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  • 1
    Electronic Resource
    Electronic Resource
    Whole cell hybridization as a tool to study Frankia populations in root nodules (1997)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 99 (1997), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Molecular methods based on DNA or rRNA hybridization are powerful tools in microbial ecology for the specific detection and enumeration of bacteria unbiased by the limitations of culturability. A promising alternative to the analysis of Frankia populations in root nodules by methods based on rRNA extraction or on DNA extraction followed by the polymerase chain reaction (PCR) is the whole cell hybridization technique. This technique includes the microscopic detection of labeled probes hybridized to specific target sequences on marker molecules such as rRNA in fixed microbial cells. The analysis of uncultured Frankia populations in root nodules can reliably be performed on a subgroup level when digoxigenin-labeled oligonucleotide probes or in vitro transcripts directed against an actinomycetes-specific insertion on the 23S rRNA are used. Digoxigenin-labeled probes are more suitable for in situ detection of Frankia than fluorescent probes since the sensitivity is higher and problems arising from the autofluorescence of cells and plant material are avoided. All these strategies, however, require pretreatments to increase the permeability of vesicles, hyhae and spores.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1997.tb05374.x
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  • 2
    Electronic Resource
    Electronic Resource
    Origins and fate of fungi and bacteria in the gut of Lumbricus terrestris L. studied by image analysis (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 28 (1999), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The effect of the passage through the gut of the earthworm Lumbricus terrestris L. on fungi and bacteria ingested with decomposing leaves of Taraxacum officinale and with soil was quantified using image analysis tools. Both leaf and soil material were labeled with fluorescent latex microbeads to allow a quantification of the food sources in the fore-, mid-, and hindgut of the earthworms. The content of leaf material in the gut varied in a range between 4 and 59% of the total gut content in different earthworms and the different parts of the intestine of individual animals. Filamentous fungi in the gut compartments were found to originate mainly from leaf material (7700±1800 μg (g leaf (dry wt.))−1), however, the major part was disrupted before arriving in the intestine. Remaining hyphae in the foregut with a biomass of up to 900±150 μg (g gut content (dry wt.))−1 were completely digested during passage through the earthworm gut. Spores of fungi were not detected in our studies. Bacterial cell numbers in the gut compartments ranged from 63±5×108 to 327±16×108 (g gut content (dry wt.))−1 and were significantly higher than the numbers found in the soil (50±1×108 cells (g soil (dry wt.))−1). Cell numbers usually increased from fore- to hindgut. This increase was not correlated to contents of organic material and only partially due to a multiplication of bacterial cells. Numbers of dividing cells accounted in total for approximately 12% of all bacteria, increasing significantly from fore- to hindgut, counts were from 10±1×108 to 25±2×108 (g gut content (dry wt.))−1, respectively. Average cell volumes of bacteria calculated from cell size distributions in leaf and soil material differed significantly, being 0.197 and 0.063 μm3, respectively. In the gut compartments, average cell volumes ranged from 0.043 to 0.070 μm3, which may indicate the disruption of large cells originating from the leaves before arriving in the foregut.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.1999.tb00579.x
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  • 3
    Electronic Resource
    Electronic Resource
    Analysis of Frankia populations in three soils devoid of actinorhizal plants (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 28 (1999), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Frankia populations were analyzed in three soils devoid of actinorhizal plants but containing monocultures of birch (Betula pendula Roth), pine (Pinus sylvestris L.) or spruce (Picea abies (L.) Karsten). Bioassays using seedlings of Alnus incana as capture plants resulted in nodulation capacities of 3160±7, 2267±13, and 2747±6 nodulation units g−1 of these soils, respectively. Comparative sequence analysis of an actinomycetes-specific insertion in domain III of the 23S rRNA allowed a grouping of isolates obtained from nodules of the capture plants into three distinct groups of the Alnus host infection group. This separation was confirmed by the analysis of genomic fingerprints of the isolates generated by rep-PCR fingerprinting with the BOX primer. Genomic fingerprints also demonstrated that all isolates differed from each other. The isolates accounted for a significant proportion of the Frankia population in root nodules of the capture plants as shown by in situ hybridization with specific probes. However, only those Frankia strains isolated from soil of the birch stand via Alnus seemed to represent the total Frankia population in root nodules. Nodules induced after inoculation with soil from the pine or spruce stand also contained Frankia populations which were not isolated during this study and which could not be identified by in situ hybridization. Depending upon whether the soil originated from a birch, pine or spruce stand, different Frankia populations were found in the nodules of the capture plants. Because a nested PCR on nucleic acids extracted from these different soils did not indicate differences in the diversity of the total Frankia populations, it was concluded that Frankia populations in nodules of the capture plants represent the fraction of physiologically active, infecting frankiae in the soils rather than the total Frankia population.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.1999.tb00556.x
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  • 4
    Electronic Resource
    Electronic Resource
    Field-scale 13C-labeling of phospholipid fatty acids (PLFA) and dissolved inorganic carbon: tracing acetate assimilation and mineralization in a petroleum hydrocarbon-contaminated aquifer (2002)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 41 (2002), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: This study was conducted to determine the feasibility of labeling phospholipid-derived fatty acids (PLFA) of an active microbial population with a 13C-labeled organic substrate in the denitrifying zone of a petroleum hydrocarbon-contaminated aquifer during a single-well push-pull test. Anoxic test solution was prepared from 500 l of groundwater with addition of 0.5 mM Br− as a conservative tracer, 0.5 mM NO3−, and 0.25 mM [2-13C]acetate. At 4, 23 and 46 h after injection, 1000 l of test solution/groundwater mixture were sequentially extracted. During injection and extraction phases we measured Br−, NO3− and acetate concentrations, characterized the microbial community structure by PLFA and fluorescent in situ hybridization (FISH) analyses, and determined 13C/12C ratios in dissolved inorganic carbon (DIC) and PLFA. Computed first-order rate coefficients were 0.63±0.08 day−1 for NO3− and 0.70±0.05 day−1 for acetate consumption. Significant 13C incorporation in DIC and PLFA was detected as early as 4 h after injection. At 46 h we measured δ13C values of up to 5614‰ in certain PLFA (especially monounsaturated fatty acids), and up to 59.8‰ in extracted DIC. Profiles of enriched PLFA and FISH analysis suggested the presence of active denitrifiers. Our results demonstrate the applicability of 13C labeling of PLFA and DIC in combination with FISH to link microbial structure and activities at the field scale during a push-pull test.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.2002.tb00987.x
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  • 5
    Electronic Resource
    Electronic Resource
    Sulfate-reducing bacterial community response to carbon source amendments in contaminated aquifer microcosms (2002)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 42 (2002), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Microbial sulfate reduction is an important metabolic activity in many reduced habitats. However, little is known about the sulfate-reducing communities inhabiting petroleum hydrocarbon (PHC)-contaminated freshwater aquifer sediments. The purpose of this study was to identify the groups of sulfate-reducing bacteria (SRB) selectively stimulated when sediment from a PHC-contaminated freshwater aquifer was incubated in sulfate-reducing aquifer microcosms that were amended with specific carbon sources (acetate, butyrate, propionate, lactate, and citrate). After 2 months of incubation, the SRB community was characterized using phospholipid fatty acid (PLFA) analysis combined with multivariate statistics as well as fluorescence in situ hybridization (FISH). Molybdate was used to specifically inhibit SRB in separate microcosms to investigate the contribution of non-SRB to carbon source degradation. Results indicated that sulfate reduction in the original sediment was an important process but was limited by the availability of sulfate. Substantially lower amounts of acetate and butyrate were degraded in molybdate treatments as compared to treatments without molybdate, suggesting that SRB were the major bacterial group responsible for carbon source turnover in microcosms. All of the added carbon sources induced changes in the SRB community structure. Members of the genus Desulfobulbus were present but not active in the original sediment but an increase of the fatty acids 15:1ω6c and 17:1ω6c and FISH results showed an enrichment of these bacteria in microcosms amended with propionate or lactate. The appearance of cy17:0 revealed that bacteria affiliated with the Desulfobacteriaceae were responsible for acetate degradation. Desulfovibrio and Desulfotomaculum spp. were not important populations within the SRB community in microcosms because they did not proliferate on carbon sources usually favored by these organisms. Metabolic, PLFA, and FISH results provided information on the SRB community in a PHC-contaminated freshwater environment, which exhibited stimulation patterns similar to other (e.g. marine) environments.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.2002.tb01000.x
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  • 6
    Electronic Resource
    Electronic Resource
    Analysis of bacterial and protozoan communities in an aquifer contaminated with monoaromatic hydrocarbons (1998)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 27 (1998), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Bacterial and protozoan communities were examined in three cores (A, B and C) from an aquifer located at an abandoned refinery near Hünxe, Germany. Cores were removed along a transect bordering a plume containing various monoaromatic hydrocarbons. Monoaromatic hydrocarbons could not be detected in the unsaturated zone in any core but were present in the saturated zones of core C (between 280 and 42 600 μmol kg−1 of core material [dry wt.]) and cores A and B (between 30 and 190 μmol kg−1 of core material [dry wt.]). Xylene isomers accounted for 50–70% of monoaromatic hydrocarbons in all cores. The number of DAPI-stained bacteria was found to increase from the low-contaminated cores A and B (approx. 0.1×108 cells and 0.2×108 cells g−1 of core material [dry wt.], respectively) to the high-contaminated core C (2.4×108 cells g−1 of core material [dry wt.]). The higher bacterial numbers in core C were found to coincide with a higher detection rate obtained by in situ hybridization using probe Eub338 to target the domain Bacteria (13–42% for core C as compared to 3–25% for cores A and B, respectively). Proteobacteria of the δ-subdivision (which includes many sulfate-reducing bacteria) were the most predominant of the groups investigated (7–15% of DAPI-stained bacteria) and were followed by Proteobacteria of the γ- and β-subdivisions (4% and 1% of DAPI-stained bacteria, respectively). The total numbers of protozoa and bacteria determined by direct counting occurred in a ratio of approx. 1:103, which was independent of depth or core examined. Most probable number analysis combined with a subsequent classification of the culturable protozoa revealed nanoflagellates as the major component of the protozoan community. Naked amoebae became increasingly more encysted with depth, except in the high-contaminated core C where vegetative trophozoites were present in the saturated zone. The co-occurrence of bacteria and protozoa in association with high concentrations of monoaromatic hydrocarbons suggests the involvement of trophic interactions in the process of biodegradation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.1998.tb00532.x
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  • 7
    Electronic Resource
    Electronic Resource
    Field-scale isotopic labeling of phospholipid fatty acids from acetate-degrading sulfate-reducing bacteria (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 51 (2005), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Isotopic labeling of biomarker molecules is a technique applied to link microbial community structure with activity. Previously, we successfully labeled phospholipid fatty acids (PLFA) of suspended nitrate-reducing bacteria in an aquifer. However, the application of the method to low energy-yielding processes such as sulfate reduction, and extension of the analysis to attached communities remained to be studied. To test the feasibility of the latter application, an anoxic test solution of 500 l of groundwater with addition of 0.5 mM Br− as a conservative tracer, 1.1 mM SO2−4, and 2.0 mM [2-13C]acetate was injected in the transition zone of a petroleum hydrocarbon-contaminated aquifer where sulfate-reducing and methanogenic conditions prevailed. Thousand liters of test solution/groundwater mixture were extracted in a stepwise fashion after 2–46 h incubation. Computed apparent first-order rate coefficients were 0.31 ± 0.04 day−1 for acetate and 0.34 ± 0.05 day−1 for SO2−4 consumption. The δ13C increased from −71.03‰ to +3352.50‰ in CH4 and from −16.15‰ to +32.13‰ in dissolved inorganic carbon (DIC). A mass balance suggested that 43% of the acetate-derived 13C appeared in DIC and 57% appeared in CH4. Thus, acetate oxidation coupled to sulfate reduction and acetoclastic methanogenesis occurred simultaneously. The δ13C of PLFA increased on average by 27‰ in groundwater samples and 4‰ in sediment samples. Hence, both suspended and attached communities actively degraded acetate. The PLFA labeling patterns and fluorescent in situ hybridization (FISH) analyses of sediment and groundwater samples suggested that the main sulfate-reducing bacteria degrading the acetate were Desulfotomaculum acetoxidans and Desulfobacter sp. in groundwater, and D. acetoxidans in sediment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsec.2004.08.010
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  • 8
    Electronic Resource
    Electronic Resource
    Tracing toluene-assimilating sulfate-reducing bacteria using 13C-incorporation in fatty acids and whole-cell hybridization (2001)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 38 (2001), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Polar lipid-derived fatty acids (PLFA) commonly found in sulfate-reducing bacteria were detected in high abundance in the sediment harvested from a monitoring well of a petroleum-hydrocarbon (PHC)-contaminated aquifer. Aquifer microcosms were incubated under sulfate-reducing conditions with [methyl-14C]toluene to determine the 14C-mass balances and with [methyl-13C]toluene to follow the flow of carbon from toluene into biomarker fatty acids. An aliquot was used to establish an aquifer-derived toluene-degrading sulfate-reducing consortium, which grew well in liquid medium. Whole-cell hybridization using 16S rRNA-targeted oligonucleotide probes specific for different phylogenetic levels within the sulfate-reducing bacteria was applied in order to characterize the sulfate-reducing populations in the original sediment, the aquifer microcosms, and the aquifer-derived consortium. In the aquifer microcosms, the 14C quantification revealed that 61.6% of the [methyl-14C]toluene was mineralized and 2.7% was assimilated. Following [methyl-13C]toluene depletion (〈1 μM), the highest 13C-enrichment was found in PLFA 16:1ω5c. In addition, biomarker fatty acids characteristic for the genera Desulfobacter and Desulfobacula (cy17:0 and 10Me16:0) were also 13C-enriched, contrary to those of other sulfate-reducing genera, e.g. Desulfovibrio and Synthrophobacter (i17:1ω7c), Desulfobulbus and Desulforhabdus (15:1ω6c and 17:1ω6c). Although hybridization detection rates remained low, indicating low bacterial activities, 43% (aquifer sediment) and 30% (aquifer microcosm) of the total active bacteria belonged to the Desulfobacteriaceae thus supporting the PLFA-based results. Desulfobacter-species (42%), which belong to the Desulfobacteriaceae, dominated the community of the consortium. Our study showed that carbon stable isotope analysis in combination with whole-cell hybridization could link toluene degradation in aquifer microcosms to the metabolic activity of the Desulfobacter-like populations. These populations could play an important role in the clean up of aromatic PHC-contaminated aquifers.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.2001.tb00890.x
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