Effect of feeding pattern and storage on the sludge settleability under aerobic conditions

The selection of filamentous bacteria is often assumed to be associated with specific microbial properties such as growth rate, substrate uptake rate, substrate affinity and potential for substrate storage. In this study we aimed to verify some of these factors. Sequencing batch reactor (SBR) systems were used to scale-down aerobic activated sludge systems with an aerobic selector. Adding acetate in different aerobic feeding periods allowed us to simulate a variable relative size of aerobic selector with different bulk liquid substrate concentrations. The experiments showed that as expected, the aerobic fill time ratio (FTR(ox)) and the corresponding feast period, which can be assumed similar to contact time in an aerobic selector, had a strong effect on the sludge settleability. Promoting a strong substrate gradient in the SBR (FTR(ox)<5.4%) resulted in good sludge settleability (SVI<120mLg(-1)). Whenever acetate was added in a limiting rate (FTR(ox)>6.2%), a condition in which the acetate concentration in the reactor was always very low, the sludge settleability decreased (SVI>150mLg(-1)). Sludge settleability could be improved by changing the feeding strategy to a pulse feed. The maximum specific acetate uptake rate and poly beta-hydroxybutyrate (PHB) production rate of bad settling sludge, including bulking sludge, was similar to well-settling sludge, which is not in accordance with the general assumptions that well settling sludge have a higher maximal substrate uptake rate and better storage capacities. An alternative hypothesis for the development of filamentous structures in biological flocs has been formulated. It is hypothesized that bulking sludge originates from the presence of substrate gradients in sludge aggregates. Whereas at low bulk liquid substrate concentration filamentous bacteria give easier access to the substrate at the outside of the flocs and thereby proliferate, at high bulk liquid substrate concentration there is no substrate advantage for filamentous organisms and smooth bacterial structures predominate. In this hypothesis there is no need for an intrinsic difference in kinetic parameters between floc and filamentous bacteria. Where presence of filamentous bacteria is related to process conditions, the presence of a specific filament is likely due to presence of a specific limiting substrate.     

Effect of feeding pattern on biochemical storage by activated sludge under anoxic conditions

This study investigated the effect of feeding pattern on bacterial storage under anoxic conditions, emphasizing previous adaptation of biomass to a long term feeding condition, under steady-state operation. Storage was evaluated in a sequencing batch reactor (SBR) system operated in a sequence of anoxic/aerobic phases, fed with acetate as the sole carbon source during anoxic conditions. The experimental results indicated that biochemical storage of acetate as PHB occurred when substrate was fed as a pulse, while acetate was removed mostly through direct microbial growth under continuous feeding. For pulse feeding, the anoxic yield, Y(STOD) was calculated as 0.58mg COD (mgCOD)(-1) in two different ways, using mass balances on acetate utilization, PHB generation and nitrate consumption. This value was supported by parallel batch tests and similar results in the literature. Similarly, the rate coefficient for storage under anoxic conditions, k(STOD) was computed as 9.3day(-1) based on basic stoichiometry and model calibration of experimental data. Batch tests conducted with biomass adapted to different feeding patterns showed that substrate storage was insignificant when the feed was added continuously at low concentrations, even if the biomass was previously adapted to storage. Similarly biomass acclimated to continuous feeding could not store the excess substrate although fed instantly. For the operating conditions selected for the study, storage response was significant only with a microbial culture with metabolic activities previously adapted to storage and with short-term substrate feeding at high concentrations.     

Short-term harmful effects of ammonia nitrogen on activated sludge microfauna

The response of activated sludge microfauna in terms of abundance and diversity has been analysed to evaluate both the toxic effect of ammonia nitrogen and the acclimatisation capacity of these microorganisms to its toxicity. The harmful effect of ammonia nitrogen was studied by means of two toxicological tests. The ammonia concentrations tested were: 9, 20, 30 and 50mg NH4+-N/l in the first toxicological test and 30, 40, 50 and 80 mg NH4+-N/l in the second. The results suggest that ammonia nitrogen causes a clear but reversible toxic effect on microfauna abundance when its concentrations are around three times higher than that which the microfauna is used to. Chilodonella uncinata and Acineria uncinata were the ciliates least affected by the ammonia nitrogen toxicity. Furthermore, the majority of microfauna groups analysed (gymnamoebae and ciliates) showed capability for acclimatisation to ammonia nitrogen in terms of abundance.     

Metal toxicity in municipal wastewater activated sludge investigated by multivariate analysis and in situ hybridization

The toxicity of three heavy metals (copper, zinc and nickel) on an activated sludge biomass was studied by measuring several parameters of microbiological activity and the dynamics of microbial community composition. The effects on the microflora in the activated-sludge tank were evaluated by dosing the tank with different metal concentrations and a non-inhibiting concentration of copper for a 2-week period. The relationship between biological effects and chemical measurements was elucidated by principal component analysis (PCA). The biochemical data suggested a higher sensitivity of nitrifiers to these metals than heterotrophs. This was confirmed by fluorescent in situ hybridization (FISH) analysis on the microbial community. The impact of copper was displayed by biomass deflocculating phenomena (pin-point) and a decrease in the beta-proteo bacteria component. In addition, the zone settling velocity (ZSV) was shown not to be a good indicator of chronic toxicity phenomena. Finally, the sequence of biomass metal accumulation capability was assessed, and corresponds to Cu>Ni>Zn.     

Metabolic analysis of the removal of formic acid by unacclimated activated sludge

This paper investigates the removal of formic acid by unacclimated biomass from a municipal activated sludge wastewater treatment plant. The biomass was initially able to remove formic acid, but its removal rate and Oxygen Uptake Rate (OUR) decreased with time, until formic acid removal stopped before the formic acid had been exhausted. Formaldehyde was removed in a similar way, whereas the same biomass was simultaneously able to grow and store PHAs when acetic acid was used as substrate.Batch tests with glycine and ¹³C NMR analysis were performed, showing that unacclimated biomass was not able to synthesize all the metabolic intermediates from formic acid alone. At least glycine needed to be externally supplemented, in order to activate the serine synthesis pathway. A small amount of formic acid removal in the absence of growth was also possible through formaldehyde formation and its further conversion to formalin (1,2-formaldehyde dimer), whereas no PHAs were formed.     

Density effects on activated sludge zone settling velocities

A recently developed method to manipulate biomass density based on the addition of synthetic microspheres was used to isolate density effects on zone (hindered) settling velocities in samples from full-scale activated sludge plants over a range biomass densities and concentrations. Settling velocities increased by as much as a factor of five as density was increased by microsphere addition. The effects of density on the Vesilind sedimentation model parameters (V=V(o)e(-kX) , where V is the settling velocity, X is the biomass concentration, and V(o) and k are constants) were evaluated. The parameter V(o) was positively correlated with density for values greater than approximately 1.02 g/mL, while k values were approximately constant in this range. New models were developed for settling velocity as a function of both density and biomass concentration, and these may be useful for future incorporation with clarifier models to improve predictions of system performance, particularly when biomass characteristics known to affect density are variable, such as polyphosphate and nonvolatile suspended solids content.     

Characterization and treatability of aerobic bacterial thermophilically treated wastewater by a conventional activated sludge and granular activated carbon

An industrial wastewater that was pretreated by an aerobic thermophilic bacterial consortium (THE) was subjected to additional treatability studies by granular activated carbon (GAC) and a conventional activated sludge (CAS). The removal of dissolved organic carbon (DOC) in both systems was generally found to be similar. While GAC was able to attain better effluent concentrations of toluene and methyl isobutyl ketone (MIBK), the CAS was much more efficient at removing acetone. Furthermore, unlike the GAC, the performance of the CAS was not influenced by the high degree of variability in the influent wastewater. Characterization of the influent thermophilic wastewater using gas chromatography-mass spectroscopy (GC/MS) was performed to quantify the micropollutants as well as to evaluate removal efficiencies from the GAC and CAS systems.     

Impacts of structural characteristics on activated sludge floc stability

Activated sludge samples from seven full-scale plants were investigated in order to determine the relationship between floc structure and floc stability. Floc stability was determined by shear sensitivity and floc strength. Floc structure was considered in terms of two size scales, the micro- and macrostructure. The microstructure refers to the organization of the floc components, such as the individual microorganisms. The macrostructure refers to the overall floc. The floc macrostructure was characterized by filament index, sludge volume index, size, and fractal dimension. It had a significant impact on floc stability. Large and open flocs with low fractal dimensions containing large number of filaments were more shear sensitive and had lower floc strength compared to small and dense flocs. Fluorescent in situ hybridization analysis indicated that the organization of the bacterial cells might also have an effect on the floc stability.     

Investigation of bacterial community in activated sludge with an anaerobic side-stream reactor (ASSR) to decrease the generation of excess sludge

The goal of this study was to investigate the bacterial community in activated sludge with an anaerobic side-stream reactor (ASSR), a process permitting significant decrease in sludge production during wastewater treatment. The study operated five activated sludge systems with different sludge treatment schemes serving as various controls for the activated sludge with ASSR. Bacterial communities were analyzed by denaturing gradient gel electrophoresis (DGGE), sequencing and construction of phylogenetic relationships of the identified bacteria. The DGGE data showed that activated sludge incorporating ASSR contained higher diversity of bacteria, resulting from long solids retention time and recirculation of sludge under aerobic and anaerobic conditions. The similarity of DGGE profiles between ASSR and separate anaerobic digester (control) was high indicating that ASSR is primarily related to conventional anaerobic digesters. Nevertheless, there was also unique bacteria community appearing in ASSR. Interestingly, sludge in the main system and in ASSR showed considerably different bacterial composition indicating that ASSR allowed enriching its own bacterial community different than that from the aeration basin, although two reactors were connected via sludge recirculation. In activated sludge with ASSR, sequences represented by predominant DGGE bands were affiliated with Proteobacteria. The remaining groups were composed of Spirochaetes, Clostridiales, Chloroflexi, and Actinobacteria. Their putative role in the activated sludge with ASSR is also discussed in this study.     

Effect of low ORP in anoxic sludge zone on excess sludge production in oxic-settling-anoxic activated sludge process

This paper studied the effect of oxidation-reduction potential (ORP) in the anoxic sludge zone on the excess sludge production in the oxic-settling-anoxic process (OSA process), a modified activated sludge process. Two pilot-scale activated sludge systems were employed in this study: (1) an OSA process that was modified from a conventional activated sludge process by inserting a sludge holding tank or namely the "anoxic" tank in the sludge return line; and (2) a conventional process used as the reference system. Each was composed of a membrane bioreactor to serve the aeration tank and solid/liquid separator. Both systems were operated with synthetic wastewater for 9 months. During the operation, the OSA system was operated with different ORP levels (+100 to -250 mV) in its anoxic tank. It has been confirmed that the OSA system produced much less excess sludge than the reference system. A lower ORP level than +100 mV in the anoxic tank is in favor of the excess sludge reduction. When the ORP level decreased from +100 to -250 mV the sludge reduction efficiency was increased from 23% to 58%. It has also been found that the OSA system performed better than the reference system with respect to the chemical oxygen demand removal efficiency and sludge settleability. The OSA process may present a potential low-cost solution to the excess sludge problem in an activated sludge process because addition of a sludge holding tank is only needed.     

Molecular characterization of the microbial community structure in two activated sludge systems for the advanced treatment of domestic effluents

Although activated sludge systems with enhanced biological phosphorus removal (EBPR) represent state-of-the-art technology for phosphate removal from wastewater it is still unknown which species of bacteria are responsible for the EBPR process. The aim of this study was to compare the bacterial composition of activated sludge from two laboratory plants with different modes of operation, anoxic/oxic- (EBPR, no nitrification) and Phoredox-system (EBPR, nitrification and denitrification) with particular emphasis on microorganisms responsible for EBPR process. In addition to fluorescence in situ hybridization (FISH), we applied further rRNA-based molecular techniques like terminal restriction-fragment length polymorphism analysis and comparative 16S rDNA analysis to yield additional information and to verify the results from FISH analysis, like e.g. for the identification of polyphosphate accumulating organisms (PAO). Despite the different modes of operation only minor differences in the bacterial composition were detected by FISH analysis based on the probes used in this study. In contrast T-RFLP analysis yielded characteristic community fingerprints for each of the investigated plants and comparative 16S rDNA analysis indicated highly diverse microbial communities in both plants suggesting substantial differences in the microbial structure. The results obtained by FISH analysis with specific probes for PAOs support the presumption that not only one specific organism is responsible for the EBPR. In our case Tetrasphaera spp. dominated the PAO community, but other possible PAOs, like Microlunatus spp. and members of the Rhodocyclus group, were also detected.     

Suspended biofilm carrier and activated sludge removal of acidic pharmaceuticals

Removal of seven active pharmaceutical substances (ibuprofen, ketoprofen, naproxen, diclofenac, clofibric acid, mefenamic acid, and gemfibrozil) was assessed by batch experiments, with suspended biofilm carriers and activated sludge from several full-scale wastewater treatment plants. A distinct difference between nitrifying activated sludge and suspended biofilm carrier removal of several pharmaceuticals was demonstrated. Biofilm carriers from full-scale nitrifying wastewater treatment plants, demonstrated considerably higher removal rates per unit biomass (i.e. suspended solids for the sludges and attached solids for the carriers) of diclofenac, ketoprofen, gemfibrozil, clofibric acid and mefenamic acid compared to the sludges. Among the target pharmaceuticals, only ibuprofen and naproxen showed similar removal rates per unit biomass for the sludges and biofilm carriers. In contrast to the pharmaceutical removal, the nitrification capacity per unit biomass was lower for the carriers than the sludges, which suggests that neither the nitrite nor the ammonia oxidizing bacteria are primarily responsible for the observed differences in pharmaceutical removal. The low ability of ammonia oxidizing bacteria to degrade or transform the target pharmaceuticals was further demonstrated by the limited pharmaceutical removal in an experiment with continuous nitritation and biofilm carriers from a partial nitritation/anammox sludge liquor treatment process.     

Denitrification potential and rates of complex carbon source from dairy effluents in activated sludge system

A predictive model for the denitrification performance of complex carbon sources was proposed based on compositional data. Potential and rates of denitrification of single dairy components (lactose, lactate, proteins, fat), as well as binary and complex (modelled "process water") mixtures were assessed using test for nitrogen uptake rate (NUR). In all experiments, denitrification potential of mixtures was found to be significantly higher than the sum of individual potentials and denitrification rate with the readily biodegradable moiety of the mixtures was similar to the highest rate obtained with individual components (lactose or lactate). This work shows that activated sludge acclimated to dairy components can be modelled as a single biomass where the maximal anoxic growth rate of the biomass limits the denitrification rate with dairy components. As a consequence, lactose or lactate determine the maximal denitrification rate possible using dairy effluents.     

Bioflocculation of mesophilic and thermophilic activated sludge

Thermophilic activated sludge treatment is often hampered by a turbid effluent. Reasons for this phenomenon are so far unknown. Here, the hypothesis of the temperature dependency of the hydrophobic interaction as a possible cause for diminished thermophilic activated sludge bioflocculation was tested. Adsorption of wastewater colloidal particles was monitored on different flat surfaces as a function of temperature. Adsorption on a hydrophobic surface varied with temperature between 20 and 60 degrees C and no upward or downward trend could be observed. This makes the hydrophobic interaction hypothesis unlikely in explaining the differences in mesophilic and thermophilic activated sludge bioflocculation. Both mesophilic and thermophilic biomass did not flocculate with wastewater colloidal particles under anaerobic conditions. Only in the presence of oxygen, with biologically active bacteria, the differences in bioflocculation behavior became evident. Bioflocculation was shown only to occur with the combination of wastewater and viable mesophilic biomass at 30 degrees C, in the presence of oxygen. Bioflocculation did not occur in case the biomass was inactivated or when oxygen was absent. Thermophilic activated sludge hardly showed any bioflocculation, also under mesophilic conditions. Despite the differences in bioflocculation behavior, sludge hydrophobicity and sludge zetapotentials were almost similar. Theoretical calculations using the DLVO (Derjaguin, Landau, Verweij and Overbeek) theory showed that flocculation is unlikely in all cases due to long-range electrostatic forces. These calculations, combined with the fact that bioflocculation actually did occur at 30 degrees C and the unlikelyness of the hydrophobic interaction, point in the direction of bacterial exo-polymers governing bridging flocculation. Polymer interactions are not included in the DLVO theory and may vary as a function of temperature.     

Characterization of the heterotrophic biomass and the endogenous residue of activated sludge

The activated sludge process generates an endogenous residue (X_E) as a result of heterotrophic biomass decay (X_H). A literature review yielded limited information on the differences between X_E and X_H in terms of chemical composition and content of extracellular polymeric substances (EPS). The objective of this project was to characterize the chemical composition (x, y, z, a, b and c in C_xH_yO_zN_aP_bS_c) of the endogenous and the active fractions and EPS of activated sludge from well designed experiments. To isolate X_H and X_E in this study, activated sludge was generated in a 200 L pilot-scale aerobic membrane bioreactor (MBR) fed with a soluble and completely biodegradable synthetic influent of sodium acetate as the sole carbon source. This influent, which contained no influent unbiodegradable organic or inorganic particulate matter, allowed the generation of a sludge composed essentially of two fractions: heterotrophic biomass X_H and an endogenous residue X_E, the nitrifying biomass being negligible. The endogenous decay rate and the active biomass fraction of the MBR sludge were determined in 21-day aerobic digestion batch tests by monitoring the VSS and OUR responses. Fractions of X_H and X_E were respectively 68% and 32% in run 1 (MBR at 5.2 day SRT) and 59% and 41% in run 2 (MBR at 10.4 day SRT). The endogenous residue was isolated by subjecting the MBR sludge to prolonged aerobic batch digestion for 3 weeks, and was characterized in terms of (a) elemental analysis for carbon, nitrogen, phosphorus and sulphur; and (b) content of EPS. The MBR sludge was characterized using the same procedures (a and b). Knowing the proportions of X_H and X_E in this sludge, it was possible to characterize X_H by back calculation. Results from this investigation showed that the endogenous residue had a chemical composition different from that of the active biomass with a lower content of inorganic matter (1:4.2), of nitrogen (1:2.9), of phosphorus (1:5.3) and of sulphur (1:3.2) but a similar content of carbon (1:0.98). Based on these elemental analyses, chemical composition formulae for X_H and X_E were determined as CH_1.240O_0.375N_0.200P_0.0172S_0.0070 and CH_1.248O_0.492N_0.068P_0.0032S_0.0016, respectively. Data from EPS analyses also confirmed this difference in structure between X_E and X_H with an EPS content of 11-17% in X_Eversus 26-40% in X_H.     

Short- and long-term temperature effects on aerobic polyhydroxybutyrate producing mixed cultures

Short- and long-term temperature effects on polyhydroxybutyrate (PHB) producing mixed cultures enriched in feast-famine sequencing batch reactors (SBRs) were investigated in a temperature range of 15-35 °C and 15-30 °C, respectively. After short-term temperature changes (i.e. 1 cycle) from the steady state temperature of 20 °C, reaction rate changes in the famine phase could be described over the whole temperature range with the Arrhenius equation with one temperature coefficient. For the feast phase different temperature coefficients were identified for acetate uptake, PHB production and growth. These were only valid for temperatures 5 °C higher or lower than the steady state temperature.Long-term temperature changes (i.e. new steady states) influenced not only the reaction rates but also the selective pressure in the SBR. At higher temperatures (30 °C) the SBR feast phase was short and the rates of acetate uptake and PHB storage were very high. This culture was characterized by a storage strategy with high yields of PHB and low yields of biomass in the feast phase. The PHB storage capacity of this culture was 84 wt% as evaluated in fed-batch experiments. At lower temperatures (15 °C) the feast phase was longer due to a lower rate of acetate uptake and the culture followed a strategy of direct growth on acetate rather than on PHB. This culture had a low maximal PHB storage capacity (about 35 wt%). The SBR culture enriched at 20 °C was able to store up to about 70 wt% PHB. The temperature at which fed-batch experiments were conducted did not influence the maximal PHB storage capacity. The SBR temperature was found to be an important factor to consider when designing a mixed culture PHB production process.     

Effect of ZnO particles on activated sludge: role of particle dissolution

The release of nanoparticles (NPs) into the environment, including wastewater treatment plants, is expected to increase in the future. Therefore, it is important to understand the potential effects of these NPs on activated sludge treatment processes. A pulse-flow respirometer was used to study the toxicity of nano-ZnO on activated sludge endogenous respiration, BOD biodegradation, and nitrification. In addition, toxicities of bulk ZnO particles and Zn ion (e.g. soluble Zn) were also studied. All three Zn forms were found to adversely impact the activity of activated sludge, with soluble Zn exhibited the greatest toxicity. The effects of nano-ZnO and bulk ZnO on activated sludge were caused by soluble Zn resulting from ZnO particle dissolution. The IC₅₀ values of soluble Zn on activated sludge endogenous respiration, BOD biodegradation, ammonia oxidation, and nitrite oxidation were 2.2, 1.3, 0.8, and 7.3 mg-Zn/L, respectively. Therefore, the first step of nitrification was most sensitive to Zn.     

Fine-scale bacterial community dynamics and the taxa-time relationship within a full-scale activated sludge bioreactor

In activated sludge bioreactors, aerobic heterotrophic communities efficiently remove organics, nutrients, toxic substances, and pathogens from wastewater, but the dynamics of these communities are as yet poorly understood. A macroecology metric used to quantify community shifts is the taxa-time relationship, a temporal analog of the species-area curve. To determine whether this metric can be applied to full-scale bioreactors, activated sludge samples were collected weekly over a one-year period at a local municipal wastewater treatment plant. Bacterial community dynamics were evaluated by monitoring 16S rRNA genes using Terminal Restriction Fragment Length Polymorphism (T-RFLP), corroborated by clone libraries. Observed taxa richness increased with time according to a power law model, as predicted by macroecological theory, with a power law exponent of w = 0.209. The results reveal strong long-term temporal dynamics during a period of stable performance (BOD removal and nitrification). Community dynamics followed a gradual succession away from initial conditions rather than periodicity around a mean “equilibrium”, with greater within-month then among-month community similarities. Changes in community structure were significantly associated via multivariate statistical analyses with dissolved oxygen, temperature, influent silver, biomass (MLSS), flow rate, and influent nitrite, cadmium and chromium concentrations. Overall, our results suggest patterns of bacterial community dynamics likely regulated in part by operational parameters and provide evidence that the taxa-time relationship may be a fundamental ecological pattern in macro- and microbial systems.     

Impact of the repetition of oxygen deficiencies on the filamentous bacteria proliferation in activated sludge

Cases of low stress can frequently occur in a wastewater treatment plant (WWTP). As they have no visible impact on the plant operation, they generally go unnoticed. Nevertheless, it would appear that an accumulation of such cases can result in serious operational problems. The impact of the repeated application of oxygen deficiency on the proliferation of the filamentous bacteria Sphaerotilus natans, Haliscomenobacter hydrossis, Eikelboom type 021N and Thiothrix spp has been studied on WWTP pilots. Two different oxygen deficiencies series were tested. Filamentous bacteria evolution in activated sludge was monitored using fluorescent in situ hybridization. For the weak deficiencies, no significant variation in the filamentous bacteria response level was recorded, whereas with more severe deficiencies the filamentous bacteria response increased considerably with the increase in stress number. From the results obtained, it would appear that there is an intensity and frequency threshold beyond which the filamentous bacteria response levels increase when the stress is applied in series. This "oxygen deficiencies repetition" appears to be an important factor in the appearance of bulking.     

Enzymatic extraction of activated sludge extracellular polymers and implications on bioflocculation

This study examines enzyme hydrolysis, a mild, effective, but a rarely used method of extracellular polymer extraction, in removing polymers from mixed culture activated sludge flocs. Two carbohydrate specific enzymes (alpha-amylase and cellulase) and a protein specific enzyme (proteinase) are used during the study. First, the kinetic aspect is investigated, then enzyme dose optimization is carried out on laboratory grown activated sludge samples cultured at solids retention times (SRT) of 4 and 20 days. A more commonly used cation exchange resin (CER) extraction technique is also employed for comparison purposes. Results indicate that the extraction of extracellular polymers by enzymes is a rather quick process reaching equilibrium within only a few hours. As the doses of enzymes are increased, the extracted polymer quantities increase up to a certain dose, beyond which not much extraction is observed. The method does not cause any significant cell lysis as measured by the viable cell counts. Carbohydrate-hydrolyzing enzymes extract small amount of proteins along with the carbohydrates and protein-hydrolyzing enzyme extracts some carbohydrates together with the proteins, indicating that proteins and carbohydrates exist bound to each other in the extracellular polymer network of sludge. Enzyme extraction generally gives a lower estimate of polymers compared to the CER method, but correctly detects the trends in the polymer quantity.