Pilot-scale Membrane Bioreactor Research Articles

Membrane biofilm communities in full-scale membrane bioreactors are not randomly assembled and consist of a core microbiome

Finding efficient biofouling control strategies requires a better understanding of the microbial ecology of membrane biofilm communities in membrane bioreactors (MBRs). Studies that characterized the membrane biofilm communities in lab-and pilot-scale MBRs are numerous, yet similar studies in full-scale MBRs are limited. Also, most of these studies have characterized the mature biofilm communities with very few studies addressing early biofilm communities. In this study, five full-scale MBRs located in Seattle (Washington, U.S.A.) were selected to address two questions concerning membrane biofilm communities (early and mature): (i) Is the assembly of biofilm communities (early and mature) the result of random immigration of species from the source community (i.e. activated sludge)? and (ii) Is there a core membrane biofilm community in full-scale MBRs? Membrane biofilm (early and mature) and activated sludge (AS) samples were collected from the five MBRs, and 16S rRNA gene sequencing was applied to investigate the bacterial communities of AS and membrane biofilms (early and mature). Alpha and beta diversity measures revealed clear differences in the bacterial community structure between the AS and biofilm (early and mature) samples in the five full-scale MBRs. These differences were mainly due to the presence of large number of unique but rare operational taxonomic units (∼13% of total reads in each MBR) in each sample. In contrast, a high percentage (∼87% of total reads in each MBR) of sequence reads was shared between AS and biofilm samples in each MBR, and these shared sequence reads mainly belong to the dominant taxa in these samples. Despite the large fraction of shared sequence reads between AS and biofilm samples, simulated biofilm communities from random sampling of the respective AS community revealed that biofilm communities differed significantly from the random assemblages (P < 0.001 for each MBR), indicating that the biofilm communities (early and mature) are unlikely to represent a random sample of the AS community. In addition to the presence of unique operational taxonomic units in each biofilm sample (early or mature), comparative analysis of operational taxonomic units and genera revealed the presence of a core biofilm community in the five full-scale MBRs. These findings provided insight into the membrane biofilm communities in full-scale MBRs. More comparative studies are needed in the future to elucidate the factors shaping the core and unique biofilm communities in full-scale MBRs.

Application of powdered activated carbon (PAC) for membrane fouling control in a pilot-scale MBR system.

Membrane fouling is considered to be the most serious drawback in wastewater treatment when using membrane bioreactors (MBRs), leading to membrane permeability decrease and efficiency deterioration. This work aims to develop an integrated methodology for membrane fouling control, using powdered activated carbon (PAC), which will enhance the adsorption of soluble microbial products (SMP) and improve membrane filterability, by altering the mixed liquor's characteristics. Reversible fouling was assessed in terms of sludge filterability measurements, according to the standard time-to-filter (TTF) method, while irreversible fouling was assessed in terms of SMP removal. Results showed that the addition of PAC at the concentration of 3 g/L in the mixed liquor reduced SMP concentration and enhanced substantially the sludge filterability. Furthermore, the TTFPAC/TTFno PAC ratios were lower, than the corresponding SMPPAC./SMPno PAC ratios, indicating that the batch-mode, short-term addition of PAC promotes the reversible, rather than the irreversible fouling mitigation.

Activated Sludge Production Parameters and Nutrient Content of Organic Sludge Components.

An important part of biological treatment system design is quantifying the sludge production and the nutrient removal capacity. Influent wastewater COD fractionation, biomass growth and endogenous respiration directly impacts the composition of the mixed liquor solids in activated sludge systems. The objectives of this project were to determine the model kinetic and stoichiometric parameters associated with activated sludge production and the nutrient content (N and P) of unbiodegradable organic matter components. A complete sludge retention experiment was conducted over 70 days in a pilot-scale membrane bioreactor fed with a real municipal wastewater, and operated with alternating growth and famine periods. Experimental results were simulated and compared using the default values from two well-accepted model parameter sets. The General ASDM parameter set was found to better fit the experimental data than the Metcalf and Eddy parameter set, mainly to characterize endogenous respiration and the heterotrophic biomass concentration. An influent unbiodegradable organic particulate fraction (fXU,Inf) value of 0.16 g COD/g COD was determined by calibration of the accumulated sludge total COD, suspended solids and heterotrophic biomass concentrations. The nutrient content of the accumulated endogenous residue (XE) and influent unbiodegradable organic particulate (XU,Inf) components were calibrated to 0.030 and 0.100 g N/g COD and 0.035 and 0.008 g P/g COD, respectively. These values are in the range of those reported in the literature except for the high P content found in the endogenous residue, possibly due to the presence of coagulants added for P removal in the accumulated sludge. These results were consistent under the wide range of dynamic conditions tested and could improve model prediction of sludge production and composition.

Application of quorum quenching bacteria entrapping sheets to enhance biofouling control in a membrane bioreactor with a hollow fiber module

Quorum quenching (QQ) has been recognized as an innovative approach for biofouling control in membrane bioreactors (MBRs). Recently, QQ bacteria entrapping beads (QQ-beads) have been developed and verified to have excellent anti-biofouling potential in a pilot-scale MBR with flat-sheet membrane modules. In this study, considering the dense structure of hollow fiber (HF) bundles into which QQ-beads can hardly penetrate, QQ bacteria entrapping sheets (QQ-sheets) were developed as a new shape of QQ-media suitable for MBRs with HF modules. In a lab-scale MBR, QQ-sheets with a thickness of 0.5mm exhibited a greater physical washing effect than did QQ-beads with a diameter of 3.5mm because the former collided with membrane surfaces at the inner as well as the outer part of HF bundles, whereas the latter only made contact with the outer part. Moreover, QQ-sheets showed 2.5-fold greater biological QQ activity than did QQ-beads due to their greater total surface area at a fixed volume of QQ-media. These results suggest high potential for QQ-sheets to be used in MBRs with HF modules.

Assessing membrane fouling and the performance of pilot-scale membrane bioreactor (MBR) to treat real municipal wastewater during winter season in Nordic regions.

In this study, the performance of a pilot-scale membrane bioreactor (MBR) to treat real municipal wastewater was assessed at low temperatures (7 to 20°C) in Nordic regions. First, the effect of low temperatures on membrane fouling was evaluated by monitoring trans-membrane pressure. A significant membrane fouling was observed when the sludge temperature inside the MBR unit was below 10°C with a 75% permeability drop, thus indicating high deterioration of the membrane performance at low temperatures. Moreover, increasing values of sludge volume index (SVI) during low temperatures showed high deterioration of sludge settleability. As for the pollution removal, MBR achieved high performances primarily for pathogens and emerging micropollutants. The average log reductions of 1.82, 3.02, and 1.94 log units were achieved for norovirus GI, norovirus GII, and adenoviruses, respectively. Among the four trace organic compounds (TrOCs), the average removal efficiencies of bisoprolol, diclofenac and bisphenol A were 65%, 38%, and >97%, respectively. However, carbamazepine was not efficiently removed (-89% to 28%). Regarding trace metals, an average removal of >80% was achieved for Cd, Pb, and V. For the rest of the metals, the removal capacities were between 30 and 60%.

Batch and continuous dosing of conventional and composite coagulation agents for fouling control in a pilot-scale MBR

In the present study, conventional and composite, laboratory prepared coagulation agents were added in a fully automatic pilot-scale Membrane Bioreactor unit, both in batch-mode addition (‘Process I’) and in continuous-flow addition (‘Process II’) experiments, aiming to improve the overall process operability, i.e. as a fouling control method. A systematic effort was made to: i) correlate the recorded trans-Membrane Pressure with two novel, easily generated fouling indices (Ratio a and Ratio b) and ii) elucidate the relationship between the coagulant equilibrium concentration in the activated sludge and the optimal amount of coagulant added/L of incoming wastewater, by applying the corresponding mass balance equation. In both processes, the ranking trend among the optimal coagulants can be classified as: FO 4350 SSH<PSiCAFPAC-18-10-15<PAC A9-M, in increasing order of Soluble Microbial Products (SMP) removal, and as: PAC A9-M<PSiCAFPAC-18-10-15<FO 4350SSH, in increasing order of sludge filterability enhancement. Among the three coagulation agents, the cationic polyelectrolyte FO 4350SSH was identified as the optimal one, since its continuous-flow addition was found to cause the largest TMP decrease (almost 40%), at the optimal dosage of 0.16mg/L of incoming wastewater, which was 63 times lower than its equilibrium concentration in the bioreactor (10mg/L). The respective low values of Ratio b and the short-term nature of continuous-flow experiments (6days) indicate that the mitigation of reversible fouling was mainly responsible for this.

Spatiotemporal dynamics and correlation networks of bacterial and fungal communities in a membrane bioreactor

To systematically study biofilm communities responsible for biofouling in membrane bioreactors (MBRs), we characterized the spatiotemporal dynamics of bacterial and fungal biofilm communities, and their networks, in a pilot-scale flat-sheet MBR treating actual municipal wastewater. Activated sludge (AS) and membrane samples were collected on days 4 and 8. The membranes were cut into 18 tiles, and bacterial and fungal communities were analyzed using next generation sequencing. Nonmetric multidimensional scaling (NMDS) plots revealed significant temporal variations in bacterial and fungal biofilm communities due to changes in the abundances of a few dominant members. Although the experimental conditions and inoculum species pools remained constant, variogram plots of bacterial and fungal communities revealed decay in local community similarity with geographic distance at each sampling time. Variogram modeling (exponential rise to maximum, R2 ≥ 0.79) revealed that decay patterns of both communities were different between days 4 and 8. In addition, networks of bacteria or fungi alone were distinct in network composition between days 4 and 8. The day-8 networks were more compact and clustered than those of the earlier time point. Bacteria-fungi networks show that the number of inter-domain associations decreased from 113 to 40 with time, confirming that membrane biofilm is a complex consortium of bacteria and fungi. Spatiotemporal succession in biofilm communities may be common on MBR membranes, resulting from different geographic distributions of initial microbial populations and their priority effects.

Numerical simulations of impact of membrane module design variables on aeration patterns in membrane bioreactors

Computational Fluid Dynamics (CFD) models incorporating sludge rheology model and porous media sub-models were used to simulate the impact of eight design variables, including fibre orientation, filtration tank geometry and aeration design, on the hydrodynamics and aeration patterns in a pilot scale membrane bioreactor (MBR) fitted with commercially available membrane modules. Comparison of simulated with experimental grid averaged flow velocities measured using Particle Image Velocimetry (PIV) in an aerated bench scale MBR differed by approximately 6%. The assessment of different turbulence models revealed that it was difficult to accurately simulate local variations in turbulence induced by air bubbles due to limitations of Reynolds averaging Navier Stokes (RANS) models. Simulation results revealed that MBR's fitted with hollow fibres in a vertical orientation in the filtration zone experienced 25% more membrane surface shear than horizontally oriented fibres at the same aeration intensity. The inclusion of baffles around the membrane modules promoted turbulence and increased shear in the upper section of the membrane module by approximately 30%. This is important for control of fouling along sections of the hollow fibre membrane that experience the highest transmembrane pressure and are more susceptible to fouling. Rotating the nozzle aperture to face the bottom of the tank increased the homogeneity of shear stress on the lower half of the module and increased shear on the upper half of the membrane surface. This effect was amplified by locating the air diffusers 100mm below the bottom of the module and aligning the aeration pipe in parallel with the direction of the fibre bundle. This research demonstrates the capability of using Computational Fluid Dynamics to optimise the design of the filtration zone comprising the membrane module, aeration system and MBR tank dimensions.

Dynamics of the Fouling Layer Microbial Community in a Membrane Bioreactor.

Membrane fouling presents the greatest challenge to the application of membrane bioreactor (MBR) technology. Formation of biofilms on the membrane surface is the suggested cause, yet little is known of the composition or dynamics of the microbial community responsible. To gain an insight into this important question, we applied 16S rRNA gene amplicon sequencing with a curated taxonomy and fluorescent in situ hybridization to monitor the community of a pilot-scale MBR carrying out enhanced biological nitrogen and phosphorus removal with municipal wastewater. In order to track the dynamics of the fouling process, we concurrently investigated the communities of the biofilm, MBR bulk sludge, and the conventional activated sludge system used to seed the MBR system over several weeks from start-up. As the biofilm matured the initially abundant betaproteobacterial genera Limnohabitans, Hydrogenophaga and Malikia were succeeded by filamentous Chloroflexi and Gordonia as the abundant species. This study indicates that, although putative pioneer species appear, the biofilm became increasingly similar to the bulk community with time. This suggests that the microbial population in bulk water will largely determine the community structure of the mature biofilm.

Assessment of genomic rearrangements in Chinese Hamster Ovary (CHO) cells

Evolution of irreversible fouling in pilot-scale membrane bioreactors (MBRs) treating municipal wastewater was studied. Three membrane modules were sacrificed to analyze foulants after they had been used for different durations (1 day, 8 days, 24 days). The module used for 1 day reflected fouling occurring at the initial stage, whereas the module used for 24 days reflected the later stage of fouling. After each module had been used for the predetermined period, it was disassembled and subjected to powerful chemical analysis such as Fourier transform infrared (FTIR) analysis. It was clearly shown that components involved in evolution of irreversible fouling gradually changed depending on the duration. The initial fouling (conditioning of membrane) was caused by humics, whereas later fouling was mainly caused by polysaccharides. A model for evolution of irreversible fouling in MBRs and implications for fouling control are discussed.

Comparison of EDCs removal in full and pilot scale membrane bioreactor plants: Effect of flux rate on EDCs removal in short SRT

Currently, it is estimated that over 1 billion people are short of adequate portable water and this is expected to increase to 2.5 billion in the year 2025. Membrane Bioreactors (MBR) are now accepted as important tools to extend the availability of water by facilitating the reuse of wastewaters. Sludge Retention Time (SRT) and liquid flux rate are the two most important parameters for controlling the MBR process. In this study, the removal of selected endocrine disrupter compounds (EDCs), diltiazem, progesterone, estrone (E1), carbamazepine (Cbz) and acetaminophen (Acet), by one full scale (VRM) and one pilot scale (clear-box) MBR plants was investigated. During the study, sludge age was set at 10 days and the sludge concentration was fixed at about 5.0 g/L. The transmembrane pressure (TMP) got higher with either increasing flux or sludge concentration in the membrane chamber. Therefore, changing the flux from 13 to 30 L/m2-h in both plants caused enhancement in TMP from −25 to −300 mbar in the clear-box and from −160 to over −350 in the full scale MBRs. It was understood that flux had very little effect on the removal of EDCs at very low concentrations. Moreover, diltiazem was completely removed in the full scale whereas no removal was achieved in the pilot scale. Estrone and progesterone were completely removed by biodegradation in both plants. Acetaminophen was completely removed in the full scale plant whereas over 95% removal was achieved in the pilot scale MBR.

Wastewater Treatment in Membrane Bioreactors: The Use of Polyelectrolytes to Control Membrane Fouling

The present study investigates the addition of several commercially available cationic (FO 4000 SSH and ADIFLOC series) and anionic (LT-25 Magnafloc) polyelectrolytes in a fully automated, pilot-scale Membrane Bioreactor (MBR), aiming to the development of an integrated methodology for the control of membrane fouling in MBR treatment systems. For this purpose, a series of batch experiments were conducted in order to evaluate their effectiveness on membrane fouling mitigation, using a typical hollow fiber, commercially available microfiltration membrane. Reversible fouling was assessed in terms of sludge filterability tests, whereas irreversible fouling was assessed in terms of SMPs (Soluble Microbial Products) concentration measurements. Results showed that the addition of cationic polyelectrolytes FO 4000 SSH series and ADIFLOC series were found capable to reduce both reversible and irreversible fouling. More specifically, the FO 4350 SSH type exhibited a remarkable performance, regarding both sludge filterability enhancement and SMP removal, when added at the concentration of 10 mg/L. Further comparison with the anionic polyelectrolyte LT-25 Magnafloc reinforced the allegation that cationic polyelectrolytes are preferable for control of membrane fouling, mainly due to charge neutralization mechanism.

Membrane fouling potentials and cellular properties of bacteria isolated from fouled membranes in a MBR treating municipal wastewater

Membrane fouling remains a major challenge for wider application of membrane bioreactors (MBRs) to wastewater treatment. Membrane fouling is mainly caused by microorganisms and their excreted microbial products. For development of more effective control strategies, it is important to identify and characterize the microorganisms that are responsible for membrane fouling. In this study, 41 bacterial strains were isolated from fouled microfiltration membranes in a pilot-scale MBR treating real municipal wastewater, and their membrane fouling potentials were directly measured using bench-scale cross-flow membrane filtration systems (CFMFSs) and related to their cellular properties. It was found that the fouling potential was highly strain dependent, suggesting that bacterial identification at the strain level is essential to identify key fouling-causing bacteria (FCB). The FCB showed some common cellular properties. The most prominent feature of FCB was that they formed convex colonies having swollen podgy shape and smooth lustrous surfaces with high water, hydrophilic organic matter and carbohydrate content. However, general and rigid biofilm formation potential as determined by microtiter plates and cell surface properties (i.e., hydrophobicity and surface charge) did not correlate with the fouling potential in this study. These results suggest that the fouling potential should be directly evaluated under filtration conditions, and the colony water content could be a useful indicator to identify the FCB.

Functional maintenance and structural flexibility of microbial communities perturbed by simulated intense rainfall in a pilot-scale membrane bioreactor.

Intense rainfall is one of the most serious and common natural events, causing the excessive inflow of rainwater into wastewater treatment plants. However, little is known about the impacts of rainwater dilution on the structure and function of the sludge microorganisms. Here, high-throughput sequencing of 16S ribosomal RNA (rRNA) genes was implemented to describe the microbial community dynamics during the simulated intense rainfall situation (event i) in which approximately 45% of the sludge biomass was artificially overflowed by massive water supply in a pilot-scale membrane bioreactor. Thereafter, we investigated the functional and structural responses of the perturbed microbial communities to subsequent conditional changes, i.e., an increase in organic loading rate from 225 to 450mg chemical oxygen demand (COD)l(-1)day(-1) (event ii) and an addition of a microbiota activator (event iii). Due to the event i, the COD removal declined to 78.2%. This deterioration coincided with the decreased microbial diversity and the proliferation of the oligotrophic Aquabacterium sp. During the succeeding events ii and iii, the sludge biomass increased and the COD removal became higher (86.5-97.4%). With the apparent recovery of the reactor performance, microbial communities became diversified and the compositions dynamically changed. Notably, various bacterial micropredators were highly enriched under the successive conditions, most likely being involved in the flexible reorganization of microbial communities. These results indicate that the activated sludge harbored functionally redundant microorganisms that were able to thrive and proliferate along with the conditional changes, thereby contributing to the functional maintenance of the membrane bioreactor.

Application of fluorescent particles as a tracer to detect the membrane surface damage in a pilot scale membrane bioreactor

In this study, a fluorescent silica nano particle is used as the surrogate for challenging test of membrane surface integrity. The particles are functionalized by a fluorescent dying agent so that as an ultraviolet light is imposed a bright fluorescent image from the particles can be taken. If a membrane surface is damaged and has a compromised part larger than the size of surrogate the fluorescent particles would pass through and contained in the permeate. An operator can directly notice whether the membrane surface is damaged or not by detecting a fluorescent image taken from the permeate. Additionally, the size of compromised part is estimated through analysing the fluorescent image in which we surmise the mass of particles included in the permeate by calculating an average RGB value of the image. The pilot scale experiments showed that this method could be applied successfully to determine if a membrane surface had a damaged parts regardless of the test condition. In the testing on the actual damaged area of , the lowest error of estimating the damaged area was -1.32% with the surrogate concentration of 80 mg/L, flux of for 25 minutes of detection. A further study is still going on to increase the lowest detection limit and thus decrease the error of estimation.

Removals of pharmaceutical compounds from hospital wastewater in membrane bioreactor operated under short hydraulic retention time

Pilot-scale membrane bioreactor (MBR) was operated at a short hydraulic retention time (HRT) of 3 h for the treatment of hospital wastewater. The removals of eleven pharmaceutical compounds in MBR operated at different mixed liquor suspended solids (MLSS) level were investigated during which nitrification degree was differed. The results experiments revealed the importance of immediate adsorption onto the colloidal particles in supernatant of MBR sludge and subsequently removed by membrane filtration for the recalcitrant pharmaceutical compounds. Nevertheless, the removals through biodegradation during short HRT were also found significant for some compounds. DGGE profile revealed the development of pharmaceutical degrading microorganisms in MBR.

Particle counting as surrogate measurement of membrane integrity loss and assessment tool for particle growth and regrowth in the permeate of membrane bioreactors

Abstract Wastewater is increasingly used as a viable water source. Reclamation schemes rely on membranes technologies, such as Membrane Bioreactors (MBR) and subsequent membrane filtration steps to achieve the discharge or reuse limits. However, membrane technology is only effective if the membranes are intact. Moreover, growth or regrowth of particles between membrane filtration steps should be restricted, to guarantee a stable water production. Membrane integrity tools are being developed for the drinking water industry with high sensitivity and accuracy. However, such standards are not required for MBRs, where the produced permeate is not used for consumption without further treatment. In this research, we focused on permeate quality and particle counting measurements in the range 2–100 μm. A total of 433 samples of permeate and process water were measured at 8 full-scale and pilot-scale MBR locations, which were compared with 43 de-mineralized water samples measured at TU Delft. Only at one full-scale MBR the membrane integrity was compromised, which was successfully assessed by counts and shapes of the permeate particle counting distributions. All permeate samples had particles about a 100 times larger than the membrane pore size. Our results allowed us to define the relevant steps of a methodology to assess membrane integrity, particle or biomass growth and aggregation in MBR permeate. The latter two are of particular interest to determine whether growth in permeate lines is still acceptable or whether a cleaning action of the permeate collection system is required.

Membrane Bioreactor and Promising Application for Textile Industry in Vietnam

A pilot-scale membrane bioreactor (MBR) was developed in order to run two membrane modules in parallel for the treatment of model textile wastewater (MTDW). Two independently operated commercially available ultrafiltration membrane modules called UP150 from Microdyn-Nadir where tested in the same activated sludge tank over a period of 70 days for their removal efficiency of the MTDW. In general the results of both membrane modules are in very good agreement. The water permeability ranged between 20 – 50 L/(m2.h.bar). Typically, the chemical oxygen demand (COD) removal efficiency indicated good biodegradation performance above 95%. The nitrification rate depended on the food to microorganism (F/M) ratio i.e. below 0.2kg COD/(kg MLSS.d) the system showed complete nitrification. However, the color rejection for the model dyes was only around 20% to 60% what can be attributed to the low biodegradability of these chemicals. The next step is to run the MBR with novel nanostructured membranes in parallel with the commercially available membrane to compare their performances. This study contributes to sustainable development in the textile industry by improving water quality of treated textile wastewater what helps to reduce fresh water consumption and pollutant discharge.

Effects of Organic-Loading-Rate Reduction on Sludge Biomass and Microbial Community in a Deteriorated Pilot-Scale Membrane Bioreactor.

The effects of a precipitous decrease in the inlet organic loading rate on sludge reductions and the microbial community in a membrane bioreactor were investigated. The sludge biomass was markedly reduced to 47.4% of the initial concentration (approximately 15,000 mg L−1) within 7 d after the organic loading rate was decreased by half (450 to 225 mg chemical oxygen demand L−1 d−1). An analysis of the microbial community structure using high-throughput sequencing revealed an increase in the abundance of facultative predatory bacteria-related operational taxonomic units as well as microorganisms tolerant to environmental stress belonging to the classes Deinococci and Betaproteobacteria.

Fine-scale monitoring of shifts in microbial community composition after high organic loading in a pilot-scale membrane bioreactor

In biological wastewater treatment, municipal wastewater sometimes undergoes unexpected changes in physicochemical parameters, such as organic carbon concentration. The aim of this study was to understand how microbial communities in activated sludge in a membrane bioreactor (MBR) adapt to high organic loading and maintain their degradation ability during reactor operation. A pilot-scale MBR was operated for 19 days. On day 8, the concentration of organic matter in the synthetic wastewater increased from 450 to 900 mg chemical oxygen demand (COD)/L. Even under conditions of high organic loading, COD removal rates were high, ranging from 85.3 to 91.4%. High-throughput sequencing of 16S rRNA genes revealed that microbial communities changed drastically with increased organic loading. After day 8, Aquabacterium- and Azospira-related operational taxonomic units (OTUs) belonging to the class β-proteobacteria became dominant; this potentially enhanced the degradation of organic substances and decreased activated sludge microbial diversity. Due to the use of dissolved oxygen (DO) for degradation of organic substances, DO levels in the reactor decreased. This led to an increase in a subset of OTUs related to not only aerobic but also anaerobic bacteria, e.g., those in the class Clostridia. During this period, anaerobic microorganisms may have contributed to the degradation of organic substances to maintain MBR performance. On the other hand, high-throughput sequencing also made it possible to identify yet-to-be cultured or minor microorganisms affiliated with the candidate phylogenetic division SR1 and ammonia-oxidizing archaea in activated sludge.