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PubMed2004

Molecular profiling of microbial communities associated with seeds of Beta vulgaris subsp. Vulgaris (sugar beet).

The composition of microbial communities on and within seeds may effect their storage and field performance, whether they are indigenous or applied as biocontrol agents. In this study, we have explored the usefulness of profiling small subunit ribosomal (SSR) gene fragments for studying the microflora associated with seeds. DNA was amplified by the polymerase chain reaction (PCR) and the amplicons separated using denaturing gradient gel electrophoresis (DGGE). Primers targeting eukaryotic SSRs were used to investigate fungal communities, and primers targeting bacterial SSRs were employed to study the eubacterial microflora. As a case study, we attempted to profile the fungi and bacteria associated with seeds of Beta vulgaris (sugar beet) to permit an insight into the varying field performance of several well-characterised commercial seed lots. Serious interference with the microbial signals was observed from the plant's own nuclear 18S rRNA genes and chloroplast 16S rRNA genes using standard PCR conditions and DNA extracted from whole seeds as template. Hot-start and touchdown PCR made no appreciable improvement to these signals. Seed imbibition and dissection into operculum and fruit wall and true seed prior to DNA extraction improved signal recovery in the fruit fraction. With primer modification, bacteria and fungi were detected in an excess of plant DNA of 100:1 and 10:1, respectively. With this method, microbial communities on seeds could be profiled, however, it is likely that targeted depletion of plant rDNA targets will be a necessary extra step before this approach can be used to screen seeds routinely.

Beta vulgaris↗

PubMed2002

Microbial community dynamics during the Scamorza Altamurana cheese natural fermentation.

The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests. Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening. RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus. The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic. Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z dominated all through the fermentation process. Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo. The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products.

Animals↗

PubMed2003

Microbial communities in activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor.

Microbial communities of activated sludge in an anaerobic/aerobic sequencing batch reactor (SBR) supplied with acetate as sole carbon source were analyzed to identify the microorganisms responsible for enhanced biological phosphorus removal. Various analytical methods were used such as electron microscopy, quinone, slot hybridization, and 16S rRNA gene sequencing analyses. Electron photomicrographs showed that coccus-shaped microorganisms of about 1 microm diameter dominated the microbial communities of the activated sludge in the SBR, which had been operated for more than 18 months. These microorganisms contained polyphosphate granules and glycogen inclusions, which suggests that they are a type of phosphorus-accumulating organism. Quinones, slot hybridization, and 16S rRNA sequencing analyses showed that the members of the Proteobacteria beta subclass were the most abundant species and were affiliated with the Rhodocyclus-like group. Phylogenetic analysis revealed that the two dominating clones of the beta subclass were closely related to the Rhodocyclus-like group. It was concluded that the coccus-shaped organisms related to the Rhodocyclus-like group within the Proteobacteria beta subclass were the most dominant species believed responsible for biological phosphorus removal in SBR operation with acetate.

Bacteria, Aerobic↗

PubMedDate not supplied

Molecular characterization of anaerobic microbial communities from benzene-degrading sediments under methanogenic conditions.

Anaerobic benzene degradation was confirmed in microbial communities enriched from Baltimore Harbor (Baltimore, MD) sediments under methanogenic conditions. Molecular characterization based on 16S rDNA gene sequences revealed that the strains in the communities were diversely affiliated with such phylogenetic branches as the Bacteroidetes, Euryarchaeota, Firmicutes, and Thermotogae phyla. Of interest was that the majority of the microbial populations detected in these cultures were closely related to the members of dechlorinating microbial communities. Further, some of those species were previously found in naphthalene- or phenanthrene-degrading methanogenic communities. Finally, this result could be used to design targeted isolation strategies for anaerobic benzene-degrading strains under methanogenic conditions.

Bacteria, Anaerobic↗

PubMed1999

Characterization of microbial communities of biofilters by phospholipid fatty acid analysis and rRNA targeted oligonucleotide probes.

The microbial community of a biofilter for waste gas treatment of an animal rendering plant was characterized by the analyses of the phospholipid fatty acids (PLFAs) of the filter material. For these analyses five samples of one filter were taken in intervals between one and two months. The main components of the PLFA profiles were straight chain saturated, monounsaturated and cyclopropyl fatty acids. Terminally branched and 10-methyl branched fatty acids were present in minor amounts. The structure and succession of the microbial community was interpreted by the presence and quantitative changes of diagnostic fatty acids. The stability of diagnostic fatty acids in relation to varying incubation parameters was tested for a number of bacterial isolates from biofilters representing different phylogenetic branches. For two samples, the data from the PLFA-analyses were compared with data obtained by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes specific for the alpha-, beta- and gamma-subclass of the Proteobacteria, the Actinobacteria (Firmicutes with high G+C content) and the Firmicutes with low G+C content. These data indicated a dominating number of Proteobacteria (54% and 35% of DAPI-stained cells), in which the gamma-Proteobacteria represented the main fraction. Actinobacteria were detected in minor amounts, the number of Firmicutes with low G+C content was near the detection limit of the method. About half of the cells detected with a probe specific for Bacteria did not hybridize with the probes specific for the alpha-, beta- and gamma subclass of the Proteobacteria and the two subgroups of the Firmicutes. The results of both methods, the fluorescence in situ hybridization (FISH) and the PLFA analyses corresponded well and were best suited to confirm and complement each other.

Air Pollutants↗

PubMed1998

Molecular phylogenetic identification of the intestinal anaerobic microbial community in the hindgut of the termite, Reticulitermes speratus, without cultivation.

A termite maintains an anaerobic microbial community in its hindgut, which seems to be the minimum size of an anaerobic habitat. This microbial community consists of bacteria and various anaerobic flagellates, and it is established that termites are totally dependent on the microbes for the utilization of their food. The molecular phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy that does not rely on cultivation of the resident microorganisms. Small subunit ribosomal RNA (ssrRNA) genes were directly amplified from the mixed-population DNA of the termite gut by polymerase chain reaction (PCR) and clonally isolated. Most sequenced clones were phylogenetically affiliated with the four major groups of the domain Bacteria: the Proteobacteria group, the Spirochete group, the Bacteroides group, and the Low G + C gram-positive bacteria. The 16S rRNA sequence data show that the majority of the intestinal microflora of the termite consists of new species that are yet to be cultured. The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite (R. speratus) was analyzed without cultivation. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order. The diversity of nitrogen-fixing organisms was also investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the mixed-population DNA of the termite gut and were clonally isolated. The phylogenetic analysis of the nifH amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. The molecular phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of R. speratus was also examined by the same strategy. The whole-cell hybridization experiments indicated that the sequence originated from a large hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that the hypermastigote and trichomonads shared a recent common ancestry.

Animals↗

PubMed2001

Microbial community dynamics and evaluation of bioremediation strategies in oil-impacted salt marsh sediment microcosms.

Microbial community dynamics in wetlands microcosms emended with commercial products (surfactant, a biological agent, and nutrients) designed to enhance bioremediation was followed for 3 months. The effectiveness of enhanced degradation was assessed by determining residual concentrations of individual petroleum hydrocarbons by GC/MS. The size and composition of the sediment microbial community was assessed using a variety of indices, including bacterial plate counts, MPNs, and DNA hybridizations with domain- and group-specific oligonucleotide probes. The addition of inorganic nutrients was the most effective treatment for the enhancement of oil degradation, resulting in marked degradation of petroleum alkanes and a lesser extent of degradation of aromatic oil constituents. The enhanced degradation was associated with increases in the amount of extractable microbial DNA and Streptomyces in the sediment, although not with increased viable counts (plate counts, MPN). Bacteria introduced with one of the proprietary products were still detected in the microcosms after 3 months, but were not a major quantitative constituent of the community. The biological product enhanced oil degradation relative to the control, but to a lesser extent than the nutrient additions alone. In contrast, application of the surfactant to the oil-impacted sediment decreased oil degradation.

Biodegradation, Environmental↗

PubMed2003

Multi-scale variation in spatial heterogeneity for microbial community structure in an eastern Virginia agricultural field.

To better understand the distribution of soil microbial communities at multiple spatial scales, a survey was conducted to examine the spatial organization of community structure in a wheat field in eastern Virginia (USA). Nearly 200 soil samples were collected at a variety of separation distances ranging from 2.5 cm to 11 m. Whole-community DNA was extracted from each sample, and community structure was compared using amplified fragment length polymorphism (AFLP) DNA fingerprinting. Relative similarity was calculated between each pair of samples and compared using geostatistical variogram analysis to study autocorrelation as a function of separation distance. Spatial autocorrelation was found at scales ranging from 30 cm to more than 6 m, depending on the sampling extent considered. In some locations, up to four different correlation length scales were detected. The presence of nested scales of variability suggests that the environmental factors regulating the development of the communities in this soil may operate at different scales. Kriging was used to generate maps of the spatial organization of communities across the plot, and the results demonstrated that bacterial distributions can be highly structured, even within a habitat that appears relatively homogeneous at the plot and field scale. Different subsets of the microbial community were distributed differently across the plot, and this is thought to be due to the variable response of individual populations to spatial heterogeneity associated with soil properties.

Bacteria↗

PubMed2001

Characterization of microbial community in an activated sludge process treating domestic wastewater using quinone profiles.

The dynamics of microbial community structure of activated sludges in a small-scale domestic wastewater treatment process were examined using a novel approach of quinone profiles. The composition and content of quinones in the activated sludges were analyzed monthly over a period of one year. More than 4 types of ubiquinones and 12 types of menaquinones were observed in the activated sludges, with the dominant quinones being ubiquinone (UQ)-8, menaquinone (MK)-7, followed by UQ-10, MK-8 and MK-6. The total quinone contents in the activated sludges varied from 0.93 to 2.68 mumol per gram of particle organic carbon. The molar ratio of ubiquinones to menaquinones (UK/MK) changed from 0.38 to 0.98, indicating that anaerobic bacteria dominated the microbial community of the activated sludges examined. The ratio of UQ/MK varied similar to that of dissolved oxygen in the bulk. The microbial diversity of the activated sludges calculated from the quinone compositions was 13.4-16.8. The diversity of menaquinones was much higher than that of ubiquinones, and increased slightly with increasing temperature. The microorganisms containing menaquinones appear to be sensitive to the change in temperature than those containing ubiquinones.

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↗

PubMed2001

Impact of fumigants on soil microbial communities.

Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity, H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.

Allyl Compounds↗

PubMed1988

Adaptation of aquatic microbial communities to pollutant stress.

The importance of microbial adaptation in the removal of environmental pollutants and in maintaining active microbial communities in impacted ecosystems is discussed using the biodegradation of p-nitrophenol and the volatilization of mercuric mercury as examples. A molecular mechanism of adaptation is suggested by enrichment of mercury resistance (mer) genes in some communities upon exposure to mercury.

Adaptation, Biological↗

PubMed1999

The Distribution of Microbial Communities in Anaerobic and Aerobic Zones of a Shallow Coastal Plain Aquifer.

Randomly amplified polymorphic DNA (RAPD) fingerprinting was used to determine the genetic similarity of whole-community DNA extracts from unattached microorganisms in several groundwater wells. The study site was a shallow coastal plain aquifer on the Eastern Shore of Virginia that contains distinct regions of anaerobic and aerobic groundwater. Several wells in each region were sampled, and principal component and cluster analyses showed a clear separation of the microbial communities from the two chemical zones of the aquifer. Within these zones, there was no relationship between the genetic relatedness of a pair of communities and their spatial separation. Two additional sets of samples were taken at later times, and the same clear separation between communities in the different zones of the aquifer was observed. The specific relationships between wells within each zone changed over time, however, and the magnitude and direction of these changes corresponded to concurrent changes in the groundwater chemistry at each well. Together, these results suggest that local variation in groundwater chemistry can support genetically distinct microbial communities, and that the composition of the microbial communities can follow seasonal fluctuations in groundwater chemistry.

Journal Article↗

PubMed2005

Plasmid donor affects host range of promiscuous IncP-1beta plasmid pB10 in an activated-sludge microbial community.

Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression was repressed in the donors by chromosomal lacI(q). The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the alpha- and gamma-Proteobacteria. The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443, Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.

Conjugation, Genetic↗

PubMed1995

Structural differences of the microbial community in two sites of the Terra Nova Bay (Ross Sea, Antarctica): a statistical analysis.

The microbial community structure was investigated in a one month survey at two sampling stations located in the Terra Nova Bay (Antarctica). The microbial assemblage was studied by direct counts with epifluorescent microscopy (picoplankton and picophytoplankton), fractionated adenosine-triphosphate, lipopolysaccarides and bacterial growth on cultural medium. The results underwent the Spearman rank correlation and the principal component analysis together with other environmental parameters (temperature, salinity, nutrient salts and chlorophyll a) with the aim of revealing differences in the microbial community structure between the in-shore and off-shore sites.

Antarctic Regions↗

PubMed2000

Use of substrate responsive-direct viable counts to visualize naphthalene degrading bacteria in a coal tar-contaminated groundwater microbial community.

A microscopy-based method was developed to distinguish naphthalene-degrading bacteria within the microbial community of a coal tar-contaminated groundwater system. Pure cultures of Pseudomonas putida NCIB 9816-4 were used to develop the substrate responsive-direct viable count (SR-DVC) method. Cells were concentrated on membrane filters, placed on agar plates of Stanier's minimal basal salts media containing antibiotics (nalidixic acid, piromidic acid, pipemidic acid, and cephalexin), and exposed to vapors of naphthalene. Following brief incubation, samples were fixed in 2% formaldehyde and examined by epifluorescent microscopy. Pure cultures displayed the expected cell elongation response to the SR-DVC assay and required a minimum incubation time of 9 h for differentiation of elongated cells. When applied to groundwater samples from the study site, naphthalene responsive cells in the groundwater community were easily distinguished from unresponsive cells and debris (350+/-180 substrate responsive cells/ml, relative to negative controls with no added growth substrate). In an attempt to reduce background counts of elongated bacteria and fungi, the SR-DVC procedure was modified by adding a wash step prior to incubation and a fungal inhibitor, cyclohexamide, to the plates. When groundwater samples were subjected to the modified procedure, only cells in washed samples showed a significant response to naphthalene (150+/-25 cells/ml), indicating the presence of inhibitory substances in the groundwater. Variations in response of the groundwater microbial community to the two SR-DVC procedures suggest that subsurface conditions (microbial and chemical composition) vary temporally. SR-DVC allows the phenotypes of individual naturally occurring cells to be assessed.

Bacteria↗

PubMed2006

Microbial community of biological phosphorus removal process fed with municipal wastewater under different electron acceptor conditions.

The microbial community in a biological phosphorus removal process under different electron acceptor conditions was estimated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) assay and principal-component analysis (PCA). For this purpose, a lab-scale sequencing batch reactor (SBR) fed with municipal wastewater was operated under anaerobic-aerobic, anaerobic-anoxic-aerobic and anaerobic-anoxic conditions. The results of PCR-DGGE targeting the 16S rRNA gene indicated a significant shift in the microbial community with electron acceptor conditions. From the 16S rRNA-based PCA, the microbial shift implies that little oxygen supply caused the deterioration of aerobic bacteria, including aerobic polyphosphate-accumulating organisms (PAOs). Moreover, it also reflects the existence of nitrate-utilizing denitrifiers. On the other hand, although the band patterns of DGGE targeting a functional gene of denitrification (nirS) also showed the microbial shift, the result of PCA differed from that of 16S rRNA-based analysis. There is no conclusive proof that the bacteria represented as the dominant bands detected in the present study are denitrifying-PAOs so far, it should be worthwhile to identify the detected bacteria and to examine their traits as new denitrifying-PAO candidates.

Bacteria↗

PubMed2004

A previously unexposed forest soil microbial community degrades high levels of the pollutant 2,4,6-trichlorophenol.

2,4,6-trichlorophenol (2,4,6-TCP) is a hazardous pollutant that is efficiently degraded by some aerobic soil bacterial isolates under laboratory conditions. The degradation of this pollutant in soils and its effect on the soil microbial community are poorly understood. We report here the ability of a previously unexposed forest soil microbiota to degrade high levels of 2,4,6-TCP and describe the changes in the soil microbial community found by terminal restriction fragment length polymorphism (T-RFLP) analysis. After 30 days of incubation, about 50% degradation of this pollutant was observed in soils amended with 50 to 5,000 ppm of 2,4,6-TCP. The T-RFLP analysis showed that the soil bacterial community was essentially unchanged after exposure to up to 500 ppm of 2,4,6-TCP. However, a significant decrease in richness was found with 2,000 and 5,000 ppm of 2,4,6-TCP, even though the removal of this pollutant remained high. The introduction of Ralstonia eutropha JMP134 or R. eutropha MS1, two efficient 2,4,6-TCP degraders, to this soil did not improve degradation of this pollutant, supporting the significant bioremediation potential of this previously unexposed, endogenous forest soil microbial community.

Bacteria↗