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PubMed2003

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

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

Bacteria↗

PubMed2002

Detecting structural and functional differences in activated sludge bacterial communities originating from laboratory treatment of elementally and totally chlorine-free bleaching effluents.

The ability to differentiate functional and structural diversity of bacterial communities present in activated sludges adapted to elementally (ECF) and totally (TCF) chlorine-free bleaching effluents was evaluated. Community function was evaluated through substrate utilization patterns in BiologGN microplates, and taxonomic structure was evaluated by fluorescent in situ hybridization using probes targeting the Eubacteria; the alpha, beta, and gamma subclasses of the Proteobacteria; and gram-positive bacteria with high GC content. Over 6-week sampling periods, ECF-and TCF-adapted sludge bacterial communities presented reproducible substrate utilization patterns that through principal components (PCs) analysis, separated the ECF samples from the TCF samples. Application of the fluorescent in situ hybridization technique was complicated by the intense autofluorescence of the bleaching effluent sludge samples that interfered with detection of specific hybridization signals. The most notable difference in community structure detected using the chosen set of probes was the relatively greater proportion of cells of the alpha subclass in TCF sludge (27%) than in ECF sludge (6%). Nonspecific hybridization with beta and gamma probes was relatively high, but both sludges appeared to have similar proportions of cells of the beta (20-22%) and gamma (11-12%) subclasses. The two sludges presented relatively few gram-positive cells with high GC content (<0.2% of eubacterial counts). Differences in both metabolic potential and taxonomic structure of the microbial communities in the ECF- and TCF-activated sludges were detected. The kinetics of the development of these differences in treatment plants and their relationships with treatment efficiency and production process conditions should now be evaluated.

Chlorine↗

PubMed2004

Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model.

Molecular techniques previously used for genome comparisons of closely related bacterial species could prove extremely valuable for comparisons of complex microbial communities, or metagenomes. Our study aimed to determine the breadth and value of suppressive subtractive hybridization (SSH) in a pilot-scale analysis of metagenomic DNA from communities of microorganisms in the rumen. Suppressive subtractive hybridization was performed using total genomic DNA isolated from rumen fluid samples of two hay-fed steers, arbitrarily designated as tester or driver. Ninety-six subtraction DNA fragments from the tester metagenome were amplified, cloned and the DNA sequences were determined. Verification of the isolation of DNA fragments unique to the tester metagenome was accomplished through dot blot and Southern blot hybridizations. Tester-specific SSH fragments were found in 95 of 96 randomly selected clones. DNA sequences of subtraction fragments were analysed by computer assisted DNA and amino acid comparisons. Putative translations of 26 (32.1%) subtractive hybridization fragments exhibited significant similarity to Bacterial proteins, whereas 15 (18.5%) distinctive subtracted fragments had significant similarity to proteins from Archaea. The remainder of the subtractive hybridization fragments displayed no similarity to GenBank sequences. This metagenomic approach has exposed an unexpectedly large difference in Archaeal community structure between the rumen microbial populations of two steers fed identical diets and housed together. 16S rRNA dot blot hybridizations revealed similar proportions of Bacteria and Archaea in both rumen samples and suggest that the differences uncovered by SSH are the result of varying community structural composition. Our study demonstrates a novel approach to comparative analyses of environmental microbial communities through the use of SSH.

Animals↗

PubMed2006

Population genomics in natural microbial communities.

Little is known about the evolutionary processes that structure and maintain microbial diversity because, until recently, it was difficult to explore individual-level patterns of variation at the microbial scale. Now, community-genomic sequence data enable such variation to be assessed across large segments of microbial genomes. Here, we discuss how population-genomic analysis of these data can be used to determine how selection and genetic exchange shape the evolution of new microbial lineages. We show that once independent lineages have been identified, such analyses enable the identification of genome changes that drive niche differentiation and promote the coexistence of closely related lineages within the same environment. We suggest that understanding the evolutionary ecology of natural microbial populations through population-genomic analyses will enhance our understanding of genome evolution across all domains of life.

Archaea↗

PubMedDate not supplied

[Distribution and activity of microorganisms in the deep repository for liquid radioactive waste at the Siberian Chemical Combine].

The physicochemical conditions, composition of microbial communities, and the rates of anaerobic processes in the deep sandy horizons used as a repository for liquid radioactive wastes (LRW) at the Siberian Chemical Combine (Seversk, Tomsk oblast), were studied. Formation waters from the observation wells drilled into the production horizons of the radioactive waste disposal site were found to be inhabited by microorganisms of different physiological groups, including aerobic organotrophs, anaerobic fermentative, denitrifying, sulfate-reducing, and methanogenic bacteria. The density of microbial population, as determined by cultural methods, was low and usually did not exceed 10(4) cells/ml. Enrichment cultures of microorganisms producing gases (hydrogen, methane, carbon dioxide, and hydrogen sulfide) and capable of participation in the precipitation of metal sulfides were obtained from the waters of production horizons. The contemporary processes of sulfate reduction and methanogenesis were assayed; the rates of these terminal processes of organic matter destruction were found to be low. The denitrifying bacteria from the underground repository were capable of reducing the nitrates contained in the wastes, provided sources of energy and biogenic elements were available. Biosorption of radionuclides by the biomass of aerobic bacteria isolated from groundwater was demonstrated. The results obtained give us insight into the functional structure of the microbial community inhabiting the waters of repository production horizons. This study indicates that the numbers and activity of microbial cells are low both inside and outside the zone of radioactive waste dispersion, in spite of the long period of waste discharge.

Bacteria↗

PubMed2003

Microbial composition and structure of a rotating biological contactor biofilm treating ammonium-rich wastewater without organic carbon.

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in Kölliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.

Bacteria, Aerobic↗

PubMed2005

Changes in enzymes activity, substrate utilization pattern and diversity of soil microbial communities under cadmium pollution.

Heavy metal pollution has received increasing attention in recent years mainly because of the public awareness of environmental issues. In this study we have evaluated the effect of cadmium (Cd) on enzymes activity, substrate utilization pattern and diversity of microbial communities in soil spiked with 0, 20, 40, 60, 80, and 100 mg/kg Cd, during 60 d of incubation at 25 degrees C. Enzyme activities determined at 0, 15, 30, 45, and 60 d after heavy metal application (DAA) showed marked declines for various Cd treatments, and up to 60 DAA, 100 mg/kg Cd resulted in 50.1%, 47.4%, and 39.8% decreases in soil urease, acid phosphatase and dehydrogenase activities, respectively to control. At 60 DAA, substrate utilization pattern of soil microbial communities determined by inoculating Biolog ECO plates indicated that Cd addition had markedly inhibited the functional activity of soil microbial communities and multivariate analysis of sole carbon source utilization showed significantly different utilization patterns for 80 and 100 mg/kg Cd treatments. The structural diversity of soil microbial communities assessed by PCR-DGGE method at 60 DAA, illustrated that DGGE patterns in soil simplified with increasing Cd concentration, and clustering of DGGE profiles for various Cd treatments revealed that they had more than 50% difference with that of control.

Acid Phosphatase↗

PubMed1999

Copper speciation and microbial activity in long-term contaminated soils.

Most soil quality guidelines do not distinguish among the various forms of metals in soils; insoluble, nonreactive, and nonbioavailable forms are deemed as hazardous as highly soluble, reactive, and toxic forms. The objective of this study was to better understand the long-term effects of copper on microorganisms in relation to its chemical speciation in the soil environment. Carbon mineralization processes and the global structure of different microbial communities (fungi, eubacteria, actinomycetes) are still affected after more than 50 years of copper contamination in 20 soils sampled from two different agricultural sites. The microbial respiration lag period (LP) preceding the beginning of mineralization process increases with the level of soil copper contamination and is not significantly affected by other environmental factors such as soil pH and soil organic matter (SOM) content. The total copper concentration showed the best correlation with the LP when each site is considered separately. However, when considering the whole set of data, soil solution free Cu2+ activity (pCu2+) is the best predictor of Cu toxicity determined by LP (quite likely because pCu2+ integrates the soil physicochemical variability). The maximum mineralization rate (MMR), even if well correlated with the pCu2+, appears not to be a good biomonitor of copper contamination in soils since it is highly sensitive to soil characteristics such as SOM content. This study emphasizes the importance of the physicochemical properties of the environment on soil heavy metal toxicity and on soil toxicological measurements. These properties must be characterized in soil toxicological studies with respect to (1) their interactions with heavy metals, and (2) their direct impact on the selected biological test. The measurement of pCu2+ to characterize the level of soil contamination and of lag period as a bioindicator of metal effects in the soil are recognized as useful tools for the evaluation of the biological quality of soils.

Copper↗

PubMed2005

The effects of organic carbon, ammoniacal-nitrogen, and oxygen partial pressure on the stratification of membrane-aerated biofilms.

The purpose of this study was to examine the effects of different nutrient (carbon, nitrogen, oxygen) concentrations on the microbial activity and community structure in membrane-aerated biofilms (MABs). MABs were grown under well-defined conditions of fluid flow, substrate concentration, and membrane oxygen partial pressure. Biofilms were then removed and thin-sliced using a cryostat/microtome parallel to the membrane. Individual slices were analyzed for changes with depth in biomass density, respiratory activity, and the population densities of ammonia-oxidizing and denitrifying bacteria populations. Oxygen-sensing microelectrodes were used to determine the depth of oxygen penetration into each biofilm. Our results demonstrated that ammonia-oxidizing bacteria grow near the membrane, while denitrifying bacteria grow a substantial distance from the membrane. However, nitrifying and denitrifying bacteria did not grow simultaneously when organic concentrations became too high or ammonia concentrations became too low. In conclusion, membrane-aerated biofilms exhibit substantial stratification with respect to community structure and activity. A fundamental understanding of the factors that control this stratification will help optimize the performance of full-scale membrane-aerated biofilm reactors for wastewater treatment.

Ammonia↗

PubMed2005

Principles of biological adaptation of organisms in artificial ecosystems to changes of environmental factors.

Studying material transformations and biotic cycling in artificial ecosystems (AES), we need to know the principles of biological adaptation of active organisms to change in the environment. Microorganisms in AES for water purification are the most active transforming organisms and consumers of the organic substances contained in wastes. Utilization of organic substances is directly connected with the energy fluxes used by AES. According to energy criteria, the energy fluxes used by a biological system tend to reach maximum values under stable conditions. Unutilized substrate concentration decreases as a result of biological adaptations. After a dramatic change in environmental factors, for example, after a sharp increase in the flow rate of organic substances, the biological system is not able to react quickly. The concentration of unutilized substrate increases and the energy flux used by the biological system decreases. The structure of the microbial community also changes, with a decrease in biological diversity. The efficiency of energy use by simple terrestrial ecosystems depends on the energetic intensity and interactions between plants and rhizospheric microorganisms.

Biodegradation, Environmental↗

PubMed2006

Biodegradation of nonylphenol in soil.

We investigated the effects of various factors (brij 30, brij 35, yeast extract, hydrogen peroxide and compost) on the aerobic degradation of nonylphenol (NP) in soil and characterized the structure of the microbial community in that soil. Residues of NP were measured using gas chromatography-mass spectrometry (GC-MS) and a change of microbial communities was demonstrated using denaturing gradient gel electrophoresis (DGGE). The results showed that Taichung sandy clay loam had higher NP degradation rate than Kaoshiung silty clay. The addition of compost, yeast extract (0.5 mg/l), brij 30 (55 microM), or brij 35 (91 microM) enhanced NP degradation, while the addition of hydrogen peroxide (1.0 mg/l) inhibited its degradation. We also found that the addition of various substrates changed the microbial community in the soils. Cytophaga sp. and Ochrobactrum sp. were constantly dominant bacteria under various conditions in the soil.

Analysis of Variance↗

PubMed2006

Molecular techniques in wastewater: Understanding microbial communities, detecting pathogens, and real-time process control.

Traditionally, the detection of pathogens in water, wastewater, and other environmental samples is restricted by the ability to culture such organisms from complex environmental samples. During the last decade the use of molecular methods have supplied the means for examining microbial diversity and detecting specific organisms without the need for cultivation. The application of molecular techniques to the study of natural and engineered environmental systems has increased our insight into the vast diversity and interaction of microorganisms present in complex environments. In this paper, we will review the current and emerging molecular approaches for characterizing microbial community composition and structure in wastewater processes. Recent studies show that advances in microarray assays are increasing our capability of detecting hundreds and even thousands of DNA sequences simultaneously and rapidly. With the current progress in microfluidics and optoelectronics, the ability to automate a detection/identification system is now being realized. The status of such a system for wastewater monitoring is discussed.

DNA Fingerprinting↗

PubMed2001

Comparison of microbial and meiofaunal community analyses for determining impact of heavy metal contamination.

The impact of long-term heavy metal contamination on soil communities was assessed by a number of methods. These included plate counts of culturable bacteria, community level physiological profiling (CLPP) by analysis of the utilization of multiple carbon sources in BIOLOG plates, community fatty acid methyl ester (C-FAME) profiling and dehydrogenase enzyme activity measurements. These approaches were complemented with microscopic assessments of the diversity of the nematode community. Samples from two sites with different histories of heavy-metal input were assessed. Major differences in microbial and meiofaunal parameters were observed both between and within the sites. There was a large degree of congruence between each of the microbiological approaches. In particular, one sample appeared to be distinguished by a reduction in culturable bacteria (especially pseudomonads), limited response to carbon sources in CLPP, and major differences in extracted fatty acid profiles. The use of multivariate analysis to examine the relationship between microbial and physicochemical measurements revealed that CLPP and plate counts were useful for determining the gross effect of metals on soil microbial communities, whereas proportions of metal-resistant bacteria and dehydrogenase activity differentiated between the two sites. Copper and zinc concentrations and pH all showed significant correlation with the microbial parameters. Nematode community structure was affected to a greater extent by soil pH than by metal content, but the within-site rankings were the same as those achieved for microbiological analyses. The use of these methods for field evaluation of the impact of industrial pollution may be possible provided care is taken when interpreting the data.

Animals↗

PubMed2000

rRNA operon copy number reflects ecological strategies of bacteria.

Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability. Soil bacteria that formed colonies rapidly upon exposure to a nutritionally complex medium contained an average of 5.5 copies of the small subunit rRNA gene, whereas bacteria that responded slowly contained an average of 1.4 copies. In soil microcosms pulsed with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), indigenous populations of 2,4-D-degrading bacteria with multiple rRNA genes ( = 5.4) became dominant, whereas populations with fewer rRNA genes ( = 2.7) were favored in unamended controls. These findings demonstrate phenotypic effects associated with rRNA gene copy number that are indicative of ecological strategies influencing the structure of natural microbial communities.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2004

Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-melting-temperature (T(m(L))) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to T(m(L)). The average T(m(L)) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.

Animals↗

PubMed2006

Identification and specific detection of a novel pseudomonadaceae cluster associated with soils from winter wheat plots of a long-term agricultural field experiment.

The genus Pseudomonas (sensu stricto) represents a group of microorganisms directly involved in functions conferring plant health. We performed a study in the DOK long-term agricultural field experiment on the basis of previously published Pseudomonas-selective PCR primers in order to investigate the community structure of the microbial groups defined by the target range of these primers. Three different agricultural management systems, i.e., conventional, biodynamic, and bio-organic, along with mineral and unfertilized controls were investigated, with each system planted with either winter wheat or a grass-clover ley. Amplified small-subunit rRNA gene fragments were analyzed using the genetic profiling techniques restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE), revealing distinct differences between soils planted with winter wheat and grass clover but only minor differences between the management systems. Phylogenetic analyses of 59 clone sequences retrieved from bio-organic and unfertilized systems identified sequences related to Pseudomonas fluorescens and a novel cluster termed Cellvibrio-related Pseudomonadaceae (CRP). The CRP clones were exclusively isolated from winter wheat soil samples and were responsible for the crop-specific differences observed in RFLP and DGGE profiles. New primers were designed for the amplification of CRP targets directly from soil DNA, yielding strong signals exclusively for winter wheat soils. We concluded that crop-associated CRP exist in agricultural soils and that genetic profiling followed by specific probe design represents a valuable approach for identification as well as sensitive and rapid monitoring of novel microbial groups in the environment.

Agriculture↗

PubMedDate not supplied

In situ bioremediation through mulching of soil polluted by a copper-nickel smelter.

Bioremediation of a heavy metal-polluted soil was investigated in a 3-yr field experiment by adding mulch to a polluted forest floor. The mulch consisted of a mixture of compost and woodchips. The remediation treatment decreased the toxicity of the soil solution to bacteria as determined by the [3H]-thymidine incorporation technique, that is, by measuring the growth rate of soil bacteria extracted from unpolluted humus after exposing them to soil solution containing heavy metals from the experimental plots. Canonical correlation analysis was performed in order to identify the chemical and microbiological changes in the soil. The pH of the mulched organic layer increased by one unit. The concentration of complexed Cu increased and that of free Cu2+ decreased in the soil solution from the mulch treatment. According to basal respiration and litter decomposition, microbial activity increased during the 3 yr following the remediation treatment. The [3H]-thymidine incorporation technique was also used to study the growth rate and tolerance of bacteria to Cu. The bacterial growth rate increased and the Cu tolerance decreased on the treated plots. The structure of the microbial community, as determined by phospholipid fatty acid (PLFA) analysis, remained unchanged. The results indicate that remediation of the polluted soil had occurred, and that adding a mulch to the forest floor is a suitable method for remediating heavy metal-polluted soil.

Bacteria↗

PubMedDate not supplied

[Influence of different ecological groups of earthworms on the intensity of nitrogen fixation].

The results of studying nitrogen fixation in organic substrates processed by different ecological groups of earthworms suggest that the earthworms Aporrectoidea caliginosa actively stimulate nonsymbiotic nitrogen fixation. In this respect, they exceed the manure worms Eizenia foetida tens of times due to the formation in the organic substrate of conditions favorable for the nitrogen fixing bacteria, and namely: low content of nitrogen easily accessible for microorganisms, changes in the structure of the microbial community of the substrate in favor of non-spore forms of bacteria, and suppression of the growth of saprophyte bacilli, the main competitors of nitrogen fixing bacteria for carbon nourishment sources.

Animals↗