Introduction

As a critical urban infrastructure, the efficient operation of wastewater treatment plants (WWTPs) is essential for maintaining environmental quality and public health1. The conventional activated sludge process remains the dominant wastewater treatment technology due to its high adaptability, operational flexibility, and stable performance2. However, inherent limitations of activated sludge and operational constraints pose substantial challenges, including increasing volumetric load, enhancing treatment efficiency, and reducing energy consumption. These bottlenecks hinder conventional process from meeting the escalating demands for water quality and quantity in rapidly urbanizing regions3. Addressing these challenges requires the development of more efficient and low-carbon wastewater treatment technologies4,5.

Activated sludge plays a central role in biological wastewater treatment, where microbial quantity, diversity, and activity directly influence treatment efficiency and capacity6. Biofilm technology has demonstrated considerable advantages in enhancing microbial biomass, boosting microbial diversity, and improving microbial activity7. However, the formation of a mature and stable biofilm on carriers typically requires several months8, and the commonly used suspended carriers necessitate high aeration strength to maintain the fluidization process. Additionally, centimeter-scale carriers tend to form localized stacks and may cause structural abrasion in concrete tanks. To overcome these limitations, powder carriers with a high specific surface area have been proposed as an alternative9. These carriers are expected to accelerate microbial aggregation while maintaining fluidization under lower aeration conditions10. Functional powder materials, such as biochar, zeolite, and iron-carbon composites, have been shown to effectively enrich functional microorganisms due to their larger surface area and higher porosity11. Despite these advantages, powder carriers are prone to loss during treatment, either through effluent or sludge discharge. Consequently, the powder carrier still has the limited application in continuous-flow systems in municipal wastewater treatment. Recent advancements in hydrocyclone separator technology offer a promising solution to this challenge12,13. By integrating hydrocyclone separators with powder carrier systems, carriers can be effectively recovered, minimizing loss and improving long-term operational stability. This integration represents a notable step toward enhancing wastewater treatment efficiency while reducing energy and material consumption.

Hydrocyclone separator technology generates a vortex within the chamber, where heavier particles are forced toward the outer wall and gradually accumulate in a settling zone14. In contrast, lighter particles remain near the center, where the central vortex propels them upward and directs them out through the outlet pipe. In the context of activated sludge mixed liquor, the hydrocyclone vortex selectively removes lighter components from sludge floc surfaces while concentrating heavier, carrier-loaded fractions15. This mechanism suggests that integrating powder carriers with hydrocyclone separators can alter sludge floc microstructure and facilitate micro-granular sludge formation, with powder carriers serving as nuclei9. The formation of micro-granules is critical for enriching functional microorganisms, as the carrier nuclei provide attachment sites for microbial colonization and enhance the retention of carrier-supported functional bacteria16. Furthermore, the coexistence and dynamic interaction between micro-granules and flocs likely optimize the spatial distribution and ecological niches of functional bacteria within the sludge matrix. This spatial organization may enhance microbial synergy, improving overall system performance and treatment efficiency.

From a scientific research perspective, while previous studies have explored the characteristics and mechanisms of micro-granular sludge-based processes, gaps remain in understanding the structural features of the sludge system and the micro-spatial distribution of functional microorganisms17,18. In particular, the mechanisms governing microbial synergy and metabolic responses within this sludge microstructure require further investigation. From an engineering application standpoint, aerobic granular sludge technology has encountered challenges in promoting microbial aggregation and granulation, largely due to the operational and hydraulic constraints of continuous-flow processes19. Similarly, the influence of continuous-flow hydraulic conditions on micro-granule formation and microstructure development induced by powder carriers remains insufficiently understood. To bridge these knowledge gaps, further pilot-scale validation is essential to assess the performance, stability, and feasibility of this process in large-scale, real-world wastewater treatment applications.

This study proposed and implemented a micron-medium biofilm composite sludge (MMBCS) system at a pilot-scale for municipal wastewater treatment, with a treatment capacity of 338 m3 per day (Fig. 1). The pilot-scale system was operated under continuous-flow hydraulic conditions using the anaerobic–anoxic–oxic (AAO) process. The research investigated the formation and characteristics of this composite sludge system, driven by the integration of powder carriers and hydrocyclone separator. The composition, structure, and functional characteristics of the sludge within the pilot system were analyzed. Microbial analysis revealed the abundance and the distribution difference of functional microorganisms in carrier-laden biofilm and suspended flocs. Furthermore, the mechanisms of microbial synergy in micro-granules and flocs for nitrogen and phosphorus removal were explored, providing an in-depth analysis of the underlying metabolic processes. These results elucidated the scientific mechanisms responsible for the enhanced nitrogen and phosphorus removal capability of the MMBCS system. The findings of this study offer a promising technological approach for upgrading biological wastewater treatment, contributing to more efficient and sustainable wastewater management.

Fig. 1: Schematic diagram of the pilot-scale micron-medium biofilm composite sludge (MMBCS) system.
figure 1

Pilot-scale installation (top) and schematic representation of the pilot system (bottom).

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Results and discussion

Overall performance of pilot-scale system

Long-term nutrient removal performance

The long-term carbon, nitrogen, and phosphorus removal performance of the pilot-scale MMBCS system is illustrated in Fig. 2. In phase Ⅰ, the influent load was set at 1.5 times that of the actual WWTP. The effluent NH4+-N and total nitrogen (TN) concentrations were 0.42 ± 0.21 mg L−1 and 4.78 ± 0.63 mg L−1, respectively, achieving removal efficiencies of 98.43 ± 0.82% and 83.08 ± 2.87% (Fig. 2a, b). Meanwhile, effluent of total phosphorus (TP) and chemical oxygen demand (COD) concentrations were 0.28 ± 0.05 mg L−1 and 15.18 ± 8.87 mg L−1, respectively (Fig. 2c, d). These results demonstrated that the MMBCS process could rapidly enhance wastewater treatment capacity in situ, ensuring full compliance with discharge standards. In phase Ⅱ, the hydraulic retention time (HRT) was further decreased to 4.85 h, and the influent load was increased to twice that of the WWTP. After a brief performance dip, the pilot-scale MMBCS system swiftly recovered and maintained stable operation. Effluent TN, TP, and COD concentrations were maintained at 4.98 ± 1.37 mg L−1, 0.28 ± 0.11 mg L−1, and 19.10 ± 8.84 mg L−1, respectively. Compared to the actual WWTP, the TN removal efficiency improved from 68.84 ± 4.75% to 84.01 ± 2.93%, with a TN removal rate of 0.14 ± 0.02 mg L−1 d−1 (Supplementary Figs. S1 and S2). The actual WWTP continued to experience effluent NH4+-N and TP exceedances.

Fig. 2: Long-term operational performance of the pilot-scale micron-medium biofilm composite sludge system.
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a Influent and effluent NH4+-N concentrations and NH4+-N removal efficiency. b Influent and effluent total nitrogen (TN) concentrations and TN removal efficiency. c Influent and effluent total phosphorus (TP) concentrations and TP removal efficiency. d Influent and effluent chemical oxygen demand (COD) and COD removal efficiency. The shaded area and error bars represent the standard deviation.

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These findings suggested that the in-situ addition of micron-sized diatomite carriers allowed the system to double its treatment capacity without production downtime or facility expansion. Furthermore, the system demonstrated robust stability and resilience in response to increased influent loads. Notably, the influent COD concentration ranged from 123.7 to 181.3 mg L−1, with approximately 50–70% being biodegradable carbon sources, typical of low-carbon urban wastewater4. This also highlighted the capability of the MMBCS system to achieve stable operation and efficient nitrogen and phosphorus removal, even in carbon-limited municipal wastewater.

Nutrient transformation and mass balance in the treatment process

Nutrient concentrations were measured across each treatment unit, and mass balances were calculated to assess pollutant removal dynamics (Fig. 3). In the anaerobic zones, influent COD was considerably consumed, decreasing to 56.2 mg L−1 in the WWTP and 37.5 mg L−1 in the pilot-scale MMBCS system (Fig. 3a, b). Influent organic matters could be taken up by polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) and stored as the internal carbon source20. Also, the influent organics drove the denitrification process in the anaerobic zone. The remaining organic matter was further decomposed and consumed in the aerobic zones. At a high sludge concentration of 8 g L−1 and a short HRT of 0.72 h, the composite sludge exhibited a marked instantaneous adsorption capacity, leading to a rapid decrease in NH4+-N and TN concentrations within the anaerobic zone21,22. The porous and loosely structured sludge, along with the extracellular polymeric substances (EPS) layer formed under the induction of micron-scale carriers, was capable of adsorbing both organic matter and NH4+-N from the wastewater23. Due to the internal recirculation of mixed liquor from the aerobic zone, dissolved oxygen (DO) was introduced into the front section of the anoxic zone, promoting partial oxidation of NH4+-N in the pilot-scale MMBCS system. Consequently, NH4+-N concentrations in the anoxic effluent decreased from 7.77 mg L−1 to 0.78 mg L−1, while NO3−-N increased from 0.57 mg L−1 to 4.39 mg L−1, which was lower than the reduction of NH4+-N (Supplementary Fig. S3). Also, TN concentrations declined to 5.45 mg L−1, accounting for 28.4% of total effluent nitrogen (Fig. 3c). These results indicated that a portion of the NO3−-N (e.g., portion of NO3−-N resulted from the oxidation of NH4+-N) was removed through denitrification and also suggested the occurrence of simultaneous nitrification and denitrification (SND) in the anoxic zone. In the aerobic zones, the TN concentration was further reduced in MMBCS system, suggesting aerobic denitrification as an additional removal pathway. In contrast, the concentrations of NH4+-N and NO3−-N in the aerobic zones of the WWTP exhibited an inverse relationship. Phosphorus uptake by PAOs was most pronounced in the anoxic zones, with higher phosphorus assimilation in the pilot-scale MMBCS system compared to the WWTP. This showed that PAOs played a more active role in phosphorus cycling under the modified sludge conditions.

Fig. 3: Comparison of nutrients removal performance in different zones.
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a, b Changes in nutrient concentrations in different areas of the wastewater treatment plant (WWTP) and pilot-scale micron-medium biofilm composite sludge (MMBCS) system. Data are presented as mean ± standard deviation (samples n  =  3). c Nutrient removal contributions of each treatment unit.

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These findings highlighted that the pilot-scale MMBCS system achieved most pollutant removal within the anaerobic and anoxic zones, whereas the WWTP relied more on aerobic treatment for complete reduction. The observed SND and aerobic denitrification played a crucial role in nitrogen removal, while phosphorus release and uptake were notably more efficient. The composite sludge system, induced by micron-sized carriers, likely enhanced material transfer and microbial cooperation24, thereby coupling denitrification with biological phosphorus removal and improving overall treatment efficiency.

Composite sludge system driven by powder carrier and hydrocyclone separator

Sludge micro-structure dominated by powder carriers

The pilot-scale MMBCS system comprised a mixture of carrier-based micro-granules and suspended flocs, which were separated using a 200-mesh sieve for structural analysis (Fig. 4a, b). The micron-sized diatomite carriers, characterized by high specific surface area and porosity, exhibited strong adsorption to suspended floc sludge and zoogloea, promoting bio-aggregate formation10. Notably, the morphological characteristics of these micron-medium-based bio-aggregates differed from traditional biofilms and granular sludge. In conventional biofilm systems, carriers act as attachment points, supporting the development of layered biofilms on their surfaces7. In granular sludge systems, powder carriers typically function as nucleation sites, facilitating granulation under shear forces in the flow9. However, in the MMBCS system, micron-sized carriers were embedded within zoogloea in a disordered and random manner, rather than serving as cores for sludge aggregation. Instead, they appeared to function as connectors or bridges between flocs, resulting in the formation of a loosely structured bio-aggregate (Supplementary Fig. S4). This unique sludge microstructure may not only enhance the retention of functional microorganisms but also potentially promote mass transfer and intercellular communication25. This sludge structure provided deep insights into biofilm technology, offering a distinct pathway for enhancing microbial aggregation and process stability in wastewater treatment.

Fig. 4: Sludge characteristics of pilot-scale micron-medium biofilm composite sludge (MMBCS) system.
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a Images of microscopy. b Scanning electron microscopy photograph. c Mixed liquor suspended solids (MLSS). d Sludge volume index (SVI). e Particle size distribution and fractal dimension. f Size interval distribution. For (c and d), the shaded area represents the standard deviation. Data are presented as mean ± standard deviation (samples n  =  3).

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Micro-granule formation under continuous-flow conditions

Long-term monitoring of sludge concentration revealed that, apart from a slight decrease during startup, mixed liquor suspended solids (MLSS) remained stable at 7500–8000 mg L−1, even under increased loading and flow rates (Fig. 4c and Supplementary Fig. S5). This stability suggested that the hydrocyclone effectively recovered micron-sized carriers, preventing carrier loss. The hydrocyclone separator achieved a recovery efficiency of over 90% for the diatomite powder, requiring only a small amount of daily supplementation (Supplementary Text S1). While a reduction in carrier size and an increase in specific surface area theoretically enhance microbial adsorption and could lead to increased sludge concentration24, no substantial increase in biomass or EPS content was observed (Supplementary Fig. S6). This indicated that carriers likely did not function solely as microbial attachment sites but may also influence sludge structure and microbial metabolism26. The consumption of endogenous substances may have constrained microbial growth. With increasing influent load, SVI5 increased from 96.3 ± 2.2 mL g−1 to 126.1 ± 1.8 mL g−1, while SVI30 rose from 75.8 ± 2.5 mL g−1 to 93.3 ± 3.1 mL g−1 (Fig. 4d). Consequently, the SVI5/SVI30 ratio increased from approximately 1.28 to 1.35, suggesting that higher influent flow rates in the continuous-flow process may have affected sludge aggregation, leading to a slight decline in settling performance19. Previous studies indicate that when SVI5 is close to SVI30, granular sludge settleability improves5. In the MMBCS system, the SVI5/SVI30 ratio remained stable between 1.28 and 1.35, which was likely due to the presence of suspended floc sludge. Despite this, the system exhibited good settleability and effectively prevented sludge bulking associated with excessive microbial growth. Although standalone hydrocyclone separators have previously been applied in municipal wastewater treatment to separate and recirculate dense sludge and improve settling performance27, they do not fundamentally alter the characteristics of suspended flocs in conventional activated sludge systems. In contrast, the integration of micron-scale powder carriers with hydrocyclone separation formed a micro-granule–floc composite system, which simultaneously enhanced sludge settleability and fundamentally transforms the microstructure of the sludge.

The particle size distribution of sludge in the pilot-scale MMBCS system is shown in Fig. 4e, f. The inoculated sludge from the WWTP had an average particle size of 57.1 μm, with 92.02% of particles smaller than 100 μm. In contrast, under the adsorption and connection effects of micron-sized diatomite carriers, the particle size distribution shifted clearly to the right, indicating sludge aggregation. The average particle size increased to 99.3 μm (day 65) and 115.8 μm (day 120), with particles >100 μm comprising 53.68%. This suggested that the MMBCS system effectively maintained sludge stability, even under continuous-flow conditions and increased influent load. Fractal dimension analysis was conducted to assess sludge structural characteristics (Fig. 4e). In conventional activated sludge systems, fractal dimensions typically range from 1.07 to 1.6828, where higher values indicate denser sludge flocs and lower values suggest more porous structures. In the MMBCS system, the fractal dimension increased from 1.25 (inoculated sludge) to 1.78 (day 65), indicating denser sludge aggregation. However, by day 120, the fractal dimension decreased slightly to 1.72, suggesting that influent load variations led to a looser structure, which correlated with observed settling performance trends. Previous studies have shown that granular sludge typically has a fractal dimension exceeding 229,30, higher than the values observed in the MMBCS system. This discrepancy likely arises from the presence of suspended floc sludge and the carrier-mediated aggregation mechanism, which differs from traditional granular sludge formation. The loosely aggregated microbial structures induced by micron-scale powder carriers enable the enrichment of functional microorganisms while maintaining efficient mass transfer31, thereby integrating the respective advantages of both activated sludge and granular sludge.

Microbial community composition and metabolic pathways

Differential spatial distribution of functional microorganisms in sludge micro-structure

The pilot-scale MMBCS system, driven by micron-sized carriers, cultivated a unique microbial community. Principal coordinates analysis (PCoA) revealed significant differences in community structure during the long-term operation of the pilot-scale MMBCS system (R² = 0.8930, P = 0.001) (Fig. 5a). The microbial community in the pilot-scale system underwent gradual succession, showing an increasing divergence from the inoculated sludge of the WWTP. Moreover, a more compact microbial cluster was observed in the pilot-scale system on day 120, implying a more stable and resilient community structure32. With the formation of bio-aggregates and the increased influent load, the number of unique species in the pilot-scale system considerably decreased (Fig. 5b). This reduction in diversity suggested that microbial diversity was constrained within the MMBCS system, indicating a shift from a more diverse to a more specialized microbial community. These findings demonstrated that, unlike traditional processes, the MMBCS system could foster a uniquely structured and functionally specialized microbial community, likely contributing to its enhanced treatment efficiency and process stability.

Fig. 5: Microbial diversity and community dynamics.
figure 5

a Principal coordinates analysis (PCoA) on amplicon sequence variant (ASV) level. b Venn diagram on ASV level. c The main phyla in pilot-scale system. d Changes in abundance of functional bacteria at genus level. e The relative abundance of nitrogen- and phosphorus-removing microbial taxa in the mixed sludge of the pilot-scale system. f Differential distribution of functional microbial groups between granular sludge and suspended flocs. Granule micro-granule sludge in pilot-scale system, Floc suspended sludge in pilot-scale system, GAOs glycogen-accumulating organisms, DNB denitrifying bacteria, PAOs polyphosphate-accumulating organisms, AOB ammonia-oxidizing bacteria, NOB nitrite-oxidizing bacteria, DPAOs denitrifying phosphorus-accumulating organisms.

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The phylum-level microbial composition of the pilot-scale MMBCS system revealed that Proteobacteria, Patescibacteria, Bacteroidota, Chloroflexi, and Actinobacteriota were the dominant phyla (Fig. 5c and Supplementary Table S1). At the genus level, the relative abundance of Candidatus_Competibacter, a typical GAO known for competing with PAOs for carbon sources in the anaerobic phase, was considerably reduced from 7.96% (inoculated sludge) to 5.67% (day 120) (Fig. 5d and Supplementary Table S2). This decline implied that the formation of composite sludge effectively controlled GAOs, preventing negative impacts on anaerobic phosphorus release while potentially enhancing endogenous denitrification33. Maintaining a balanced GAO population is essential for high-efficiency nitrogen and phosphorus removal.4 In terms of nitrification, norank_f_NS9_marine_group and Nitrospira were identified as ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), respectively. While AOB abundance remained stable, NOB abundance decreased after the addition of powder carriers (Fig. 5e). This indicated that the formation of the composite sludge system can promote AOB enrichment and NOB suppression, showing a potential for achieving shortcut nitrification7. Furthermore, Candidatus_Microthrix and Tetrasphaera were identified as denitrifying phosphorus-accumulating organisms (DPAOs)34. Their abundance increased from 1.60% (inoculated sludge) to 8.16% (day 120), indicating that the composite sludge system notably enriched DPAOs, thereby enhancing the simultaneous removal of nitrogen and phosphorus. Additionally, autotrophic denitrifying bacteria (DNB), including Iamia, Hyphomicrobium, Rhodobacter, and Romboutsia35, exhibited an increasing trend over the course of operation.

Sludge samples collected from the pilot-scale system on day 120 were fractionated to obtain micro-granule and suspended floc sludge. Further analysis revealed the spatial distribution of functional microorganisms within the sludge microstructure (Fig. 5f). DNB and DPAOs were predominantly located in the micro-granule sludge, with abundances reaching 17.40% and 4.46%, respectively—higher than in the suspended floc sludge (8.59% and 2.52%). This showed that the formation of bio-aggregates, driven by the micron-sized carriers, played a crucial role in enhancing denitrification performance in the pilot-scale MMBCS system. In contrast, AOB and PAOs were primarily enriched in the floc sludge, with relative abundances of 1.25% and 5.88%, respectively. This distribution facilitated efficient substrate and DO utilization while promoting phosphorus-laden sludge discharge. The coexistence of micro-granule and floc sludge in the pilot-scale MMBCS system optimized the spatial organization of functional microorganisms, fostering microbial synergy. The enrichment of DNB and DPAOs, coupled with the effective control of GAOs, contributed to enhanced nitrogen and phosphorus removal. This microbial synergy was key to improving overall system performance, demonstrating the effectiveness of the MMBCS process in wastewater treatment.

Optimization of metabolic pathways mediated by micron-sized carriers

Metagenomic analysis was conducted to assess the abundance of key metabolic pathways and functional genes involved in carbon, nitrogen, and phosphorus transformations. As shown in Supplementary Fig. S7, the analysis of nitrogen-related metabolic modules revealed distinct characteristics of nitrogen conversion in the pilot-scale MMBCS system. The M00528 module, associated with nitrification36, was approximately twice as abundant on day 120 compared to the inoculated sludge, implying the enhancement of shortcut nitrification with the formation of the composite sludge system (Supplementary Table S3). In contrast, the M00804 module, linked to complete nitrification37, exhibited slightly higher abundance in the inoculated sludge, suggesting that this process was primarily occurring in floc sludge rather than micro-granule sludge. Furthermore, the M00529 module, related to denitrification24, was more abundant in micro-granule sludge, highlighting the critical role of bio-aggregate formation in facilitating efficient nitrogen removal.

Differential analysis of key metabolic and functional genes involved in nitrogen, carbon, and phosphorus transformations further supported these findings (Fig. 6a and Supplementary Table S4). Several nitrogen metabolism-related genes (narGHI, napA, nosZ, norBC, amoABC) were enriched in the composite sludge system. Notably, the denitrification-related gene nosZ was 39.5% more abundant in the pilot-scale system than in the inoculated sludge, with a 64.3% increase in micro-granule sludge, suggesting enhanced nitrous oxide reduction. The amoABC genes, associated with nitrification24, increased by 43.2% in floc sludge, reinforcing the differential distribution of nitrifiers between sludge types. These findings supported the conclusion that the composite sludge system, driven by micron-sized carriers, facilitated the selective enrichment of DNB and promoted the differential distribution of functional genes. Metatranscriptomic analysis further revealed the differential expression of key genes involved in nitrogen transformation across samples collected on day 120 (Fig. 6b). Consistent with the metagenomic results, the expression levels of most nitrogen-related functional genes were higher in the pilot-scale MMBCS system compared to the full-scale WWTP. NosZ, the most highly expressed gene associated with denitrification, accounted for 13.15% and 16.93% of expression in the WWTP and the MMBCS system, respectively, reaching 18.29% in micro-granule sludge. Similarly, pmoC-amoC and narG were the most abundantly expressed genes associated with ammonia oxidation and nitrate reduction, reaching 15.31% and 5.10% in the floc sludge of the MMBCS system, respectively (Supplementary Table S5). The spatial differentiation in the expression of nitrogen-transforming genes between micro-granules and flocs also indicated the shortcut nitrification-denitrification process in MMBCS system. In addition, the MMBCS system promoted the simultaneous removal of nitrogen and phosphorus, with DPAOs playing a key (Fig. 6c). The biosynthesis of polyhydroxyalkanoates (PHAs), glycogen, and pyruvate generated critical energy and electron donors (ATP, NADH) required for denitrification and phosphorus accumulation20,38. In micro-granule sludge, key genes involved in PHA synthesis and hydrolysis (phaC, phaZ, phbB) exhibited a considerable increase in abundance. Similarly, genes related to glycogen metabolism (glgA, glgB) and pyruvate metabolism (pdhAB, acs) were notably enriched. Metatranscriptomic analysis also indicated that the genes involved in regulating PHA biosynthesis were highly expressed in the MMBCS system, particularly within the micro-granule sludge (Fig. 6b). These findings demonstrated that bio-aggregate formation, driven by micron-sized carriers, promoted endogenous carbon metabolism, enhancing carbon source utilization in the nitrogen and phosphorus removal process. Additionally, the abundance of genes encoding phosphate transport proteins, specifically pst and pho, as well as the gene encoding polyphosphate kinase (ppk)39, was found to be elevated after the long-term operation. Notably, the ppa and ppk2 genes exhibited prominent expression in the floc sludge of the pilot-scale system, reaching 1.04% and 1.24%, respectively (Supplementary Table S5). These genes exhibited differential enrichment between micro-granule and floc sludge, suggesting that both sludge types contributed to the phosphorus removal process. This differential distribution further emphasized the distinct roles of micro-granular and floc sludge in optimizing phosphorus accumulation and removal in the system.

Fig. 6: Functional genes and metabolic pathways.
figure 6

a Abundance differences of key genes involved in carbon, nitrogen, and phosphorus metabolism in different sludge samples (based on gene abundance in inoculated sludge). b Changes in gene abundance of key metabolic pathways at the transcriptional level. c Metabolic map depicting the flow of carbon, nitrogen, and phosphorus nutrients in pilot-scale system, alongside the key coding genes. WWTP activated sludge in wastewater treatment plant, CS composite sludge in pilot-scale on day 120, Granule granular sludge from the pilot-scale system on day 120, Floc suspended floc sludge from the pilot-scale system on day 120, PHAs polyhydroxyalkanoates, TCA cycle tricarboxylic acid cycle.

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Compared to the traditional activated sludge process, the micron-sized carriers in the pilot-scale MMBCS system enhanced the enrichment and expression of key genes related to carbon, nitrogen, and phosphorus metabolism role (Fig. 6c). The coexistence of micro-granule and floc sludge, coupled with the unique bio-aggregate structure, facilitated microbial interactions and communication (Supplementary Fig. S8). The increased abundance of genes associated with endogenous carbon synthesis and metabolism in micro-granule sludge provided essential electron donors and energy, supporting simultaneous denitrification and phosphorus removal. The enrichment and differential distribution of functional genes were pivotal in achieving high-efficiency nutrient removal, closely aligning with the spatial organization of functional microorganisms within the system.

Potential mechanisms and application prospects

Microbial synergy in micro-granules and flocs for nitrogen and phosphorus removal

In the pilot-scale MMBCS system, the integration of micron-sized powder carriers and a hydrocyclone separator facilitated the formation of a composite sludge system, where micro-granules and suspended floc sludge coexisted (Fig. 7). This system effectively promoted the selective enrichment and differential distribution of functional microorganisms, enhancing microbial synergy. The suspended flocs, with their high mass transfer efficiency, supported ammonia oxidation, leading to the enrichment of nitrifying bacteria while simultaneously supplying nitrate for denitrification in micro-granules. Meanwhile, the longer sludge age of the micro-granules favored the retention of DPAOs, and the discharge of floc sludge contributed to phosphorus removal. Sludge characterization revealed that bio-aggregates induced by micron-sized diatomite formed a unique structure, distinct from both traditional activated sludge and granular sludge. Unlike conventional biofilm technology, where carriers primarily serve as attachment sites or granulation cores7,9, the powder carriers in the MMBCS system were randomly dispersed between zoogloea in a connected or embedded form (Fig. 4a, b). This structural feature suggested that, despite the increased specific surface area of the powder carriers, the biomass did not experience a proportional increase. The loose aggregation structure facilitated not only the enrichment of DNB and DPAOs but also improved mass transfer and microbial communication, thereby optimizing key metabolic functions. Additionally, EPS analysis revealed that the sludge EPS content did not correlate with changes in particle size but instead showed a slight decrease (Supplementary Fig. S6). This reduction likely reflects the consumption of endogenous carbon sources, suggesting that more carbon and energy were directed toward denitrification and phosphorus removal40. Consequently, the micron-sized carriers and hydrocyclone separator played a pivotal role in shaping a sludge micro-structure, which mediated the optimization of microbial metabolic pathways. These findings highlight the key role of composite sludge in promoting functional microbial enrichment, improving microbial synergy, and optimizing metabolic processes.

Fig. 7: Micro-granule and floc composite sludge system driven by micron-medium and microbial synergy mechanism.
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AAO-MMBCS system anaerobic–anoxic–oxic micron-medium biofilm composite sludge system, GAOs glycogen-accumulating organisms, DNB denitrifying bacteria, PAOs polyphosphate-accumulating organisms, AOB ammonia-oxidizing bacteria, NOB nitrite-oxidizing bacteria, DPAOs denitrifying phosphorus-accumulating organisms, EPS extracellular polymeric substances, PolyP polyphosphate, ATP adenosine triphosphate. ADP adenosine diphosphate.

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Engineering significance and implications

This study confirmed the stability of the powder carriers and hydrocyclone integration for modifying sludge micro-structure under continuous-flow hydraulic conditions. The composite sludge system successfully doubled its treatment capacity while ensuring compliance with effluent standards, without requiring system expansion or operational downtime. At a short HRT of 4.85 h, the effluent TN and TP concentrations were 4.98 ± 1.37 mg L−1 and 0.28 ± 0.11 mg L−1, respectively, with removal efficiencies of 84.01 ± 2.93% and 92.49 ± 3.26% (Fig. 2). Following the addition of micron-sized diatomite, the pilot system immediately adapted to a 1.5-fold increase in influent load while maintaining efficient nitrogen and phosphorus removal. This highlights the MMBCS system’s potential for WWTP upgrades, considerably shortening construction timelines, enhancing treatment capacity, and offering flexible compatibility with various activated sludge processes. Furthermore, during the pilot-scale system operation, no exogenous carbon source was added, yet functional microorganisms involved in endogenous carbon metabolism and their associated genes exhibited considerable enrichment (Fig. 6). This suggests that the MMBCS system is not only efficient in utilizing carbon sources from the influent but also offers advantages in terms of energy savings, further enhancing the sustainability and cost-effectiveness of this system. The integrated system of micron-scale powder carriers and hydrocyclone separator addresses several key challenges in continuous-flow processes, including the difficulty of granular sludge formation, the slow development of carrier-attached biofilms, and the high energy consumption associated with fluidized aeration. Feasibility analysis further supports the practicality of full-scale implementation (Supplementary Text S1). Full-scale application would only require adjusting the powder carrier dosage and the number of hydrocyclone units in proportion to the treatment capacity. Importantly, the cost of diatomite carriers can be fully offset by the savings from reduced external carbon source dosing. Therefore, this technology is both technically and economically viable for upgrading existing WWTPs. These findings provide strong evidence for the application of the MMBCS system in the technological upgrading of WWTPs, implying its potential to enhance treatment capacity, improve efficiency, and reduce energy consumption.

Conclusions

This study developed a pilot-scale MMBCS system integrating powder carriers and hydrocyclone separator, effectively establishing a composite sludge system composed of micro-granules and suspended flocs. This system facilitated the selective enrichment and spatial differentiation of functional bacteria, enhancing microbial synergy for simultaneous nitrogen and phosphorus removal. Under a short HRT of 4.85 h, the system achieved high TN and TP removal efficiencies of 84.01 ± 2.93% and 92.49 ± 3.26%, respectively. The micron-sized carriers promoted sludge aggregation in an unordered, embedded, or connected form, leading to loosely structured bio-aggregates distinct from traditional activated sludge and granular sludge. This structural configuration enhanced sludge settleability and improved material and oxygen transfer efficiency. Microbial analysis revealed the enrichment of Candidatus Microthrix and Tetrasphaera in the micro-granules, where they played critical roles in denitrification and phosphorus removal. In contrast, nitrifying bacteria and related genes (amoABC) were predominantly concentrated in suspended flocs, supporting ammonia oxidation. Furthermore, the enhancement of endogenous carbon metabolism pathways, including the synthesis of polyhydroxyalkanoates, glycogen, and pyruvate, provided essential electron donors and energy, further supporting simultaneous nitrogen and phosphorus removal. Notably, the pilot system successfully doubled treatment capacity without requiring plant expansion or operational downtime, while maintaining effluent quality within discharge standards. These findings validate the stability and effectiveness of integrating powder carriers and hydrocyclone separator in modifying sludge microstructure under continuous-flow hydraulic conditions. This study offers deep insights into the role of powder carriers in wastewater treatment and presents a promising approach for upgrading biological wastewater treatment systems.

Materials and methods

Pilot-scale AAO-MMBCS system

A WWTP in Wuhan employs the AAO process for nitrogen and phosphorus removal, with a treatment capacity of 30 × 104 m3 per day. As wastewater volume continues to rise, there is a need to enhance its treatment capacity by 1.5 times (to 45 × 104 m3 per day) without expanding the plant’s land area. Currently, the effluent concentrations of NH4+-N and TP fall short of regulatory standards (Supplementary Fig. S1). With increasing treatment loads and the implementation of more stringent discharge limits, there is a potential risk of effluent non-compliance. To address these challenges, a MMBCS process is proposed for technical optimization, aiming to both increase treatment capacity and ensure compliance with discharge standards. By simulating the operational conditions of a real WWTP based on equivalent HRT, this composite sludge system based on powder carrier and hydrocyclone separator was established within the AAO system (Fig. 1).

The pilot-scale AAO-MMBCS system consisted of a biological treatment unit (68.3 m3), a secondary sedimentation tank (31.2 m3), and a carrier recovery unit. The inoculated sludge was sourced from the biochemical tank of the actual WWTP, with an initial MLSS concentration of 4235 ± 123 mg L−1. Micron-sized diatomite carriers were introduced into the aerobic section of the system. The initial carrier dosage was set at 4000 mg L−1, followed by a daily supplementation of 1.5 kg to maintain the MLSS within the range of 7000–8000 mg L−1 in the system. The particle size of the diatomite carrier was 19.54 ± 5.39 μm, with an average surface area of 21.64 m2 g−1. The pilot-scale system was operated with the same influent characteristics as the full-scale WWTP. System performance was evaluated in two phases, each designed to simulate the existing process configurations and operational parameters at the plant (Table 1). In phase Ⅰ, the HRT was set to 6.31 h, with the anaerobic section lasting 0.93 h, the anoxic section 1.88 h, and the aerobic section 3.50 h. The treatment capacity was 260 m3 per day, corresponding to the actual WWTP treatment scale of 45 × 104 m3 per day. In phase Ⅱ, the HRT was further reduced to 4.85 h, with the anaerobic section lasting 0.71 h, the anoxic section 1.44 h, and the aerobic section 2.70 h. The treatment capacity was increased to 338 m3 per day, simulating a treatment scale of 58.5 × 104 m3 per day. The nitrifying liquid reflux rate was set to 150%, and the sludge reflux rate was 100%. The sludge retention time (SRT) was approximately 20 days, with sludge discharge controlled to maintain a consistent sludge concentration within the system. Simultaneously, a portion of secondary settling tank sludge (3.13 m3/d) was directed to the hydrocyclone separator. Following separation, the underflow, which accounted for 30% of the inflow and contained dense sludge and powder carriers, was recovered and returned to the aerobic tank. Details of the sludge discharge procedure and operational parameters of the hydrocyclone separator are provided in Supplementary Text S2. In the aerobic section, the DO concentration ranged from 2.5 to 3.0 mg L−1 at the front end to 0.5–1.0 mg L−1 at the end.

Table 1 Operation conditions of pilot-scale micron-medium biofilm composite sludge system
Full size table

Methods for chemical analysis

Influent and secondary sedimentation tank effluent samples from both the WWTP and the pilot-scale system were collected daily. Water quality indicators, including NH4+-N, TN, COD, and TP, were analyzed following standard methods41. Sludge samples from the pilot-scale system were collected to measure the MLSS and the SVI to assess sludge concentration and settleability. The particle size distribution of the sludge was determined using a laser particle size analyzer (Partica LA-960, HORIBA, Japan). The fractal dimension of the sludge was calculated using a light scattering method28. The morphology and structure of the sludge were observed under an optical microscope, while it was further examined by scanning electron microscopy (Zeiss Gemini 300, Germany) following dewatering and freeze-drying (Supplementary Text S3). EPS were extracted during the stable phase following previously reported methods42, and the content of polysaccharides and proteins in the EPS was quantified.

Microbial analysis

Sludge samples were collected from the pilot-scale system on days 0, 65, and 120. The mixed sludge samples (day 120) from the pilot-scale system were separated into micro-granule and floc sludge fractions using a 200-mesh sieve. Metagenomic analysis was performed to examine the microbial diversity and community. Genomic DNA was extracted from the sludge samples using the FastDNA SPIN Kit for Soil (Bio 101, CA). DNA integrity was assessed by 1% agarose gel electrophoresis, while DNA concentration and purity were measured using NanoDrop and Qubit to ensure that the samples met the requirements for downstream sequencing. The DNA was fragmented into 300–500 bp fragments via sonication, followed by the addition of adapter sequences. Library construction was carried out using the Illumina library construction kit, and paired-end sequencing was performed on the Illumina HiSeq platform to generate raw data. The sequences were processed with FastQC and Trimmomatic software to remove adapter sequences, low-quality bases, and short reads, resulting in high-quality, valid sequence data. Predicted gene sequences were clustered using the CD-HIT software, and the longest sequence from each cluster was selected as the representative sequence to create a non-redundant gene set. Total RNA was extracted using TRIzol® reagent following the manufacturer’s protocol. RNA integrity and concentration were assessed via an Agilent 5300 Bioanalyzer and NanoDrop ND-2000. RNA purification, library construction, and sequencing were carried out by Majorbio Bio-Pharm Technology (Shanghai, China). Raw reads were trimmed with fastp, then aligned to the reference genome using HISAT2. Transcript assembly was performed with StringTie, and transcript abundance was quantified using the Transcripts Per Million method. PCoA and Venn diagrams were used to assess species differences and similarities at the amplicon sequence variant (ASV) level across the different sludge samples. Statistical analysis of the species composition and key gene abundance in the samples was conducted. Gene sequences were aligned with KEGG to analyze the metabolic pathways and functional features of the microbial community (Supplementary Text S4). A co-occurrence network of microbes was constructed for different samples based on Spearman correlation analysis, and the topological properties of the network were quantitatively assessed to identify changes in microbial interaction patterns24. The raw sequence data have been deposited in the NCBI database (PRJNA1207416).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Calculations

Water samples were collected from various units of the WWTP and the pilot-scale system to assess the pollutant removal efficiency of each biochemical unit and to evaluate the pathways of pollutant removal. The mass balance calculations were performed using the following Eqs. (1–4):

$${R}_{{Anaerobic}}\left( \% \right)=\frac{Q\times {C}_{{Inf}}+Q\times {C}_{{Eff}}-2Q\times {C}_{{Anaerobic}}}{Q\times {C}_{{Inf}}}\times 100 \%$$
(1)
$${R}_{{Anoxic}}\left( \% \right)=\frac{2Q\times {C}_{{Anaerobic}}+1.5Q\times {C}_{{Oxic}}-3.5Q\times {C}_{{Anoxic}}}{Q\times {C}_{{Inf}}}\times 100 \%$$
(2)
$${R}_{{Oxic}}\left( \% \right)=\frac{3.5Q\times {C}_{{Anoxic}}-3.5Q\times {C}_{{Oxic}}}{Q\times {C}_{{Inf}}}\times 100 \%$$
(3)
$${R}_{{Sediment}}\left( \% \right)=\frac{2Q\times {C}_{{Oxic}}-2Q\times {C}_{{Eff}}}{Q\times {C}_{{Inf}}}\times 100 \%$$
(4)

where RAnaerobic, RAnoxic, ROxic, and RSediment represent the removal ratios for carbon, nitrogen, and phosphorus in the anaerobic, anoxic, oxic, and sedimentation tanks, respectively. CInf and CEff are the pollutant concentrations in the influent and effluent, while CAnaerobic, CAnoxic, and COxic refer to the pollutant concentrations in the effluent from the anaerobic, aerobic, and anoxic tanks. Q is the flow rate of the treatment system.