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General regularities in the structure of the microbial communities of southern taiga soil ecosystems and taxonomic differences between the microbial communities of soils with different hydrothermal characteristics are discussed with reference to the main types of soils of the Central State Forest Biosphere Reserve.
Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number-PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site's capacity for anaerobic benzene degradation.
Total DNA was directly extracted from environmental samples and amplified with polymerase chain reaction (PCR) technique. The PCR products were fingerprinted via denaturing gradient gel electrophoresis (DGGE). Significant differences were observed in the microbial community structures between traditional treatment process and chem-bioflocculation process. The microbial community structure shift at different sampling locations in chem-bioflocculation process and on two typical operational conditions was studied. 16S rDNA V3 regions of some dominant species were sequenced and the species were identified. The microbial communities were stable in both the chem-bioflocculation process and the activated sludge process under various experimental conditions presented in this work. The attached growth treatment process was less stable when operational conditions changed.
The vertical and temporal changes in microbial communities were investigated throughout the water column and sediment of the saline meromictic Lake Kaiike by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Marked depth-related changes in microbial communities were observed at the chemocline and the sediment-water interface. However, no major temporal changes in the microbial community below the chemocline were observed during the sampling period, suggesting that the ecosystem in the anoxic zone of Lake Kaiike was nearly stable. Although the sequence of the most conspicuous DGGE band throughout the anoxic water and in the top of the microbial mat was most similar to that of an anoxic, photosynthetic, green sulphur bacterium, Pelodyction luteolum DSM273 (97% similarity), it represented a new phylotype. A comparison of DGGE banding patterns of the water column and sediment samples demonstrated that specific bacteria accumulated on the bottom from the anoxic water layers, and that indigenous microbial populations were present in the sediment. The measurements of bicarbonate assimilation rates showed significant phototrophic assimilation in the chemocline and lithoautotrophic assimilation throughout the anoxic water, but were not clearly linked with net sulphide turnover rates, indicating that sulphur and carbon metabolisms were not directly correlated.
Molecular genetic methods were used to analyze the remnants of microbial ecosystems contained within an ancient oceanic microbial habitat that was recovered from a continental drilled core of black shale approximately 100 million years in age. Bacterial ribosomal RNA genes were vertically amplified from the six different depths of a black shale core associated with a phosphate-rich stratum, defined as one of the mid-Cretaceous oceanic anoxic events (OAEs). Although the black shale core was recovered from a terrestrial coring effort, the recovered 16S rRNA gene sequences showed affinity to microbial communities previously seen in deep-sea sedimentary environments (i.e., the microbial assemblage was easily recognizable as a marine community). In particular, a number of 16S rRNA gene clones of oceanic sulfate-reducing bacteria within the delta-Proteobacteria predominated at the OAE layer. The recovered bacterial DNA signatures are consistent with the interpretation that the sequences are derived from the past microbial communities buried in either sea-bottom or subseafloor environments during the sedimentation process and, after ceasing growth, preserved until the present.
The effects of heavy-metal-containing sewage sludge on the soil microbial community were studied in two agricultural soils of different textures, which had been contaminated separately with three predominantly single metals (Cu, Zn, and Ni) at two different levels more than 20 years ago. We compared three community-based microbiological measurements, namely, phospholipid fatty acid (PLFA) analysis to reveal changes in species composition, the Biolog system to indicate metabolic fingerprints of microbial communities, and the thymidine incorporation technique to measure bacterial community tolerance. In the Luddington soil, bacterial community tolerance increased in all metal treatments compared to an unpolluted-sludge-treated control soil. Community tolerance to specific metals increased the most when the same metal was added to the soil; for example, tolerance to Cu increased most in Cu-polluted treatments. A dose-response effect was also evident. There were also indications of cotolerance to metals whose concentration had not been elevated by the sludge treatment. The PLFA pattern changed in all metal treatments, but the interpretation was complicated by the soil moisture content, which also affected the results. The Biolog measurements indicated similar effects of metals and moisture to the PLFA measurements, but due to high variation between replicates, no significant differences compared to the uncontaminated control were found. In the Lee Valley soil, significant increases in community tolerance were found for the high levels of Cu and Zn, while the PLFA pattern was significantly altered for the soils with high levels of Cu, Ni, and Zn. No effects on the Biolog measurements were found in this soil.
Both PCR-TGGE (temperature gradient gel electrophoresis, TGGE) and 16S rRNA gene clone library construction were used to comparatively analyze the microbial communities of a water injection well (WW) and an oil well (OW) in Dagang oilfield. TGGE analysis of the PCR amplified 16S rDNA V3 region products showed great difference between these two microbial communities. Six major bands were detected in the TGGE profile of the WW sample, while only one predominant band in the OW sample was found. Two 16S rRNA gene clone libraries were also constructed, and 108 and 50 clones were selected from the WW and OW library respectively for amplified ribosomal DNA restriction analysis (ARDRA). 33 taxanomic operational units (OTUs) were found in the WW library with 6 major OTUs, while only 8 OTUs were found in the OW library with one OTU predominant. The results of TGGE and clone library profiling analysis both indicated that microbial community of the WW had higher diversity than the OW. Sequence analysis of the representative clone of each OTU showed that most bacteria of the WW were affiliated with alpha, beta, and gamma Proteobacteria and Actinobacteria, especially Rhodobacter (47%). Most bacteria of the OW were affiliated with alpha, beta, and gamma Proteobacteria, especially Pseudomonas (62%). Molecular analysis of the microbial diversity in oilfield provides foundation for better application of MEOR (Microbial Enhanced Oil Recovery).
Anaerobic manure storage systems are one of the major contributors to the odor and environmental pollution associated with swine (Sus scrofa) production systems. The microbial ecology of manure storage systems and the relationships between microbial communities and odor production are largely unknown. In this study, we used community fatty acid methyl ester (FAME) analysis to generate lipid profiles to assess seasonal differences among microbial communities inhabiting various types of outdoor swine manure storage systems. Concurrently, we measured manure concentrations of several malodor indicator compounds as well as pH, temperature, and solids content. Principal components analysis (PCA) showed that there are differences in FAME profiles among the swine manure storage systems examined and most of the variation was in the relative abundance of 18:0, 18:1omega7t, 18:1omega7c/omega9t/omega12t, and 16:1omega7t/i15:0 2OH FAMEs. The PCA of the FAME profiles revealed that the phototrophic systems were more similar to each other and that the nonphototrophic systems were more similar to each other than they were to phototrophic lagoons. There were seasonal changes in the FAME profiles in the phototrophic systems and the concrete nonphototrophic basin (CNPB), and in one phototrophic system, the FAME profiles more closely resembled a CNPB FAME profile during the winter than the other phototrophic lagoons. In the phototrophic lagoon systems, there was a direct correlation between the abundance of the FAMEs identified in the PCA and manure concentrations of phenol, p-cresol, and 4-ethyl phenol. In the CNPB, there was a negative correlation between the total phenolics concentration and the 18:1omega7t FAME. Our results indicate that community FAME profiles could be used as a diagnostic tool for obtaining preliminary evidence that management practices are altering the system's microbial community to one that favors less air pollution potential.
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.
AIMS: Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum. METHODS AND RESULTS: An experimental harbour seawater microcosm, supplemented with nutrients and crude oil, was monitored above 15 days in comparison with unpolluted ones (control microcosms). Bacterial cells were counted with a Live/Dead BacLight viability kit; leucine aminopeptidase, beta-glucosidase, alkaline phosphatase, lipase and esterase enzymes were measured using fluorogenic substrates. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil addition stimulated an increase of the total bacterial abundance, leucine aminopeptidase and phosphatase activity rates, as well as a change in the community structure. This suggested a prompt response of micro-organisms to the load of petroleum hydrocarbons. CONCLUSIONS: The present study on the viability, specific composition and metabolic characteristics of the microbial community allows a more precise assessment of oil pollution. Both structural and functional parameters offer interesting perspectives as indicators to monitor changes caused by petroleum hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of microbial structural successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in microcosm studies improve our understanding of natural processes occurring during oil spills.
Fatty acids are a widely studied group of lipids of sufficient taxonomic diversity to be useful in defining microbial community structure. The extraordinary resolution of glass capillary gas-liquid chromatography can be utilized to separate and tentatively identify large numbers of fatty acid methyl esters derived from the lipids of estuarine detritus and marine benthic microbiota without the bias of selective methods requiring culture or recovery of the microbes. The gas-liquid chromatographic analyses are both reproducible and highly sensitive, and the recovery of fatty acids is quantitative. The analyses can be automated, and the diagnostic technique of mass spectral fragmentation analysis can be readily applied. Splitless injection on glass capillary gas chromatographic columns detected by mass spectral selective ion monitoring provides an ultrasensitive and definitive monitoring system. Reciprocal mixtures of bacteria and fungi, when extracted and analyzed, showed progressive changes of distinctive fatty acid methyl esters derived from the lipids. By manipulating the environment of an estuarine detrital microbial community with antibiotics and culture conditions, it was possible to produce a community greatly enriched in eucaryotic fungi, as evidenced by scanning electron microscopic morphology. The fatty acid methyl esters from the lipids in the fungus-enriched detritus showed enrichment of the C(18) dienoic and the C(18) and C(20) polyenoic esters. Manipulation of the detrital microbiota that increased the procaryotic population resulted in an absence of large structures typical of fungal mycelia or diatoms, as evidenced by scanning electron microscopy, and a significantly larger proportion of anteiso- and isobranched C(15) fatty acid esters, C(17) cyclopropane fatty acid esters, and the cis-vaccenic isomer of the C(18) monoenoic fatty acid esters. As determined by these techniques, a marine settling community showed greater differences in bacterial as contrasted to microeucaryotic populations when compared with the microbial communities of benthic cores.
PCR-single-strand conformation polymorphism (SSCP) and Southern blotting techniques were adopted to investigate microbial community dynamics in a sulfate-reducing bioreactor caused by decreasing influent alkalinity. Experimental results indicated that the sulfate-removal rate approached 87% in 25 d under the conditions of influent alkalinity of 4000 mg/L (as CaCO3) and sulfate-loading rate of 4.8 g/(L*d), which indicated that the bioreactor started up successfully. The analysis of microbial community structure in this stage showed that Lactococcus sp., Anaerofilum sp. and Kluyvera sp. were dominant populations. It was found that when influent alkalinity reduced to 1000 mg/L, sulfate-removal rate decreased rapidly to 35% in 3 d. Then influent alkalinity was increased to 3000 mg/L, the sulfate-removal rate rose to 55%. Under these conditions, the populations of Dysgonomonas sp., Sporobacte sp., Obesumbacterium sp. and Clostridium sp. got to rich, which predominated in the community together with Lactococcus sp., Anaerofilum sp. and Kluyvera sp. However, when the alkalinity was decreased to 1500 mg/L, the sulfate-removal rate rose to and kept stable at 70% and populations of Dysgonomonas sp., Sporobacter sp. and Obesumbacterium sp. died out, while some strains of Desulfovibrio sp. and Clostridium sp. increased in concentration. In order to determine the minimum alkalinity value that the system could tolerate, the influent alkalinity was decreased from 1500 to 400 mg/L secondly. This resulted in the sulfate-removal rate, pH value and effluent alkalinity dropping quickly. The amount of Petrotoga sp., Prevotella sp., Kluyvera sp. and Neisseria sp. reduced obviously. The result data from Southern blotting indicated that the amount of sulfate-reducing bacteria (SRBs) decreased with influent alkalinity dropping. Analysis of the microbial community structure and diversity showed that the SRBs populations were very abundant in the inoculated activated sludge and the alkalinity decrease caused the reduction of the populations noted. Most of resident populations in the bioreactor were fermentative acidogenic bacteria (FABs), among which the phylum Firmicute was in the majority, but SRBs were very few. This community structure demonstrates the cooperation between SRBs and FABs, which sustains the system's high sulfate-removal and operation stability.
Scanning and transmission electron microscopy were used to examine the adherence and in situ morphology of the microbial community colonizing the anal and vulvar pores of the subfamily Cyathostominae (Nematoda: Strongylidae) from the colon of Burchell's zebra (Equus burchelli antiquorum). Two different morphological types of asporogenous rod were prominent in the microbial community. One was a thin, septate, filamentous organism (0.4 to 0.5 micron by 2 to 3 microns) with blunt ends, which was more prominent at the site of attachment. The other was a larger (1.8 to 2.4 microns by 5 to 10 microns) multicellular rod with round ends in the form of a trichome. Spiral- and vibrio-shaped bacteria were also present in the thin sections. The septate filaments were shown to contain a cell spacer similar to those described in Methanospirillum hungatei. Attachment to the cuticle was by means of an amorphous electron-dense material with fibrillar appearance and not by specialized holdfast segments. Ten isolates were obtained from a habitat-simulating medium on which a homogenate from the posterior region was plated. Antibodies were raised to whole cells of five rod-shaped isolates in rabbits and fluorescein isothiocyanate labeled. Positive bright-yellow fluorescence was obtained with one of the clostridial isolates. The results are discussed with reference to other bacteria with similar morphology, the nature of this unique interrelationship between the microbial community and its parasitic host inside the equine hindgut, and the possibility of biological control of parasitic helminths.
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.
Changes in microbial numbers and activities in a soil in response to bentazon applied at 10 and 100 ppm were studied after 4 and 30 weeks of incubation in laboratory conditions. As regards the eight general and functional microbial groups studied (aerobic and anaerobic bacteria, fungi, aerobic and anaerobic N2-fixing bacteria, nitrifiers, aerobic and anaerobic cellulolytic microorganisms), only the number of anaerobic N2-fixing bacteria significantly decreased, in the presence of the highest herbicide concentration for 30 weeks. At both the incubation times, only the higher dose of bentazon markedly inhibited soil nitrification and CO2 emission. Methanogenesis was inhibited by 1000 ppm bentazon added to anaerobic liquid cultures containing 5% soil for at least 2 weeks. There was an incomplete recovery of the herbicide at the two incubation times: < 5% of 10 ppm after 4 weeks and about 30% of 100 ppm after 30 weeks. No biodegradation of the compound was observed in liquid cultures under aerobic or anaerobic conditions. It is concluded that a bentazon concentration no higher than the field rate distributed within a 2-cm layer of soil does not considerably affect the microflora even in the absence of microbial degradation.
Twenty-eight sediment samples collected from Osaka Bay, Japan, were analyzed for phospholipid ester-linked fatty acids (PLFA) to determine regional differences in microbial community structure of the bay. The abundance of three major groups of C(10) to C(19) PLFA (saturated, branched, and monounsaturated PLFA), which accounted for 84 to 97% of the total PLFA, indicated the predominance of prokaryotes in the sediment. The distribution of six clusters obtained by similarity analysis in the bay revealed a marked regional distribution in the PLFA profiles. Total PLFA concentrations (0.56 to 2.97 mug/g [dry weight] of the sediment) in sediments also showed marked variation among the stations, with higher concentrations of total PLFA in the central part of the bay. The biomass, calculated on the basis of total PLFA concentration, ranged from 0.25 x 10 to 1.35 x 10 cells per g (dry weight) of the sediment. The relative dominance of microbial groups in sediments was described by using the reported bacterial biomarker fatty acids. Very small amounts of the characteristic PLFA of microeukaryotes in sediments indicated the restricted distribution of microeukaryotes. By examining the distribution of clusters and groups of microorganisms in the bay, there were two characteristics of the distribution pattern: (i) the predominance of anaerobic bacteria and gram-positive prokaryotes, characterized by the high proportions of branched PLFA in the eastern and northeastern sides of the bay, where the reported concentrations of pollutants were also high, and (ii) the predominance of aerobic prokaryotes and eukaryotes, except for a few stations, in the western and southwestern sides of the bay, as evidenced by the large amounts of monounsaturated PLFA. Such significant regional differences in microbial community structure of the bay indicate shifts in microbial community structure.
This investigation introduces the application of a relatively rapid technique to obtain information about the dynamic nature of microbial communities in activated sludge. The objective has been to consider variability due to measurement errors and protocol changes within the same quantitative framework as the analysis of systematic differences in microbial communities in large-scale aerobic activated sludge secondary wastewater treatment systems. Adjustments to the methodology were considered due to their potential for simplifying and shortening the analysis procedure. All modifications to the protocols used to assay the composition of microbial fatty acids (MFAs) of activated sludge imposed some bias to the chromatographic data. This methodological bias was similar in magnitude to the level of discrimination between activated sludge microbial community structures that were considered as part of the present study. MFA analysis supported the expectations of subtle but systematic community structure differences and shifts in activated sludge based on the current understanding of these wastewater treatment systems. A standardized MFA methodology was shown to be sensitive to minor systematic changes in activated sludge communities due the anticipated underlying factors of selective pressures from the process configuration, history, operational conditions and/or nutrient status. The chemometric approach of fatty acid isopropyl ester analysis of activated sludge can provide a routine tool for meaningful and quantitative information of changes in activated sludge quality in full-scale treatment systems.
Electrokinetic techniques have been used to stimulate the removal of organic pollutants within soil, by directing contaminant migration to where remediation may be more easily achieved. The effect of this and other physical remediation techniques on the health of soil microbial communities has been poorly studied and indeed, largely ignored. This study reports the impact on soil microbial communities during the application of an electric field within ex situ laboratory soil microcosms contaminated with pentachlorophenol (PCP; 100mg kg(-1) oven dry soil). Electrokinetics reduced counts of culturable bacteria and fungi, soil microbial respiration and carbon substrate utilisation, especially close to the acidic anode where PCP accumulated (36d), perhaps exacerbated by the greater toxicity of PCP at lower soil pH. There is little doubt that a better awareness of the interactions between soil electrokinetic processes and microbial communities is key to improving the efficacy and sustainability of this remediation strategy.