Activated Sludge Wastewater Treatment Research Articles

Heterogeneous multi-population competitive algorithm and its application in ASM1 parameter estimation.

Activated sludge model 1 (ASM1) is a biokinetic model of the activated sludge wastewater treatment process, which has several uncertain parameters and complex chemical reaction processes. This model is often used in simulations to assess whether the effluent quality of wastewater meets discharge standards. Accurate and rapid estimation of the parameters of ASM1 is a necessary prerequisite for its successful application in industrial practice. However, ASM1-based parameter estimation is difficult to implement in practical operations due to the slow convergence rate and low convergence accuracy of most parameter estimation algorithms as well as the non-uniqueness of parameter estimation. In view of this, a novel metaheuristic optimization algorithm combining Legendre function network and dynamic partitioning strategy, named heterogeneous multi-group competitive algorithm (HMCA), is proposed to overcome the problem of low algorithmic efficiency and poor practicality in parameter estimation. The performance of the algorithm in terms of convergence speed, convergence accuracy and applicability is verified by two sets of test functions and eight state-of-the-art comparison algorithms. The superior performance of the algorithm in parameter estimation is then validated in conjunction with Benchmark Simulation Model no. 1 (BSM1) and four sets of operational data.

Gordonia pseudamarae sp. nov., a home for novel actinobacteria isolated from stable foams on activated sludge wastewater treatment plants.

The taxonomic status of two Gordonia strains, designated BEN371 and CON9T, isolated from stable foams on activated sludge plants was the subject of a polyphasic study which also included the type strains of Gordonia species and three authenticated Gordonia amarae strains recovered from such foams. Phylogenetic analyses of 16S rRNA gene sequences showed that these isolates formed a compact cluster suggesting a well-supported lineage together with a second branch containing the G. amarae strains. A phylogenomic tree based on sequences of 92 core genes extracted from whole genome sequences of the isolates, the G. amarae strains and Gordonia type strains confirmed the assignment of the isolates and the G. amarae strains to separate but closely associated lineages. Average nucleotide index (ANI) and digital DNA-DNA hybridisation (dDDH) similarities showed that BEN371 and CON9T belonged to the same species and had chemotaxonomic and morphological features consistent with their assignment to the genus Gordonia. The isolates and the G. amarae strains were distinguished using a range of phenotypic features and by low ANI and dDDH values of 84.2 and 27.0 %, respectively. These data supplemented with associated genome characteristics show that BEN371 and CON9T represent a novel species of the genus Gordonia. The name proposed for members of this taxon is Gordonia pseudamarae sp. nov. with isolate CON9T (=DSM 43602T=JCM 35249T) as the type strain.

Strategies for Controlling Filamentous Bulking in Activated Sludge Wastewater Treatment Plants: The Old and the New

Filamentous bulking and foaming are the most common settling problems experienced in activated sludge (AS) wastewater treatment plants (WWTPs). The quality of the final effluent is poor during episodes of bulking and foaming, which is an environmental, human health and economic burden. Remedial measures are often ineffective, and traditional non-specific methods such as chlorination may also negatively impact important functional bacterial species such as nitrifiers. Modifications to older methods as well as new strategies are required for controlling filamentous bulking. Laboratory testing needs to be followed by testing at scale in WWTPs. This review describes the filamentous bacteria responsible for filamentous bulking, with a focus on their global distribution and known factors which are selective for the growth of specific filaments. Traditional and new non-specific and biological control strategies are reviewed and discussed. Research gaps are identified with the aim of promoting continued efforts to establish effective control strategies for filamentous sludge bulking.

Biological control of problematic bacterial populations causing foaming in activated sludge wastewater treatment plants-phage therapy and beyond.

The production of a stable foam on the surfaces of reactors is a global operating problem in activated sludge plants. In many cases, these foams are stabilized by hydrophobic members of the Mycolata, a group of Actinobacteria whose outer membranes contain long-chain hydroxylated mycolic acids. There is currently no single strategy which works for all foams. One attractive approach is to use lytic bacteriophages specific for the foam stabilizing Mycolata population. Such phages are present in activated sludge mixed liquor and can be recovered readily from it. However, no phage has been recovered which lyses Gordonia amarae and Gordonia pseudoamarae, probably the most common foaming Mycolata members. Whole genome sequencing revealed that both G. amarae and G. pseudoamarae from plants around the world are particularly well endowed with genes encoding antiviral defence mechanisms. However, both these populations were lysed rapidly by a parasitic nanobacterium isolated from a plant in Australia. This organism, a member of the Saccharibacteria, was also effective against many other Mycolata, thus providing a potential agent for control of foams stabilized by them.

Negative impacts of cleaning agent DEPTAL MCL® on activated sludge wastewater treatment system.

DEPTAL MCL® is a professional cleaning agent approved by the Portuguese Food Regulatory Authority and is used in agro-food industries, namely in fish canning industries in the north of Portugal. Its extensive use during cleaning procedures results in potential significant negative impacts on the performance of the downstream municipal wastewater treatment plant (WWTP). A lab-scale extended areation activated sludge wastewater treatment system, continuously fed by influent collected at a municipal WWTP, was used to assess the impact of a range of DEPTAL MCL® concentrations during 72 h. Despite distinct activated sludge community composition (due to its dynamic nature) and variations in real influent characteristics, a relevant impact was observed. DEPTAL MCL® effect was underscored through the use of a multivariate analysis using seventeen physicochemical operational factors and nineteen quantitative image analysis (QIA) parameters. DEPTAL MCL® exerted a severe negative impact on phosphorous (P-PO4) removal, total nitrogen (TN) removal and sludge volume index (SVI). With increasing DEPTAL MCL® concentrations, both P-PO4 and TN removal were affected and diminished proportionally. Moreover, several QIA parameters indicate defloculation when DEPTAL MCL® was present, in particular for intermediate size aggregates with significant impacts. Optical density of the effluent (Ode), displayed an increase of effluent turbidity. Percentage of area covered by small aggregates (%Areasml) was also significantly higher for the intermediate and higher DEPTAL MCL® concentrations tested. Principal component analysis exhibited 3 distinct ordenations: (i) control without addition of DEPTAL MCL®; (ii) addition of 0.03% and 0.06% and of (iii) 0.13 and 0.26% (v DEPTAL MCL®/v aeration tank). Canonical correspondence analysis (CCA) was used to correlate the physicochemical data, QIA and the filamentous bacteria species prevalence to DEPTAL MCL® concentration and incubation time. A time persistent DEPTAL MCL® effect was observed, underscoring the need of a pretreatment of wastewater containing this cleaning agent.

Bioaerosols emission characteristics from wastewater treatment aeration tanks and associated health risk exposure assessment during autumn and winter

Aeration tanks from activated sludge wastewater treatment plants (WWTPs) can release a large amount of bioaerosols that can pose health risks. However, risk characterization of bioaerosols emissions form wastewater treatment plants is currently not systematically carried out and still in its infancy. Therefore, this study investigated emission characteristic of two indicator model bioaerosols Staphylococcus aureus and Escherichia coli, emitted from aeration tanks of a municipal WWTP. Monte Carlo simulation was then used to quantitatively assess microbial risk posed by different aeration modes under optimistic and conservative estimates. Further to this, two different exposure scenarios were considered during 3 days sampling campaign in autumn and winter. Results showed that the bioaerosol concentration from microporous aeration tank (20–262 CFU m−3) was one order of magnitude lower than rotating disc aeration tank. Average aerosolization rate was 7.5 times higher with mechanical aeration mode. Health risks of exposed populations were 0.4 and 9.6 times higher in winter than in autumn for E. coli and S. aureus bioaerosol, respectively. Health risks of staff members were 10 times higher than academic visitors. Interesting results were observed for academic visitors without personal protective equipment (PPE) respectively exposed to S. aureus and E. coli bioaerosol in autumn and winter: while the derived infection risk met the United States Environmental Protection Agency (U.S. EPA) benchmark under optimistic estimation, the disease risk burden was over the World Health Organization (WHO) benchmark under conservative estimation. These revealed that only satisfying one of the two benchmarks didn't mean absolute acceptable health risk. This study could facilitate the development of better understanding of bioaerosol quantitative assessment of risk characterizations and corresponding appropriate risk control strategies for wastewater utilities.

A probabilistic assessment of microbial infection risks due to occupational exposure to wastewater in a conventional activated sludge wastewater treatment plant

Exposure to pathogens during wastewater treatment could result in significant health risks. In this paper, a probabilistic approach for assessing the risks of microbial infection for workers in an activated sludge wastewater treatment plant is presented. A number of exposure routes were modelled, including hand-to-mouth and droplet ingestion of untreated wastewater, droplet ingestion and inhalation of aerosols after secondary treatment, and ingestion of sludge during drying. Almost all workers exposed to untreated wastewater could be infected with the three selected potential pathogens of pathogenic E. coli, Norovirus and Cryptosporidium spp. Hand-to-mouth ingestion is the single most significant route of exposure at the head of works. There is also a risk of infections resulting from ingestion of droplets or inhalation of aerosols at the aeration tanks or contaminated hands at the clarifiers during secondary wastewater treatment. For sludge, the risks of infection with Norovirus was found to be the highest due to accidental ingestion (median risks of 2.2 × 10−2(±3.3 × 10−3)). Regardless of the point and route of exposure, Norovirus and Cryptosporidium spp. presented the highest risks. The study finds that occupational exposure to wastewater at wastewater treatment plants can result in significant viral and protozoan infections. This risk assessment framework can be used to establish and measure the success of risk reduction measures in wastewater treatment plants. These measures could include the use of personal protective equipment and adherence to strict personal hygiene.

A Comprehensive Derivation and Application of Reference Values for Benchmarking the Energy Performance of Activated Sludge Wastewater Treatment

Wastewater treatment plants (WWTPs) are facing challenges concerning the service’s effectiveness and reliability, as well as the efficiency and sustainability of resource utilization, where energy represents one of the higher costs in activated sludge (AS) treatment. This paper presents the latest developments in the new energy performance indices (PXs) we have been developing for benchmarking, i.e., assessing and improving the performance of this widely used treatment. PXs compare the energy consumption with the energy requirements for the carbon and nitrogen removals needed for the plant’s compliance with the discharge consents (the closer they are, the better the performance). PXs are computed by applying to the state variables a performance function that is defined by the reference values for excellent, acceptable, and unsatisfactory performance. This paper shows the rationale for selecting the state variables for the AS energy performance and the comprehensive derivation of the equations to determine the reference values for energy consumption, which incorporate the effect of key parameters (flows, concentrations, and operating conditions). Reference values for the operating conditions affecting the energy performance are also proposed. A sensitivity analysis identified the key parameters for improving the aeration performance: α, F, and SOTE for air diffusers, and α and N0 for mechanical aerators. Fourteen Portuguese urban WWTPs (very diverse in size and inflows) were analyzed, and aeration (0.08–1.03 kWh/m3) represented 25–80% of total energy consumption (0.23–1.30 kWh/m3). The reference values for excellent performance were 0.23–0.39 kWh/m3 (P25–P75) for AS systems with air diffusers and 0.33–0.80 kWh/m3 for those with mechanical aerators. A comprehensive application in one WWTP (16–18 d solids retention time) showed the system’s ability at identifying which operating conditions to adjust (to F/M ratio lower than 0.09 d−1 and decreasing aeration during the low season) to improve the energy performance/savings while maintaining the treatment’s effectiveness and reliability.

Formation of algal flocs from activated sludge in synthetic medium and secondary treated wastewater

AbstractBACKGROUNDPhotogranules seem to be an alternative solution for wastewater treatment and biomass harvesting. The aim of our work was to investigate the formation of algal flocs as an efficient method for the treatment of wastewater and separation of algal cells.RESULTSThe progressive change of activated sludge to algal flocs was evaluated under static and stirring conditions using a synthetic substrate (modified Blue Green‐11 medium) and the secondary effluent from an activated sludge wastewater treatment plant. Algal flocs were observed in all cultures by day 10 and a high chlorophyll concentration was detected in all cases reaching values up to 610–630 μg L−1 at the end of the experiment after 30 days. The size of flocs in all cultures at either static or mixing conditions ranged from 330 to 994 μm, resulting in biomass concentration from 330 to 660 mg L−1. The cultures in secondary effluent at static conditions presented the greatest size, and the mean diameter on days 10 and 30 was 250 and 994 μm, respectively. The cultures were efficient in nutrient removal and, by the end of the experiment, nitrates and phosphates were completely consumed.CONCLUSIONSAlgal flocs were developed from activated sludge without adding any other flocculant and a highly efficient biomass was developed, which can easily settle and remove nutrients from the medium. © 2022 Society of Chemical Industry (SCI).

Comparative Genomics of Members of the Genus Defluviicoccus With Insights Into Their Ecophysiological Importance.

Members of the genus Defluviicoccus occur often at high abundances in activated sludge wastewater treatment plants designed to remove phosphorus, where biomass is subjected to alternating anaerobic feed/aerobic famine conditions, believed to favor the proliferation of organisms like Ca. Accumulibacter and other phosphate-accumulating organisms (PAO), and Defluviicoccus. All have a capacity to assimilate readily metabolizable substrates and store them intracellularly during the anaerobic feed stage so that under the subsequent famine aerobic stage, these can be used to synthesize polyphosphate reserves by the PAO and glycogen by Defluviicoccus. Consequently, Defluviicoccus is described as a glycogen-accumulating organism or GAO. Because they share a similar anaerobic phenotype, it has been proposed that at high Defluviicoccus abundance, the PAO are out-competed for assimilable metabolites anaerobically, and hence aerobic P removal capacity is reduced. Several Defluviicoccus whole genome sequences have been published (Ca. Defluviicoccus tetraformis, Defluviicoccus GAO-HK, and Ca. Defluviicoccus seviourii). The available genomic data of these suggest marked metabolic differences between them, some of which have ecophysiological implications. Here, we describe the whole genome sequence of the type strain Defluviicoccus vanusT, the only cultured member of this genus, and a detailed comparative re-examination of all extant Defluviicoccus genomes. Each, with one exception, which appears not to be a member of this genus, contains the genes expected of GAO members, in possessing multiple copies of those for glycogen biosynthesis and catabolism, and anaerobic polyhydroxyalkanoate (PHA) synthesis. Both 16S rRNA and genome sequence data suggest that the current recognition of four clades is insufficient to embrace their phylogenetic biodiversity, but do not support the view that they should be re-classified into families other than their existing location in the Rhodospirillaceae. As expected, considerable variations were seen in the presence and numbers of genes encoding properties associated with key substrate assimilation and metabolic pathways. Two genomes also carried the pit gene for synthesis of the low-affinity phosphate transport protein, pit, considered by many to distinguish all PAO from GAO. The data re-emphasize the risks associated with extrapolating the data generated from a single Defluviicoccus population to embrace all members of that genus.

Analysis of Some Properties of an Activated Sludge Wastewater Treatment Model

The aim of the paper is to study some properties of a model describing the wastewater treatment process with activated sludge. The process configuration consists of an aeration tank and of a secondary settler. The mathematical model is described by three nonlinear ordinary differential equations. The paper presents important results on existence and uniqueness of positive model solutions. Numerical computations establish the stability properties of the equilibrium points with respect to two important control model parameters and present the corresponding input-output static characteristics. The dynamic behaviour of the model solutions is illustrated through computer experiments carried out in the computer algebra system Maple. The results can be useful in designing real control strategies of the wastewater treatment processes with activated sludge.

Smart energy savings for aeration control in wastewater treatment

This paper describes control algorithm of minimum energy loss in the aeration process of wastewater treatment in the activated sludge system. The basic equations of the aeration process have been proposed in this paper with the dissolved oxygen concentration (DO) and the amount of sludge discharge (QW) being dual-parameter control variables and the organic matter concentration S and the microbial concentration X in the aeration tank being the state variables. Based on the present study on optimization control of wastewater treatment, the multivariable optimal control model with restriction factors have been presented in the paper. The model with modern control theory and system analysis are introduced into the field of activated sludge wastewater treatment. It takes the two most important control parameters (DO &QW), effluent quality as restriction factor. The minimum energy consumption in the aeration process is used as the objective function. This approach has been carried out and the results show that adopting optimal control strategy in the aeration process can effectively conserve energy with the assurance of effluent water quality.

Seasonal microbial community dynamics complicates the evaluation of filamentous bulking mitigation strategies in full-scale WRRFs

The activated sludge wastewater treatment process has been thoroughly researched in more than 100 years, yet there are still operational challenges that have not been fully resolved. Such a challenge is the control of filamentous bulking caused by the overgrowth of certain filamentous bacteria. In this study, we tested different mitigation strategies to reduce filamentous bulking, caused by two common filamentous genera found in full-scale water resource recovery facilities (WRRF), Candidatus Microthrix and Candidatus Amarolinea. PAX dosing, ozone addition, hydrocyclone implementation, and the addition of nano-coagulants were tested as mitigation strategies in four parallel treatment lines in a full-scale WRRF over three consecutive years. Unexpectedly, the activated sludge settleability was not affected by any of the mitigation strategies. Some of the strategies appeared to have a strong mitigating effect on the two filamentous species. However, detailed analyses of the microbial communities revealed strong recurrent seasonal variations in all four lines, including the control line which masked the real effect. After removing the effect of the seasonal variation by using a time-series decomposition approach, it was clear that the filamentous bacteria were mostly unaffected by the mitigation strategies. Only PAX dosing had some effect on Ca. Microthrix, but only on one species, Ca. Microthrix subdominans, and not on the most common Ca. Microthrix parvicella. Overall, our study shows the importance of long-term monitoring of microbial communities at species level to understand the normal seasonal pattern to effectively plan and execute full-scale experiments. Moreover, the results highlight the importance of using parallel reference treatment lines when evaluating the effect of mitigation strategies in full-scale treatment plants.

Performance assessment of ultrasonic sludge disintegration in activated sludge wastewater treatment plants under nutrient-deficient conditions

Although biological oxidation is a commonly applied technique for the removal of pollutants in industrial and municipal wastewater, one major drawback is its excess sludge production, having both an environmental and economic impact. The integration of ultrasonic sludge disintegration in a conventional biological wastewater treatment plant was explored experimentally via assessing the long-term effects on excess sludge reduction by conducting on-site pilot tests at a food processing company and a biodiesel production plant. Two sequencing batch reactors, each with a maximum active volume of 700 L, were operated in parallel for the treatment of industrial wastewater with nutrient deficient characteristics (COD/N/P ratio of 100/1.4/0.12) for over 6 months. One of the reactors was equipped with a side-stream recirculation system to enable the ultrasonic treatment of part of the settled sludge, whereas the other was operated in a conventional way as a reference. The experimental results show a reduction of the observed sludge production yield by 15% to 45% for the ultrasonically treated sequencing batch reactor compared to the conventional one, at a relatively low specific energy of less than 6,000 kJ/kg DS when 15 to 30% of the total sludge mass is treated per day. It was proven that the sludge reduction efficiency is directly proportional to the applied sludge retention time, suggesting that the ultrasonic treatment converts part of the hardly biodegradable particulate material in the activated sludge and improves its biodegradability. During the long-term pilot experiment at the biodiesel production plant, the electrical energy needed for decreasing the excess sludge production by 1 ton could be reduced by more than 80% when the sludge retention time was increased from 11 to 44 days. In addition, the sludge settling characteristics at the food processing company were considerably improved by the ultrasonic treatment, enabling to reduce or even avoid the need for external addition of expensive nutrients to prevent sludge bulking. Finally, an improved effluent quality was observed. Improvements of 7 to 17%, 9 to 20% and 17 to 30% for COD, nitrogen and phosphorus removal, respectively, were established.

Activated Sludge Wastewater Treatment Systems Based on Parallel Intelligence

As a typical biochemical treatment method, the activated sludge is used in various wastewater treatments. Activated wastewater treatment process is hard to control because of the complex nonlinear behavior, however, the stability of dissolved oxygen concentration (DOC) in the reaction tank is related to the quality and efficiency of processing. In this paper, a parallel wastewater treatment system based on the parallel intelligence theory is proposed to deal with the time-varying, nonlinear, and delay in the wastewater treatment process. Besides, computational experiments are designed and conducted to explore and verify the control strategy, which will guide the actual process through virtual and real interaction, so as to adapt to the complex and changeable wastewater treatment environment.

Segmentation of Activated Sludge Flocs in Microscopic Images for Monitoring Wastewater Treatment

The proposed work describes an approach for segmentation of activated sludge flocs from the microscopic images for monitoring wastewater treatment. The morphological features of flocs (microbial aggregates) and filaments are related to the state of an activated sludge wastewater treatment plant and must be monitored for proper functioning. Hence, image processing and analysis could be a time‐saving monitoring tool. To address this challenge, we propose a novel framework involving a multiphase edge detection algorithm based on information theory. The proposed framework is evaluated and scrutinized critically considering the artifacts found in the photographs tested. To evaluate the segmentations, gold approximation of estimated truth images is created. In addition, the performance was subjectively evaluated for its potential for segmenting activated sludge images. Experimental results show that the proposed framework exhibits the good results and demonstrates its effectiveness.

Development of a Nonlinear Model Predictive Controller of Flexible Structure for Greenhouse Gas Emissions Minimisation in Pulp and Paper Wastewater Treatment Processes

It is a well-established fact that activated sludge Wastewater Treatment Plants (WWTP) are placed among the most significant contributors of Greenhouse Gas (GHG) emissions. The task of intelligent emission control of such plants is a great challenge, mainly due to the highly nonlinear dynamics and associated uncertainty of its constituent bioprocesses. The need for more efficient and environmentally conscious control is ever increasing, due to the upsurge of industrial and urban water usage. In the current work, a nonlinear Model Predictive Control (MPC) scheme that considers an activated sludge model is developed for pulp and paper industry. The nonlinear MPC employs a flexible controller input structure through a process superstructure formulation that enables the adaptive utilisation of the most suitable set of manipulated variables based on the prevailing operating conditions. A generalised framework of this flexible controller type along with a nonlinear dynamic model for the plant accounting for the simultaneous satisfaction of stringent control objectives on the effluent quality and the GHG minimisation is developed. The proposed controller incorporates manipulated variables for all admissible aeration intensities, internal flow recirculation streams, external carbon sources, influent flow distribution in each bioprocessing unit, unit volume variations and recirculation flow percentage. Accurate simulations indicate the achievement of a superior emission reduction performance compared to state-of-the-art MPC implementations in WWTP. Every consideration of a manipulated variable set is associated with respect to its contribution to the total GHG reduction for each mode of operation.

A Practical Methodology for Forecasting the Impact of Changes in Influent Loads and Discharge Consents on Average Energy Consumption and Sludge Production by Activated Sludge Wastewater Treatment

This paper proposes a simple and easy-to-use methodology for forecasting the impact of changes in influent chemical oxygen demand (COD) and in the emission limit values (ELVs) of COD and total nitrogen on average energy requirements for aeration and sludge production by activated sludge wastewater treatment plants (WWTPs). The methodology is based on mass balances of sludge production and oxygen requirements for carbonaceous material biodegradation and/or nitrification, oxygen transfer and aeration equipment efficiency. Using average values of historical data of regular monitoring (water quality and operating conditions) WWTP-specific equations of oxygen requirements, energy consumption and sludge production are derived as a function of influent COD and influent N-total, which may be used to quantify the impact of influent and ELV changes. The methodology was tested in five extended aeration WWTPs for three scenarios established by the utility. The results show that increasing influent COD, from 900 to 1300 mg/L, for example, significantly increases the energy consumption by 49% and sludge production by 53%. For influent 54–68 mg/L N-total, imposing 15 mgN/L ELV results in a 9–26% increase in energy consumption. The COD ELV change studied (season-specific, from 150 mg/L 12 months/year to 125 mg/L 8 months/year to 100 mg/L 4 months/year) increases the energy consumption by 1.8–2.6% and the sludge production by 4.3–5.4%.

Biogas, Solar and Geothermal Energy—The Way to a Net-Zero Energy Wastewater Treatment Plant—A Case Study

Wastewater treatment plants designed to meet the requirements of discharging wastewater to a receiving water body are often not energy optimised. Energy requirements for conventional activated sludge wastewater treatment plants are estimated to range from 0.30 to 1.2 kWh/m3, with the highest values achieved using the nitrification process. This article describes the energy optimisation process of the wastewater treatment plant in Gubin (Poland) designed for 90,000 PE (population equivalent) using renewable energy sources: solar, biogas, and geothermal. At the analysed wastewater treatment plant electricity consumption for treating 1 m3 of wastewater was 0.679 kWh in 2020. The combined production of electricity and heat from biogas, the production of electricity in a photovoltaic system, and heat recovery in a geothermal process make it possible to obtain a surplus of heat in relation to its demand in the wastewater treatment plant, and to cover the demand for electricity, with the possibility of also selling it to the power grid.

Activity-Based Cell Sorting Reveals Resistance of Functionally Degenerate Nitrospira during a Press Disturbance in Nitrifying Activated Sludge.

ABSTRACTManaging and engineering activated sludge wastewater treatment microbiomes for low-energy nitrogen removal requires process control strategies to stop the oxidation of ammonium at nitrite. Our ability to out-select nitrite-oxidizing bacteria (NOB) from activated sludge is challenged by their metabolic and physiological diversity, warranting measurements of their in situ physiology and activity under selective growth pressures. Here, we examined the stability of nitrite oxidation in activated sludge during a press disturbance induced by treating a portion of return activated sludge with a sidestream flow containing free ammonia (FA) at 200 mg NH3-N/liter. The nitrite accumulation ratio peaked at 42% by day 40 in the experimental bioreactor with the press disturbance, while it did not increase in the control bioreactor. A subsequent decrease in nitrite accumulation within the experimental bioreactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). We applied bioorthogonal noncanonical amino acid tagging (BONCAT) coupled with fluorescence-activated cell sorting (FACS) to investigate changes in the translational activity of NOB populations throughout batch exposure to FA. BONCAT-FACS confirmed that the single Nitrospira ASV washed out of the experimental bioreactor had reduced translational activity following exposure to FA, whereas the two Nitrospira ASVs that emerged after process acclimation were not impacted by FA. Thus, the coexistence of functionally degenerate and physiologically resistant Nitrospira populations provided resilience to the nitrite-oxidizing function during the press disturbance. These results highlight how BONCAT-FACS can resolve ecological niche differentiation within activated sludge and inform strategies to engineer and control microbiome function.IMPORTANCE Nitrogen removal from activated sludge wastewater treatment systems is an energy-intensive process due to the large aeration requirement for nitrification. This energy footprint could be minimized with engineering control strategies that wash out nitrite-oxidizing bacteria (NOB) to limit oxygen demands. However, NOB populations can have a high degree of physiological diversity, and it is currently difficult to decipher the behavior of individual taxa during applied selective pressures. Here, we utilized a new substrate analog probing approach to measure the activity of NOB at the cellular translational level in the face of a press disturbance applied to the activated sludge process. Substrate analog probing corroborated the time series reactor sampling, showing that coexisting and functionally degenerate Nitrospira populations provided resilience to the nitrite oxidation process. Taken together, these results highlight how substrate analog approaches can illuminate in situ ecophysiologies within shared niches, and can inform strategies to improve microbiome engineering and management.