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Sludge granulation in continuous-flow systems is an emerging technology to intensify existing activated sludge infrastructure for nutrient removal. In these systems, the nutrient removal contributions and partitioning of microbial functions between granules and flocs can offer insights into process implementations. To this end, a reactor system that simulates the continuous-flow environment using an equal amount of initial granule and floc biomass was investigated. The two operational strategies for maintaining granule growth in the continuous-flow system were (a) the higher solids retention time (SRT) for the granules versus flocs, as well as (b) selective feeding of carbon to the granules. The SRT of the large granule fractions (>425 µm, LG) and floc/small granule fractions (<425 µm, FSG) were controlled at 20 and 2.7-6.0 days, respectively. Long term operation of the hybrid granule/floc system achieved high PO 4 3- and NH 4 + removal efficiencies. Higher polyphosphate-accumulating organisms (PAO) activity was observed in the FSG than LG, while ammonia-oxidizing bacteria (AOB) activities were similar in the two biomass fractions. Nitrite shunt was observed in the FSG, possibly due to out-competition by the high NOB activity in LG. More importantly, washing out the FSG caused a reduction in LG's AOB and PAO activity, indicating a possible dependency of LG on FSG for maintaining its nutrient removal capacity. Our findings highlighted the partitioning and potential competition/cooperation of key microbial functional groups between LG and FSG, facilitating nutrient removal in a hybrid granular activated sludge system, as well as implications for practical application of the treatment platform., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

An aerobic granular sludge (AGS) pilot plant fed with a mixture of acetate amended centrate and secondary effluent was used to investigate the optimal granule size range for simultaneous nitrification and denitrification (SND) and ortho-phosphate removal. The anaerobic phase was mixed to understand how AGS will perform if integrated with a continuous flow activated sludge system that cannot feed the influent through the settled sludge bed. Five different granule size fractions were taken from the pilot (operated at DO setpoint of 2mgO 2 /L) and each size was subjected to activity tests in a well-controlled lab-scale AGS reactor at four dissolved oxygen (DO) concentrations of 1, 2, 3, and 4 mgO 2 /L. The size fractions were: 212 - 600 µm, 600 - 1000 µm, 1000 - 1400 µm, 1400 - 2000 µm, and >2000 µm. The smallest size range (212 - 600 µm) had the highest nitrification and phosphate removal rates at DO setpoints from 1 - 3 mgO 2 /L, which was attributed to the higher aerobic volume fraction in small granules and hence a higher abundance of phosphorus accumulating organisms (PAO) and ammonia oxidizing bacteria (AOB). In comparison, large granules (>1000 µm) had 1.4 - 4.7 times lower ammonia oxidation rates than the smallest size range, which aligned with their lower AOB abundance relative to granule biomass. The granules with the highest anoxic volume fraction had the highest abundance of nitrite reductase genes (nir gene) but did not show the highest specific nitrogen removal rate. Instead, smaller granules (212 - 600 and 600 - 1000 µm), which had a lower nir gene abundance, had the highest specific nitrogen removal rates (1.2 - 3.1 times higher than larger granules) across all DO values except at 4 mgO 2 /L. At a DO setpoint of 4 mgO 2 /L, nitrite production by ammonia oxidation (ammonia monooxygenase) exceeded nitrite reduction by nitrite reductase in granules smaller than 1000 µm, in addition, some denitrifying heterotrophs switched to oxygen utilization in deeper layers hence suppressing denitrification activity. At the DO range of 2 - 4 mg/L, granular size had a greater effect on nutrient removal than DO. Therefore, for AGS developed at an average DO setpoint of 2 mgO 2 /L, selecting for size fractions in the range of 212 - 1000 µm and avoiding DO values higher than 3 mgO 2 /L can achieve both a higher nitrogen removal capacity and energy savings. This study is the first to investigate the influence of different DO values on SND and biological phosphorus removal performance of different aerobic granular sludge sizes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The relationship between ammonia oxidation rate, nitrifiers population, and modelled aerobic zone volume in different granule sizes was investigated using aerobic granular sludge from a pilot-scale reactor. The pilot was fed with centrate and secondary effluent amended with acetate as the main carbon source. The maximum specific ammonia oxidation rates and community composition of different aerobic granular sludge size fractions were evaluated by batch tests, quantitative PCR, and genomic analysis. Small (331µm) granules had a 4.72 ± 0.09 times higher maximum specific ammonia oxidizing rate per 1 gVSS, and a 4.05 ± 0.17 times higher specific amoA gene copy number than large (2225µm) granules per 1 gram of wet biomass. However, when related to surface area, small granules had 1.43 ± 0.01 times lower maximum specific ammonia oxidation rate and a 1.66 ± 0.04 times lower specific amoA gene copy number per unit surface than large granules. Experimental results aligned with modeling results in which smaller granules had a higher specific aerobic zone volume to biomass and lower specific aerobic zone volume to surface area. Aerobic granular sludge reactors having the same average diameter of granules may have very different proportions of granule size fractions and hence possess different nitrification rates. Therefore, instead of the commonly reported average granule diameter, a new method was proposed to determine the aerobic volume density per sample, which correlated well with the nitrification rate. This work provides a roadmap to control nitrification capacity by two methods: (a) crushing larger granules into smaller fractions, or (b) increasing the mixed liquor suspended solid concentration to increase the total aerobic zone volume of the system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To date, high performance of full-scale aerobic granular sludge (AGS) technology has been demonstrated on a global scale. Its further integration with existing continuous flow activated sludge (CFAS) treatment plants is the next logical step. All granular sludge reactors operated in sequencing batch reactors (SBR) mode with anaerobic feeding conditions select for growth of phosphorus and glycogen accumulating organisms (PAO and GAO, respectively), which are known to enhance sludge settling characteristics. Therefore, we hypothesized that AGS are commonly present at full-scale CFAS processes with enhanced biological phosphorus removal (EBPR) and low sludge volume index (SVI). This hypothesis was confirmed at 13 EBPR plants, where granules were found present (at plants where SVI was lower than 100 ml/g) with a strong correlation between high granule abundance and low SVI. A wide range of granule abundance was found among the plants, ranging from 0.5% to as high as 80%. Evaluations of the EBPR plant process configurations showed that high granule abundances may be related to selector design features such as high anaerobic food to mass (F/M) ratios, unmixed in-line fermentation, and high influent soluble COD fraction. Granules were also observed at a non-EBPR plant with an aerobic selector receiving high F/M feeds. Quantitative PCR and 16S rRNA gene sequencing analyses revealed higher relative gene abundance of Accumulibacter PAO and Competibacter GAO in the granules over flocs, as well as a correlation between granule abundance and some possible EPS producers such as Flavobacterium and Competibacter. Our results indicated that process configurations that select for slow-growing or EPS-producing heterotrophs play an important role for granule formation in full-scale CFAS systems as previously shown in SBR configurations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nitrifying granules were grown in a sidestream reactor fed municipal anaerobic digestion centrate and added in an initial slug dose and subsequent smaller daily doses to a non-nitrifying mainstream activated sludge system at 12 °C and 2.5-day aerobic solids retention time (SRT) to increase its nitrification capacity. Effluent NH3-N concentrations less than 1 mg/L were achieved with bioaugmentation, and nitrification was immediately lost when granules were removed after 30 days of bioaugmentation. Molecular microbial analyses indicated that nitrifying organisms remained attached to granules in the mainstream system with little loss to the flocculent sludge. Maximum specific nitrification activity of the bioaugmented granules decreased in mainstream treatment but the nitrification capacity remained due to new granule growth in the mainstream. This study demonstrated that bioaugmentation with sidestream nitrifying granules can intensify nitrification capacity in low-SRT, low-temperature flocculent activated sludge systems to achieve low effluent NH3-N concentrations and nitrogen removal.

Three types of nitrifying granules were grown on media simulating anaerobic digestion dewatering reject water and compared for their potential to increase nitrification capacity when added to mainstream flocculent activated sludge treatment. An advantage of nitrification bioaugmentation with sidestream granules instead of flocculent biomass is that the granules can be selectively maintained at longer retention times than flocs and thus provide higher nitrification capacity from bioaugmentation. The three granule types and feeding conditions were: nitrifying granules with aerobic feeding, nitrifying-denitrifying granules with anoxic feeding, and nitrifying-denitrifying/phosphate-accumulating (NDN-PAO) granules with anaerobic feeding. NDN-PAO granular sludge showed the highest potential for nitrification bioaugmentation due to its better treatment performance, granule physical characteristics, and much greater production of granular mass and nitrification capacity. Dechloromonas-associated organisms were dominant in these granules; Candidatus Accumulibacter-related organisms were also present. Nitrosomonas was the dominant ammonia-oxidizing bacteria, while Candidatus Nitrotoga was an abundant nitrite-oxidizer in all granule types., (Copyright © 2017. Published by Elsevier Ltd.)

Fats, oils and greases (FOG) are energy-dense wastes that can be added to anaerobic digesters to substantially increase biomethane recovery via their conversion through long-chain fatty acids (LCFAs). However, a better understanding of the ecophysiology of syntrophic LCFA-degrading microbial communities in anaerobic digesters is needed to develop operating strategies that mitigate inhibitory LCFA accumulation from FOG. In this research, DNA stable isotope probing (SIP) was coupled with metagenomic sequencing for a genome-centric comparison of oleate (C 18:1 )-degrading populations in two anaerobic codigesters operated with either a pulse feeding or continuous-feeding strategy. The pulse-fed codigester microcosms converted oleate into methane at over 20% higher rates than the continuous-fed codigester microcosms. Differential coverage binning was demonstrated for the first time to recover population genome bins (GBs) from DNA-SIP metagenomes. About 70% of the 13 C-enriched GBs were taxonomically assigned to the Syntrophomonas genus, thus substantiating the importance of Syntrophomonas species to LCFA degradation in anaerobic digesters. Phylogenetic comparisons of 13 C-enriched GBs showed that phylogenetically distinct Syntrophomonas GBs were unique to each codigester. Overall, these results suggest that syntrophic populations in anaerobic digesters can have different adaptive capacities, and that selection for divergent populations may be achieved by adjusting reactor operating conditions to maximize biomethane recovery.

This study investigated the impacts of long-chain fatty acid (LCFA) feeding frequencies on microbial community structure, bioconversion kinetics, and process stability during anaerobic codigestion. Parallel laboratory-scale anaerobic codigesters fed with dairy cattle manure were either pulse-fed every two days or continuously-fed daily, respectively, with oleate (C 18:1 ) in incremental step increases over 200 days up to 64% of the influent chemical oxygen demand (COD). The effluent acetate concentration exceeded 3000 mg/L in the continuous-fed codigester at the highest oleate loading rate, but remained below 100 mg/L in the pulse-fed codigester at the end of its 48-hr oleate feed cycle. Maximum substrate conversion rates of oleate (q max, oleate ) and acetate (q max, acetate ) were significantly higher in the pulse-fed codigester compared to the continuous-fed codigester. 16S rRNA gene amplicon sequencing showed that Bacteria and Archaea community profiles diverged based on the codigester LCFA feeding pattern and loading rate. LCFA-degrading Syntrophomonas bacteria were significantly enriched in both LCFA codigesters relative to the control digester. The pulse-fed codigester had the highest community fraction of Syntrophomonas 16S rRNA genes by the end of the experiment with 43% of Bacteria amplicon sequences. q max, oleate and q max, acetate values were both significantly correlated to absolute concentrations of Syntrophomonas and Methanosaeta 16S rRNA genes, respectively. Multiple-linear regression models based on the absolute abundance of Syntrophomonas and Methanosaeta taxa provided improved predictions of oleate and acetate bioconversion kinetics, respectively. These results collectively suggest that pulse feeding rather than continuous feeding LCFA during anaerobic codigestion selected for higher microbial bioconversion kinetics and functional stability, which were related to changes in the physiological diversity and adaptive capacity of syntrophic and methanogenic communities., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Codigesting fats, oils, and greases with municipal wastewater sludge can greatly improve biomethane recovery at wastewater treatment facilities. Process loading rates of fats, oils, and greases have been previously tested with little knowledge of the digester microbial community structure, and high transient fat loadings have led to long chain fatty acid (LCFA) accumulation and digester upsets. This study utilized recently-developed quantitative PCR assays for syntrophic LCFA-degrading bacteria along with 16S amplicon sequencing to relate changes in microbial community structure to LCFA accumulation during transient loading increases to an anaerobic codigester receiving waste restaurant oil and municipal wastewater sludge. The 16S rRNA gene concentration of the syntrophic β-oxidizing genus Syntrophomonas increased to ∼15% of the Bacteria community in the codigester, but stayed below 3% in the control digester that was fed only wastewater sludge. Methanosaeta and Methanospirillum were the dominant methanogenic genera enriched in the codigester, and together comprised over 80% of the Archaea community by the end of the experimental period. Constrained ordination showed that changes in the codigester Bacteria and Archaea community structures were related to measures of digester performance. Notably, the effluent LCFA concentration in the codigester was positively correlated to the specific loading rate of waste oil normalized to the Syntrophomonas 16S rRNA concentration. Specific loading rates of 0-1.5 × 10(-12) g VS oil/16S gene copies-day resulted in LCFA concentrations below 30 mg/g TS, whereas LCFA accumulated up to 104 mg/g TS at higher transient loading rates. Based on the community-dependent loading limitations found, enhanced biomethane production from high loadings of fats, oils and greases can be achieved by promoting a higher biomass of slow-growing syntrophic consortia, such as with longer digester solids retention times. This work also demonstrates the potential for controlling the loading rate of fats, oils, and greases based on the analysis of the codigester community structure, such as with quantitative PCR measurements of syntrophic LCFA-degrading bacteria abundance., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The removal of the potent endocrine-disrupting estrogen hormone, 17α-ethinylestradiol (EE2), in municipal wastewater treatment plant (WWTP) activated sludge (AS) processes can occur through biodegradation by heterotrophic bacteria growing on other organic wastewater substrates. Different kinetic and metabolic substrate utilization conditions created with AS bioselector processes can affect the heterotrophic population composition in AS. The primary goal of this research was to determine if these changes also affect specific EE2 biodegradation kinetics. A series of experiments were conducted with parallel bench-scale AS reactors treating municipal wastewater with estrogens at 100-300 ng/L concentrations to evaluate the effect of bioselector designs on pseudo first-order EE2 biodegradation kinetics normalized to mixed liquor volatile suspended solids (VSS). Kinetic rate coefficient (kb) values for EE2 biodegradation ranged from 5.0 to 18.9 L/g VSS/d at temperatures of 18 °C to 24 °C. EE2 kb values for aerobic biomass growth at low initial food to mass ratio feeding conditions (F/Mf) were 1.4 to 2.2 times greater than that from growth at high initial F/Mf. Anoxic/aerobic and anaerobic/aerobic metabolic bioselector reactors achieving biological nutrient removal had similar EE2 kb values, which were lower than that in aerobic AS reactors with biomass growth at low initial F/Mf. These results provide evidence that population selection with growth at low organic substrate concentrations can lead to improved EE2 biodegradation kinetics in AS treatment.

An evaluation of costs, rate, and environmental impacts of upgrading publically owned treatment works (POTWs) in the State of Utah to four levels of nutrient control allowed a variety of nutrient control policies to be assessed. Upgrade costs and rate impacts indicated that costs would be within a defined range for many POTWs, especially with design capacities greater than 40,000 m3/day (-10 mgd). However, costs were significantly higher for some POTWs with lower design capacities, and nutrient upgrades to the most stringent levels would not be affordable for these communities, representing about 15 percent of the service population. The resulting equity issues can be addressed through hardship grants program and/or regulations based on a trading scheme. Analysis demonstrated that trading offers advantages, including cost efficiency and flexibility to accommodate further nutrient reductions and population growth, and greater ability to interface with urban and rural nonpoint nutrient control. Currently, the State of Utah is establishing technology-based nutrient limits that can be affordably implemented at all POTWs in phases. Additionally, a multi-faceted approach is being evaluated that will consider prioritized watershed-scale strategies, point and nonpoint sources of pollution, ecological and socioeconomic implications, and stakeholder participation in nutrient reduction programs.

The aim of this study was to expand the International Water Association Activated Sludge Model No. 2d (ASM2d) to account for a newly defined readily biodegradable substrate that can be consumed by polyphosphate-accumulating organisms (PAOs) under anoxic and aerobic conditions, but not under anaerobic conditions. The model change was to add a new substrate component and process terms for its use by PAOs and other heterotrophic bacteria under anoxic and aerobic conditions. The Gdansk (Poland) wastewater treatment plant (WWTP), which has a modified University of Cape Town (MUCT) process for nutrient removal, provided field data and mixed liquor for batch tests for model evaluation. The original ASM2d was first calibrated under dynamic conditions with the results of batch tests with settled wastewater and mixed liquor, in which nitrate-uptake rates, phosphorus-release rates, and anoxic phosphorus uptake rates were followed. Model validation was conducted with data from a 96-hour measurement campaign in the full-scale WWTP. The results of similar batch tests with ethanol and fusel oil as the external carbon sources were used to adjust kinetic and stoichiometric coefficients in the expanded ASM2d. Both models were compared based on their predictions of the effect of adding supplemental carbon to the anoxic zone of an MUCT process. In comparison with the ASM2d, the new model better predicted the anoxic behaviors of carbonaceous oxygen demand, nitrate-nitrogen (NO3-N), and phosphorous (PO4-P) in batch experiments with ethanol and fusel oil. However, when simulating ethanol addition to the anoxic zone of a full-scale biological nutrient removal facility, both models predicted similar effluent NO3-N concentrations (6.6 to 6.9 g N/m3). For the particular application, effective enhanced biological phosphorus removal was predicted by both models with external carbon addition but, for the new model, the effluent PO4-P concentration was approximately one-half of that found from ASM2d. On a PO4-P removal percentage basis, the difference was small, that is, 94.1 vs. 97.1%, respectively, for the ASM2d and expanded ASM2d.

The aim of this study was to investigate the occurrence and fate of colloidal and dissolved organic nitrogen (CON and DON) across biological nutrient removal (BNR) activated sludge bioreactors. Primary and secondary effluent total nitrogen (TN) measurements and component fractionation, CON and DON concentration profiles across BNR bioreactors, and laboratory batch experiments with the process mixed liquor were carried out at several full-scale BNR plants in northern Poland. The organic nitrogen (ON) components were divided into high CON, low CON, and DON based on sequential filtration through 1.2, 0.45 and 0.1 μm pore-size filters. The average influent DON(0.1 μm) (<0.1 μm) concentrations ranged from 1.1 g N/m(3) to 3.9 g N/m(3) and accounted for only 4-13% of total organic nitrogen. In the effluents, however, this contribution increased to 12-45% (the DON(0.1 μm) concentrations varied in a narrow range of 0.5-1.3 g N/m(3)). Conversions of ON inside the bioreactors were investigated in more detail in two largest plants, i.e. Gdansk (565,000 PE) and Gdynia (516,000 PE). Inside the two studied bioreactors, the largest reductions of the colloidal fraction were found to occur in the anaerobic and anoxic compartments, whereas an increase of DON(0.1 μm) concentrations was observed under aerobic conditions in the last compartment. Batch experiments with the process mixed liquor confirmed that DON(0.1 μm) was explicitly produced in the aerobic phase and significant amounts of ON were converted in the anoxic phase of the experiments., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Two estrogen fate and transformation models were integrated with a comprehensive activated sludge model (ASM) to predict estrogen removal based on biomass and solids production. Model predictions were evaluated against published full-scale plant data as well as results from a laboratory-scale sequencing batch reactor (SBR) fed synthetic wastewater. The estrogen fate model relating the rate of total estrogen degradation to soluble estrogen concentrations successfully predicted estrogen removals when compared with measured concentrations. Model fit 17α-ethinylestradiol (EE2) biodegradation rate constant was 19 to 43% of the estrone (E1) value and 31 to 72% of the 17β-estradiol (E2) value.

The increasingly more stringent phosphorus (P) discharge limits, which are below the concentrations reliably achievable with currently available technologies, demand for better understanding of phosphorus removal mechanisms. This study investigated the compositional fractions of phosphorus (P) in various effluents as well as the efficacy of different levels of treatment processes for removing different fractions of P in wastewater. The results showed that BNR can effectively remove most fractions of P, with relatively higher efficiencies (>93%) towards bioavailable forms of P including soluble reactive P (sRP), particulate reactive P (pRP) portion and particulate acid hydrolysable P (pAHP) and, it showed relatively lower efficiency (78%) towards organic P. Soluble acid hydrolysable P (sAHP) was not effectively removed (<40%). Chemical P removal process was more effective for elimination of sRP, sAHP and particulate organic P (pOP), but was not as effective for removing pAHP and, it exhibited nearly no removal of dissolved organic P (DOP). We found that chemical P removal process led to a significant increase in the concentration of pRP by up to 255%, indicating that these pRP (presumably as chemically bounded P) are likely formed through chemical precipitation/co-adsorption. Only 22% and 64% of the pRP was removed through tertiary clarifier and filtration, respectively. This implies that chemical addition converts sRP into particulate-associated P, mostly as pRP that was not easily removed by sedimentation and filtration, therefore, the efficacy of chemical P removal highly depends on the effectiveness of solid and liquid separation process. As more sRP and particulate P were removed through the series of treatment processes, the percentage contribution from organic P increases with the level of treatment due to its recalcitrant nature. Our results indicated that in order to achieve extremely low effluent P levels, technologies and processes that can enhance pRP and DOP removal will be required.

For biological nutrient removal (BNR) systems designed to maximize nitrogen removal, the effluent total nitrogen (TN) concentration may range from 2.0 to 4.0 g N/m(3) with about 25-50% in the form of organic nitrogen (ON). In this study, current approaches to modeling organic N conversions (separate processes vs. constant contents of organic fractions) were compared. A new conceptual model of ON conversions was developed and combined with Activated Sludge Model No. 2d (ASM2d). The model addresses a new insight into the processes of ammonification, biomass decay and hydrolysis of particulate and colloidal ON (PON and CON, respectively). Three major ON fractions incorporated are defined as dissolved (DON) (<0.1 µm), CON (0.1-1.2 µm) and PON (41.2 µm). Each major fraction was further divided into two sub-fractions - biodegradable and non-biodegradable. Experimental data were collected during field measurements and lab experiments conducted at the ''Wschod'' WWTP (570,000 PE) in Gdansk (Poland). The accurate steady-state predictions of DON and CON profiles were possible by varying ammonification and hydrolysis rates under different electron acceptor conditions. With the same model parameter set, the behaviors of both inorganic N forms (NH4-N, NOX-N) and ON forms (DON, CON) in the batch experiments were predicted. The challenges to accurately simulate and predict effluent ON levels from BNR systems are due to analytical methods of direct ON measurement (replacing TKN) and lack of large enough database (in-process measurements, dynamic variations of the ON concentrations) which can be used to determine parameter value ranges.

The WERF Nutrient Challenge is a multi-year collaborative research initiative established in 2007 to develop and provide current information about wastewater treatment nutrients (specifically nitrogen and phosphorus in wastewater), their characteristics, and bioavailability in aquatic environments to help regulators make informed decisions. The Nutrient Challenge will also provide data on nutrient removal so that treatment facilities can select sustainable, cost-effective methods and technologies to meet permit limits. To meet these goals, the Nutrient Challenge has teamed with a wide array of utilities, agencies, consultants, universities and other researchers and practitioners to collaborate on projects that advance these goals. The Nutrient Challenge is focusing on a different approach to collaborating and leveraging resources (financial and intellectual) on research projects by targeting existing projects and research that correspond with its goals and funding those aspects that the Nutrient Challenge identified as a priority. Because the Nutrient Challenge is focused on collaboration, outreach is an absolutely necessary component of its effectiveness. Through workshops, webinars, a web portal and online compendium, published papers, and conference lectures, the Nutrient Challenge is both presenting important new information, and soliciting new partnerships.

Estrogens from anthropogenic and livestock sources are a serious concern for aquatic ecosystems at concentrations less than 1 ng/L Fundamental process parameters to reduce estrogenic activity were investigated for two biotreatment methods: heterotrophic bacterial degradation in municipal activated sludge (AS) and a nitration process that is applicable to high NH4-N wastewaters. Batch tests with estrogen and nitro-estrogen compounds were conducted at nanogram per liter concentrations with mixed liquor from an AS wastewater treatment facility (WWTF) operating at a 3 day solids retention time (SRT) and a membrane bioreactor (MBR) WWTF operating at a 30-40 day SRT. The estrogenic activities of estrone (E1), 17beta-estradiol (E2), and 17alpha-ethinylestradiol (EE2) were reduced 80-97% following nitration. First-order biological degradation rate coefficients (kb) of the nitrated estrogens were 10-50% lower than the parent estrogen compounds. The kb values for EE2 in MBR and AS mixed liquors were similar, 1.67 and 1.63 L/gVSS-day respectively, indicating that the bacteria responsible for EE2 degradation were present at long and short SRTs. The kb values for E1 and E2 were 2 orders of magnitude greater than for EE2. EE2 degradation was 7.5 times faster in the presence of E1 and E2, and no effect was observed with other estrogen mixtures.

Understanding estrogen-removal mechanisms in wastewater treatment is imperative, as estrogens have environmental effects at trace concentrations. Previous research investigating co-metabolic degradation of 17alpha-ethinylestradiol (EE2) by ammonia-oxidizing bacteria (AOB) revealed that, in batch tests where high nitrite-nitrogen (NO2-N) concentrations occurred as a result of ammonia-nitrogen (NH4-N) oxidation by AOB, an abiotic estrogen nitration reaction actually was occurring--not co-metabolic degradation. This paper addresses nitration kinetics. A first-order abiotic nitration model was developed that predicts nitration of EE2, 17beta-estradiol (E2), and estrone (El) as a function of temperature, pH, estrogen (EE2, E2, and E1), and NO2-N concentration. A contact time of 3.6 to 4.1 days is required for 90% estrogen nitration at 500 mg/L NO2-N and pH 6.4. At 20 degrees C and pH 6.4, the threshold NO2-N concentration for nitration to occur is 9 mg/L; therefore, estrogen nitration is not likely in activated sludge treatment of domestic wastewater, but has potential for high-NH4-N-strength wastewaters.

The aim of this study was to investigate nitrogen mass balances occurring inside full-scale BNR activated sludge systems, with special attention to colloidal and dissolved organic nitrogen (CON and DON) transformations. For this purpose, laboratory experiments were carried out using process biomass from two large BNR plants in northern Poland. Two parallel batch reactors were operated in a 3-phase (anaerobic/anoxic/aerobic) cycle. In one reactor, the settled wastewater without any pretreatment was used, whereas the settled wastewater after coagulation-flocculation (to remove colloidal and particulate fractions) was added to another reactor. The chemical pretreatment of settled wastewater with ZnSO(4) did not adversely affect the observed nitrification rates in the (last) aerobic phase. It caused, however, a reduction of denitrification rates in the anoxic phase. Moreover, the chemical pretreatment did not appear to generally decrease DON but decreased CON. DON was explicitly produced in the aerobic phase and organic nitrogen conversion also occurred at a significant rate in the anoxic phase with biodegradable COD consumption and solids hydrolysis. The inorganic N mass balances revealed N losses up to approximately 10% which could be attributed to a few novel pathways of nitrogen removal, most likely aerobic denitrification or simultaneous nitrification/denitrification.

The effect of single-stage and 4-stage anoxic selectors and an anoxic/aerobic sequencing batch reactor (SBR) on the removal of readily biodegradable chemical oxygen demand (rbCOD) and slowly biodegradable COD (sbCOD) and on filamentous growth and sludge settleability was studied. Microbial community fingerprinting in the three selector configurations was done using automated ribosomal intergenic spacer analysis. Nostocoida limicola II and type 1851 filamentous bacteria were observed in all systems. The diluted sludge volume index (DSVI) decreased with increased selector staging. The rbCOD was almost completely removed in all selectors, and the lower DSVI values with increased selector staging were the result of greater sbCOD removal in the selector. The plug-flow kinetics in the SBR aerobic phase also improved DSVI. The bacteria community composition in the 4-stage selector system was found to be more similar to that for the SBR than for the single-stage selector.

Aceticlastic methanogens are seen as a key to digester capacity and stability. This paper develops and applies an assay to measure digester stability by measuring the maximum aceticlastic methane production rate (Vmax,ac). The Vmax,ac in combination with acetate concentrations was found to be an effective digestion monitoring tool to indicate process upsets. At steady state, thermophilic, first stage and short SRT digesters generally had a greater Vmax,ac than mesophilic, second stage or long SRT digesters. The ratio of the Vmax,ac to the plant aceticlastic methane production rate, termed the Acetate Capacity Number (ACN), is a measure of the excess capacity of the digester. Either Vmax,ac or ACN can be used to estimate the capability to handle higher organic loading rates. Monod modeling was used to predict Vmax,ac, ACN and maximum VS loading rates for mesophilic and thermophilic digestion and for staged digesters to better understand expected digestion capacity and stability.

Impacts of trace concentrations of estrogens on aquatic ecosystems are a serious environmental concern, with their primary source being wastewater treatment facility effluents. Increased removal of 17alpha-ethinylestradiol (EE2) has been reported for activated sludge treatment with long enough solids retention time for nitrification. Previous work based on batch tests with Nitrosomonas europaea and nitrifying activated sludge at high EE2 concentrations (>300 000 ng/L) and high NH4-N concentrations (>200 mg/L) has led to the hypothesis that ammonia oxidizing bacteria cometabolically degrade EE2. This work investigated EE2 transformation with N. europaea and Nitrosospira multiformis at environmentally relevant EE2 concentrations and LC-MS-MS to observe transformation products. Degradation of EE2 was not observed in batch tests with no NH4-N addition or with 10 mg/L NH4-N fed daily. At increased NH4-N concentrations (200-500 mg/L) EE2 transformation was observed, but the only detected products were nitrated EE2. Abiotic assays with growth medium confirmed EE2 removal by nitration, which is enhanced at low pH and high NO2-N concentrations. These results suggest that EE2 removal at low concentrations found in municipal treatment activated sludge systems is not due to cometabolic degradation by ammonia oxidizing bacteria, or to abiotic nitration, but most likely due to heterotrophic bacteria.

The abundance and relevance ofAccumulibacter phosphatis (presumed to be polyphosphate-accumulating organisms [PAOs]), Competibacter phosphatis (presumed to be glycogen-accumulating organisms [GAOs]), and tetrad-forming organisms (TFOs) to phosphorus removal performance at six full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants were investigated. Coexistence of various levels of candidate PAOs and GAOs were found at these facilities. Accumulibacter were found to be 5 to 20% of the total bacterial population, and Competibacter were 0 to 20% of the total bacteria population. The TFO abundance varied from nondetectable to dominant. Anaerobic phosphorus (P) release to acetate uptake ratios (P(rel)/HAc(up)) obtained from bench tests were correlated positively with the abundance ratio of Accumulibacter/(Competibacter +TFOs) and negatively with the abundance of (Competibacter +TFOs) for all plants except one, suggesting the relevance of these candidate organisms to EBPR processes. However, effluent phosphorus concentration, amount of phosphorus removed, and process stability in an EBPR system were not directly related to high PAO abundance or mutually exclusive with a high GAO fraction. The plant that had the lowest average effluent phosphorus and highest stability rating had the lowest P(rel)/HAc(up) and the most TFOs. Evaluation of full-scale EBPR performance data indicated that low effluent phosphorus concentration and high process stability are positively correlated with the influent readily biodegradable chemical oxygen demand-to-phosphorus ratio. A system-level carbon-distribution-based conceptual model is proposed for capturing the dynamic competition between PAOs and GAOs and their effect on an EBPR process, and the results from this study seem to support the model hypothesis.

This paper reports on the design, implementation, and results of a course focused on decentralized and onsite wastewater treatment in global contexts. Problem-based learning was the primary pedagogical method, with which students tackled real-world problems and designed systems to meet the needs of diverse populations. Both learning and course evaluations demonstrated that the course was successful in fulfilling learning objectives, increasing student design skills, and raising awareness of global applications. Based on this experience a list of recommendations was created for co-developing and team-teaching multidisciplinary design courses. These recommendations include ideas for aligning student and teacher goals, overcoming barriers to effective group-work, and imbedding continuous course assessments., (Copyright IWA Publishing 2008.)

The stability, capacity, and solids destruction efficiency of single versus two-stage anaerobic digestion was studied in bench-scale reactors using combined waste activated and primary sludge. Laboratory staged mesophilic digesters showed an improved volatile solids and volatile suspended solids destruction efficiency over a single-stage system (at the same total solids retention time [SRT]) of approximately 3.2 and 5.8 percentage points, respectively. To quantify stability and capacity, a new digester monitoring method was introduced that measured the digester maximum acetate utilization capacity, V(max,ac), and was used to investigate the potential for digester instability at different transient loadings. The ratio of the V(max,ac) value to the estimated acetate production rate for a given digester loading was termed the acetate capacity number (ACN). Values greater than 1.0 indicate excess acetate utilization capacity. The first stage of the laboratory two-stage mesophilic system (10-day SRT for each stage) had an ACN number of 1.3 compared with a value of 1.8 for the single-stage 20-day SRT digester. Thus, while a staged mesophilic system can improve solids destruction efficiency, it demonstrates a lower capacity for metabolizing highly variable loads.

Recuperative thickening of anaerobic digester sludge (thickening with solids return) yields increased digester capacity. Common thickening methods cause oxygen exposure to the digester sludge. This study evaluated the effects of various levels of oxygen exposure on the acetoclastic methanogens. Gravity belt thickening had no detrimental effect on the acetoclastic activity. From a 7-day batch test with continuous oxygen exposure of digester sludge, a 12% loss in acetoclastic activity was predicted for a digester with a 20-day solids retention time (SRT) and 100% recycle with recuperative thickening via dissolved air flotation thickening. However, a greater loss (27%) was found from a long-term, bench-scale digester operated under similar conditions. This loss did not affect the digester performance, as measured by volatile solids destruction. This research suggests that recuperative thickening may not affect digester performance at a long SRT with constant operation, but may change the reserve capacity of the anaerobic community.

Methanosarcina species with a high maximum specific growth rate (mumax) and high half-saturation coefficient (KS) and Methanosaeta species with a low mumax and low KS are the only known aceticlastic methanogens. Because of Methanosaeta's low KS, the low acetate concentrations in conventional, mesophilic anaerobic digestion yield Methanosaeta dominance. However, Methanosarcina absorbs increases in acetate more efficiently and thus promotes more stable digestion. This paper tests the hypothesis that decreasing digester feeding frequencies can increase Methanosarcina predominance. Two acetate-fed reactors were established at a 17-day solids retention time. One reactor was fed hourly, and one was fed once daily. Microscopic and molecular methods were used to verify that the hourly fed reactor enriched for Methanosaeta, while the daily fed reactor enriched for Methanosarcina. Growth and substrate-use kinetics were measured for each reactor. A digester overload condition was simulated, and the Methanosarcina-enriched reactor was found to perform better than the Methanosaeta-enriched reactor. These findings indicate that Methanosarcina dominance can be achieved with infrequent feedings, leading to more stable digestion.

The ADM1 was employed to assess the effect of variations in solids hydrolysis and acetoclastic methanogen process characterizations on municipal digester stability relating to excess acetate utilization capacity. First-order single- and dual-pathway hydrolysis rate functions and single and competitive acetoclastic methanogen rate functions were implemented in the ADM1. The acetate capacity number (ACN), defined as the ratio between the maximum acetate utilization rate and the average acetate production rate, was used to index digester instability. Simulations of a single CSTR at steady state indicate a similar ACN can be obtained with a 12-day SRT digester dominated by Methanosarcina sp and a 24-day SRT digester dominated by Methanosaeta sp. An increase in ACN with a decrease in SRT representing Methanosarcina sp. selection was observed for particulate feed loadings from 40 g COD/L to 90 g COD/L. Feeding frequency and dual-pathway hydrolysis were found to have less effect on the ACN than the competitive acetoclastic model structure.

A bacterial strain, SL-1T, capable of degrading trichloroethene was isolated from a laboratory enrichment in the Department of Civil and Environmental Engineering, University of Washington, USA. The material in the enrichments was derived from a soil sample from Seattle, WA, USA. Strain SL-1T was capable of using phenol as a source of carbon and energy. Chemotaxonomic, morphological, physiological and phylogenetic analyses showed that strain SL-1T is a member of the genus Pseudonocardia. The ability of strain SL-1T to utilize phenol and degrade trichloroethene, as well as other phenotypic properties and the results from a 16S rRNA phylogenetic analysis, led to the proposal of a novel species, Pseudonocardia chloroethenivorans sp. nov. The type strain is SL-1T (=ATCC BAA-742T=DSM 44698T). Trichloroethene and other chloroethenes are major pollutants at many environmental sites, and P. chloroethenivorans has biodegradation properties that should be of interest to environmental microbiologists and engineers.

In order to study the effect of different chloroethenes (electron acceptors) on the bacterial composition of dechlorinating communities, two reductive dechlorinating enrichment cultures were developed that were able to reduce trichloroethene (TCE) and cis-1,2-dichloroethene (cis-DCE) to ethene using hydrogen as electron donor, respectively. The inoculum for the cultures was material from a methanogenic fluidized bed reactor (FBR), which was originally seeded with digester sludge and showed a stable capacity for tetrachloroethene (PCE) reduction to ethene for over six years. Molecular methods were used to determine and compare the microbial communities of these two enrichment cultures. A clone library of bacterial 16S rRNA genes was generated for each enrichment. The clones were screened into different groups by restriction fragment length polymorphism (RFLP) analysis using two different four base pair recognition restriction enzymes. A total of 12 sequence types were identified by phylogenetic analysis of nearly complete 16S rDNA sequences ( approximately 1450 bp). The sequences were affiliated with six recognized phyla of the domain Bacteria: Firmicutes (low G+C Gram-positives), Chloroflexi (green non-sulphur bacteria), Actinobacteria (high G+C Gram-positives), Bacteroidetes (Cytophaga-Flexibacter-Bacteroides), Nitrospira and Spirochaetes. The results led to the identification of an organism closely related to Dehalococcoides ethenogenes to be the presumptive dechlorinator in both enrichments. Different electron acceptors affected the bacterial diversity and the community profiles of the two enrichments. Most of the sequences identified in our dechlorinating enrichments shared high similarities with sequences previously obtained from other enriched dechlorinating cultures and chlorinated-compound-contaminated sediments or aquifers, suggesting these bacteria may have direct or indirect roles in reductive dechlorination.

The steady-state performance of thermophilic (55 degrees C) and mesophilic (35 degrees C) anaerobic digestion as a function of solids retention time (SRT) was evaluated in laboratory digesters at SRTs ranging from 4 to 15 days, and in pilot-plant digesters at a 20-day SRT. All of the digesters were fed the same source of municipal combined primary and secondary waste sludge. The destruction efficiency of volatile solids increased from 53% to 66% as the SRT was increased from 6 days to 20 days. The average destruction efficiency of volatile solids was 3 percentage points higher for the thermophilic digester at the 6-day SRT and approximately 1 percentage point higher for the higher SRTs, but the difference was only statistically significant at the 15-day SRT. Based on volatile suspended solids measurements, the thermophilic solids destruction efficiency was approximately 4 percentage points higher at the 10- and 15-day SRTs. At a 4-day SRT, methanogenic activity could only be maintained in the thermophilic digester. The pH, alkalinity, ammonia, volatile fatty acid, and soluble chemical oxygen demand concentrations were higher for the thermophilic digester at each SRT. At SRTs of 10 days and less, the thermophilic digester had a much higher propionate and slightly higher butyrate concentration. Carbohydrates were readily degraded by both digesters, protein was the major component in the sludge at the long SRTs, and lipid degradation increased with increasing SRT.

Site evaluation for bioremediation of chlorinated ethenes may need treatability studies to assess the reductive dechlorination potential of vinyl chloride (VC). Dehalogenation of vinyl bromide (VB) was investigated as a surrogate measurement for the dechlorination potential of VC. VB dehalogenation rates and kinetics were studied and compared with those of VC by a methanogenic reductive dechlorinating enrichment culture that was dominated by Dehalococcoides species and by microcosms from two chloroethene-contaminated sites. The enrichment culture dehalogenated VB to ethene at higher rates than VC at similar concentrations. VB was dehalogenated with a higher enzyme affinity than was VC, as indicated by their half-velocity constants, 240 +/- 150 and 21 +/- 8 microM, for VC and VB, respectively. Cross-inhibition study exhibited some evidence for competitive inhibition between VC and VB, suggesting that their degradation might be catalyzed by the same enzyme in the culture. Laboratory microcosm studies using subsurface soil and groundwater from two contaminated sites demonstrated that the production of the reductive dehalogenation product (ethene) could be detected faster with VB as a substrate than with VC. As a result, a substantially shorter (up to 5-10 times) incubation time would be required to detect the same level of reductive dehalogenation activity using VB as a surrogate for VC in treatability assessments.

The Michaelis-Menten biodegradation kinetics (k and Ks) of aromatic compounds and trichloroethene (TCE) by an aerobic enrichment culture grown on phenol and dominated by a unique filamentous bacterium were measured. The average k and Ks values for phenol, benzene (B), toluene (T), ethylbenzene (E), o-xylene (oX), p-xylene (pX), naphthalene and TCE in g per g VSS-d and mg L-1 were 5.72 and 0.34, 1.20 and 0.51, 2.09 and 0.47, 0.77 and 0.23, 0.61 and 0.16, 0.73 and 0.23, 0.17 and 0.18, and 0.16 and 0.18, respectively. Significant variability in these measured kinetics was noted between tests conducted over the 5-month period during which the fed-batch culture with a 5-day solids retention time was maintained; the coefficient of variation of the k and Ks values ranged from 11-43% and 4-50%, respectively. This variation was significantly greater than the method measurement error on a given date. Degradation of BTEoXpX mixtures could be described by a basic competitive inhibition model. Batch tests during which the culture was fed individual BTEX compounds showed the culture grew poorly on the xylenes and had poor subsequent xylene degradation rates. This work indicates the potential to simultaneously treat a mixture of volatile organic compounds using this consortium, and the ability to predict the mixture biodegradation rates on the basis of the individual compound biodegradation kinetics.