antarctic and 2 of which were novel. Collectively, these Antarctic sequences together with all the other polar sequences were distributed in 22 lineages, 9 of which were exclusively Antarctic, including the 2 novel lineages observed in this study. The cultured BGC mat had lower diversity than the field mat. However, the two samples shared three morphotypes and three phylotypes. Moreover, the BGC mat allowed enrichment of one additional phylotype. ITS sequence analysis revealed a complex signal that was difficult to interpret. Finally, this study provided evidence of molecular diversity of cyanobacteria in Antarctica that is much greater than the diversity currently known based on traditional microscopic analysis. Furthermore, Antarctic endemic species were more abundant than was estimated on the basis of morphological features. Decisive arguments concerning the global geographic distribution of cyanobacteria should therefore incorporate data obtained with the molecular tools described here.

Acclimatization↗
PubMed2006

[An efficient method for DNA extraction from compost].

An efficient method for obtaining DNA from compost which contained high levels of organic matter was developed. The protocol consisted of washing with phosphate-EDTA before extraction, cell lysis with hot-SDS and enzymes (lysozyme, lywalzyme, proteinase K), removing humic acid and other inhibitors with PVPP and precipitation with PEG-8000. The compost total DNA was extracted from four different composts, the DNA yield was 63.54 +/- 12.08 to approximately 106.50 +/- 28.36 microg/g of dry compost. Molecular size of DNA obtained using this protocol was about 23kb and contained low protein and humic acid contamination with the A260/A280 ratios exceeding 1.6 and A260/A230 ratios reaching 1.8. Usually, additional purification steps such as agarose gel electrophoresis, gel permeation chromatography, or affinity chromatography were needed to get PCR-amplifiable DNA, but the DNA obtained using this protocol could directly be used to PCR-amplification and restriction enzyme digestion. Just like purity of DNA template, lower DNA yield also appears to introduce a bias towards lower community diversity. In this study compared the purified DNA the direct DNA reveals higher microbial community diversity assessed by denaturing gradient gel electrophoresis(DGGE) of amplified V3 region of 16S rDNA.

DNA, Bacterial↗