Fate of natural estrogens in batch mixing experiments using municipal sewage and activated sludge

Since natural estrogens such as 17beta-estradiol (E2) and estrone (E1) are excreted daily by humans, E2 and E1, which are classified as inevitable endocrine-disrupting chemicals, are always present in sewage wastewater. For several years, the monitoring and removal of natural estrogens at sewage treatment plants have been examined by many investigators. However, little is known regarding the exact behavior of estrogens in actual sewage when in contact with activated sludge. In this study, the fate of E2 and E1 as a result of adsorption and decomposition in batch mixing experiments using municipal wastewater and activated sludge collected from an actual municipal sewage treatment plant was investigated. Estrogen concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) kit. E2 and E1 in sewage were removed from the liquid phase in contact with activated sludge, and E2 and E1 adsorbed on the sludge were decomposed in 4h. Significant changes in the adsorption and decomposition of E2 and E1 on the sludge were not observed at low temperatures or when different sludge samples were used such as those acclimatized to low-loading and high-loading conditions. In contrast, the processes leading to the removal of estrogens, such as the adsorption and decomposition of estrogens in contact with activated sludge, were inactivated by sterilizing the sludge. Natural estrogens adsorb onto the activated sludge, and are therefore easy to be biodegraded. In a biological reaction process, therefore, the estrogens will be rapidly removed at the initial stage, when the sewage is satisfactorily mixed with the sludge.     

Effect of solids retention time on structure and characteristics of sludge flocs in sequencing batch reactors

The effect of solids retention time (SRT) (4-20 d) on sludge floc structure, size distribution and morphology in laboratory-scale sequencing batch reactors receiving a glucose-based synthetic wastewater was studied using image analysis in a long-term experiment over one year. Floc size distribution (>10 microm) could be characterized by a log-normal model for no bulking situations, but a bi-modal distribution of floc size was observed for modest bulking situations. In each operating cycle of the SBRs, the variation in food /microorganisms ratio (0.03-1.0) had no significant influence on floc size distribution and morphology. The results from a long-term study over one year showed that no clear relationship existed between SRT and median floc size based on frequency. However, sludge flocs at the lower SRTs (4-9 d) were much more irregular and more variable in size with time than those at higher SRTs (16 and 20 d). The level of effluent-suspended solids at lower SRTs was higher than that at higher SRTs.     

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.     

Comparison of natural estrogen removal efficiency in the conventional activated sludge process and the oxidation ditch process

The presence of natural estrogens, 17beta-estradiol (E2), estrone (E1) and estriol (E3), as well as estrogenic activity in wastewater influents and secondary effluents were investigated in 20 full-scale wastewater treatment plants in Japan. In all of the influent samples, natural estrogens were detected at concentrations above the minimum limits of detection (0.5ng/L). The concentrations of natural estrogens detected in the effluent of oxidation ditch plants were generally lower than previously reported values. On the other hand, in the conventional activated sludge plants, increments of E1 during biological treatment were frequently observed although E2 and E3 were removed effectively in the process. The removal rates of natural estrogens or estrogenic activity show no observed statistical relationship with the solids retention time (SRT) and the hydraulic retention time (HRT). However, the plants with high SRT or HRT generally showed high and stable removal of both natural estrogens and estrogenic activity.     

Production of polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater: effect of pH,sludge retention time (SRT), and acetate concentration in influent

In this paper, the production of biodegradable plastics polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater was investigated. The effect of three operational factors, i.e. the acetate concentration in influent, pH, and sludge retention time (SRT) were studied. Sludge acclimatized with municipal wastewater supplemented with acetate could accumulate PHA up to 30% of sludge dry weight, while sludge acclimatized with only municipal wastewater achieved 20% of sludge dry weight. It was found that activated sludge with an SRT of 3 days possessed better PHA production capability than sludge with an SRT of 10 days. Sludge acclimatized under pH 7 and 8 conditions in sequencing batch reactors (SBRs) exhibited similar PHA production capability. However, in PHA production batch experiments, pH value influenced significantly the PHA accumulation behavior of activated sludge. When pH of batch experiments was controlled at 6 or 7, a very low PHA production was observed. The production of PHA was stimulated when pH was kept at 8 or 9.     

A thermodynamic analysis on adsorption of estrogens in activated sludge process

The adsorption behaviors of estrone (E1), 17beta-estradiol (E2), estriol (E3), 17 alpha-ethinylestradiol (EE2), and equol were studied with a deactivated sludge subjected to heat treatment at 80 degrees C for 30 min. The heat-treatment hardly changed the adsorption features of activated sludge (AS). The adsorption equilibrium of all estrogens was approached within 10 min at 20 degrees C, and a high removal of estrogens was achieved simultaneously. The equilibrium data were well fitted by a Freundlich isotherm. The adsorption behaviors of E1, E2, E3 and EE2 in the AS system were independent of their Kow values. Thermodynamic analysis revealed that the adsorption behaviors of E1, E2, E3 and EE2 could be considered as an exothermic, physical and reversible process, resulting in their higher adsorption capacities at lower temperature. Regarding equol, its adsorption was an endothermic, chemical and irreversible process.     

Characterizing the extracellular and intracellular fluorescent products of activated sludge in a sequencing batch reactor

Three-dimensional excitation-emission-matrix (EEM) fluorescence spectrometry was used to characterize the extracellular and intracellular substances of activated sludge in a sequencing batch reactor (SBR). Parallel factor analysis (PARAFAC) was applied to extract the pure spectra from the overlapped spectra. Three main components, proteins, fulvic- and humic-like substances, were identified from the extracellular substances. Their fluorescence peaks were at an excitation/emission (Ex/Em) of 280/350, 340/400 and 390/450 nm, respectively. The fluorescence of the extracellular proteins had a similar changing pattern with the wastewater chemical oxygen demand, the fulvic-like substance did not vary significantly in a cycle and the humic-like substances accumulated in the substrate uptake phase but decreased later. Proteins and nicotinamide adenine dinucleotide, reduced form (NADH), were identified as the two main intracellular fluorophores, and their fluorescence peaks (Ex/Em) were at 280/340 and 350/450 nm, respectively. The fluorescence intensity scores of the intracellular fluorophores were closely related to the bioreactor performance. Thus, the results of this work provide a foundation for potential utilization of the EEM fluorescence spectroscopy to monitor the activated sludge systems for wastewater treatment.     

Aerobic batch degradation of 17-beta estradiol (E2) by activated sludge: effects of spiking E2 concentrations, MLVSS and temperatures

Aerobic batch degradation of 17beta estradiol (E2) spiked into the activated sludge liquor from a sewage treatment plant was studied; and the likely impacts of E2's initial concentrations (C0), microbial population densities (MLVSS) and temperatures (TEMPT) were examined for a variety of combinations of these three factors: C0 = 10, 30 and 50 microgl(-1); MLVSS = 1750, 875 and 435 mgl(-1); and TEMPT = 5, 20 and 35 degrees C. The results, together with those obtained through two control runs performed using a killed sludge sample, demonstrated clearly that E2 was eliminated from the aqueous phase readily under appropriate MLVSS and temperature levels, with the role of sorption by biomass being less significant. By fitting observed concentration data with a first-order rate expression, the degradation rate constants (k) under all experimental conditions were estimated. The magnitude of k changed markedly in the range of 0.23-4.79 h(-1), following a general order that the higher the MLVSS was, the higher the rate constant, and that the higher the temperature, the higher the rate constant. An obvious increasing trend of the biomass-modified average rate constant (k') with increases in the temperature was also presented: the k' values at 5, 20 and 35 degrees C were 0.79, 1.77 and 3.29l MLVSS g(-1)h(-1), respectively. Furthermore, based upon the estimated k values, the temperature coefficients (theta) over the ranges of 5-20 and 20-35 degrees C were determined. In similarity with the magnitude of theta reported for ordinary BOD-based organic matrices in domestic wastewater, the theta values of E2 varied in the range of 1.026-1.09, suggesting that the temperature impacts on the degradation rates of E2 and BOD constituents are probably similar.     

2-fluorophenol degradation by aerobic granular sludge in a sequencing batch reactor

Aerobic granular sludge is extremely promising for the treatment of effluents containing toxic compounds, and it can economically compete with conventional activated sludge systems. A laboratory scale granular sequencing batch reactor (SBR) was established and operated during 444 days for the treatment of an aqueous stream containing a toxic compound, 2-fluorophenol (2-FP), in successive phases. Initially during ca. 3 months, the SBR was intermittently fed with 0.22 mM of 2-FP added to an acetate containing medium. No biodegradation of the target compound was observed. Bioaugmentation with a specialized bacterial strain able to degrade 2-FP was subsequently performed. The reactor was thereafter continuously fed with 0.22 and 0.44 mM of 2-FP and with 5.9 mM of acetate (used as co-substrate), for 15 months. Full degradation of the compound was reached with a stoichiometric fluoride release. The 2-FP degrading strain was successfully retained by aerobic granules, as shown through the recovering of the strain from the granular sludge at the end of the experiment. Overall, the granular SBR has shown to be robust, exhibiting a high performance after bioaugmentation with the 2-FP degrading strain. This study corroborates the fact that bioaugmentation is often needed in cases where biodegradation of highly recalcitrant compounds is targeted.     

Biodegradability of activated sludge organics under anaerobic conditions

From an experimental and theoretical investigation of the continuity of activated sludge organic (COD) compounds along the link between the fully aerobic or N removal activated sludge and anaerobic digestion unit operations, it was found that the unbiodegradable particulate organics (i) originating from the influent wastewater and (ii) generated by the activated sludge endogenous process, as determined from response of the activated sludge system, are also unbiodegradable under anaerobic digestion conditions. This means that the activated sludge biodegradable organics that can be anaerobically digested can be calculated from the active fraction of the waste activated sludge based on the widely accepted ordinary heterotrophic organism (OHO) endogenous respiration/death regeneration rates and unbiodegradable fraction. This research shows that the mass balances based steady state and dynamic simulation activated sludge, aerobic digestion and anaerobic digestion models provide internally consistent and externally compatible elements that can be coupled to produce plant wide steady state and dynamic simulation WWTP models.     

Biodegradation of natural and synthetic estrogens by nitrifying activated sludge and ammonia-oxidizing bacterium Nitrosomonas europaea

This report describes the uses of nitrifying activated sludge (NAS) and ammonia-oxidizing bacterium Nitrosomonas europaea to significantly degrade estrone (E1), 17beta-estradiol (E2), estriol (E3), and 17alpha-ethynylestradiol (EE2). Using NAS, the degradation of estrogens obeyed first-order reaction kinetics with degradation rate constants of 0.056 h(-1) for E1, 1.3 h(-1) for E2, 0.030 h(-1) for E3, and 0.035 h(-1) for EE2, indicating that E2 was most easily degraded. Then, we confirmed that E2 was degraded via E1 by NAS. With/without the ammonia oxidation inhibitor, it was observed that ammonia-oxidizing bacteria in conjunction with other microorganisms in NAS degraded estrogens. Using N. europaea, the degradation of estrogens reasonably obeyed zero-order reaction kinetics, and no remarkable difference is present among the four estrogens degradation rates and it was found that E1 was not detected during E2 degradation period. We suggested that E2 was degraded to E1 in NAS could be caused by other heterotrophic bacteria, not by ammonia-oxidizing bacteria.     

Role of extracellular polymeric substances in bioflocculation of activated sludge microorganisms under glucose-controlled conditions

Extracellular polymeric substances (EPS) secreted by suspended cultures of microorganisms from an activated sludge plant in the presence of glucose were characterized in detail using colorimetry, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy. EPS produced by the multi-species community were similar to literature reports of pure cultures in terms of functionalities with respect to C and O but differed subtly in terms of N and P. Hence, it appears that EPS produced by different microorganisms maybe homologous in major chemical constituents but may differ in minor components such as lipids and phosphodiesters. The role of specific EPS constituents on microbial aggregation was also determined. The weak tendency of microorganisms to bioflocculate during the exponential growth phase was attributed to electrostatic repulsion when EPS concentration was low and acidic in nature (higher fraction of uronic acids to total EPS) as well as reduced polymer bridging. However, during the stationary phase, polymeric interactions overwhelmed electrostatic interactions (lower fraction of uronic acids to total EPS) resulting in improved bioflocculation. More specifically, microorganisms appeared to aggregate in the presence of protein secondary structures including aggregated strands, β-sheets, α- and 3-turn helical structures. Bioflocculation was also favored by increasing O-acetylated carbohydrates and overall C-(O,N) and functionalities.     

Enumeration of protozoan ciliates in activated sludge: Determination of replicate number using probability

A new approach to the enumeration of ciliate protozoa in activated sludge is described. A 25 μL sub-sample volume is optimal for routine analysis using a standard slide and 24 × 24 mm cover slip requiring between 20 and 40 min per sub-sample for full enumeration and identification of species. However, to achieve high probability (≥95%) of recovering all species large numbers of replicates are required (i.e. 23-47). To achieve high probabilities of recovery using less replicates it is necessary to neglect rare species with low densities (<0.5%); based on the assumption that they do not play a significant role in plant performance. The precise number of replicates required for different probabilities of recovering species is determined by conducting an initial pilot survey analysing a minimum of 8 replicates and using a probability equation to determine the optimum replicate number for that particular plant. Six replicate 25 μL sub-samples provided excellent species recovery (90-95% excluding up to 3 rare species), while analysing just two or three replicates, as commonly used in previous wastewater studies, only gave probabilities of 25 and 50% respectively for the same recovery. Ciliate analysis should be completed within 8 h of collection with significant changes in community structure occurring beyond this period.     

Biological hydrolysis and acidification of sludge under anaerobic conditions: the effect of sludge type and origin on the production and composition of volatile fatty acids

New wastewater treatment processes resulting in considerably reduced sludge production and more effective treatment are needed. This is due to the more stringent legislations controlling discharges of wastewater treatment plants (WWTPs) and existing problems such as high sludge production. In this study, the feasibility of implementing biological hydrolysis and acidification process on different types of municipal sludge was investigated by batch and semi-continuous experiments. The municipal sludge originated from six major treatment plants located in Denmark were used. The results showed that fermentation of primary sludge produced the highest amount of volatile fatty acids (VFAs) and generated significantly higher COD- and VFA-yields compared to the other sludge types regardless of which WWTP the sludge originated from. Fermentation of activated and primary sludge resulted in 1.9-5.6% and 8.1-12.6% COD-yields, soluble COD (SCOD)/total COD (TCOD), in batch experiments, respectively. The COD-yields for primary, activated and mixed sludge were 19.1%, 6.5% and 21.37%, respectively, in semi-continuous experiments operated at solids retention time (SRT) of 5d and temperature of 37 degrees C. The benefit of fermentation for full-scale application was roughly estimated based on the experiments performed in semi-continuous reactors. The results revealed that even though the VFA production of primary sludge was higher compared to activated sludge, substantial amounts of VFA could be produced by fermentation of activated sludge due to the substantially higher production of activated sludge in WWTPs.     

Separate estimation of adsorption and degradation of pharmaceutical substances and estrogens in the activated sludge process

The removal of three estrogens such as 17beta-estradiol, two endocrine disruptors like bisphenol A, and 10 pharmaceutical substances like ibuprofen (IBP) by activated sludge was experimentally examined. The contribution of adsorption and degradation to the overall removal was estimated separately and successfully. At the neutral pH condition, the target pharmaceutical substances showed little tendency of adsorption to the sludge and their water-sludge partition coefficients were lower than those of the target estrogens. On the other hand, the increasing tendency of adsorption was observed in the lower pH condition. A linear relationship between the log of the partitioning coefficient and logK(ow) was observed when pH was lowered to keep the pharmaceuticals neutral solutes. The acidic operational condition was preferable for the removal of acidic pharmaceutical substances because the limiting stage for the removal was not biodegradation but the transfer of substances from the water phase to the sludge phase.     

Biodegradation of organonitriles by adapted activated sludge consortium with acetonitrile-degrading microorganisms

A microbial process for the degradation of three types of structurally distinct organonitriles (i.e., saturated and unsaturated aliphatic nitrile and aromatic nitrile) was studied. Microorganisms were enriched from the activated sludge of a pharmaceutical wastewater treatment plant and adapted through providing acetonitrile as the sole carbon and nitrogen source for their growth. The adapted mixed culture was then examined for their capability of degrading acetonitrile, acrylonitrile and benzonitrile under various operational conditions. The performance of biodegradation and the metabolic intermediate- and end-products in the process were monitored. The results show that an average removal rate of 0.083 g acetonitrile g(-1)-VSS h(-1), 0.0074 g acrylonitrile g(-1)-VSS h(-1) or 0.0029 g benzonitrile g(-1)-VSS h(-1) was achieved in the batch bioreactor under the common operational condition of 25 degrees C and pH 7. The biodegradation of acetonitrile and acrylonitrile showed a two-step pathway, with the generation of acetamide followed by acetic acid and ammonia for acetonitrile or acrylamide followed by acrylic acid and ammonia for acrylonitrile. However, the biodegradation of benzonitrile appeared to have only one step, with the direct production of benzoic acid and ammonia, but without benzamide being detected in the process. The results suggest that, depending on the substrates, the adapted mixed culture can develop very different degradation pathways, such as nitrile hydratase plus amidase for acetonitrile or acrylonitrile and nitrilase for benzonitrile. Therefore, the adapted mixed culture has a great potential and flexibility for actual applications in biodegradation of various organonitrile compounds.     

Waste activated sludge fermentation: Effect of solids retention time and biomass concentration

Laboratory scale, room temperature, semi-continuous reactors were set-up to investigate the effect of solids retention time (SRT, equal to HRT hydraulic retention time) and biomass concentration on generation of volatile fatty acids (VFA) from the non-methanogenic fermentation of waste activated sludge (WAS) originating from an enhanced biological phosphorus removal process. It was found that VFA yields increased with SRT. At the longest SRT (10 d), improved biomass degradation resulted in the highest soluble to total COD ratio and the highest VFA yield from the influent COD (0.14 g VFA-COD/g TCOD). It was also observed that under the same SRT, VFA yields increased when the biomass concentration decreased. At a 10 d SRT the VFA yield increased by 46%, when the biomass concentration decreased from 13 g/L to 4.8 g/L. Relatively high nutrient release was observed during fermentation. The average phosphorus release was 17.3 mg PO₄-P/g TCOD and nitrogen release was 25.8 mg NH₄-N/g TCOD.     

Comparison of control strategies for nitrogen removal in an activated sludge process in terms of operating costs: a simulation study

In this paper several control strategies for nitrogen removal are proposed and evaluated in a benchmark simulation model of an activated sludge process. The goal is to determine which control strategy delivers better performance with respect to plant operating costs. In the study, constant manipulated variables and various PI and feedforward control strategies are tested and compared with predictive control, which uses an ideal process model. The control strategies differ in the information used about the process (number of sensors and sensor location) and in the complexity of the control algorithms. To determine the set-points that yield optimal operating costs, an operational map is constructed for each control strategy. Results of the simulation show that with PI and feedforward controllers almost the same optimal operating costs can be achieved as with more advanced MPC algorithms under various plant operating conditions. More advanced control algorithms are advantageous only in cases where the plant is highly loaded and if stringent effluent fines are imposed by legislation.     

Removal of resin acids and sterols from pulp mill effluents by activated sludge treatment

The wastewater treatment plant of an elemental chlorine free bleaching kraft pulp mill located in eastern Finland was sampled in order to study the fate of wood extractives and the toxicity to luminescence bacteria (Vibrio fischeri) in different parts of the plant. Resin acids and sterols were analyzed from water, particles and sludge samples during three different runs. Waters before biotreatment and primary sludge were found to be toxic; but in the activated sludge treatment toxicity was removed. During wastewater treatment, concentrations of wood extractives were reduced over 97%. In activated sludge treatment, over 94% of the resin acids and over 41% of the sterols were degraded or transformed to other compounds. Furthermore, in general, less than 5% of the resin acids and over 31% of the sterols were removed in biosludge to the sludge thickener. Most of the extractives were discharged attached to particles. Although some disturbing factors increased the load of wood extractives during samplings, these factors did not affect the operational efficiency of the secondary treatment system.