Search

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the 'MiDAS 4' database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus. Microbial communities are responsible for biological wastewater treatment. Here, Dueholm et al. generate more than 5 million high-quality, full-length 16S rRNA gene sequences from wastewater treatment plants across the world to construct a database with a comprehensive taxonomy, providing insights into diversity and function of these microbial communities. [ABSTRACT FROM AUTHOR]

Human urine accounts for only a fraction of the sewage volume, but it contains the majority of valuable nutrient load in wastewater. In this study, synthetic urine was nitrified in a closed photo-bioreactor through photosynthetic oxygenation by means of a consortium of microalgae and nitrifying bacteria. In situ production of oxygen by photosynthetic organisms has the potential to reduce the energy costs linked to conventional aeration. This energy-efficient strategy results in stable urine for further nutrient recovery, while part of the nutrients are biologically recovered in the form of valuable biomass. In this study, urine was nitrified for the first time without conventional aeration at a maximum photosynthetic oxygenation rate of 160 mg O2 gVSS-1 d-1 (VSS: volatile suspended solids). A maximum volumetric nitrification rate of 67 mg N L-1 d-1 was achieved on 12% diluted synthetic urine. Chemical oxygen demand (COD) removal efficiencies were situated between 44% and 83% at a removal rate of 24 mg COD gVSS-1 d-1 . After 180 days, microscopic observations revealed that Scenedesmus sp. was the dominant microalga. Overall, photosynthetic oxygenation for urine nitrification is promising as a highly electricity efficient approach for further nutrient recovery. [ABSTRACT FROM AUTHOR]

Wastewater is typically treated by the conventional activated sludge process, which suffers from an inefficient overall energy balance. The high-rate contact stabilization (HiCS) has been proposed as a promising primary treatment technology with which to maximize redirection of organics to sludge for subsequent energy recovery. It utilizes a feast-famine cycle to select for bioflocculation, intracellular storage, or both. We optimized the HiCS process for organics recovery and characterized different biological pathways of organics removal and recovery. A total of eight HiCS reactors were operated at 15°C at short solids retention times (SRT; 0.24-2.8 days), hydraulic contact times (tc; 8 and 15 min), and stabilization times (ts; 15 and 40 min). At an optimal SRT between 0.5 and 1.3 days and tc of 15 min and ts of 40 min, the HiCS system oxidized only 10% of influent chemical oxygen demand (COD) and recovered up to 55% of incoming organic matter into sludge. Storage played a minor role in the overall COD removal, which was likely dominated by aerobic biomass growth, bioflocculation onto extracellular polymeric substances, and settling. The HiCS process recovers enough organics to potentially produce 28 kWh of electricity per population equivalent per year by anaerobic digestion and electricity generation. This inspires new possibilities for energy-neutral wastewater treatment. [ABSTRACT FROM AUTHOR]

Biorefineries allow the production of value-added chemicals, yet this also causes the formation of considerable amounts of wastewater that require suitable treatment. These biorefinery wastewaters often contain a high salinity, which inhibits methanogenesis. In this research, molasses were used to mimic these waste streams to evaluate their treatability by anaerobic digestion. Two different carrier materials, i.e., carbon felt and biochar, with similar surface properties were evaluated for their potential to stabilize anaerobic digestion of these wastewaters via active enrichment of the methanogenic community. Initial stable methane production values between 620 and 640 mL CH L day were reported in each treatment. At the end of the experiment, methane production decreased with more than 50 %, while VFA increased to values up to 20 g COD L, indicating severe process failure, due to the high potassium concentration in these wastewaters, irrespective of the presence of carrier material. However, an increased relative abundance of Methanosaetaceae both on the biochar and carbon felt was observed. In conclusion, this research demonstrated that carbon felt and biochar are both suitable carrier materials for selective enrichment of Methanosaetaceae, yet this did not lead to stable anaerobic digestion of a potassium-rich molasses waste stream. The increased relative abundance of Methanosaetaceae on both carrier materials can, nonetheless, be considered valuable in terms of alternative applications and warrants further research. [ABSTRACT FROM AUTHOR]

Microalgal bacterial flocs (MaB-flocs) in sequencing batch reactors are a novel and promising technology to treat aquaculture wastewater. To improve the economics of this technology, the harvested MaB-flocs should be valorized. Therefore, we investigated if MaB-flocs from an outdoor pilot reactor treating pikeperch ( Sander lucioperca L.) wastewater can be used as a colour enhancing ingredient for shrimp diets. Considering the nutritional composition and high ash content of MaB-flocs, five iso-nitrogenous and iso-lipidic shrimp diets were formulated by replacing 2-8% of the basal diet ingredients by MaB-flocs. These diets were continuously fed to juvenile Pacific white shrimps Litopenaeus vannamei (Boone) cultured in a hybrid recirculating aquaculture system. The addition of these relatively low amounts of MaB-flocs to shrimp diets significantly increased the pigmentation (redness and yellowness) of cooked shrimp tails. The diet modifications did not affect the shrimp survival, weight gain, size distribution and food conversion rate, nor did they affect the proximate composition and fatty acid profile of the raw shrimp muscle. This shows that MaB-flocs originating from treatment of pikeperch wastewater can substitute 8% of diet ingredients of Pacific white shrimp while enhancing its pigmentation. [ABSTRACT FROM AUTHOR]

The conventional activated sludge process is widely used for wastewater treatment, but to progress toward energy self-sufficiency, the wastewater treatment scheme needs to radically improve energy balances. We developed a high-rate contact stabilization (HiCS) reactor system at high sludge-specific loading rates (>2 kg bCOD kg −1 TSS d −1 ) and low sludge retention times (<1.2 d) and demonstrate that it is able to recover more chemical energy from wastewater organics than high-rate conventional activated sludge (HiCAS) and the low-rate variants of HiCS and HiCAS. The best HiCS system recovered 36% of the influent chemical energy as methane, due to the combined effects of low production of CO 2 , high sludge yield, and high methane yield of the produced sludge. The HiCS system imposed a feast-famine cycle and a putative selection pressure on the sludge micro-organisms toward substrate adsorption and storage. Given further optimization, it is a promising process for energy recovery from wastewater. [ABSTRACT FROM AUTHOR]

Resources in used water are at present mainly destroyed rather than reused. Recovered nutrients can serve as raw material for the sustainable production of high value bio-products. The concept of using hydrogen and oxygen, produced by green or off-peak energy by electrolysis, as well as the unique capability of autotrophic hydrogen oxidizing bacteria to upgrade nitrogen and minerals into valuable microbial biomass, is proposed. Both axenic and mixed microbial cultures can thus be of value to implement re-synthesis of recovered nutrients in biomolecules. This process can become a major line in the sustainable “water factory” of the future. [ABSTRACT FROM AUTHOR]

Nitrification is a well-studied and established process to treat ammonia in wastewater. Although thermophilic nitrification could avoid cooling costs for the treatment of warm wastewaters, applications above 40 °C remain a significant challenge. This study tested the effect of salinity on the thermotolerance of mesophilic nitrifying sludge (34 °C). In batch tests, 5 g NaCl L increased the activity of aerobic ammonia-oxidizing bacteria (AerAOB) by 20-21 % at 40 and 45 °C. For nitrite-oxidizing bacteria (NOB), the activity remained unaltered at 40 °C, yet decreased by 83 % at 45 °C. In a subsequent long-term continuous reactor test, temperature was increased from 34 to 40, 42.5, 45, 47.5 and 50 °C. The AerAOB activity showed 65 and 37 % higher immediate resilience in the salt reactor (7.5 g NaCl L) for the first two temperature transitions and lost activity from 45 °C onwards. NOB activity, in contrast to the batch tests, was 37 and 21 % more resilient in the salt reactor for the first two transitions, while no difference was observed for the third temperature transition. The control reactor lost NOB activity at 47.5 °C, while the salt reactor only lost activity at 50 °C. Overall, this study demonstrates salt amendment as a tool for a more efficient temperature transition for mesophilic sludge (34 °C) and eventually higher nitrification temperatures. [ABSTRACT FROM AUTHOR]

Abstract: In this lab-scale study, domestic wastewater is subjected to a chemical biological adsorption (A-stage), followed by treatment in an innovative roof installed parallel plate microalgae biofilm reactor for nutrient immobilization (I-stage). The A-stage process was operated at a hydraulic retention time (HRT) of 1h and a solid retention time of 1day (FeSO4 as flocculant). The I-stage, which consequently received the effluent of the A-stage process, was operated at an HRT of 1day and exposed to natural light. The overall system removed on average 74% of the total chemical oxygen demand, 82% of the total suspended solids, 67% of the total nitrogen and 96% of the total phosphorous in the wastewater. The design involves a relatively low capital and operating cost which is in the order of 0.5€/m3 wastewater treated. These aspects suggest that the A/I process can be used as a decentralized domestic wastewater treatment system. [Copyright &y& Elsevier]

The occurrence of a range of recalcitrant organic micropollutants in our aquatic environment has led to the development of various tertiary wastewater treatment methods. In this study, biogenic manganese oxides (Bio-MnOx), biogenic silver nanoparticles (Bio-Ag0) and ionic silver were used for the oxidative removal of the frequently encountered drug diclofenac and its dechlorinated form, 2-anilinophenylacetate (APA). Diclofenac was rapidly degraded during ongoing manganese oxidation by Pseudomonas putida MnB6. Furthermore, whereas preoxidized Bio-MnOx, Bio-Ag0 and Ag+ separately did not show any removal capacity for diclofenac, an enhanced removal occurred when Bio-MnOx and silver species were combined. Similar results were obtained for APA. Finally, a slow removal of diclofenac but more rapid APA degradation was observed when silver was added to manganese-free P. putida biomass. Combining these results, three mechanisms of diclofenac and APA removal could be distinguished: (i) a co-metabolic removal during active Mn2+ oxidation by P. putida; (ii) a synergistic interaction between preoxidized Bio-MnOx and silver species; and (iii) a (bio)chemical process by biomass enriched with silver catalysts. This paper demonstrates the use of P. putida for water treatment purposes and is the first report of the application of silver combined with biogenic manganese for the removal of organic water contaminants. [ABSTRACT FROM AUTHOR]

In order to improve wastewater treatment processes, a need exists for tools that rapidly give detailed insight into the community structure of activated sludge, supplementary to chemical and physical data. In this study, the advantages of microarrays and quantitative polymerase chin reaction (PCR) methods were combined into a real-time PCR assay that allows the simultaneous quantification of phylogenetic and functional genes involved in nitrification and denitrification processes. Simultaneous quantification was possible along a 5-log dynamic range and with high linear correlation ( R 2 > 0.98). The specificity of the assay was confirmed by cloning and sequencing analyses of PCR amplicons obtained from activated sludge. The real-time assay was validated on mixed liquid samples of different treatment plants, which varied in nitrogen removal rate. The abundance of ammonia oxidizers was in the order of magnitude of 106 down to 104 ml−1, whereas nitrite oxidizers were less abundant (103–101 order of magnitude). The results were in correspondence with the nitrite oxidation rate in the sludge types. As for the nirS, nirK, and nosZ gene copy numbers, their abundance was generally in the order of magnitude of 108–105. When sludge samples were subjected to lab-scale perturbations, a decrease in nitrification rate was reflected within 18 h in the copy numbers of nitrifier genes (decrease with 1 to 5 log units), whereas denitrification genes remained rather unaffected. These results demonstrate that the method is a fast and accurate tool for the analysis of the (de)nitrifying community structure and size in both natural and engineered environmental samples. [ABSTRACT FROM AUTHOR]

Genes that encode the degradation of both naturally occurring and xenobiotic organic compounds are often located on plasmids, transposons or other mobile and/or integrative elements. The list of published reports of such mobile genetic elements (MGEs) keeps growing as researchers continue to isolate and characterize new degrading bacteria and their corresponding degradative genes. There is also growing evidence that horizontal exchange of catabolic (degradative) genes among bacteria in microbial communities plays an important role in the evolution of catabolic pathways. Around 10 years ago the hypothesis was raised that we might be able to accelerate this natural gene exchange and pathway construction by introducing and subsequently spreading degradative genes, located on MGEs, into well established, competitive indigenous microbial populations as a means of bioaugmentation of polluted soils and waters. During the last decade, only a few reports on successful MGE- mediated bioaugmentation have been published. After summarizing the diversity of degradative MGEs, this review presents an overview of studies that have monitored the transfer of degradative genes in soil microcosms and in activated sludge and other wastewater treatment reactors, with emphasis on those that have clearly shown a direct effect of gene transfer on accelerated biodegradation. A few successful cases suggest that the strategy could indeed work under specific conditions, such as when the in situ degradation potential is absent and the pollutant degrading transconjugants can grow and become numerically dominant populations in the bacterial community. Further studies in this area are obviously needed to improve our current knowledge on the efficiency of gene dissemination as a tool in bioremediation. [Copyright &y& Elsevier]

Graphical abstract Highlights • Aerobic and anoxic carbon removal were fast in start-up at all temperatures. • Increasing temperature lowered aerobic sludge yield, mainly due to lower µ max. • High thermophilic denitritation/denitrification rates up to 3.3 g N g−1 VSS d−1. • Thermophilic denitritation as cost-saving alternative to denitrification. • Increasing temperature yielded ambiguous trends in aerobic and anoxic settling. Abstract In specific municipal and industrial cases, thermophilic wastewater treatment (>45 °C) might bring cost advantages over commonly applied mesophilic processes (10–35 °C). To develop such a novel process, one needs sound parameters on kinetics, sludge yield and sludge settleability of three heterotrophic conversions: aerobic carbon removal, denitritation and denitrification. These features were evaluated in acetate-fed sequencing batch reactors (30, 40, 50 and 60 °C). Higher temperatures were accompanied by lower sludge production and maximum specific removal rates, resulting mainly from lower maximum growth rates. Thermophilic denitritation was demonstrated for the first time, with lower sludge production (18–26%), higher nitrogen removal rates (24–92%) and lower carbon requirement (40%) compared to denitrification. Acceptable settling of thermophilic aerobic (60 °C) and anoxic biomass (50 and 60 °C) was obtained. Overall, this parameter set may catalyze the establishment of thermophilic nitrogen removal, once nitritation and nitratation are characterized. Furthermore, waters with low COD/N ratio might benefit from thermophilic nitritation/denitritation. [ABSTRACT FROM AUTHOR]

So far, researchers investigated microbially induced CaCO 3 precipitation (MICP) for soil reinforcement, self-repairing concrete and Ca 2+ removal from industrial waste streams. Reported MICP yields were mainly achieved under nutrient-rich conditions. However, creating the tested nutrient-rich conditions in intended applications is both an economical and a practical issue. Therefore, investigation of MICP in more realistic conditions is necessary. This study presents optimization of MICP through denitrification in minimal nutrient conditions. To optimize their MICP performances, we isolated two strains, Pseudomonas aeruginosa and Diaphorobacter nitroreducens , by following an application oriented selection procedure. Upon performance optimization, in 2 days, D. nitroreducens and P. aeruginosa precipitated 14.1 and 18.9 g CaCO 3 /g NO 3 -N, respectively. Repetitive CaCO 3 precipitation was also achieved from a single inoculum in both 2 days and 3 weeks intervals. Selected strains and the process were further evaluated for three MICP applications: (1) Ca 2+ removal from paper mill wastewater (2) soil reinforcement, (3) crack repair in concrete. Overall, denitrification was found to be an effective process to remove Ca 2+ from paper mill wastewater. P. aeruginosa and D. nitroreducens could be introduced as potential candidates for soil and concrete applications due to their enhanced precipitation yields, resilience and performance under minimal nutrient conditions. [ABSTRACT FROM AUTHOR]

High-rate activated sludge processes allow for the recovery of organics and energy from wastewaters. These systems are operated at a short sludge retention time and high sludge-specific loading rates, which results in a higher sludge yield and better digestibility than conventional, low-rate activated sludge. Little is known about the microbial ecology of high-rate systems. In this work, we address the need for a fundamental understanding of how high-rate microbial communities differ from low-rate communities. We investigated the high-rate and low-rate communities in a sewage treatment plant in relation to environmental and operational variables over a period of ten months. We demonstrated that (1) high-rate and low-rate communities are distinctly different in terms of richness, evenness and composition, (2) high-rate community dynamics are more variable and less shaped by deterministic factors compared to low-rate communities, (3) sub-communities of continuously core and transitional members are more shaped by deterministic factors than the continuously rare members, both in high-rate and low-rate communities, and (4) high-rate community members showed a co-occurrence pattern similar to that of low-rate community members, but were less likely to be correlated to environmental and operational variables. These findings provide a basis for further optimization of high-rate systems, in order to facilitate resource recovery from wastewater. [ABSTRACT FROM AUTHOR]

Cost-efficient biological treatment of warm nitrogenous wastewaters requires the development of thermophilic nitrogen removal processes. Only one thermophilic nitrifying bioreactor was described so far, achieving 200 mg N L −1 d −1 after more than 300 days of enrichment from compost samples. From the practical point of view in which existing plants would be upgraded, however, a more time-efficient development strategy based on mesophilic nitrifying sludge is preferred. This study evaluated the adaptive capacities of mesophilic nitrifying sludge for two linear temperature increase patterns (non-oscillating vs. oscillating), two different slopes (0.25 vs. 0.08 °C d −1 ) and two different reactor types (floc vs. biofilm growth). The oscillating temperature pattern (0.25 °C d −1 ) and the moving bed biofilm reactor (0.08 °C d −1 ) could not reach nitrification at temperatures higher than 46 °C. However, nitrification rates up to 800 mg N L −1 d −1 and 150 mg N g −1 volatile suspended solids d −1 were achieved at a temperature as high as 49 °C by imposing the slowest linear temperature increase to floccular sludge. Microbial community analysis revealed that this successful transition was related with a shift in ammonium oxidizing archaea dominating ammonia oxidizing bacteria, while for nitrite oxidation Nitrospira spp. was constantly more abundant than Nitrobacter spp.. This observation was accompanied with an increase in observed sludge yield and a shift in maximal optimum temperature, determined with ex-situ temperature sensitivity measurements, predicting an upcoming reactor failure at higher temperature. Overall, this study achieved nitrification at 49 °C within 150 days by gradual adaptation of mesophilic sludge, and showed that ex-situ temperature sensitivity screening can be used to monitor and steer the transition process. [ABSTRACT FROM AUTHOR]

The development of thermophilic nitrogen removal strategies will facilitate sustainable biological treatment of warm nitrogenous wastewaters. Thermophilic denitrification was extensively compared to mesophilic denitrification for the first time in this study. Two sequential batch reactors (SBR) at 34 °C and 55 °C were inoculated with mesophilic activated sludge (26 °C), fed with synthetic influent in a first phase. Subsequently, the carbon source was switched from acetate to molasses, whereas in a third phase, the nitrate source was fertilizer industry wastewater. The denitrifying sludge maintained its activity at 55 °C, resulting in an immediate process start-up, obtaining nitrogen removal rates higher than 500 mg N g-1 VSS d-1 in less than one week. Although the mesophilic SBR showed twice as high specific nitrogen removal rates, the maximum thermophilic denitrifying activity in this study was nearly 10 times higher than the activities reported thus far. The thermophilic SBR moreover had a 73% lower sludge volume index, a 45% lower sludge production and a higher resilience towards a change in carbon source compared with the mesophilic SBR. The higher resilience was potentially related to a higher microbial diversity and evenness of the thermophilic community at the end of the synthetic feeding period. The thermophilic microbial community showed a higher similarity over the different feeding periods implying a more stable community. Overall, this study showed the capability of mesophilic denitrifiers to maintain their activity after a large temperature increase. Existing mesophilic process systems with cooling for the treatment of warm wastewaters could thus efficiently be converted to thermophilic systems with low sludge production and good settling properties. [ABSTRACT FROM AUTHOR]

Highlights: [•] MaB-floc SBRs were up-scaled from indoor reactors to outdoor raceway pond. [•] Up-scaling lowered nutrient removal and biomass productivity. [•] Outdoors, flue gas sparging was needed to obtain a dischargeable effluent pH. [•] Outdoor operation improved MaB-floc settling and ash content. [•] MaB-flocs were dewatered by filtering with 99% recovery to 43% TS. [ABSTRACT FROM AUTHOR]

Microbial processes are central elements in wastewater treatment plants (WWTPs) to mineralize the organic matter, to degrade pollutants and to decrease the amount of suspended solids. This activity can be disrupted by organic and inorganic pollutants present in wastewater streams. Hence, it is of primary importance to investigate and monitor the structure and functionality of the sludge-resident microbial communities. We simulated a 3-chloroaniline (3-CA) shock load in 3-CA adapted and non-adapted semi-continuous activated-sludge (SCAS) reactors to selectively stress the Ammonia Oxidizing Bacteria (AOB). Recently developed setting-independent theoretical interpretation of molecular DNA and RNA fingerprinting patterns were used to evaluate the responses of the microbial populations. Ammonium accumulation in treated reactors upon 3-CA addition confirmed the disruption of the functionality under stress conditions. Molecular analyses coupled to their interpretation highlighted that shock loaded reactors clustered separately from non-treated ones, especially when AOBs community was specifically targeted. Furthermore, the interpretation of RNA-based analyses, as compared to DNA-based ones, allowed to more promptly depicting shifts in a stressed community. We showed that the use of RNA-based molecular tools in combination with a new set of parameters is a powerful tool to link functional failures with microbial structure modifications in WWTPs, providing a potential tool for a rational optimization of the processes (Microbial Resource Management – MRM). [ABSTRACT FROM AUTHOR]

To decrease the load of pharmaceuticals to the environment, decentralized wastewater treatment has been proposed for important point-sources such as hospitals. In this study, a microbial electrolysis cell (MEC) was used for the dehalogenation of the iodinated X-ray contrast medium diatrizoate. The presence of biogenic palladium nanoparticles (bio-Pd) in the cathode significantly enhanced diatrizoate removal by direct electrochemical reduction and by reductive catalysis using the H2 gas produced at the cathode of the MEC. Complete deiodination of 3.3 μM (2 mg L-1) diatrizoate from a synthetic medium was achieved after 24 h of recirculation at an applied voltage of -0.4 V. An equimolar amount of the deiodinated metabolite 3,5-diacetamidobenzoate (DAB) was detected. Higher cell voltages increased the dehalogenation rates, resulting in a complete removal after 2 h at -0.8 V. At this cell voltage, the MEC was also able to remove 85% of diatrizoate from hospital effluent containing 0.5 μM (292 μg L-1), after 24 h of recirculation. Complete removal was obtained when the effluent was continuously fed at a volumetric loading rate of 204 mg diatrizoate m-3 total cathodic compartment (TCC) day-1 to the MEC with a hydraulic retention time of 8 h. At -0.8 V, the MEC system could also eliminate 54% of diatrizoate from spiked urine during a 24 h recirculation experiment. The final product DAB was demonstrated to be removable by nitrifying biomass, which suggests that the combination of a MEC and bio-Pd in its cathode offers potential to dehalogenate pharmaceuticals, and to significantly lower the environmental burden of hospital waste streams. [ABSTRACT FROM AUTHOR]

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species. specific physiology and by hydrodynamic shear and mixing. [ABSTRACT FROM AUTHOR]