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PubMed2003

Impact of the plant rhizosphere and augmentation on remediation of polychlorinated biphenyl contaminated soil.

This study investigated the interactive effects of bioaugmentation, biostimulation, and the rhizosphere during remediation of Aroclor 1242-contaminated soil. Treatments were repeatedly augmented with polychlorinated bipheny (PCB)-degrading bacteria, inducers (carvone and salicylic acid), surfactant (sorbitan trioleate), minimal salts medium in a 20-cm high soil column, or a combination of these elements. Soils containing a single Brassica nigra plant achieved 61% PCB removal in the 0 to 2 and 2 to 6 cm depths after 9 weeks of bioaugmentation, whereas only 43 and 14% PCB removal, respectively, was achieved in unplanted controls. Gas diffusion coefficients of 13.0 and 5.0 x 10(-7) m2 s(-1) were calculated from a methane diffusion assay for planted and unplanted soils respectively, indicating the positive effect of plant roots on gas diffusion into the soil. A second, modified column study removed 87, 73, 63, and 45% of PCB after 12 weeks in the 0 to 5, 5 to 11, 11 to 26, and 26 to 35 cm depths, respectively, in planted-bioaugmented soils, whereas 65, 54, 53, and 47% of PCB was removed from the planted-minimal salts treatment, respectively. Shifts in the soil microbial community structure were demonstrated by denaturing gradient gel electrophoresis of bacterial 16S ribosomal DNA. Results support that Brassica nigra directly contributed to accelerated PCB removal by increased oxygen diffusion, amendment infiltration, and microbial enrichment.

Biodegradation, Environmental↗

PubMed2004

Design of 16S rRNA-targeted oligonucleotide probes and microbial community analysis in the denitrification process of a saline industrial wastewater treatment system.

Three 16S rRNA-targeted oligonucleotide probes, namely, PSMg437 targeting several members of the genus Pseudomonas, Hlm474 targeting several members of the genus Halomonas, and Clw844 targeting several members of the genus Colwellia, were designed. The microbial community structure and nitrogen removal ability of nitrate-containing saline wastewater treatment systems with anaerobic packed bed and fluidized bed were monitored. Direct cell counting using fluorescence in situ hybridization (FISH) images revealed that various phylogenetic groups were evenly distributed in the anaerobic packed bed whereas members of the genus Halomonas were dominant particularly in the anaerobic fluidized bed. These results suggest that the microbial communities produced by different flow conditions correlated with denitrification ability in saline industrial wastewater treatment systems.

Bioreactors↗

PubMed2005

Semi-automated genetic analyses of soil microbial communities: comparison of T-RFLP and RISA based on descriptive and discriminative statistical approaches.

Cultivation independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. This approach is based on direct extraction of total soil DNA followed by PCR amplification of selected marker genes and subsequent genetic fingerprint analyses. Semi-automated genetic fingerprinting techniques such as terminal restriction fragment length polymorphism (T-RFLP) and ribosomal intergenic spacer analysis (RISA) yield high-resolution patterns of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring, when rapid high throughput screening for differences or changes is more important than phylogenetic identification of organisms affected. Our objective was to perform profound statistical analysis to evaluate the cultivation independent approach and the consistency of results from T-RFLP and RISA. As a model system, we used two different heavy metal treated soils from an open top chamber experiment. Bacterial T-RFLP and RISA profiles of 16S rDNA were converted into numeric data matrices in order to allow for detailed statistical analyses with cluster analysis, Mantel test statistics, Monte Carlo permutation tests and ANOVA. Analyses revealed that soil DNA-contents were significantly correlated with soil microbial biomass in our system. T-RFLP and RISA yielded highly consistent and correlating results and both allowed to distinguish the four treatments with equal significance. While RISA represents a fast and general fingerprinting method of moderate cost and labor intensity, T-RFLP is technically more demanding but offers the advantage of phylogenetic identification of detected soil microorganisms. Therefore, selection of either of these methods should be based on the specific research question under investigation.

Bacteria↗

PubMed2006

Microbial community analysis of soils contaminated with lead, chromium and petroleum hydrocarbons.

The impact on the microbial community of long-term environmental exposure to metal and organic contamination was investigated. Twenty-four soil samples were collected along a transect dug in soils contaminated with road paint and paint solvents, mainly toluene. Chemical analysis along the transect revealed a range from high to low concentrations of metals (lead and chromium) and organic solvent compounds. Principal components analysis of microbial community structure based on denaturing gradient gel electrophoresis of the V3 region of the 16S rRNA gene and fatty acid methyl esters derived from phospholipids (phospholipid fatty acid analysis) showing samples with similar fingerprints also had similar contaminant concentrations. There was also a weak positive correlation between microbial biomass and the organic carbon concentration. Results indicated that microbial populations are present despite some extreme contaminant levels in this mixed-waste contaminated site. Nucleotide sequence determination of the 16S rRNA gene indicated the presence of phylogenetically diverse bacteria belonging to the alpha-, beta-, gamma-, and delta-Proteobacteria, the high and low G + C Gram-positive bacteria, green nonsulfur, OP8, and others that did not group within a described division. This indicates that soils contaminated with both heavy metals and hydrocarbons for several decades have undergone changes in community composition, but still contain a phylogenetically diverse group of bacteria (including novel phylotypes) that warrant further investigation.

Bacteria↗

PubMed2006

Bacterial community changes in diesel-oil-contaminated soil microcosms biostimulated with Luria-Bertani medium or bioaugmented with a petroleum-degrading bacterium, Pseudomonas aeruginosa strain WatG.

The microbial community structure of diesel-oil-contaminated soil microcosms biostimulated with Luria-Bertani medium (LB-BS) or bioaugmented with a petroleum-degrading bacterium, Pseudomonas aeruginosa strain WatG (WatG-BA), was investigated by denaturing gradient gel electrophoresis (DGGE) and by monitoring diesel oil degradation. The degradation in WatG-BA (64.0% +/- 4.2%) was higher than that in LB-BS (49.5% +/- 12.0%) during the first two weeks. The microbial community in WatG-BA, which was markedly dominated by strain WatG, was much simpler than that in LB-BS, where hydrocarbon degraders occurred after a lag of 3-7 days after the addition of diesel oil. The clustering profiles of the DGGE banding patterns of the two soil microcosms were only 12% similar. This difference is probably due to antibacterial substances, such as rhamnolipids, secreted by strain WatG.

Actinobacteria↗

PubMed2005

Structural and functional diversity of microbial communities from a lake sediment contaminated with trenbolone, an endocrine-disrupting chemical.

Effects of trenbolone (TBOH), a hormone used in cattle production, on the structure and function of microbial communities in a fresh water sediment from a lake in Southern Germany were studied in a microcosm experiment. The microbial community structure and the total gene pool of the sediment, assessed by 16S rRNA/rDNA and RAPD fingerprint analysis, respectively, were not significantly affected by TBOH. In contrast, the N-acetyl-glucosaminidase activity was almost 50% lower in TBOH treated samples (P<0.05). Also, the substrate utilization potential, measured using the BIOLOG system, was reduced after TBOH treatment. Interestingly, this potential did not recover at the end of the experiment, i.e. 19 days after the addition of the chemical. Repeated application of TBOH did not lead to an additional reduction in the substrate utilization potential. Overall results indicate that microbial community function was more sensitive to TBOH treatment than the community structure and the total gene pool.

Biodegradation, Environmental↗

PubMed2002

Molecular and conventional analyses of microbial diversity in mesophilic and thermophilic upflow anaerobic sludge blanket granular sludges.

The microbial community structure of mesophilic (35 degrees C) and thermophilic (55 degrees C) methanogenic granular sludges was surveyed by using both cultivation-independent molecular approach and conventional cultivation technique in order to address the fundamental questions on the microbial populations, i.e. who are present, where they are located, and what they are doing there. To elucidate the microbial constituents within both sludges, we first constructed 16S ribosomal DNA clone libraries, and partial sequencing of the clones was conducted for phylogenetic analysis. In this experiment, we found a number of unidentifiable clones within the domain Bacteria as well as clones that were closely related with 16S rDNAs of cultured microbes. The unidentifiable clones accounted for approximately 60-70% of the total clones in both mesophilic and thermophilic libraries. 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was subsequently employed to examine where the uncultured populations were located within sludge granules. Spatial organization of uncultured microbes was visualized in thin-sections of both types of granules using fluorescent oligonucleotide probes, which were designed based on the clone sequences of certain novel clusters. This resulted in the detection of two types of uncultured cells in specific locations inside the granules. Finally, the goal-directed conventional cultivation technique was employed to recover such uncultured anaerobes and uncover their physiology and functions. In this approach, a total of five new species of thermophilic microorganisms were isolated, including several types of syntrophs and a novel sugar-fermenting bacterium. In the previous molecular approaches, all of these isolates were suggested to be significant populations within thermophilic granular sludge, hence obtaining these isolates in pure culture decreased the fraction of unknown clones in the previous thermophilic clone library from 70% to 40%. In conclusion, these approaches successfully revealed biodiversity and spatial organization of microbes of interest in sludge granules, and enlarged the fundamental knowledge of microbial constituents functioning as significant populations in the UASB processes.

Bacteria, Anaerobic↗

PubMed2006

Introducing SONS, a tool for operational taxonomic unit-based comparisons of microbial community memberships and structures.

The recent advent of tools enabling statistical inferences to be drawn from comparisons of microbial communities has enabled the focus of microbial ecology to move from characterizing biodiversity to describing the distribution of that biodiversity. Although statistical tools have been developed to compare community structures across a phylogenetic tree, we lack tools to compare the memberships and structures of two communities at a particular operational taxonomic unit (OTU) definition. Furthermore, current tests of community structure do not indicate the similarity of the communities but only report the probability of a statistical hypothesis. Here we present a computer program, SONS, which implements nonparametric estimators for the fraction and richness of OTUs shared between two communities.

Biodiversity↗

PubMed2004

Microbial diversity in soil: selection microbial populations by plant and soil type and implications for disease suppressiveness.

An increasing interest has emerged with respect to the importance of microbial diversity in soil habitats. The extent of the diversity of microorganisms in soil is seen to be critical to the maintenance of soil health and quality, as a wide range of microorganisms is involved in important soil functions. This review focuses on recent data relating how plant type, soil type, and soil management regime affect the microbial diversity of soil and the implication for the soil's disease suppressiveness. The two main drivers of soil microbial community structure, i.e., plant type and soil type, are thought to exert their function in a complex manner. We propose that the fact that in some situations the soil and in others the plant type is the key factor determining soil microbial diversity is related to the complexity of the microbial interactions in soil, including interactions between microorganisms and soil and microorganisms and plants. A conceptual framework, based on the relative strengths of the shaping forces exerted by plant and soil versus the ecological behavior of microorganisms, is proposed.

Agriculture↗

PubMed2000

Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons.

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2005

Structure of a cellulose degrading bacterial community during anaerobic digestion.

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells.

Anaerobiosis↗

PubMed2006

Quantifying the roles of immigration and chance in shaping prokaryote community structure.

Naturally occurring populations of bacteria and archaea are vital to life on the earth and are of enormous practical significance in medicine, engineering and agriculture. However, the rules governing the formation of such communities are still poorly understood, and there is a need for a usable mathematical description of this process. Typically, microbial community structure is thought to be shaped mainly by deterministic factors such as competition and niche differentiation. Here we show, for a wide range of prokaryotic communities, that the relative abundance and frequency with which different taxa are observed in samples can be explained by a neutral community model (NCM). The NCM, which is a stochastic, birth-death immigration process, does not explicitly represent the deterministic factors and therefore cannot be a complete or literal description of community assembly. However, its success suggests that chance and immigration are important forces in shaping the patterns seen in prokaryotic communities.

Archaea↗

PubMed2005

Exploration of soil bacterial communities for their potential as bioresource.

Soil is a repository of diverse microorganisms, which has frequently been used to isolate and exploit microbes for industrial, environmental and agricultural applications. Knowledge about the structure and dynamics of bacterial communities in soil has been limited as only a small fraction of bacterial diversity is accessible to culture methods. Traditional enrichment techniques and the pure culture approach for microbiological studies have offered only a narrow portal for examining the soil microbial flora due to their limited selectivity. Therefore, the morphological and nutritional criteria used to describe bacterial community failed to provide a natural taxonomic order according to evolutionary relationship. Molecular methods under an emerging discipline of biology "molecular microbial ecology" are now helping in getting these constraints removed to some extent. Nucleic acid extraction from soil is the first crucial step in the application of most of the molecular techniques, which have largely been dominated by diverse variations of PCR. Due to its rapidity, sensitivity and specificity, PCR-based finger printing techniques have proved extremely useful in assessing the changes in microbial community structure. Such techniques can yield complex community profiles and can also provide useful phylogenetic information. Fluorescent in situ hybridization (FISH) can be used to evaluate the distribution and function of bacterial population in situ. DNA microarray techniques have also been developed and being frequently used for the evaluation of ecological role and phylogenetic affiliations of bacterial populations in the soil.

Bacteria↗

PubMed1996

Phospholipid Fatty Acid Composition and Heavy Metal Tolerance of Soil Microbial Communities along Two Heavy Metal-Polluted Gradients in Coniferous Forests.

The effects of long-term heavy metal deposition on microbial community structure and the level of bacterial community tolerance were studied along two different gradients in Scandinavian coniferous forest soils. One was near the Harjavalta smelter in Finland, and one was at Ronnskar in Sweden. Phospholipid fatty acid (PLFA) analysis revealed a gradual change in soil microbial communities along both pollution gradients, and most of the individual PLFAs changed similarly to metal pollution at both sites. The relative quantities of the PLFAs br18:0, br17:0, i16:0, and i16:1 increased with increasing heavy metal concentration, while those of 20:4 and 18:2(omega)6, which is a predominant PLFA in many fungi, decreased. The fungal part of the microbial biomass was found to be more sensitive to heavy metals. This resulted in a decreased fungal/bacterial biomass ratio along the pollution gradient towards the smelters. The thymidine incorporation technique was used to study the heavy metal tolerance of the bacteria. The bacterial community at the Harjavalta smelter, exposed mainly to Cu deposition, exhibited an increased tolerance to Cu but not to Cd, Ni, and Zn. At the Ronnskar smelter the deposition consisting of a mixture of metals increased the bacterial community tolerance to all tested metals. Both the PLFA pattern and the bacterial community tolerance were affected at lower soil metal concentrations than were bacterial counts and bacterial activities. At Harjavalta the increased Cu tolerance of the bacteria and the change in the PLFA pattern of the microbial community were found at the same soil Cu concentrations. This indicated that the altered PLFA pattern was at least partly due to an altered, more metal-tolerant bacterial community. At Ronnskar, where the PLFA data varied more, a correlation between bacterial community tolerance and an altered PLFA pattern was found up to 10 to 15 km from the smelter. Farther away changes in the PLFA pattern could not be explained by an increased community tolerance to metals.

Journal Article↗

PubMed2005

Effects of community versus single strain inoculants on the biocontrol of Salmonella and microbial community dynamics in alfalfa sprouts.

Potential biological control inoculants, Pseudomonas fluorescens 2-79 and microbial communities derived from market sprouts or laboratory-grown alfalfa sprouts, were introduced into alfalfa seeds with and without a Salmonella inoculum. We examined their ability to inhibit the growth of this foodborne pathogen and assess the relative effects of the inoculants on the alfalfa microbial community structure and function. Alfalfa seeds contaminated with a Salmonella cocktail were soaked for 2 h in bacterial suspensions from each inoculant tested. Inoculated alfalfa seeds were grown for 7 days and sampled during days 1, 3, and 7. At each sampling, alfalfa sprouts were sonicated for 7 min to recover microflora from the surface, and the resulting suspensions were diluted and plated on selective and nonselective media. Total bacterial counts were obtained using acridine orange staining, and the percentage culturability was calculated. Phenotypic potential of sprout-associated microbial communities inoculated with biocontrol treatments was assessed using community-level physiological profiles based on patterns of use of 95 separate carbon sources in Biolog plates. Community-level physiological profiles were also determined using oxygen-sensitive fluorophore in BD microtiter plates to examine functional patterns in these communities. No significant differences in total and mesophilic aerobe microbial cell density or microbial richness resulting from the introduction of inoculants on alfalfa seeds with and without Salmonella were observed. P. fluorescens 2-79 exhibited the greatest reduction in the growth of Salmonella early during alfalfa growth (4.22 log at day 1), while the market sprout inoculum had the reverse effect, resulting in a maximum log reduction (5.48) of Salmonella on day 7. Community-level physiological profiles analyses revealed that market sprout communities peaked higher and faster compared with the other inoculants tested. These results suggest that different modes of actions of single versus microbial consortia biocontrol treatments may be involved.

Colony Count, Microbial↗

PubMed2005

Two episodes of microbial change coupled with Permo/Triassic faunal mass extinction.

Microbial expansion following faunal mass extinctions in Earth history can be studied by petrographic examination of microbialites (microbial crusts) or well-preserved organic-walled microbes. However, where preservation is poor, quantification of microbial communities can be problematic. We have circumvented this problem by adopting a lipid biomarker-based approach to evaluate microbial community changes across the Permo/Triassic (P/Tr) boundary at Meishan in South China. We present here a biomarker stratigraphic record showing episodic microbial changes coupled with a high-resolution record of invertebrate mass extinction. Variation in the microbial community structure is characterized by the 2-methylhopane (2-MHP) index (a ratio of the abundance of cyanobacterial biomarkers to more general bacterial biomarkers). Two episodes of faunal mass extinction were each preceded by minima in the 2-MHP index, followed by strong maxima, likely reflecting microbial responses to the catastrophic events that caused the extinction and initiated ecosystem changes. Hence, both cyanobacterial biomarker and invertebrate fossil records provide evidence for two episodes of biotic crisis across the P/Tr boundary.

Animals↗

PubMed2000

Seasonal microbial community dynamics in a flowerpot-using personal composting system for disposal of household biowaste.

Microbial community dynamics in a flowerpot-using solid biowaste composting (FUSBIC) process were monitored seasonally by quinone profiling and conventional microbiological methods. The FUSBIC system, which consisted of three flowerpots (14 L or 20 L capacity) with 5-6 kg each of a soil-compost mixture (SCM) as the primary reactors, was loaded daily with household biowaste from November 1998 to October 1999. The monthly average waste reduction rate was 88.2% for the 14-L system and 92.5% for the 20-L system on a wet weight basis. The direct total microbial count detected in the 14-L primary reactors ranged from 4.5 to 9.6x10(11) cells.g(-1) of dry wt of SCM, and the viable count of aerobic heterotrophic bacteria recovered on agar plates at 28 degrees C varied from 1.9 to 5.7x10(11) CFU.g(-1) of dry wt. The quinone content of SCM samples from the 14-L and 20-L systems ranged from 160 to 353 nmol.g(-1) of dry SCM. Ubiquinones, unsaturated menaquinones, and partially saturated menaquinones constituted 15.0-36.4, 14.8-22.0, and 41.8-61.6 mol% of the total content, respectively. The major quinone types detected were usually MK-8(H(2)), MK-9(H(2)), and Q-10. Variations in quinone profiles were evaluated numerically by using two parameters, the dissimilarity index (D) and microbial divergence index (MD(q)). The upper limit of seasonal changes in the microbial community structure was about 30% as expressed by D values. The MD(q) values calculated ranged from 18 to 22. A significant positive correlation was found between seasonal temperature and bacterial populations containing partially saturated menaquinones. These results indicated that the FUSBIC system contained highly diverse microbial populations that fluctuated to some extent depending on seasonal temperature. Members of the Actinobacteria were suggested to be the major constituents of the total population present.

Journal Article↗

PubMed1999

Effects of larval mosquitoes (Aedes triseriatus) and stemflow on microbial community dynamics in container habitats.

The dynamics of the microbial food sources for Aedes triseriatus larvae in microcosms were found to be strongly influenced by larval presence. The total abundance of bacteria in water samples generally increased in response to larvae, including populations of cultivable, facultatively anaerobic bacteria. Additionally, a portion of the community shifted from Pseudomonaceae to Enterobacteriaceae. Bacterial abundance on leaf material was significantly reduced in the presence of actively feeding larvae. Principle-component analysis of whole community fatty acid methyl ester (FAME) profiles showed that larvae changed the microbial community structure in both the water column and the leaf material. Cyclopropyl FAMEs, typically associated with bacteria, were reduced in microcosms containing larvae; however, other bacterial fatty acids showed no consistent response. Long-chain polyunsaturated fatty acids characteristic of microeukaryotes (protozoans and meiofauna) declined in abundance when larvae were present, indicating that larval feeding reduced the densities of these microorganisms. However, presumed fungal lipid markers either increased or were unchanged in response to larvae. Larval presence also affected microbial nitrogen metabolism through modification of the physiochemical conditions or by grazing on populations of bacteria involved in nitrification-denitrification. Stemflow primarily influenced inorganic ion and organic compound concentrations in the microcosms and had less-pronounced effects on microbial community parameters than did larval presence. Stemflow treatments diluted concentrations of all inorganic ions (chloride, sulfate, and ammonium) and organic compounds (total dissolved organic carbon, soluble carbohydrates, and total protein) measured, with the exceptions of nitrite and nitrate. Stemflow addition did not measurably affect larval biomass in the microcosms but did enhance development rates and early emergence patterns of adults.

Aedes↗