Novel Nitrospira-like bacteria as dominant nitrite-oxidizers in biofilms from wastewater treatment plants: diversity and in situ physiology

2000 ◽  
Vol 41 (4-5) ◽  
pp. 85-90 ◽  
Author(s):  
H. Daims ◽  
P.H. Nielsen ◽  
J.L. Nielsen ◽  
S. Juretschko ◽  
M. Wagner
Keyword(s):  
Wastewater Treatment ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Wastewater Treatment Plants ◽  
Combined Application ◽  
Fundamental Understanding ◽  
Unculturable Bacteria ◽  
Rrna Sequences

The frequency and distribution of putatively nitrite-oxidizing, Nitrospira- like bacteria in nitrifying biofilms from two reactors receiving wastewater with different ammonia and salt concentrations were observed by fluorescent in situ hybridization. For this purpose, new 16S rRNA-directed oligonucleotide probes targeting the bacterial phylum Nitrospira and the three main lineages within this phylum were developed and evaluated. The diversity of Nitrospira-like bacteria in the reactors was additionally investigated by retrieval and comparative analysis of full 16S rRNA sequences from the biofilms. We found that, despite of the differences in the influent composition, Nitrospira-like bacteria form dominant populations in both reactors. In addition, first insights into the physiology of these still unculturable bacteria were obtained by the incubation of active biofilm samples with radioactively labeled substrates followed by the combined application of fluorescent in situ hybridization and microautoradiography. The results are discussed in consideration of the frequently observed dominance of Nitrospira-like bacteria in nitrifying bioreactors. Consequently, high priority should be assigned to future studies on the ecology and physiology of these organisms in order to increase our fundamental understanding of nitrogen cycling and to enable knowledge-driven future improvements of nitrifying wastewater treatment plants.

scholarly journals Specific detection of viable Listeria monocytogenes in Spanish wastewater treatment plants by Fluorescent In Situ Hybridization and PCR

2011 ◽  
Vol 45 (15) ◽  
pp. 4634-4640 ◽  
Author(s):  
Yolanda Moreno ◽  
Lorena Ballesteros ◽  
Jorge García-Hernández ◽  
Paula Santiago ◽  
Ana González ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
In Situ Hybridization ◽  
Listeria Monocytogenes ◽  
Fluorescent In Situ Hybridization ◽  
Wastewater Treatment Plants ◽  
Specific Detection

scholarly journals Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ

1999 ◽  
Vol 65 (4) ◽  
pp. 1746-1752 ◽  
Author(s):  
Cleber C. Ouverney ◽  
Jed A. Fuhrman
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Microbial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Amino Acid Uptake ◽  
Cell Types ◽  
Black Box ◽  
Acid Uptake ◽  
Substrate Uptake

ABSTRACT We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class Proteobacteria(α-Proteobacteria) and of the Cytophaga-Flavobacteriumgroup obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.

scholarly journals In Situ Characterization ofNitrospira-Like Nitrite-Oxidizing Bacteria Active in Wastewater Treatment Plants

2001 ◽  
Vol 67 (11) ◽  
pp. 5273-5284 ◽  
Author(s):  
Holger Daims ◽  
Jeppe L. Nielsen ◽  
Per H. Nielsen ◽  
Karl-Heinz Schleifer ◽  
Michael Wagner
Keyword(s):  
Wastewater Treatment ◽  
16S Rrna ◽  
Laser Scanning ◽  
Wastewater Treatment Plants ◽  
Carbon Sources ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rrna Gene ◽  
Phylogenetic Affiliation

ABSTRACT Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of theNitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genusNitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates ofNitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospiramicrocolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources byNitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, theNitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO3 − or as CO2) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by theNitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.

16S rRNA fluorescent in situ hybridization of human colon and terminal ileum biopsies: A mucus-adherent bacterial flora does not exist

2001 ◽  
Vol 120 (5) ◽  
pp. A706
Author(s):  
Laurens A. Van der Waaij ◽  
Hermie J.M. Harmsen ◽  
Mohsen Madjipour ◽  
Frans G.M. Kroese ◽  
Hendrik M. Van Dullemen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Terminal Ileum ◽  
Fluorescent In Situ Hybridization ◽  
Bacterial Flora ◽  
Human Colon

Presence of Rhodocyclus in a full-scale wastewater treatment plant and their participation in enhanced biological phosphorus removal

2002 ◽  
Vol 46 (1-2) ◽  
pp. 123-128 ◽  
Author(s):  
J.L. Zilles ◽  
C.-H. Hung ◽  
D.R. Noguera
Keyword(s):  
Wastewater Treatment ◽  
Phosphorus Removal ◽  
Fluorescent In Situ Hybridization ◽  
Wastewater Treatment Plants ◽  
Full Scale ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Polyphosphate Accumulating Organisms ◽  
Biological Phosphorus

The objective of this research was to assess the relevance of organisms related to Rhodocyclus in enhanced biological phosphorus removal in full-scale wastewater treatment plants. The presence of these organisms in full-scale plants was first confirmed by fluorescent in situ hybridization. To address which organisms were involved in phosphorus removal, a method was developed which selected polyphosphate-accumulating organisms from activated sludge samples by DAPI staining and flow cytometry. Sorted samples were characterized using fluorescent in situ hybridization. The results of these analyses confirmed the presence of organisms related to Rhodocyclus in full-scale wastewater treatment plants and supported the involvement of these organisms in enhanced biological phosphorus removal. However, a significant fraction of the polyphosphate-accumulating organisms were not related to Rhodocyclus.

scholarly journals Feasibility of Transferring Fluorescent In Situ Hybridization Probes to an 18S rRNA Gene Phylochip and Mapping of Signal Intensities

2008 ◽  
Vol 74 (9) ◽  
pp. 2814-2821 ◽  
Author(s):  
Katja Metfies ◽  
Linda K. Medlin
Keyword(s):  
In Situ Hybridization ◽  
Secondary Structure ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Molecule ◽  
The 16S Rrna Gene

ABSTRACT DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.

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