scholarly journals Abundance and Phylogenetic Affiliation of Iron Reducers in Activated Sludge as Assessed by Fluorescence In Situ Hybridization and Microautoradiography

2002 ◽  
Vol 68 (9) ◽  
pp. 4629-4636 ◽  
Author(s):  
Jeppe Lund Nielsen ◽  
Stefan Juretschko ◽  
Michael Wagner ◽  
Per Halkjær Nielsen
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Fluorescence In Situ Hybridization ◽  
Most Probable Number ◽  
Probable Number ◽  
Iron Reducing Bacteria ◽  
Reducing Conditions ◽  
Phylogenetic Affiliation ◽  
Reducing Bacteria

ABSTRACT Microautoradiography (MAR) was used to enumerate acetate-consuming bacteria under Fe(III)-reducing conditions in activated sludge. This population is believed to consist of dissimilatory iron-reducing bacteria, because the applied incubation conditions and the use of specific inhibitors excluded consumption of radiolabeled acetate by other physiological groups such as sulfate reducers. By use of this approach, dissimilatory iron reducers were found in a concentration of 1.1 × 108 cells per ml, corresponding to approximately 3% of the total cell count as determined by DAPI (4′,6′-diamino-2-phenylindoledihydrochloride-dilactate) staining. The MAR enumeration method was compared to the traditional most-probable-number (MPN) method (FeOOH-MPN) and a modified MPN method, which contains Ferrozine directly within the MPN dilutions to determine the production of small amounts of ferrous iron (Ferrozine-MPN). The Ferrozine-MPN method yielded values 6 to 10 times higher than those obtained by the FeOOH-MPN method. Nevertheless, the MAR approach yielded counts that were 100 to 1,000 times higher than those obtained by the Ferrozine-MPN method. Specific in situ Fe(III) reduction rates per cell (enumerated by the MAR method) were calculated and found to be comparable to the respective rates for pure cultures of dissimilatory iron-reducing bacteria, suggesting that the new MAR method is most reliable. A combination of MAR and fluorescence in situ hybridization was used for phylogenetic characterization of the putative iron-reducing bacteria. All activated-sludge cells able to consume acetate under iron-reducing conditions were targeted by the bacterial oligonucleotide probe EUB338. Around 20% were identified as gamma Proteobacteria, and 10% were assigned to the delta subclass of Proteobacteria.

scholarly journals Effect of Signal Compounds and Incubation Conditions on the Culturability of Freshwater Bacterioplankton

2003 ◽  
Vol 69 (4) ◽  
pp. 1980-1989 ◽  
Author(s):  
Alke Bruns ◽  
Ulrich Nübel ◽  
Heribert Cypionka ◽  
Jörg Overmann
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Eutrophic Lake ◽  
Homoserine Lactone ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Probable Number ◽  
Culture Independent ◽  
Effective Signal

ABSTRACT The effect of signal compounds and of different incubation conditions on the culturability (i.e., the fraction of all cells capable of growth) of natural bacterioplankton from the eutrophic lake Zwischenahner Meer was investigated over a period of 20 months. Numbers of growing cells were determined by the most-probable-number technique in liquid media containing low concentrations (10 μM) of the signal compounds N-(oxohexanoyl)-dl-homoserine lactone, N-(butyryl)-dl-homoserine lactone, cyclic AMP (cAMP), or ATP. cAMP was the most effective signal compound, leading to significantly increased cultivation efficiencies of up to 10% of the total bacterial counts. Microautoradiography with [2,8-3H]cAMP, combined with fluorescence in situ hybridization, demonstrated that cAMP was taken up by 18% of all cells. The bacterial cAMP uptake systems had a very low Km value of ≤1 nM. Analysis of the cultured bacteria by 16S rRNA gene fingerprinting showed that different bacterial phylotypes were recovered in the presence and in the absence of cAMP. Consequently, the addition of cAMP caused a stimulation of otherwise nonculturable bacteria. Phylogenetically different bacteria were also recovered at different temperatures and oxygen partial pressures. Throughout the study period, mainly members of the β-subclass of the Proteobacteria were cultivated. In addition, some members of the Actinomycetales were enriched. Quantification by culture-independent fluorescence in situ hybridization demonstrated that β-Proteobacteria and Actinomycetales also dominated the natural bacterioplankton assemblage. Sequence comparison revealed that two members of the Actinomycetales which reached high numbers in the natural bacterioplankton assemblage could actually be enriched by our cultivation approach.

scholarly journals Enumeration and Detection of Anaerobic Ferrous Iron-Oxidizing, Nitrate-Reducing Bacteria from Diverse European Sediments

1998 ◽  
Vol 64 (12) ◽  
pp. 4846-4856 ◽  
Author(s):  
Kristina L. Straub ◽  
Berit E. E. Buchholz-Cleven
Keyword(s):  
In Situ Hybridization ◽  
Ferrous Iron ◽  
Anoxygenic Phototrophic Bacteria ◽  
Microbial Oxidation ◽  
Probable Number ◽  
Sediment Samples ◽  
Microbiological Methods ◽  
Reducing Bacteria ◽  
Nitrate Reducing Bacteria

ABSTRACT Anaerobic, nitrate-dependent microbial oxidation of ferrous iron was recently recognized as a new type of metabolism. In order to study the occurrence of three novel groups of ferrous iron-oxidizing, nitrate-reducing bacteria (represented by strains BrG1, BrG2, and BrG3), 16S rRNA-targeted oligonucleotide probes were developed. In pure-culture experiments, these probes were shown to be suitable for fluorescent in situ hybridization, as well as for hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns. However, neither enumeration by in situ hybridization nor detection by the DGGE-hybridization approach was feasible with sediment samples. Therefore, the DGGE-hybridization approach was combined with microbiological methods. Freshwater sediment samples from different European locations were used for enrichment cultures and most-probable-number (MPN) determinations. Bacteria with the ability to oxidize ferrous iron under nitrate-reducing conditions were detected in all of the sediment samples investigated. At least one of the previously described types of bacteria was detected in each enrichment culture. MPN studies showed that sediments contained from 1 × 105 to 5 × 108 ferrous iron-oxidizing, nitrate-reducing bacteria per g (dry weight) of sediment, which accounted for at most 0.8% of the nitrate-reducing bacteria growing with acetate. Type BrG1, BrG2, and BrG3 bacteria accounted for an even smaller fraction (0.2% or less) of the ferrous iron-oxidizing, nitrate-reducing community. The DGGE patterns of MPN cultures suggested that more organisms than those isolated thus far are able to oxidize ferrous iron with nitrate. A comparison showed that among the anoxygenic phototrophic bacteria, organisms that have the ability to oxidize ferrous iron also account for only a minor fraction of the population.

The in situ physiology of Nostocoida limicola II, a filamentous bacterial morphotype in bulking activated sludge, using fluorescence in situ hybridization and microautoradiography

2006 ◽  
Vol 54 (1) ◽  
pp. 47-53 ◽  
Author(s):  
E.M. Seviour ◽  
K. Eales ◽  
L. Izzard ◽  
M. Beer ◽  
E.L. Carr ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Fluorescence In Situ Hybridization ◽  
Filamentous Bacterium ◽  
Anaerobic Conditions ◽  
Pure Cultures

The in situ physiology of the actinobacterial bulking and foaming filamentous bacterium “Nostocoida limicola” II was studied by fluorescence in situ hybridization/microautoradiography. Substrate assimilation patterns of pure cultures of this bacterium were different to those seen in activated sludge biomass samples. There was no evidence to suggest that “N. limicola” II preferred hydrophobic substrates, but evidence was produced to support the view that it is metabolically active under anaerobic conditions in activated sludge.

scholarly journals Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

2008 ◽  
Vol 74 (16) ◽  
pp. 5068-5077 ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
L. Safak Yilmaz ◽  
Daniel R. Noguera ◽  
Holger Daims ◽  
Michael Wagner
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Activated Sludge ◽  
Fluorescence In Situ Hybridization ◽  
Oligonucleotide Probes ◽  
Pure Cultures ◽  
Technical Modifications ◽  
Catalyzed Reporter Deposition ◽  
Card Fish

ABSTRACT Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).

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