In situ imaging of biopolymers and extracellular enzymes in activated sludge flocs of a municipal wastewater treatment plant

BACKGROUND The aim of this study was to investigate biopolymers and extracellular enzymes in whole activated sludge flocs originating from a full‐scale wastewater treatment plant, by means of confocal laser scanning microscopy (CLSM) techniques. Both sectioned and whole activated sludge floc samples...

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Published inJournal of chemical technology and biotechnology (1986) Vol. 88; no. 7; pp. 1295 - 1304
Main Authors Szilveszter, Szabolcs, Ráduly, Botond, Ábrahám, Beáta, Lányi, Szabolcs
Format Journal Article
LanguageEnglish
Published Chichester, UK John Wiley & Sons, Ltd 01.07.2013
Wiley
Wiley Subscription Services, Inc
Subjects
Activated sludge
activated sludge floc
Applied sciences
Biopolymers
Chemical engineering
CLSM imaging
Exact sciences and technology
extracellular enzymes
fluorochromes
General purification processes
immunostaining
Lipids
Municipal wastewater
Pollution
Sewerage works: sewers, sewage treatment plants, outfalls
Spatial distribution
Wastewater treatment plants
Wastewaters
Water treatment and pollution
Activated sludge
extracellular enzymes
In situ
fluorochromes
immunostaining
Sewage treatment plant
Spatial distribution
Imaging
Laser
CLSM imaging
Bacteria
Urban waste water
biopolymers
activated sludge floc
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Summary:BACKGROUND The aim of this study was to investigate biopolymers and extracellular enzymes in whole activated sludge flocs originating from a full‐scale wastewater treatment plant, by means of confocal laser scanning microscopy (CLSM) techniques. Both sectioned and whole activated sludge floc samples have been stained using specific fluorochromes and immunostains in order to visualize the structural and functional characteristics of these bioaggregates. Samples were stained for visualization of lipids, sugars, total cells, esterase enzyme producing bacteria, and for β‐glycosidase (EC3.2.1.21), alkaline phosphatase (EC3.1.3.1) and trypsin (EC3.4.21.4) enzymes. Simultaneous staining schemes were applied and immunostaining specificity tests were performed. RESULTS By the CLSM imaging and the 3‐D reconstruction of the stained flocs the distributions of the targeted floc components were successfully assessed. The immunostain specificity controls gave satisfactory results in each case. The reflected total cells‐to‐enzymes ratio was repeatedly higher for the sectioned samples. CONCLUSIONS The CLSM imaging of whole sludge flocs delivers valuable information on the spatial distribution of the floc build‐up materials, with a satisfactory visualization accuracy of the individual components. The images of whole and sectioned samples showed similar distributions of the floc components, but the consistent differences revealed in cells‐to‐enzymes ratios call for further research.© 2012 Society of Chemical Industry
Bibliography:CLSM images of an activated sludge floc stained without sectioning for EPS and bacteria visualization: a) maximum intensity projections of the confocal slices of the sample stained with three different dyes (red-lipids, green-esterase producing bacteria, blue-α-polysaccharides) and the combined image; b) transmission image of the studied specimen; c) 3-D reconstruction of the floc.histograms showing the distribution of lipids,esterase-producing bacteria and α-polysaccharides within the sample presented on Fig S1.CLSM images of antibody and dye stained samples without sectioning for visualization of alkaline phosphatase (ab60317, red), α-polysaccharides (ConA488, green), total cells (Syto, blue) and the combined imageCLSM images of antibody and dye stained samples without sectioning for visualization of Trypsin (ab60317, red), α-polysaccharides (ConA488, green), total cells (Syto, blue) and the combined imageCLSM images of antibody and dye stained samples without sectioning for control purposes:Red- antibody staining specificity control (secondary antibody alone); green - α-polysaccharides (ConA488), blue - total cells (Syto).CLSM images of a whole activated sludge sample stained for the simultaneous visualization of alkaline phosphatase (ab60317, red) and β-glycosidaseenzymes (ab60313, green), for total cells (Syto63, blue) and the combined image of the stained sample.CLSM images of a whole activated sludge sample stained for the simultaneous visualization of trypsin (ab60317, red) and β-glycosidase enzymes (ab60313, green), total cells (Syto63, blue) and the merged image of the stained sample.The CLSM images of antibody and dye stained 5 µm thick sectioned sample for the visualization of β-polysaccharides (calcofluor white, blue), total cells (Syto 63, yellow), alkaline phosphatase (ab60317, red) and β-glycosidase (ab60313, green)The CLSM images of antibody and dye stained 5 µm thick sectioned sample for the visualization of β-polysaccharides (calcofluor white, blue), total cells (Syto 63, yellow), trypsin (ab60317, red) and β-glycosidase (ab60313, green)3D reconstruction of an activated sludge floc stained and imaged without sectioning. Link to the video: http://www.youtube.com/watch?v=_ywezMcMzIc
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ArticleID:JCTB3975
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ISSN:0268-2575
1097-4660
DOI:10.1002/jctb.3975