Using respirometric techniques and fluorescent in situ hybridization to evaluate the heterotrophic active biomass in activated sludge

2007 ◽  
Vol 98 (3) ◽  
pp. 561-568 ◽  
Author(s):  
A. Ismail ◽  
M.C. Wentzel ◽  
F. Bux
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Active Biomass

scholarly journals Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography

2005 ◽  
Vol 71 (12) ◽  
pp. 8683-8691 ◽  
Author(s):  
Maneesha P. Ginige ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridization ◽  
Stable Isotope ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Batch Reactor ◽  
External Source ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
The Impact

ABSTRACT The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.

scholarly journals Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology

1999 ◽  
Vol 65 (3) ◽  
pp. 1289-1297 ◽  
Author(s):  
Natuscka Lee ◽  
Per Halkjær Nielsen ◽  
Kjær Holm Andreasen ◽  
Stefan Juretschko ◽  
Jeppe Lund Nielsen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Microbial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Aerobic Incubation

ABSTRACT A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli andHerpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.

scholarly journals Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry

2007 ◽  
Vol 73 (6) ◽  
pp. 2020-2023 ◽  
Author(s):  
Jeremy Lenaerts ◽  
Hilary M. Lappin-Scott ◽  
Jonathan Porter
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Activated Sludge ◽  
River Water ◽  
Environmental Samples ◽  
Fluorescent In Situ Hybridization ◽  
Molecular Beacons ◽  
Target Cells ◽  
Linear Dna

ABSTRACT Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry.

Successful and unsuccessful bioaugmentation experiments monitored by fluorescent in situ hybridization

2000 ◽  
Vol 41 (12) ◽  
pp. 61-68 ◽  
Author(s):  
T. Bouchez ◽  
D. Patureau ◽  
P. Dabert ◽  
M. Wagner ◽  
J. P. Delgenès ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Bacterial Strain ◽  
Fluorescent In Situ Hybridization ◽  
Nitrogen Loss ◽  
Denitrifying Bacteria ◽  
Alginate Beads ◽  
Sludge Flocs ◽  
Temporary Protection

Two nitrifying reactors were operated under the same conditions except that one was twice inoculated with the aerobic denitrifying bacteria Microvirgula aerodenitrificans. The first bioaugmentation induced a transient nitrogen loss. Fluorescent in situ hybridization revealed that the bioaugmented bacteria had been rapidly eaten by protozoa. The second massive inoculation unbalanced the ecosystem and resulted in an overgrowth of protozoa and perturbations of nitrification, whereas both parameters remained stable in the non bioaugmented reactor. To enhance the incorporation of the added bacteria to indigenous flocs, two strategies were then tested. First, coagulating and flocculating substances were added to the reactor just after bioaugmentation and second, the bacteria were embedded in alginate beads before inoculation. The latter strategy gave the best results. After break-up of the beads, alginate fragments, containing microcolonies of M. aerodenitrificans, were found to be incorporated into the existing flocs. Alginate beads offer a temporary protection against grazing and favor the adhesion of the exogenous bacterial microcolonies to the existing flocs. These beads therefore constitute a suitable bioaugmentation vector to incorporate a bacterial strain into activated sludge flocs.

scholarly journals Quantitative Use of Fluorescent In Situ Hybridization To Examine Relationships between Mycolic Acid-Containing Actinomycetes and Foaming in Activated Sludge Plants

2000 ◽  
Vol 66 (3) ◽  
pp. 1158-1166 ◽  
Author(s):  
Russell J. Davenport ◽  
Thomas P. Curtis ◽  
Michael Goodfellow ◽  
Fiona M. Stainsby ◽  
Marc Bingley
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Threshold Value ◽  
Mycolic Acid ◽  
Foam Formation ◽  
Quantification Method ◽  
Nested Analysis ◽  
The Relationship

ABSTRACT The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid-containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods (P << 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m3completely mixed activated sludge plant. Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata. We tentatively estimated that the threshold foaming concentration of mycolata was about 2 × 106 cells ml−1 or 4 × 1012 cells m−2. We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.

Detecting structural and functional differences in activated sludge bacterial communities originating from laboratory treatment of elementally and totally chlorine-free bleaching effluents

2002 ◽  
Vol 48 (3) ◽  
pp. 245-255 ◽  
Author(s):  
Ann H Mounteer ◽  
Flávia ML Passos ◽  
Arnaldo Chaer Borges ◽  
Daison Olzany Silva
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Bacterial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Gc Content ◽  
Substrate Utilization ◽  
Taxonomic Structure ◽  
Gram Positive ◽  
Utilization Patterns

The ability to differentiate functional and structural diversity of bacterial communities present in activated sludges adapted to elementally (ECF) and totally (TCF) chlorine-free bleaching effluents was evaluated. Community function was evaluated through substrate utilization patterns in BiologGN®microplates, and taxonomic structure was evaluated by fluorescent in situ hybridization using probes targeting the Eubacteria; the alpha, beta, and gamma subclasses of the Proteobacteria; and gram-positive bacteria with high GC content. Over 6-week sampling periods, ECF- and TCF-adapted sludge bacterial communities presented reproducible substrate utilization patterns that through principal components (PCs) analysis, separated the ECF samples from the TCF samples. Application of the fluorescent in situ hybridization technique was complicated by the intense autofluorescence of the bleaching effluent sludge samples that interfered with detection of specific hybridization signals. The most notable difference in community structure detected using the chosen set of probes was the relatively greater proportion of cells of the alpha subclass in TCF sludge (27%) than in ECF sludge (6%). Nonspecific hybridization with beta and gamma probes was relatively high, but both sludges appeared to have similar proportions of cells of the beta (20–22%) and gamma (11–12%) subclasses. The two sludges presented relatively few gram-positive cells with high GC content (<0.2% of eubacterial counts). Differences in both metabolic potential and taxonomic structure of the microbial communities in the ECF- and TCF-activated sludges were detected. The kinetics of the development of these differences in treatment plants and their relationships with treatment efficiency and production process conditions should now be evaluated.Key words: activated sludge, bleaching effluents, Biolog, in situ hybridization.

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