Cultivation of Small Granular Flocs with Predominant Aerobic Denitrifiers and Formation of Densified Activated Sludge in a Sequencing Batch Reactor

1. Introduction

Aerobic granular sludge (AGS), characterized by compact microbial aggregates and stratified redox microenvironments, offers several advantages over conventional activated sludge systems, including superior settling properties, higher biomass retention, simultaneous nitrification, denitrification, and phosphorus removal [1,2]. Aerobic granules are spatially organized microbial structures in which different microbial guilds use available substrates and redox conditions for efficient biological nutrient removal. In engineered AGS systems, operational and nutritional conditions are managed to support the growth of slow-growing autotrophic nitrifiers in the oxygenated outer layer [3,4] while limiting the dominance of fast-growing aerobic heterotrophs that consume readily biodegradable carbon under oxic conditions. These conditions also promote carbon-storing, slow-growing heterotrophic bacteria that can use oxidized nitrogen species as electron acceptors [5,6]. However, dependence on these slow-growing functional groups requires rigorous operational control to maintain stable granular sludge performance.

In wastewater treatment systems, microbial aggregates are the primary functional and structural units for biological nutrient removal. Microbial community composition governs nutrient removal, while aggregate density enables effective separation of treated effluent from biomass [7]. Larger granules are often favoured because they settle faster than smaller flocs. During the settling phase of a sequencing batch reactor (SBR), biomass stratifies by aggregate size and density, with larger granules settling rapidly and smaller or less dense flocs remaining in the upper liquid layer. Conventional granular sludge cultivation therefore applies selection pressures that retain large granules while wasting slow-settling floc [5]. Although preferential retention of larger granules improves phase separation, increasing granule size can reduce mass transfer and nutrient diffusion rates. Furthermore, large granules may become structurally unstable when they exceed a size threshold, as internal nutrient limitations and metabolic stress can trigger cell lysis, weaken the core, and lead to granule disintegration. Additionally, blockage of internal pores can trap nitrogen gas bubbles, reduce aggregate density, and contribute to biomass washout [8,9]. Consequently, although AGS is an advanced biological nutrient removal technology, long start-up periods and concerns over long-term granule stability continue to limit broader full-scale application [5,10].

Densified activated sludge (DAS) offers an alternative strategy to improve biological nutrient removal without imposing strict granule-size requirements. The selection pressures that promote the formation and retention of dense microbial aggregates in DAS are similar to those used in AGS [11,12]. However, DAS systems retain dense aggregates across a wider size range, including small dense flocs that conventional AGS operation may selectively wash out [11,13]. This retention strategy can increase biomass concentration, improve solid-liquid separation, and support stable nutrient removal. By shifting the target from large, morphologically defined granules to broader densified biomass, DAS can support faster start-up and sustained nutrient-removal kinetics [11]. Although densified flocs settle more slowly than larger AGS granules, their smaller size can reduce mass-transfer limitations and internal substrate-diffusion constraints, helping avoid some metabolic and structural limitations associated with larger granules [11].

Conventional nitrogen removal typically relies on sequential nitrification and denitrification by slow-growing nitrifiers and denitrifiers and, in some systems, anaerobic ammonium-oxidizing bacteria. These microbial groups are sensitive to environmental fluctuations and often require complex operational strategies to maintain effective nitrogen removal [14]. Heterotrophic nitrification and aerobic denitrification (HN-AD) pathways offer an alternative route for nitrogen removal by microorganisms to perform nitrification and denitrification under oxygen-rich conditions [15]. Nitrogen assimilation by fast-growing HN-AD bacteria can also contribute to total nitrogen removal, with reported contributions of up to 40% [16]. Although pure-culture studies have shown effective HN-AD activity, pure-culture bioaugmentation remains difficult to sustain in complex, non-sterile wastewater systems [17]. Consequently, practical use of HN-AD in wastewater treatment requires strategies that enrich these functions within self-sustaining, dense microbial aggregates that retain biomass, protect niches, and resist competitive exclusion.

Several wastewater treatment systems fail to achieve stable granulation. This study initially aimed to cultivate aerobic granular sludge using selection pressures commonly applied in AGS systems, including anoxic feeding, extended anoxic mixing, increased hydrodynamic shear, and gradually reduced settling time, to cultivate dense microbial aggregates. Instead of large aerobic granules, the reactor developed DAS composed mainly of small granular flocs. We report DAS formation capable of heterotrophic nitrification, aerobic denitrification, and phosphorus removal within small granular flocs. This approach demonstrates that simultaneous nutrient removal in a compact reactor tank does not necessarily require large granular structures with strongly stratified redox conditions.

2. Materials and Methods

2.1. Experimental Setup

DAS was cultivated in a Plexiglas column-type SBR with a total volume of 7.1 L and a working volume of 4.8 L, an internal diameter of 10 cm, and a height of 90 cm. The reactor had a diffused aeration system at the base, providing a superficial air velocity (SAV) of 2.3 cm/s. The bioreactor was bottom-fed and operated for 114 days at room temperature.

The SBR used an automated control system to manage the fill, react, settle, draw, and idle phases in a 4-hour cycle (six cycles per day). The operational strategy included a 10-min feeding phase, 30 to 54 min of mixing without aeration, 150 min of aeration, 30 to 6 min of settling, 20 min of decanting, and an idle period. The hydraulic retention time (HRT) was 8 hours. The reactor was inoculated with return activated sludge from the Ashbridges Bay Wastewater Treatment Plant in Toronto, Canada. We monitored dissolved oxygen and pH during reactor operation.

During startup, we kept the settling time at 30 min to retain biomass and support acclimation to the synthetic wastewater. After the initial acclimation period, the settling time was gradually reduced from 30 min to 6 min to select for dense microbial aggregates with improved settleability.

2.2. Synthetic Wastewater and Reactor Operation

The reactor was fed acetate-based synthetic wastewater with a COD: N:P ratio of 100:5:1, as described previously ([18,19,20]. The composition of the synthetic wastewater was as follows: CH3COONa (1.28 g L-1 corresponding to a COD concentration of 1000 mg COD L-1), NH4Cl (191 mg L-1); KH2PO4 (44 mg L-1); CaCl2·2H2O (45 mg L-1); MgSO4·7H2O (25 mg L-1); FeSO4·7H2O (10 mg L-1); and 1 mL L-1 trace element solution: H3BO3 (0.05 g L-1); ZnCl2 (0.05 g L-1); (NH4)6Mo7O24·4H2O (0.05 g L-1); AlCl3 (0.05 g L-1); CoCl2·6H2O (0.05 g L-1); NiCl2·6H2O (0.05 g L-1); MnSO4·H2O (0.05 g L-1); and CuCl2·2H2O (0.03 g L-1).

2.3. Analytical Methods

Mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS), and sludge volume index after 5 minutes (SVI₅) and after 30 minutes (SVI₃₀) were measured in accordance with the Standard Methods for the Examination of Water and Wastewater [21]. Microscopic images of granular sludge samples were obtained using a LEICA microscope (Model DM1000 LED). The COD of the influent and effluent samples was measured using HACH test kits. Samples were heated in a thermostat (DRB200, HACH, Loveland, USA) to 150 °C and then analyzed using a spectrophotometer (DR 3900, HACH, USA). COD was measured after filtration through 0.45-µm Acrodisc® 32 mm filters with Supor® membrane (Cytiva, formerly Pall Corporation). Influent and effluent samples (after filtration through 0.45-µm pore-sized filters) for ammonia (NH3), nitrate (NO3), nitrite (NO2), and phosphate (PO4) were measured according to HACH methods.

2.4. DNA Extraction, Sequencing, and Microbial Community Analysis

Biomass samples were processed as described previously [22]. Briefly, samples were centrifuged, and genomic DNA was extracted from 100–150 mg of the resulting pellet. DNA extraction was automated using the PowerSoil DNA isolation kit (MoBio Laboratories Inc.) and the QIAcube Connect (Qiagen, MD, USA). The extracted DNA was submitted to the Genome Quebec Research and Testing Laboratory in Montréal, Québec, for 16S rRNA gene sequencing with a paired-end 300 kit (Illumina HiSeq-PE300).

The V3–V4 region of the 16S rRNA gene was amplified using primers 347F (GGAGGCAGCAGTRRGGAAT) and 803R (CTACCRGGGTATCTAATCC). The forward and reverse sequences were merged, and low-quality sequences were removed using the DADA2 software. The sequences were analyzed using MicrobiomeAnalyst to estimate the relative abundance of microbial communities. C3G (McGill University) performed quality control analyses of the sequencing data using the GenPipes version 4.0.0 amplicon-seq pipeline. This pipeline is based on the DADA2 package in R. First, Trimmomatic was used to trim the data, removing adapters, chimeras, and low-quality sequences before downstream analyses. MicrobiomeAnalyst was used to generate relative abundance graphs using the SILVA database. Seed biomass and densified activated sludge samples collected on day 111 were used for microbial community analysis. The datasets presented in this study are deposited in the NCBI database under accession number PRJNA1489565.

3. Results

3.1. Formation of Densified Activated Sludge

The seed biomass consisted mainly of dispersed flocs, pinpoint flocs, and filamentous bacteria. This seed biomass progressively transformed into predominantly small, granular flocs measuring 50 to 100 µm (Figure 1).

The biomass’s settling properties at the start of operation were poor. This poor settleability caused biomass washout during startup, with MLSS dropping from 6.5 g/L to 3.3 g/L by day 22 (Figure 2). During the first few days of the selection period, MLSS further decreased to 2.55 g/L due to washout. By day 44, the biomass in the SBR stabilized, as evidenced by minimal suspended solids in the effluent. The MLSS concentration gradually increased to approximately 8 g/L by day 79 and to approximately 12 g/L by day 93.

Throughout the experiment, the MLVSS/MLSS ratio remained stable at 0.889 ± 0.018 (Figure 2). This indicates that a substantial portion of the MLSS consists of organic matter and live bacterial cells involved in biological nutrient removal. This highly volatile fraction supports the accumulation of biological solids involved in nutrient removal [23].

3.2. Settling Properties and Biomass Retention

The biomass settling properties initially deteriorated when the SBR was fed synthetic wastewater using seed biomass from a full-scale municipal wastewater treatment plant. The SVI30 increased to 235 mL/g MLSS by day 23 (Figure 2). The poor settling properties of the biomass during reactor startup coincided with biomass washout, during which the MLSS concentration decreased from 6.5 g/L to 3.3 g/L.

The SVI₃₀ improved to 45 mL/g MLSS by day 65 and then decreased to 30 mL/g MLSS by day 93. From day 93 to the end of the experiment, the SVI₃₀ remained stable at approximately 30 mL/g MLSS (Figure 2). This value is comparable to typical SVI₃₀ values reported for several aerobic granular sludge (AGS) systems [24]. Consequently, at this stage of reactor operation, the small granular flocs exhibited settling properties similar to those of granular sludge, facilitating biomass retention in the reactor and promoting densification.

The SVI₃₀ to SVI₅ ratio was moderate, mostly ranging from 50% to 72% during the study (Figure 2). This was associated with the abundance of small, granular flocs ranging from 50 µm to 100 µm in size. Microscopic images indicated a limited number of filaments in the system. Despite these filaments, the dense, small, granular flocs compacted effectively and maintained an SVI₃₀ of approximately 30 mL/g MLSS. This performance indicates that selection pressure, including a shortened settling duration and elevated shear force, favoured the retention of small, granular flocs and biomass densification.

3.3. Nutrient Removal Performance

The effluent nutrient concentrations and nutrient removal efficiencies are presented in Figure 3. During the first 51 days of operation, the effluent COD ranged from 105 mg/L to 155 mg/L, corresponding to 85% to 90% COD removal efficiency. From day 58 to 79, the effluent COD ranged from 89 mg/L to 101 mg/L, corresponding to 90% to 91% removal efficiency. From day 86 to the end of operation, the effluent COD ranged from 6 mg/L to 40 mg/L, corresponding to 97.8 ± 1.7% COD removal efficiency.

After an acclimation phase, the effluent ammonia concentration ranged from 0.0 to 0.7 mg/L, corresponding to 98% to 100% ammonia removal (Figure 3). The effluent nitrite concentration ranged from 4 mg/L to 10.4 mg/L during the first 72 days of operation. The effluent nitrite concentration was zero from day 78 to the end of operation.

During the first 72 days, effluent nitrate ranged from 1.5 mg/L to 3.7 mg/L, corresponding to a nitrate removal efficiency of 93.6% ± 2.6%. From day 79 to the end of the operation, the effluent nitrate concentration increased to 11.4 ± 0.8 mg/L. Consequently, the nitrate removal efficiency declined to 77.3% ± 1.8% during this final phase of operation (Figure 3). This increase coincided with near-complete COD removal, suggesting that reduced organic carbon availability limited nitrate reduction during the final operating phase.

In addition to organic substrate and ammonia removal, we monitored phosphate removal in the bioreactor effluent. The effluent phosphate concentration ranged from 2.74 mg/L to 5.74 mg/L, yielding a removal efficiency of 57.6% ± 15% during the first 79 days of operation. Effluent phosphate concentrations subsequently declined to 0.43–0.53 mg/L by the end of operation. Phosphate removal efficiency during the last phase of bioreactor operation was 95.2% ± 0.5% (Figure 3).

3.4. Microbial Community Dynamics

Microbial community shifts between the seed biomass and densified activated sludge were evaluated using 16S rRNA gene sequencing and are presented in Figure 4. At the class level, the seed biomass contained 10.9% Alphaproteobacteria, 16.3% Gammaproteobacteria, 17% Bacteroidia, and 7.3% Actinobacteria (Figure 4). In contrast, in the densified activated sludge, Alphaproteobacteria was the predominant bacterial class, accounting for 76.7% of the total microbial community. The relative abundance of Gammaproteobacteria declined to 6.1%, Actinobacteria to 1.8%, and Bacteroidia to 10%. The seed biomass microbial community was highly diverse at the genus level. The most abundant bacterial genera in the seed biomass included Thiothrix (filamentous bacteria) and Zoogloea (Figure 4). Their high abundance corresponded to the poor settling properties of the seed biomass. In the densified activated sludge, the predominant microbial genera included Paracoccus (relative abundance 72.9%), known for heterotrophic nitrification, aerobic denitrification, and phosphorus removal [25], and Pseudazoarcus (relative abundance 5.4%). This community composition revealed enrichment of bacteria capable of heterotrophic nitrification and aerobic denitrification in small granular flocs, which can potentially intensify biological nutrient removal in a compact wastewater treatment facility.

4. Discussion

4.1. Small Granular Flocs and Nutrient Removal

Microbial aggregates, such as pinpoint flocs, flocs, small granular flocs, and granules, appear as distinct entities but are pleomorphic and can dynamically shift between structures in natural and engineered environmental systems, forming a continuum from flocs to large granules [5]. Consequently, systems designed and operated for a specific type often consist of a hybrid structure [7,26]. Granule size is a key feature of AGS reactors, as it affects settling properties and the diffusion of oxygen and substrate into the granules’ interior zone. Conventionally, stratification of redox conditions is important for the efficiency of simultaneous nitrification, denitrification, and phosphorus removal [10,27]. In this context, small granules are less likely to develop strong transport limitations for oxygen and nutrients, supporting nitrifier growth.

In this study, DAS was predominantly composed of small, granular flocs ranging from 50 to 100 µm (Figure 1). Conventionally, microbial aggregates of this size are considered less capable of simultaneous biological nutrient removal because they lack the internal redox stratification required for typical denitrification. Contrary to this expectation, this system achieved high COD removal, near-complete ammonia removal, complete nitrite removal after day 78, and high phosphate removal during the final phase of operation (Figure 3).

Conventionally, the abundance of small pinpoint flocs is correlated with poor settling properties of the biomass. However, we have previously shown that small pinpoint flocs rich in autotrophic nitrifying microbial populations exhibit granule-like settling properties, whereas the biomass settling properties gradually deteriorate when conditions that support heterotrophic bacterial growth are provided [22,28]. In this study, small flocs rich in heterotrophic bacterial populations showed granule-like settling properties and biomass accumulation. The results show that bacterial populations capable of forming compact microbial aggregates, regardless of the size of the structure, exhibit granule-like settling properties.

A distinct increase in effluent nitrate was observed during the final phase of operation, coinciding with a ~50% increase in biomass (Figure 2) and the complete removal of organic substrate (Figure 3). This pattern suggests that nitrate reduction was constrained by the availability of readily biodegradable organic carbon rather than by biomass assimilation alone. Carbon availability was a decisive constraint on denitrification. Consequently, these findings emphasize that sustained nitrate reduction, specifically for heterotrophs like Paracoccus, is contingent upon a consistent organic carbon supply to drive aerobic denitrification.

4.2. Role of HN-AD and Paracoccus

Bacteria related to the genus Paracoccus were predominant in the densified activated sludge cultivated in this study (Figure 4). The relative abundance of Paracoccus in this study was 72.9%, consistent with our other recent studies. For example, the relative abundance of Paracoccus reached up to 74% in laboratory-scale bioreactors fed similar feed, where the biomass was exposed to per- and polyfluoroalkyl substances [19]. Similarly, up to 75% relative abundance of Paracoccus was observed in a bioreactor fed a similar synthetic wastewater recipe to study the effect of microfibers on microbial aggregation [18]. Paracoccus bacteria are highly versatile, capable of heterotrophic nitrification and aerobic denitrification, and of contributing to phosphorus removal [25,29,30,31]. Unlike traditional granular sludge systems that rely on oxygen diffusion limitations to create internal anoxic zones for nitrogen removal, HN-AD bacteria may support nitrogen transformation under aerobic conditions.

Pure culture studies have reported that some Paracoccus species possess the complete set of genes required for simultaneous nitrification, denitrification, and phosphorus removal; their denitrification genes are insensitive to oxygen. Robust enzyme activity of ammonia monooxygenase, hydroxylamine oxidoreductase, nitrate reductase, nitrite reductase, nitric oxide reductase, nitrous oxide reductase, and polyphosphate kinase has been documented in pure culture studies under aerobic conditions [25]. As a fast-growing heterotroph, Paracoccus thrives on acetate, and its rapid growth may support nutrient removal through both assimilation and metabolic conversion [31]. Furthermore, some Paracoccus species have been reported to possess nitrous oxide reductase, which mitigates greenhouse gas emissions; however, this study did not measure nitrous oxide emissions [25]. Therefore, optimizing operational conditions that support the growth and enrichment of aerobic denitrifiers may support greenhouse gas mitigation, but this requires direct verification.

The prevalence of Paracoccus within small granular flocs suggests that simultaneous nutrient removal is not strictly constrained by the diffusion-limited anoxic zones typical of conventional large granular structures. Rather, these heterotrophs may contribute to nitrogen and phosphorus processing via aerobic metabolic pathways [25]. Previous studies have reported that some Paracoccus species possess genes and enzymes associated with simultaneous nitrification, denitrification, and phosphorus removal under aerobic conditions [25,32]. This metabolic capacity may reduce reliance on spatial oxygen gradients for nutrient removal in large granular structures.

Furthermore, the rapid growth kinetics of Paracoccus, driven by efficient acetate utilization, may support biomass proliferation and nutrient removal through assimilation and metabolic conversion, compared with the slower-growing autotrophic nitrifiers and obligate anoxic denitrifiers typically associated with traditional granular sludge systems. This metabolic advantage may partly explain the development of nutrient removal activity before the formation of large, diffusion-limited granules [31].

4.2. Formation of DAS

The SVI₃₀ and SVI30/SVI₅ ratios are often used to assess sludge settleability and the degree of granulation [33,34]. An SVI₃₀/SVI₅ ratio close to 100% indicates more complete granulation, whereas a value below 100% typically denotes the co-occurrence of flocs with granular sludge [34,35]. Conventional paradigms suggest that flocs can reduce overall sludge settling velocity because they have higher drag than granules [9]. However, this perspective may undervalue the functional advantages of high-density flocs. In densified systems, these smaller aggregates exhibit high specific surface areas that facilitate mass transfer and metabolic activity, which can compensate for the lower settling velocities traditionally associated with flocculated sludge [12,36]. The dynamic equilibrium, in which small flocs can mature into granules and larger structures may disintegrate, supports sustained nutrient removal and provides resilience against biomass washout [5].

In this study, an SVI₃₀ of 30 mL/g MLSS and an SVI₃₀/SVI₅ ratio of 65% to 72% were observed (Figure 2), indicating good settling compared with conventional activated sludge flocs. Microscopic analysis revealed that the small granular flocs were predominantly 50 to 100 µm in size (Figure 1). Although conventional paradigms might suggest that smaller aggregates exhibit inferior settling properties, the observed SVI metrics indicate that this DAS exhibits settling comparable to AGS. This superior performance, achieved despite the smaller aggregate size, is attributed to the compact structure of the granular flocs. Previous studies have shown that while larger granules settle faster, smaller granules may exhibit lower SVI30 and settle more compactly in the sequencing batch reactor [37]. The small granular flocs contributed to improved settling compared with conventional flocs, allowing the accumulation of approximately 12 g/L MLSS in the bioreactor (Figure 2).

MLSS in the bioreactor (Figure 2). This level of biomass retention is comparable to that of established granular sludge systems, supporting the intensification of biological nutrient removal [6]. These results suggest that, in densified activated sludge, metabolic capacity, aggregate density, and biomass retention are important performance indicators alongside aggregate size.

4.3. Granule Formation with Anoxic vs Aerobic Denitrifiers

Overall, aerobic granule formation involves two distinct approaches that underscore the complexity of microbial community adaptation to varying operational environments. The first school of thought prioritizes cultivating slow-growing bacteria by using anoxic feeding and minimizing readily available organic substrate during the aerobic phase, thereby suppressing fast-growing heterotrophs. During this anoxic feeding stage, carbon-storing bacteria convert substrates into complex polymers, fostering an organized, stratified redox structure. Consequently, nitrifying bacteria reside in the outer aerobic layers, while denitrifiers occupy the inner zones where oxygen diffusion is restricted [4,5]. This spatial stratification facilitates the synergistic exchange of substrates and metabolites, supporting the coexistence of nitrifiers and denitrifiers within an organized, functional spatial gradient [38]. Technologies based on this principle are commercially well established, using dense, spherical structures to overcome mass-transfer limitations and generate internal micro-gradients of dissolved oxygen and pH that optimize simultaneous nutrient removal [39].

Conversely, a second approach demonstrates that granulation is achievable in a completely aerobic system through aerobic feeding at high organic substrate concentrations, coupled with high hydraulic shear forces and a short settling time. These conditions facilitate the washout of filamentous bacteria and fluffy flocs and improve mass transfer within granules, promoting dense structures [40]. This mechanism suggests that high substrate availability and mechanical stressors stimulate extracellular polymeric substance production, significantly enhancing the structural integrity and hydrophobicity of microbial aggregates [38]. This strategy is advantageous for rapid granulation [40]. However, microbial interactions among relatively slow-growing nitrifiers that compete with fast-growing heterotrophs in the outer layer (which use oxygen as an electron acceptor) and slow-growing anoxic denitrifying bacteria in the core remain unclear in granules cultivated under high organic-loading conditions [5,41]. While these pathways are distinct, they are not necessarily contradictory; rather, the mechanisms governing these stochastic developmental pathways remain subjects of debate, particularly regarding how heterogeneous structural dynamics influence the spatial organization of target organisms [10,41].

In this study, a combination of these two strategies was used to cultivate densified activated sludge: the bioreactor was fed anoxically at high organic loading and then exposed to high superficial air velocity to achieve a high hydraulic shear rate. The hybrid approach leveraged the synergistic potential of both carbon-storage mechanisms and hydrodynamic selection to cultivate small granular flocs. A short settling time was applied as a selection pressure to retain dense microbial aggregates, thereby promoting the washout of slow-settling flocculent biomass and yielding densified activated sludge with favourable settling properties. This selective pressure also included a short hydraulic retention time and bottom-feeding strategies to prioritize the retention and growth of denser microbial aggregates. The strategy enriched bacteria capable of heterotrophic nitrification and aerobic denitrification, which may support nitrogen transformation under variable oxygen conditions [42].

4.4. Implications and Future Optimization

The AGS cultivation strategy, employing high organic loading, likely selected for aerobic heterotrophs capable of simultaneous nutrient removal. While conventional metabolic pathways for simultaneous heterotrophic nitrification and aerobic denitrification often require distinct oxygen-limited micro-environments within granular structures, aerobic denitrifiers such as Paracoccus can remain metabolically active across diverse oxygen conditions [42]. Consequently, the traditional assumption that large granule size is a prerequisite for creating the oxygen gradients necessary for denitrification may be overly restrictive. In this study, the compact structure of the small granular flocs (50 µm to 100 µm) appears to support efficient nutrient removal pathways, suggesting that oxygen diffusion limitations are not the only mechanism for achieving simultaneous heterotrophic nitrification and aerobic denitrification. Instead, nitrate removal efficiency declined when the organic substrate was completely removed, indicating that nitrate removal was driven by aerobic denitrifiers’ metabolic activity rather than nutrient assimilation alone.

High organic substrate availability may enable fast-growing aerobic denitrifiers to sustain metabolic activity and nutrient assimilation. A previous study with higher organic substrate levels and continuous aeration led to rapid formation of mega granules, with Paracoccus and Zoogloea bacteria predominant [43]. Conversely, under biomass washout conditions (which create a high food-to-microorganism ratio), granule formation has been linked to fluffy granules dominated by Zoogloea, resulting in poor nutrient removal [44]. Future studies are needed to optimize conditions for Paracoccus using municipal and industrial wastewater to validate the scalability and practical application of this system for improved simultaneous heterotrophic nitrification and aerobic denitrification.

5. Conclusions

Conventionally, good settling properties and simultaneous nutrient removal in a single tank are associated with granules larger than 200 µm. In this study, small granular flocs (50-100 µm) exhibited granular sludge-like settling and biomass accumulation, leading to the formation of densified activated sludge. Simultaneous nitrification, denitrification, and phosphorus removal were also observed in these small granular flocs. This outcome demonstrates that efficient biological nutrient removal is feasible without forming large granules, which typically rely on oxygen diffusion limitations to establish internal anoxic cores. Instead, a microbial community dominated by bacteria capable of heterotrophic nitrification, aerobic denitrification, and phosphorus removal, such as Paracoccus, can drive simultaneous nutrient removal within small granular flocs. By circumventing the mass transfer constraints typical of larger aggregates, this approach uses niche metabolic pathways to achieve efficient treatment in smaller, denser microbial structures. Consequently, achieving DAS and HN-AD represents an intensification of biological nutrient removal, simplifying operational requirements. Future studies are required to optimize conditions for HN-AD and to validate the scalability and practical application of this system for efficient biological nutrient removal.