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

Abstract

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 componentswere 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.

Application of bioflocculating property of Pseudomonas aeruginosa strain IASST201 in treatment of oil‐field formation water

“…The prepared bacterial cells were freeze dried for 4 h, subsequently coated with gold‐palladium powder by Sputter Coater (SC‐7625, EMITECH, India) and finally attached on to the microscope supports with silver glue. Scanning electron microscope images were taken at 5 kV using a FE‐SEM (Zeiss, ∑‐Sigma, Germany) . Ninety‐six hours older bacteria were grown upon sterile glass coupons and subjected to atomic force microscope (AFM) for visualizing bacterial cells within biofilm .…”

Activated sludge characterization through microscopy: A review on quantitative image analysis and chemometric techniques

“…The presence of EPS which have a significant influence on the physicochemical properties of microbial aggregates, such as structure, surface charge, flocculation and settling properties has also been studied using CLSM. Thorough analyses of the structural characteristics of AS flocs and spatial distribution of floc components, showing biopolymers, total cells, bacteria, and enzyme distribution, have already been published [115,116]. In spite of these advantages provided by CLSM, there are several disadvantages pointed out by Daims and Wagner [108].…”

Multivariate and Multiscale Approaches for Interpreting the Mechanisms of Nitrous Oxide Emission during Pig Manure–Wheat Straw Aerobic Composting

“…Sectioning was technically difficult due to the fragile structure of the particles. Some previous FISH–CLSM research of sludge flocs and mangrove roots did not adopt sectioning. − Szilveszter et al indicated that the CLSM images of sludge flocs without sectioning were similar to those after being sectioned (5 μm), and fluorescent probes penetrated well into the uncut flocs. The reason might be that, unlike biological tissues that are relatively dense and constitute diffusion barriers hindering the penetration of probes, the loose structure of sludge flocs would allow better penetration even though they were not sectioned.…”