Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes

1996 ◽  
Vol 32 (2) ◽  
Author(s):  
P. K�mpfer ◽  
R. Erhart ◽  
C. Beimfohr ◽  
J. B�hringer ◽  
M. Wagner ◽  
...  
1998 ◽  
Vol 64 (2) ◽  
pp. 496-503 ◽  
Author(s):  
Lorenz Adrian ◽  
Werner Manz ◽  
Ulrich Szewzyk ◽  
Helmut Görisch

ABSTRACT A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceaeand the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO2, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.


2008 ◽  
Vol 74 (16) ◽  
pp. 5068-5077 ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
L. Safak Yilmaz ◽  
Daniel R. Noguera ◽  
Holger Daims ◽  
Michael Wagner

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).


2001 ◽  
Vol 43 (6) ◽  
pp. 105-112 ◽  
Author(s):  
D. J. Oosthuizen ◽  
T. E. Cloete

Not all phosphorus removed in activated sludge systems can be accounted for by polyphosphate accumulating organisms (PAO). A method for the qualitative and quantitative in situ characterization of PAO cell clusters and closely associated extracellular polysaccharides (EPS) is described. X-ray microanalysis was performed on samples from four activated sludge plants situated in Pretoria, South Africa. Analyses were done by means of Scanning Electron Microscopy (SEM) combined with Energy Dispersive Spectrometry (EDS). Cell clusters with associated EPS on average contained between 57 and 59% phosphorus, while EPS alone contained on average between 23 and 30% phosphorus. Results suggest that phosphorus removal in activated sludge might be due not only to PAO, but also by EPS acting as a phosphorus reservoir. Extraction of EPS from two different activated sludge plants yielded different amounts of EPS, which, in combination with SEM-EDS, may shed light on different phosphate uptake abilities of different activated sludges.


2003 ◽  
Vol 69 (1) ◽  
pp. 177-185 ◽  
Author(s):  
Andrew J. McBain ◽  
Robert G. Bartolo ◽  
Carl E. Catrenich ◽  
Duane Charbonneau ◽  
Ruth G. Ledder ◽  
...  

ABSTRACT We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log10 cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log10 cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan Colpoda maupasi, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.


PLoS ONE ◽  
2015 ◽  
Vol 10 (9) ◽  
pp. e0136424 ◽  
Author(s):  
Simon Jon McIlroy ◽  
Takanori Awata ◽  
Marta Nierychlo ◽  
Mads Albertsen ◽  
Tomonori Kindaichi ◽  
...  

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