Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

2007 ◽  
Vol 53 (5) ◽  
pp. 559-571 ◽  
Author(s):  
Amy J. Pogue ◽  
Kimberley A. Gilbride
Keyword(s):  
Activated Sludge ◽  
Laser Scanning ◽  
Ammonia Oxidation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Batch Reactors ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrate Accumulation ◽  
Protozoan Grazing ◽  
Nitrite Nitrate

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.

scholarly journals Impact of Protozoan Grazing on Nitrification and the Ammonia-And-Nitrite-Oxidizing Bacterial Communities in Activated Sludge

2021 ◽  
Author(s):  
Amy Jean Pogue
Keyword(s):  
Activated Sludge ◽  
Ammonia Oxidation ◽  
Heterotrophic Bacteria ◽  
Grazing Pressure ◽  
Batch Reactors ◽  
Protozoan Grazing ◽  
Nitrite Oxidizing Bacteria ◽  
Nitrogen Concentrations ◽  
Nitrite Nitrate

The effect of protozoan grazing on nitrification rates under different conditions was examined. The spatial distribution of ammonia -and nitrite- oxidizing bacteria (AOB and NOB) in activated sludge was also examined using FISH/CSLM. Batch reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations and bacterial numbers in the presence and absence of cycloheximide, a protozoan inhibitor. In the absence of protozoan grazing, rates of nitrification were lower than in batches with protozoa. Spatially, both AOB and NOB were found clustered within the floc and neither inhibiting the protozoa or inhibiting ammonia oxidation appeared to lower the amount of AOB and NOB present or their position. These results suggest that a reduction in protozoan grazing pressure allowed the heterotrophic bacteria to proliferate which caused a corresponding decrease in the rate of nitrification. These results suggest that AOB and NOB are less active in the absence of protozoa and indicates the role of protozoa in the cycling of nitrogen.

scholarly journals Impact of Protozoan Grazing on Nitrification and the Ammonia-And-Nitrite-Oxidizing Bacterial Communities in Activated Sludge

2021 ◽  
Author(s):  
Amy Jean Pogue
Keyword(s):  
Activated Sludge ◽  
Ammonia Oxidation ◽  
Heterotrophic Bacteria ◽  
Grazing Pressure ◽  
Batch Reactors ◽  
Protozoan Grazing ◽  
Nitrite Oxidizing Bacteria ◽  
Nitrogen Concentrations ◽  
Nitrite Nitrate

The effect of protozoan grazing on nitrification rates under different conditions was examined. The spatial distribution of ammonia -and nitrite- oxidizing bacteria (AOB and NOB) in activated sludge was also examined using FISH/CSLM. Batch reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations and bacterial numbers in the presence and absence of cycloheximide, a protozoan inhibitor. In the absence of protozoan grazing, rates of nitrification were lower than in batches with protozoa. Spatially, both AOB and NOB were found clustered within the floc and neither inhibiting the protozoa or inhibiting ammonia oxidation appeared to lower the amount of AOB and NOB present or their position. These results suggest that a reduction in protozoan grazing pressure allowed the heterotrophic bacteria to proliferate which caused a corresponding decrease in the rate of nitrification. These results suggest that AOB and NOB are less active in the absence of protozoa and indicates the role of protozoa in the cycling of nitrogen.

ACTIVATED SLUDGE FLOC CHARACTERIZATION BY CONFOCAL LASER SCANNING MICROSCOPY

2012 ◽  
Vol 11 (3) ◽  
pp. 669-674 ◽  
Author(s):  
Szabolcs Szilveszter ◽  
Botond Raduly ◽  
Szilard Bucs ◽  
Beata Abraham ◽  
Szabolcs Lanyi ◽  
...  
Keyword(s):  
Activated Sludge ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

Nitrification in activated sludge batch reactors is linked to protozoan grazing of the bacterial population

2005 ◽  
Vol 51 (9) ◽  
pp. 791-799 ◽  
Author(s):  
Penny Petropoulos ◽  
Kimberley A Gilbride
Keyword(s):  
Activated Sludge ◽  
Bacterial Population ◽  
Grazing Pressure ◽  
Nitrification Rate ◽  
Batch Reactors ◽  
Ammonia Oxidizing Bacteria ◽  
Bacterial Concentration ◽  
Batch Cultures ◽  
Protozoan Grazing ◽  
Swimming Bacteria

Protozoa feed upon free-swimming bacteria and suspended particles inducing flocculation and increasing the turnover rate of nutrients in complex mixed communities. In this study, the effect of protozoan grazing on nitrification was examined in activated sludge in batch cultures maintained over a 14-day period. A reduction in the protozoan grazing pressure was accomplished by using either a dilution series or the protozoan inhibitor cycloheximide. As the dilutions increased, the nitrification rate showed a decline, suggesting that a reduction in protozoan or bacterial concentration may cause a decrease in nitrification potential. In the presence of cycloheximide, where the bacterial concentration was not altered, the rates of production of ammonia, nitrite, and nitrate all were significantly lower in the absence of active protozoans. These results suggest that a reduction in the number or activity of the protozoans reduces nitrification, possibly by limiting the availability of nutrients for slow-growing ammonia and nitrite oxidizers through excretion products. Furthermore, the ability of protozoans to groom the heterotrophic bacterial population in such systems may also play a role in reducing interspecies competition for nitrification substrates and thereby augment nitrification rates.Key words: nitrification, activated sludge, protozoan grazing, ammonia-oxidizing bacteria, cycloheximide.

scholarly journals Microbial Composition and Structure of Aerobic Granular Sewage Biofilms

2007 ◽  
Vol 73 (19) ◽  
pp. 6233-6240 ◽  
Author(s):  
S. D. Weber ◽  
W. Ludwig ◽  
K.-H. Schleifer ◽  
J. Fried
Keyword(s):  
Activated Sludge ◽  
Laser Scanning ◽  
Extracellular Polymeric Substances ◽  
Phase 1 ◽  
Simultaneous Detection ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microbial Composition ◽  
Granule Formation ◽  
Biofilm Development

ABSTRACT Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.

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.

Experimental and simulation analysis of community structure of nitrifying bacteria in a membrane-aerated biofilm

2007 ◽  
Vol 55 (8-9) ◽  
pp. 283-290 ◽  
Author(s):  
S. Matsumoto ◽  
A. Terada ◽  
Y. Aoi ◽  
S. Tsuneda ◽  
E. Alpkvist ◽  
...  
Keyword(s):  
Cluster Size ◽  
Laser Scanning ◽  
Simulation Analysis ◽  
Current Model ◽  
Membrane Surface ◽  
Laser Scanning Microscopy ◽  
Nitrifying Bacteria ◽  
Confocal Laser ◽  
Ammonia Oxidizing Bacteria ◽  
Microelectrode Measurements

Until now, only few attempts have been made to assess biofilm models simulating microenvironments in a biofilm. As a first step, we compare the microenvironment observed in a membrane aerated biofilm (MAB) to that derived from a two-dimensional computational model with individual ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) embedded in a continuum EPS matrix. Gradients of oxygen were determined by means of microelectrodes. The change in nitrifying bacterial populations with the biofilm depth was quantified using fluorescence in situ hybridization (FISH) in combination with a confocal laser scanning microscopy (CLSM). Microelectrode measurements revealed that oxic and anoxic or anaerobic regions exist within the MAB. The oxygen profile predicted by the model showed good agreement with that obtained by microelectrode measurements. The oxic part of the biofilm was dominated by NSO190 probe-hybridized AOB, which formed relatively large clusters of cells directly on the membrane surface, and by the NOB belonging to genus Nitrobacter sp. On the other hand, NOB belonging to genus Nitrospira sp. were abundant at the oxic-anoxic interface. The model prediction regarding AOB and Nitrobacter sp. distribution was consistent with the experimental counterpart. Measurements of AOB cluster size distribution showed that colonies are slightly larger adjacent to the membrane than at the inner part of the biofilm. The sizes predicted by the current model are larger than those obtained in the experiment, leading to the arguments that some factors not contained in the model would affect the cluster size.

Evaluation of the extracellular polymeric substances by confocal laser scanning microscopy in conventional activated sludge and advanced membrane bioreactors treating hospital wastewater

2014 ◽  
Vol 69 (11) ◽  
pp. 2287-2294 ◽  
Author(s):  
Mousaab Alrhmoun ◽  
Claire Carrion ◽  
Magali Casellas ◽  
Christophe Dagot
Keyword(s):  
Activated Sludge ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Extracellular Polymeric Substances ◽  
Membrane Bioreactors ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Conventional Activated Sludge

Confocal laser scanning microscopy (CLSM) combined with fluorescent viability indicators, was used in this study to investigate the impact of hospital wastewaters on floc structure and composition. In this work, three pilot-scale projects, two membrane bioreactors (MBRs) with a submerged or external membrane bioreactor and a conventional activated sludge, were installed and operated for 65 days. They were fed with an influent sampled directly from the hospital drainage system, which contained micropollutant concentrations ranging from ng/L to mg/L. Samples of flocs were observed using CLSM to characterize the extracellular polymeric substances (EPS) stained with concanavalin A–tetra methylrhodamine and fluorescein isothiocyanate solution and combined with a fluorescent viability indicator (Baclight® Bacterial Viability Kit, Molecular Probes), allowing visualization of isolated stained cells in the three-dimensional structure of flocs (damaged or not). The results of CLSM of the sludge composition were compared with classical biochemical analysis of EPS made through a thermal extraction method. The results showed a good relation between these analyses and the statistical treatment of microscopic pictures.

scholarly journals Protozoan impact on bacterial biofilm formation

2010 ◽  
Vol 47 (1) ◽  
pp. 3-10 ◽  
Author(s):  
Krzysztof Rychert ◽  
Thomas Neu
Keyword(s):  
Activated Sludge ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Grazing Pressure ◽  
Bacterial Biofilm ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Protozoan Community ◽  
Biofilm Development ◽  
The Impact

Protozoan impact on bacterial biofilm formationConfocal laser scanning microscopy in combination with digital image analysis was used to assess the impact of protozoa on bacterial colonisation of surfaces. Bacterial biofilms were developed from activated sludge in microscope flow cells and were exposed to the grazing pressure of protozoa. The protozoan community from healthy activated sludge and a culture of flagellateBodo saltanswere used as grazers. Experiments comprised 48-h incubations in 3 treatment variants: bacteria with protozoa, bacteria with protozoa added after some time and bacteria without protozoa. When necessary, the elimination of protozoa from the inoculum was carried out with cycloheximide and NiSO4. Experiments demonstrated that protozoa from healthy activated sludge initially disturbed the biofilm development but later they could stimulate its growth. Similar results could be established in the experiment withBodo saltans(inoculum: 1000 cells/ml), however differences were not statistically significant. The finding that protozoa support biofilm development during specific stages may be relevant for biofilm studies with mixed environmental biofilm communities.

scholarly journals On the Occurrence of Anoxic Microniches, Denitrification, and Sulfate Reduction in Aerated Activated Sludge

1999 ◽  
Vol 65 (9) ◽  
pp. 4189-4196 ◽  
Author(s):  
Andreas Schramm ◽  
Cecilia M. Santegoeds ◽  
Helle K. Nielsen ◽  
Helle Ploug ◽  
Michael Wagner ◽  
...  
Keyword(s):  
Activated Sludge ◽  
Sulfate Reduction ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Sulfate Reducing ◽  
Dissimilatory Sulfite Reductase ◽  
Sulfate Reduction Rates ◽  
Anoxic Zones

ABSTRACT A combination of different methods was applied to investigate the occurrence of anaerobic processes in aerated activated sludge. Microsensor measurements (O2, NO2 −, NO3 −, and H2S) were performed on single sludge flocs to detect anoxic niches, nitrate reduction, or sulfate reduction on a microscale. Incubations of activated sludge with15NO3 − and35SO4 2− were used to determine denitrification and sulfate reduction rates on a batch scale. In four of six investigated sludges, no anoxic zones developed during aeration, and consequently denitrification rates were very low. However, in two sludges anoxia in flocs coincided with significant denitrification rates. Sulfate reduction could not be detected in any sludge in either the microsensor or the batch investigation, not even under short-term anoxic conditions. In contrast, the presence of sulfate-reducing bacteria was shown by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes and by PCR-based detection of genes coding for the dissimilatory sulfite reductase. A possible explanation for the absence of anoxia even in most of the larger flocs might be that oxygen transport is not only diffusional but enhanced by advection, i.e., facilitated by flow through pores and channels. This possibility is suggested by the irregularity of some oxygen profiles and by confocal laser scanning microscopy of the three-dimensional floc structures, which showed that flocs from the two sludges in which anoxic zones were found were apparently denser than flocs from the other sludges.

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