Preserving Denitrification in High-Dissolved-Oxygen Environments: A Novel Sulfur–Polycaprolactone–Polyurethane Sponge Composite Carrier with Multi-Omics Insights

Abstract

To address the denitrification challenges in the high-dissolved-oxygen (DO) aquatic environment system caused by the secondary effluent of wastewater treatment plants, this study innovatively developed a sulfur–polycaprolactone (S⁰-PCL) dual-electron donor composite carrier embedded in polyurethane sponge to mitigate high DO stress. The system demonstrated remarkable resilience, with a denitrification efficiency decline of only 11.3% under a DO gradient (0–6.5 mg/L), significantly outperforming previously reported efficiency losses exceeding 90%. Metatranscriptomic analysis revealed that the millimeter-scale pores in the sponge created potential anoxic microzones, providing a microenvironment conducive to maintaining key denitrification gene expression. The anoxic stage was dominated by the sulfur-autotrophic denitrification (SAD) microorganism Sulfurovum (abundance 68.1%, expression 55.1%). In high-DO conditions, Alicycliphilus (55.3, 41.1%) and Paracoccus (31.6%, 31.8%) synergistically drove denitrification, while Hyphomonas (8.7%, 24.1%) assisted in DO and organics consumption. Gene expression data further revealed that Alicycliphilus hydrolyzed PCL to enter the tricarboxylic acid cycle, while Paracoccus simultaneously utilized S⁰ for SAD, establishing a dual-electron donor transfer pathway to sustain denitrification activity. The S⁰-PCL–polyurethane sponge provided resilience via oxygen isolation, sustained carbon release, and metabolic diversification, enabling microbial and functional gene reconfiguration. This study provides a novel technological pathway for deep nitrogen removal in oxygen-rich environments.

1. Introduction

Nitrate pollution in aquatic systems has emerged as a critical challenge in global water environmental management [1]. Excessive nitrogen inputs from urban, agricultural, and industrial activities have severely disrupted natural nitrogen cycling, resulting in widespread nitrate concentration exceedances in both surface and groundwater [2]. This phenomenon triggers eutrophication processes and poses significant threats to human health [3]. Sulfur-autotrophic denitrification (SAD) utilizing elemental sulfur (S⁰) as an inorganic electron donor has garnered extensive scholarly attention due to its distinctive advantages [4]. Compared to autotrophic systems relying on hydrogen or iron-based materials, the SAD process employs S⁰ as the primary electron donor, offering not only readily available materials and low operational costs, but also enabling simultaneous sludge minimization and greenhouse gas mitigation, thereby demonstrating remarkable potential for sustainable development in wastewater treatment technologies [1,5].

However, the efficiency of SAD is significantly impaired under high-dissolved-oxygen (DO) conditions [6]. In such scenarios, oxygen preferentially acts as the terminal electron acceptor, suppressing nitrate reductase activity and inhibiting the metabolic functions of sulfur-oxidizing bacteria (SOB) [7,8]. Recent studies have demonstrated that when DO exceeds 6 mg/L, the nitrate removal efficiency of SAD systems declines by over 90% compared to that under anoxic conditions [9]. This limitation restricts the applicability of SAD in oxygen-rich aquatic systems. For instance, effluent from secondary clarifiers in activated sludge processes frequently maintains DO levels at >3 mg/L due to the waterfall effect [5,6]. Similarly, high-DO conditions prevail when utilizing wastewater treatment plant secondary effluent for ecological replenishment of surface waters, presenting significant nitrogen removal challenges [10]. Consequently, there is an urgent need for innovative electron donor delivery strategies and microenvironmental regulation approaches to overcome the oxygen sensitivity bottleneck of SAD technology.

To address this challenge, mixotrophic dual-electron donor pathway denitrification strategies integrating autotrophic and heterotrophic mechanisms have garnered increasing attention [11,12]. However, conventional heterotrophic denitrification (HD) relies on intermittent dosing of soluble organic carbon. This non-steady-state substrate supply mode not only increases operational costs but also elevates risks of dissolved organic matter (DOM) accumulation and subsequent secondary pollution due to mismatched kinetics between instantaneous carbon surplus and microbial utilization rates [13]. Polycaprolactone (PCL), as a biodegradable polymer, has been explored as a slow-release carbon source for denitrification [13]. Its enzymatically controlled hydrolysis enables precise carbon release, maintaining residual DOM concentrations below 13 mg/L and achieving an optimized balance between carbon supply efficiency and ecological safety [14]. Furthermore, recent advances in microscale oxygen transport mechanisms have revealed that the physical barrier effect of three-dimensional porous matrices can spontaneously form persistent anoxic microzones at the millimeter scale [15]. For instance, polyurethane sponges have demonstrated millimeter-scale porous structures that create additional anoxic microenvironments while promoting microbial colonization and metabolism [16].

Building upon these findings, this study developed a novel composite-functionalized carrier system: S⁰ and PCL were co-immobilized within a polyurethane sponge matrix through a low-temperature melting–cooling process, constructing a sulfur–carbon dual-electron donor interface while simultaneously establishing anoxic microenvironments. Currently, research gaps persist regarding the denitrification efficiency optimization mechanisms of such composite carriers under high-DO conditions. Therefore, this study aims to (1) evaluate the coupling effects of the novel carrier on nitrogen removal efficiency under varying DO concentrations, (2) elucidate the synergistic metabolic mechanisms between autotrophic and heterotrophic microbial communities, and (3) decipher the regulatory patterns of anoxic–aerobic transitions on the expression profiles of key functional genes.

2. Materials and Methods

2.1. Preparation of the Novel Microbial Carrier

The base material of the novel carrier was polyurethane sponge with an average pore size of 60 PPI and an edge length of 1.5 cm. PCL powder (Natural Works, USA, MN), sieved to pass through a 300-mesh sieve, had an average molecular weight of 80 kDa. S⁰ was ground using a pulverizer and sieved through a 300-mesh sieve to obtain fine powder. The PCL and S⁰ powders were uniformly adsorbed onto the polyurethane sponge following the method of Song et al. [17]. Specifically, the sponge was loaded into a 5 L beaker at a 10% filling density, and equal proportions of the two solid powders were added. The mixture was stirred at 150 rpm for 4 h to achieve saturation. Excess surface powder was removed by sieving through a 300-mesh sieve, and the composite was placed in a constant-temperature oven at 90 °C (above the 62 °C melting point of PCL) to melt the PCL. The molten PCL acted as a binder to adhere both itself and S⁰ to the inner and outer surfaces of the carrier. After 20 min, the composite was removed and cooled to ambient temperature, allowing the PCL to resolidify and immobilize the adhered S⁰ within the polyurethane sponge pores. A series of elution experiments were conducted to remove unbound particles until no impurities were detected, followed by drying at 30 °C for subsequent use. The initial weight of the polyurethane sponge was 0.21 ± 0.01 g, and the final weight of the prepared carrier was 0.97 ± 0.12 g.

Scanning electron microscopy (SEM) images showing the porous microstructure of the composite carrier before use. SEM–Energy Dispersive Spectroscopy (SEM-EDS) results showed that the mass percentages of S, C, and O elements in the deposits on the composite carrier were 76.96%, 13.12%, and 9.92%, respectively, demonstrating the effective immobilization of both sulfur and carbon sources on the carrier (see Supplementary Materials Figure S3).

After carrier preparation, a series of elution experiments were conducted to quantitatively assess the immobilization efficacy of S⁰ and PCL. Specifically, a known mass (e.g., 10.0 g) of the prepared composite carrier was placed in a conical flask with a known volume (e.g., 500 mL) of ultrapure water and shaken in a constant-temperature shaker (30 °C, 150 rpm) for 24 h. The eluate was then collected, filtered through a 0.22 μm membrane, and quantitatively analyzed using gravimetric analysis (determining the mass of suspended solids in the eluate to assess the physical detachment of PCL or S⁰ powder) and ion chromatography (determining SO₄²⁻ concentration in the eluate to assess the chemical oxidative dissolution of S⁰). The experimental results clearly showed that after 3 elution cycles, the cumulative mass loss rates of both PCL and S⁰ were less than 1.5% of their initial loaded mass. This quantitatively demonstrates that the low-temperature melting–cooling process, with PCL acting as a binder, successfully immobilized both itself and S⁰ powder firmly onto the inner and outer surfaces of the polyurethane sponge skeleton, resulting in excellent initial fixation.

2.2. Experimental Setup and Operation

A moving bed biofilm reactor packed with the novel carrier was employed for the experiments (Figure S1). The reactor was constructed from plexiglass with an effective working volume of 3 L. Inoculum was sourced from a prolonged-operation SAD system. The synthetic medium was prepared using tap water, KNO₃, NaHCO₃, KH₂PO₄, K₂HPO₄, MgCl₂, CaCl₂, and trace elements [18], with influent nitrate (NO₃⁻-N) concentration controlled at 20.3 ± 1.2 mg/L. A peristaltic pump (BT100, LongerPump, China, Hangzhou) was used for influent supply and effluent discharge, maintaining a hydraulic retention time (HRT) of 0.5 h. A temperature-controlled fluid loop, connected to an external jacket via a thermostatic bath, sustained the reactor’s internal temperature at 30 °C. DO was regulated using a DO controller (SC5103, Nuosai, China, Hangzhou) and an air pump [19], with DO levels set to three groups: 0–0.5 mg/L (anoxic), 2.5–3.5 mg/L (low oxygen), and 5.5–6.5 mg/L (high oxygen), following the protocol of Kou et al. [8]. The biofilm reactor in this study was operated continuously for 92 days and was divided into three distinct stages based on DO concentration. Stage I (days 1–30, anoxic conditions): In this initial phase, the DO concentration was strictly maintained at 0–0.5 mg/L by sparging nitrogen gas (N₂) into the reactor and precisely controlling it with a DO controller. This stage aimed to initiate and stabilize the microbial community for both SAD and PCL-driven heterotrophic denitrification. Stage II (days 31–60, low-oxygen conditions): Starting on day 31, the gas supply was switched to air, and the aeration rate of the air pump was adjusted to raise and maintain the DO concentration at 2.5–3.5 mg/L. The system operated under these conditions for 30 days to evaluate the adaptation and performance of the microbial community to a microaerobic environment. Stage III (days 61–92, high-oxygen conditions): From day 61 onwards, the aeration rate was increased further to elevate and stabilize the DO concentration at 5.5–6.5 mg/L. The system continued to run for 32 days under this high-DO stress to test the resilience of the composite carrier in maintaining denitrification functionality.

2.3. Sampling, Analysis, and Bioinformatic Processing

Influent and effluent samples were collected daily, filtered through 0.22 μm filters, and analyzed for NO₃⁻-N and SO₄²⁻-S concentrations using an ion chromatograph (Dionex ICS-2100, Thermo Scientific, USA, MA). Nitrite and thiosulfate ions were undetected throughout the reactor operation. Chemical oxygen demand (COD) was measured via a quick-analysis apparatus (D60, Dtsxy, China, Quzhou) [20]. Scanning electron microscopy–Energy Dispersive Spectroscopy (SEM-EDS, Gemini300, ZEISS, Germany, Oberkochen) was employed to characterize the surface microstructure of the novel composite carrier and to observe microbial colonization on the carrier during the later experimental stages. All analytical measurements were conducted in triplicate.

Biological membrane samples were collected on days 30, 60, and 90 of reactor operation, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent DNA/RNA extraction and metagenomic/metatranscriptomic analysis. Sample processing followed the protocols detailed in the Supplementary Materials (Text S1). Raw DNA and RNA sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) database under accession number PRJNA1243562. Raw sequencing data were quality-checked using Trimmomatic (v0.36) [21] and assembled into contigs with MEGAHIT (v1.1.1-2-g02102e1) [22]. Based on metagenomic alignment results, MetaBAT2 (v2.11.1) [23] was employed for binning, followed by CheckM (v1.1.3) [24] to filter high-quality metagenome-assembled genomes (MAGs) with coverage > 70% and contamination < 10%. Binned sequences were further refined via BWA (v0.7.17-r1188) [25] and SPAdes (v3.12.0) [26] for secondary assembly. Final MAGs were evaluated with CheckM (v1.1.3) and clustered based on average nucleotide identity (ANI ≥ 95%) using fastANI. Taxonomic classification of MAGs was performed using GTDB-Tk (v1.6.0) [27]. Clean reads were mapped to non-redundant, high-quality MAGs via BWA (v0.7.17-r1188) to calculate relative abundance and expression levels. Functional genes involved in nitrogen/sulfur/carbon metabolism and electron transfer pathways were annotated using KOBAS (v3.0) [28] against the KEGG database, while gene expression levels were quantified as transcripts per million (TPM) using Salmon (v0.8.2) [29].

3. Results and Discussion

3.1. System Performance Evolution

This study systematically investigated the stage-specific performance evolution of a microbial denitrification system through multi-parameter dynamic monitoring over 92 continuous days of operation (Stage I: days 1–30, Stage II: days 31–60, and Stage III: days 61–92) (Figure 1). Under influent NO₃⁻-N concentrations fluctuating at 20.3 ± 1.5 mg/L, Stage I (anoxic conditions) exhibited a progressive enhancement in denitrification performance, with removal efficiency increasing from an initial 54.5% to 76.8%, averaging 64.7 ± 9.2%. Concurrently, SO₄²⁻-S production remained stable at 17–23 mg/L (mean: 19.2 ± 2.5 mg/L), while effluent COD concentrations were maintained between 5 and 9 mg/L (mean: 7.4 ± 1.8 mg/L). These results suggest the presence of potential synergistic interactions between heterotrophic and SAD pathways, contributing to robust denitrification efficiency.

In Stage II (microaerobic conditions), DO levels increased to 2.5–3.5 mg/L, yet NO₃⁻-N removal efficiency remained stable at 45.5–73.7% (mean: 63.7 ± 7.6%). Notably, SO₄²⁻-S production significantly increased (mean: 24.7 ± 4.1 mg/L), accompanied by a slight rise in effluent COD (mean: 8.1 ± 2.0 mg/L). This indicates that the denitrification performance was not substantially impaired under this DO regime. The elevated SO₄²⁻-S concentrations likely originated from direct oxidation of S⁰ by oxygen, aligning with findings from previous studies [8].

Stage III (high-oxygen conditions) marked a significant shift in the system behavior. During the initial period (days 61–70), the NO₃⁻-N removal efficiency sharply declined to a minimum of 20.1% (mean: 30.5 ± 6.1%), with concurrent reductions in SO₄²⁻-S production (mean: 17.6 ± 3.8 mg/L). Effluent COD concentrations, however, remained relatively stable (mean: 8.0 ± 1.8 mg/L). This decline in performance under high DO (5.5–6.5 mg/L) strongly suggests the inhibition of SAD processes. However, as the system continued to operate, the denitrification efficiency gradually recovered in the later stage (days 71–92), with the NO₃⁻-N removal efficiency peaking at 70.1% (mean: 57.4 ± 9.6%). SO₄²⁻-S production in this stage exceeded that of the preceding stages, averaging 31.0 ± 5.7 mg/L, while effluent COD remained consistently stable at 8.0 ± 1.9 mg/L.

These results demonstrate that the polyurethane sponge composite system, integrating S⁰ and PCL as dual-electron donors, can sustain effective secondary effluent NO₃⁻-N removal even under high-DO conditions. Notably, under a DO gradient (0–6.5 mg/L), effluent COD concentrations remained tightly controlled within a narrow range (mean: 8.0 mg/L), indicating that the sustained carbon release characteristics of PCL were insensitive to DO fluctuations. This stability effectively buffered the metabolic stress on denitrifying microbial communities, particularly during the high-oxygen stage (Stage III), where SAD pathways were suppressed. The persistent availability of PCL-derived carbon as an alternative electron donor was critical in maintaining a peak denitrification efficiency of 70.1% under these challenging conditions.

3.2. Microbial Community Structure

Sequencing reads from Stages I, II, and III were assembled to generate 29 high-quality metagenome-assembled genomes (MAGs) per sample (completeness > 70%, contamination < 10%). Taxonomic classification of each MAG was performed to evaluate their functional roles (Table S1), while the contribution of each MAG to the microbial community was assessed via DNA read mapping, and their transcriptional activity was quantified through RNA read mapping. Multidimensional analysis of the MAGs and transcriptomic data revealed the dynamic response mechanisms of microbial community structure and function under the dual-electron donor system (S⁰ and PCL) and DO gradient regulation (Figure 2).

In Stage I (DO: 0–0.5 mg/L), Sulfurovum dominated the community, accounting for 68.1% of the total abundance and 55.1% of the transcriptional activity. As previously reported, Sulfurovum is a canonical sulfur-autotrophic denitrifier [30]. This indicates that under anoxic conditions, microbial oxidation of S⁰ to generate SO₄²⁻ for electron-driven denitrification was the predominant pathway in the dual-electron donor system of S⁰ and PCL, aligning with findings from prior studies [13]. Paracoccus emerged as a secondary component in Stage I, contributing 15.1% of the total abundance and 18.9% of the transcriptional activity. Paracoccus species are metabolically versatile, capable of utilizing diverse electron donors (e.g., organic carbon, reduced sulfur compounds, and H₂) to drive denitrification [31]. This enables Paracoccus to simultaneously perform SAD using S⁰ and HD via PCL, forming a functional coupling with Sulfurovum for nitrogen removal. While less abundant, other microbes in the Stage I community also contributed to system stability. Giesbergeria (abundance 4.3%, transcriptional activity 6.9%) facilitated the removal of nitrogenous compounds and the degradation of recalcitrant organic matter [32]. Hyphomonas (abundance 0.4%, transcriptional activity 4.7%), a potent organic matter degrader [33], further enhanced carbon turnover. Alicycliphilus (abundance 1.6%, transcriptional activity 3.0%) could degrade PCL for HD and provide organics intermediates for other heterotrophic denitrifiers [13]. Castellaniella (abundance 1.7%, transcriptional activity 3.2%) and Comamonas (abundance 1.9%, transcriptional activity 1.7%), both heterotrophic denitrifiers [34,35], played roles in the organic matter breakdown, diversifying denitrification pathways and ensuring effluent COD compliance with discharge standards.

With the increase in DO concentration to 2.5–3.5 mg/L in Stage II, the microbial community structure underwent significant reorganization. The sulfur-autotrophic denitrifier Sulfurovum, previously dominant in Stage I, exhibited a marked decline in both abundance (1.9%) and functional activity (0.9%). In contrast, Paracoccus, a microorganism capable of utilizing both S⁰ and organic carbon as electron donors, emerged as the predominant species (abundance 57.1%, transcriptional activity 66.4%). This shift suggests that under microaerobic conditions, obligate sulfur-autotrophic denitrifiers (which rely solely on S⁰ as an electron donor) experienced severe metabolic inhibition due to competitive electron between oxygen and nitrate. As a result, these specialists lost their community dominance. The mixotrophic metabolism of Paracoccus, enabled by the supplemental electron supply from PCL, partially mitigated the adverse effects of elevated DO, allowing it to become the primary functional driver in this stage. Alicycliphilus, distinguished by its capacity for direct PCL degradation and HD, demonstrated notable proliferation and high functional activity in Stage II (abundance 25.6%, transcriptional activity 24.9%). This organism likely contributed to both organic carbon electron flux and denitrification efficiency through its metabolic versatility. Additionally, Hyphomonas showed a significant increase in community abundance, rising from 0.4% in Stage I to 3.7% in Stage II. While its transcriptional activity slightly decreased (from 4.7% to 4.2%), its persistence suggests a potential role in alleviating DO-induced inhibition. Hyphomonas may have mitigated the suppressive effects of elevated DO by consuming oxygen to degrade complex organic matter, thereby synergistically maintaining the system’s average nitrate removal efficiency of 63.7 ± 7.6% in conjunction with the aforementioned microorganisms.

The microbial community structure in Stage III exhibited a more pronounced response to elevated DO concentrations (5.5–6.5 mg/L). Despite a decline in both abundance (31.6%) and transcriptional activity (31.8%), Paracoccus, a mixotrophic denitrifier, remained a dominant member of the microbial community. Meanwhile, Hyphomonas further enhanced its active role within the microbial consortium, contributing 8.7% of the total abundance and 24.1% of the transcriptional activity. Most notably, Alicycliphilus emerged as the predominant microbial member, accounting for 55.3% of the community abundance and 41.1% of the transcriptional activity. These three functional groups established a synergistic advantage through metabolic role partitioning: Alicycliphilus dominated PCL hydrolysis and executed HD, with its degradation products supplying additional organic electron donors to Paracoccus. Paracoccus, in turn, adopted a mixotrophic metabolic strategy, simultaneously utilizing S⁰ and organic substrates to sustain denitrification activity. Hyphomonas further contributed by leveraging DO to degrade organic matter, thereby reducing electron competition while facilitating the maintenance of low effluent COD concentrations. This functional interplay among Alicycliphilus, Paracoccus, and Hyphomonas underscores the system’s capacity to adapt to high-oxygen stress through metabolic flexibility and resource partitioning. The transition from sulfur-autotrophic dominance in Stage I to heterotrophic/mixotrophic pathways in Stage III highlights the critical role of dual-electron donor systems in sustaining denitrification efficiency under fluctuating DO regimes.

3.3. Key Functional Genes

Normalized quantitative analysis of transcriptomic profiles for nitrogen, sulfur, carbon metabolism, and electron transfer pathways related to enzyme-encoding genes revealed the metabolic patterns of microbial communities across different stages (Figure 3). Membrane-bound nitrate reductases and periplasmic nitrate reductases, responsible for the reduction of NO₃⁻ to nitrite (NO₂⁻), are encoded by narGHIJ and napA, respectively (Figure 3a) [1]. Their total transcriptional abundances across the three phases were 2951 ± 168, 2817 ± 258, and 2456 ± 181 TPM. nirK and nirS, encoding copper-containing nitrite reductase and cytochrome cd1 nitrite reductase, respectively, catalyze the reduction of NO₂⁻ to nitric oxide (NO) [36]. The total expression levels of nirKS genes were 1439 ± 92, 1260 ± 82, and 1217 ± 117 TPM. Membrane-bound nitric oxide reductases, which convert NO to nitrous oxide (N₂O), are encoded by norB and norC [37], with total expression levels of 632 ± 49, 544 ± 78, and 506 ± 38 TPM. In the final denitrification step, the functional enzyme responsible for catalyzing the reduction of N₂O to nitrogen (N₂) is nitrous oxide reductase, encoded by the gene nosZ [38], which exhibited expression levels of 1052 ± 102, 1037 ± 97, and 1005 ± 44 TPM across the three phases. With increasing DO concentrations, the expression of denitrification-related genes decreased relative to Stage I by 4.6%, 12.4%, 13.9%, and 1.4% in Stage II and 16.8%, 15.4%, 20.0%, and 4.5% in Stage III. This aligns with previously reported trends demonstrating DO-induced inhibition of nitrate reductases and denitrification gene expression [6,7]. Notably, this study did not observe the severe 94% decline in denitrification efficiency reported when DO increased from 0.3 to 6.2 mg/L [9], despite a 11.3% reduction in efficiency from Stage I to III. This moderate decline correlates well with the observed downregulation of denitrification genes. These findings suggest that the anoxic microzones potentially created by the polyurethane sponge’s porous structure [15], combined with the coupled electron donor system (S⁰-PCL), partially mitigated the suppressive effects of high DO on denitrification gene activity, thereby maintaining effective denitrification performance in the system.

The SAD pathway relies on the oxidation of S⁰ to SO₄²⁻ as the electron donor (Figure 3b). The sulfur oxidation (SOX) system catalyzes a one-step oxidation of S⁰ to SO₄²⁻, encoded by the soxABC gene cluster [39], with total expression levels of 929 ± 86 (Stage I), 2030 ± 148 (Stage II), and 4137 ± 198 TPM (Stage III). Notably, the expression of soxABC increased with rising DO concentrations, contrasting the downregulation observed for denitrification genes. This suggests that under high-DO conditions, a portion of S⁰ was utilized for denitrification, while the remainder was directly oxidized to SO₄²⁻ via DO as the terminal electron acceptor. This observation aligns with the progressively increasing SO₄²⁻-S effluent concentrations across the stages (Figure 1b). Furthermore, dissimilatory sulfite reductase (DSR) facilitates a two-step oxidation pathway: S⁰ is first oxidized to sulfite (SO₃²⁻), which is subsequently converted to SO₄²⁻ via the SOX system [40]. The corresponding gene cluster for DSR is dsrAB, with total expression levels of 1221 ± 115, 713 ± 60, and 185 ± 21 TPM. The declining expression of dsrAB with increasing DO concentrations indicates a diminishing role for the two-step oxidation pathway. This trend implies that elevated DO concentrations provided sufficient electron acceptors, driving microbial populations to favor the energetically favorable one-step oxidation pathway for S⁰, while diminishing the contribution of the dsrAB-mediated two-step oxidation mechanism [41].

This study focuses on the mixed nutritional mode of SAD and HD driven by PCL degradation. Investigating the transcriptional activity of carbon metabolic pathways offers critical insights into adaptive changes in microbial nutrient utilization strategies. The key route for microbial inorganic carbon assimilation involves the Calvin–Benson–Bassham (CBB) cycle as a primary pathway [42] (Figure 3c). Within this cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) serves as the direct gateway for the metabolism and transformation of inorganic carbon [43]. The expression levels of rbcLS, the gene encoding Rubisco, decreased progressively from Stage I to Stage III (13180 ± 2938, 4637 ± 658, and 3194 ± 501 TPM). Autotrophic activity in microbial communities is positively correlated with rbcLS expression [44]. In addition, other key genes involved in the CBB cycle, such as tktA, prkB, and fbp, also exhibited elevated expression levels in Stage I. These results indicate that microbial communities in Stage I were predominantly autotrophic, with autotrophic activity declining gradually as DO concentrations increased. This decline is attributed to the suppression of SAD by high DO concentrations, which provides energy for the CBB cycle’s operation [45]. For organic carbon metabolism in microorganisms, the primary pathway is the tricarboxylic acid (TCA) cycle [18], which exhibits distinct functional gene expression patterns (Figure 3d). While inorganic carbon metabolism genes exhibited heightened activity in Stage I, the 16 TCA cycle-associated genes within the same phase demonstrated reduced transcript expression, ranging from 0.1 ± 0.03 to 86 ± 11 TPM. With increasing DO concentrations, TCA cycle genes in Stages II and III exhibited 0.6 to 12 and 1.4 to 58 times upregulation compared to that in Stage I, respectively, with Stage III showing an additional 0.01 to 3.7 times increase over Stage II. These findings suggest that under high DO stress, microorganisms utilize PCL as an organic carbon source to enter the TCA cycle, thereby enhancing TCA metabolic activity and supplying electrons for denitrification.

In this study, PCL functions as a slow-release carbon source and electron donor, and its metabolism is a multi-step, enzymatically controlled process. Initially, the PCL polymer is hydrolyzed by extracellular depolymerases (e.g., esterases and lipases) secreted by hydrolyzing bacteria such as Alicycliphilus, cleaving its ester bonds into water-soluble monomers, primarily 6-hydroxyhexanoic acid [46]. This slow, enzyme-mediated hydrolysis effectively maintains a low dissolved organic carbon concentration, thereby avoiding secondary pollution [14]. Following cellular uptake, 6-hydroxyhexanoic acid is progressively metabolized via the β-oxidation pathway, yielding acetyl-CoA [47]. Acetyl-CoA enters the TCA cycle, where it is oxidized, generating substantial reducing power in the form of NADH and FADH₂ [18]. These electron carriers subsequently donate electrons to the electron transport chain; under anoxic conditions, these electrons are ultimately transferred through a series of denitrifying reductases to progressively reduce nitrogen oxides to dinitrogen gas, thereby completing the heterotrophic denitrification process [18]. Consequently, PCL essentially acts as an enzymatically controlled solid-phase hydrogenase, providing a stable electron flux to the biofilm. Under high-dissolved-oxygen conditions, it not only supports the metabolism of obligate heterotrophic denitrifiers but also supplies organic carbon for mixotrophs, collectively establishing a metabolically redundant system resilient to dissolved oxygen stress.

Further analysis of the expression activity of genes encoding cytochrome c oxidase (CcO), a key component of the microbial electron transfer system, provides insights into the system’s electron utilization efficiency under high-DO conditions (Figure 3e) [48]. The ccoNOPQ gene cluster encodes the cbb3-type CcO, which plays a critical role in enhancing microbial environmental adaptability and energy production [49]. These genes exhibited a gradually decreasing but comparable total expression across the three stages, with values of 2948 ± 207, 2688 ± 303, and 2393 ± 156 TPM, respectively. Similarly, the cytochrome c oxidase subunit-encoding genes coxABC showed expression patterns consistent with ccoNOPQ, with TPM values of 1182 ± 146, 1102 ± 152, and 1061 ± 133 across the three stages. These results indicate that the system maintained effective electron transfer capacity and energy metabolism levels under high-DO conditions, despite the progressive decline in gene expression. The sustained activity of CcO pathways suggests that microbial populations adapted to oxygen stress by optimizing electron transfer pathways, thereby supporting continuous denitrification performance.

The observed gene expression patterns in this study diverge significantly from traditional findings, which emphasize the strong inhibitory effects of DO on nitrate reductase and the denitrification gene [7]. While DO concentrations in Stage III of this experiment reached 5.5–6.5 mg/L, the system demonstrated partial resistance to DO suppression through the synergistic integration of a polyurethane sponge’s millimeter-scale three-dimensional porous structure and the S⁰-PCL dual-electron-donor strategy. Specifically, the physical barrier effect of the polyurethane sponge maintained localized anoxic microenvironments, providing environmental stability for denitrification gene expression. The metabolic redundancy established by sulfur-autotrophic S⁰ oxidation and heterotrophic PCL degradation further mitigated DO-induced perturbations by distributing electron transfer risks across two complementary pathways (Figure 4). Microorganisms in this system could degrade PCL to release soluble organic carbon sources, which entered the TCA cycle to generate energy and electrons. These electrons were subsequently channeled via the CcO respiratory chain to key denitrification pathways, including nitrate reductase (narGHIJ) and nitrite reductase (nirKS), enabling stepwise nitrate reduction. This mechanism not only prevented a catastrophic decline in critical gene expression under high-DO conditions but also explains the relatively modest 11.3% decrease in nitrate removal efficiency in Stage III, far lower than the 94% decline reported in conventional systems [9]. The findings reveal a novel regulatory strategy for sustaining denitrification performance in high-DO environments, offering insights into engineering microbial systems with enhanced resilience to oxygen stress.

3.4. Community Genomic Analysis

Analysis of key functional gene expression levels in major MAGs within the system provides insights into the evolutionary dynamics of microbial functionality (Figure 5). In Stage I, Sulfurovum (Bin.303) dominated through high-expression S⁰ oxidation genes, supplying electrons for a complete and active denitrification process. The energy generated by this process further fueled the CBB cycle, enabling high-activity inorganic carbon metabolism. The elevated expression of CcO-encoding genes confirmed this efficient material and energy metabolism. These systemic activities established Bin.303 as the dominant microbial population in the community. Compared to Bin.303, Paracoccus (Bin.292) possessed a similarly complete set of metabolic genes but exhibited lower expression levels, making it the second dominant species in the community. The gene expression profiles of Bin.292 further demonstrated its dual metabolic capabilities: not only performing SAD but also utilizing organic matter via the TCA cycle for energy and electron generation. Other MAGs, including Giesbergeria (Bin.203), Hyphomonas (Bin.371), Castellaniella (Bin.463), Alicycliphilus (Bin.301), and Comamonas (Bin.343), represented strictly heterotrophic taxa. Among these, Bin.203, Bin.463, Bin.301, and Bin.343 exhibited complete denitrification gene clusters with detectable expression levels and unexpectedly displayed a weaker capacity for S⁰ oxidation, suggesting limited but functional sulfur-oxidizing activity in addition to HD. In contrast, Bin.371 lacked both denitrification and S⁰ oxidation pathways, relying solely on organic carbon via a fully expressed TCA cycle. The anoxic conditions in Stage I, however, limited Bin.371’s access to sufficient DO for organic carbon oxidation, impairing its energy metabolism and electron transfer capacity. This likely explains its low CcO gene expression despite a 4.7% expression level relative to its 0.4% abundance in the community. These findings further confirm that Stage I was characterized by a SAD-dominated system, with HD via PCL serving as a complementary pathway under low-DO conditions.

The increase in DO concentration during Stage II significantly altered the metabolic capabilities of the major MAGs. Bin.303, which relied exclusively on SAD, experienced near-complete suppression of its metabolic activity due to the elevated DO levels. This loss of functionality ultimately led to its decline in competitive dominance within the microbial community. In contrast, Bin.292 demonstrated superior resistance to DO stress, with high expression levels of denitrification genes, S⁰-oxidation genes, CBB cycle genes, TCA cycle genes, and CcO-encoding genes. These integrated metabolic pathways established Bin.292 as the dominant species in Stage II, capable of both sulfur-autotrophic and heterotrophic denitrification. This dual metabolic flexibility enabled it to mitigate the negative impacts of high DO through complementary electron transfer strategies. The second most abundant species in Stage II was Bin.301, which exhibited an elevated TCA cycle and denitrification gene expression. The high activity of its CcO genes further corroborated its role in HD via PCL degradation. The ability of Bin.301 to directly utilize PCL as an electron donor for denitrification, supported by its gene expression profile, highlights the effectiveness of PCL as a carbon source for microbial denitrification under high-DO conditions. Bin.371, a strictly heterotrophic organism, benefited from the elevated DO levels, which provided sufficient electron acceptors for organic carbon oxidation. This resulted in an increased TCA cycle and CcO gene expression, and to achieve a concomitant increase in community abundance (3.7%) and expression (4.2%) under the elevated DO conditions. Conversely, other heterotrophic denitrifiers observed in Stage I (Bin.203, Bin.463, and Bin.343) lost their ecological relevance in Stage II. The likely reason for their decline is the absence of mixotrophic metabolic capability, combined with an insufficient competitive capacity for PCL and its hydrolyzed carbon sources, which ultimately prevented them from sustaining the required denitrification metabolism. These findings underscore that elevated DO concentrations not only suppress SAD but also inhibit heterotrophic denitrifiers unable to couple PCL degradation with efficient electron transfer pathways. The differential responses of MAGs to DO stress highlight the critical role of metabolic versatility and substrate accessibility in sustaining denitrification performance under fluctuating oxygen conditions.

The gene expression patterns of MAGs in Stage III more clearly reflect functional adaptability to the elevated DO environment (5.5–6.5 mg/L). Within the microbial community, Bin.292 (a mixotrophic denitrifier), Bin.371 (a strong organic degrader), and Bin.301 (a heterotrophic denitrifier directly degrading PCL) collectively accounted for 95.6% abundance and 97.0% expression levels, establishing them as the core functional units of the system. Bin.292 sustained denitrification activity through dynamic switching between mixotrophic metabolic pathways, coupling sulfur-autotrophic and heterotrophic electron transfer strategies to adapt to fluctuating DO levels. Bin.301 contributed to the system stability by efficiently degrading PCL for denitrification while simultaneously providing a sustained carbon source to support microbial metabolism. Under high-DO conditions, Bin.371 preferentially utilizes organic matter as an electron donor, with a specialized focus on oxidative removal of organic substrates. In contrast, the other heterotrophic denitrifiers were gradually outcompeted due to DO-induced denitrification inhibition and limited ecological versatility. Their restricted nutritional requirements (e.g., dependence on specific carbon sources) and weaker organic substrate utilization capacity rendered them unable to compete effectively for resources under high-DO stress. Consequently, only the aforementioned three MAGs remained dominant, shaping the system’s functional resilience in Stage III.

A comprehensive analysis of MAG gene expression revealed that under high-DO conditions, Bin.301 degraded PCL to release soluble organic carbon, serving as both an energy source for its own metabolism and a carbon supply for Bin.292 to support its mixotrophic denitrification. This synergistic mechanism enabled Bin.292 to maintain nitrate reduction activity via sulfur-autotrophic–heterotrophic coupling, even under DO-inhibitory stress. Simultaneously, Bin.371, a strong organic matter degrader, prioritized the utilization of residual organic substrates as electron donors under high DO. This not only effectively reduced effluent COD but also partially consumed DO through oxidative metabolism, indirectly alleviating oxygen-induced inhibition on denitrifiers. The cooperative roles of these three microbial groups (PCL degradation, carbon source provisioning, denitrification support, and organic matter removal) established a carbon source–electron transfer–DO regulation functional feedback loop. This integrative metabolic network enabled the system to sustain denitrification performance under high-DO stress, demonstrating the critical role of microbial functional complementarity in maintaining nitrogen removal efficiency in fluctuating oxygen environments.

4. Explanation, Comparison, and Expansion

4.1. Long-Term Stability, Regeneration, and Engineering Considerations of the Carrier

Discussion on physical and chemical stability: Existing studies indicate that high-performance polyurethane sponges exhibit excellent hydrolysis resistance and mechanical resilience in aerobic/anoxic aquatic environments, with a potential service life of several years [16,50]. S⁰ is chemically stable, and the enzymatic hydrolysis of PCL is slow and controllable. No structural collapse or significant physical deterioration of the carriers was observed during the 92-day continuous operation of this study, indicating good short-term stability.

Functional longevity and failure criteria: The “failure” of the carrier refers not to physical disintegration but to the depletion of its electron-donating capacity. Its service life is determined by the initial loading of S⁰ and PCL and their consumption rates under target operating conditions. In this study, the S⁰ consumption rate can be back-calculated based on SO₄²⁻ production; however, the consumption rate of polymeric PCL cannot be reliably inferred from COD release dynamics due to its distinct degradation mechanism. The actual “replacement frequency” must be established through long-term pilot-scale studies, developing practical criteria based on either performance decline (e.g., sustained denitrification efficiency below a set threshold) or monitoring of key components (e.g., carrier weight loss to a certain level).

Online replacement strategy: The continuous-flow regime and free-moving nature of the carriers in a Moving Bed Biofilm Reactor (MBBR) offer a inherent advantage for online, non-stop replacement. During operation, pre-prepared fresh carriers can be periodically added to the system, while depleted carriers with low activity and exhausted electron donors can be simultaneously removed and collected via screened outlets. This strategy sustains long-term stability in reactor biomass and treatment performance without requiring shutdowns, making it particularly suitable for wastewater treatment scenarios demanding ultra-high operational continuity.

Offline regeneration and recycling strategy: Retrieved spent carriers constitute not solid waste but a renewable resource. We propose a circular technical pathway based on ultrasonic cleaning–drying–remanufacturing: Firstly, mild ultrasonic treatment is applied to effectively dislodge aged biofilm and residual impurities from the carrier surface, restoring its porous structure. Subsequently, after low-temperature drying, the carriers retain their mechanical integrity and structural completeness. Finally, these cleaned carriers can be reintroduced into the preparation process. Through the low-temperature melting and embedding of S⁰ and PCL, they are reformed into novel composite carriers with high denitrification capacity. This strategy aims to maximize the material’s lifecycle, reduce solid waste generation, lower operational costs, and significantly enhance the environmental and economic sustainability of the entire technology.

4.2. Potential for Integrated Nitrogen Removal Processes

While this study focused on overcoming the bottleneck of denitrification under high-DO conditions, a comprehensive nitrogen removal process typically requires the integration of nitrification with denitrification or the anaerobic ammonium oxidation (Anammox) process. Although our experimental design did not directly investigate nitrification, the intrinsic properties of our composite carrier system present intriguing possibilities for future development towards single-stage integrated nitrogen removal.

The millimeter-scale pore structure of the polyurethane sponge carrier, which creates anoxic microzones for denitrification, could potentially facilitate the co-existence of diverse functional microbes at different spatial scales within the same carrier particle. Theoretically, ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) could thrive in the aerobic outer shell of the carrier for nitrification, while the anoxic core could provide a habitat for denitrifiers (e.g., Paracoccus in this study) or Anammox bacteria. This architecture provides the necessary physicochemical gradients for achieving simultaneous nitrification–denitrification (SND) or even simultaneous partial nitrification–Anammox (PN/A).

The potential coupling with the Anammox process is particularly noteworthy. Although Anammox does not require organic carbon, it is highly sensitive to DO and requires a stable NO₂⁻ supply. The oxygen isolation capability of our carrier is paramount for protecting Anammox bacteria from DO inhibition. Furthermore, the heterotrophic denitrifiers present (e.g., Alicycliphilus), while consuming hydrolyzed PCL products, could also effectively scavenge penetrating oxygen, further reinforcing the internal anoxic environment. In the future, by introducing ammonium into the influent and controlling DO levels to suppress NOB (achieving partial nitrification), it might be possible to establish a multifunctional microbial consortium comprising AOB, Anammox bacteria, and denitrifiers on this carrier platform for synergistic carbon, nitrogen, and sulfur removal.

Certainly, realizing this vision faces challenges, primarily concerning the precise control of DO gradients, managing the competitive and collaborative spatial relationships between functional microbes (nitrifiers, denitrifiers, and Anammox bacteria), and understanding the potential influence of the electron donors (S⁰, PCL) on the Anammox process. This points the way for future research to explore this composite carrier as a platform for synergistically enriching multiple nitrogen-cycle functional microbes in more complex wastewater matrices, paving the way for developing efficient, compact novel single-reactor nitrogen removal processes.

4.3. Comparative Analysis with Other Denitrification Strategies

Zero-Valent Iron (ZVI) denitrification systems: As noted, the performance of ZVI systems is highly dependent on the DO concentration. DO significantly promotes the formation of an oxidative passivation layer on the ZVI surface [51] and triggers direct competition for electrons between nitrate ions and DO [52]. Consequently, numerous studies have indicated that nitrate reduction by ZVI is inefficient and unstable under high-DO conditions, with some even recommending its application strictly in anoxic media [53]. This stands in sharp contrast to the S⁰-PCL–sponge carrier system developed in our study. Our system does not require the avoidance of DO; instead, it leverages its physical structure (creating anoxic microzones) and chemical composition (dual–electron donors) to actively adapt to and maintain stable denitrification in high-DO environments. This inherent adaptability to high DO represents a key potential advantage of our system over ZVI technology.

Solid-phase denitrification systems using other biodegradable polymers: Regarding the comparison with other biodegradable polymers (e.g., Polyhydroxybutyrate (PHB), Polylactide (PLA)), we note that currently there are no reported studies in the open literature investigating denitrification performance specifically under high-DO conditions within a mixotrophic system constructed from S⁰ and a biodegradable polymer. Therefore, although the application of materials like PCL, PHB, and PLA in traditional solid-phase denitrification has been explored, a direct, one-to-one performance comparison is currently challenging due to the lack of experimental data under identical “high DO + mixotrophic” conditions. This also underscores the uniqueness and innovativeness of the “S⁰-PCL-sponge” system investigated in our study for treating high-DO wastewater. In addition, having a lower glass transition temperature and melting point (~60 °C) allows PCL to be co-immobilized with S⁰ onto the sponge carrier using our low-temperature melting–cooling process, acting as an excellent binder. In contrast, PLA has a higher melting point (~175 °C), and PHB has poorer thermal stability, degrading near its melting point (~170–180 °C), making them unsuitable for this simple, mild carrier fabrication strategy.

In summary, the selection of PCL for this study was deliberate, grounded in its unique physicochemical properties—which are ideally suited to the employed carrier construction method—and its superior biodegradation performance, ensuring the provision of a stable carbon source. The innovation of this work therefore resides not in the inaugural use of PCL, but in its first co-immobilization with S⁰ within a macroporous sponge carrier, coupled with the systematic demonstration of this composite material’s exceptional performance and associated microbial mechanisms in driving mixotrophic denitrification under high dissolved-oxygen stress.

4.4. Biodegradability of PCL: Validation Under Standardized Frameworks

The effectiveness of PCL as a slow-release carbon source and electron donor in this study is fundamentally predicated on its environmental biodegradability. This property has been extensively studied and validated within internationally recognized standardized testing frameworks. As reviewed by Strotmann et al. [54], the Organisation for Economic Co-operation and Development (OECD) and the International Organization for Standardization (ISO) have established a complex and rigorous tiered testing hierarchy (e.g., Ready Biodegradability Tests) for assessing the biodegradability of chemical substances. These methods are fully integrated into regulations such as the EU REACH and are considered the gold standard globally for evaluating environmental persistence.

Specifically, the enzymatically controlled hydrolysis and subsequent mineralization of PCL observed in our study align strongly with empirical findings obtained using methods resembling these OECD/ISO standards. The work by César et al. [55] demonstrated that a PCL and starch blend (PCL/S) exhibited significant biodegradation in soil, with carbon mineralization reaching up to 72.5% and 60.5% in clayey and sandy soil, respectively, after a 120-day incubation period. This was in stark contrast to polyethylene, which was nearly non-biodegradable (<5% mineralization). Their study also confirmed that the degradation of PCL/S did not elicit ecotoxicological effects on soil microbial biomass-C/N nor on the emergence and development of rice (Oryza sativa L.) seedlings. This research provides strong corroborative evidence for PCL as an environmentally benign carbon source material.

Therefore, the selection of PCL for this study was based not only on its physicochemical properties (e.g., low melting point and function as a binder) but also on its well-established biodegradability. Our results—including the consistently low effluent COD concentrations, the upregulation of TCA cycle gene expression, and the dominance of hydrolyzing bacteria like Alicycliphilus—provide microbial and molecular-level confirmation that synergistically reinforces the macro-scale conclusions drawn from standardized methods. This collective evidence firmly establishes the reliability and environmental safety of PCL for constructing sustainable wastewater treatment technologies.

4.5. Refinements in Future Experimental Design

Sulfate accumulation risks and mitigation strategies: Although sulfate itself exhibits low toxicity, its excessive accumulation in receiving water bodies may lead to negative ecological impacts. Under anaerobic conditions (e.g., in sediments or sewer pipelines), sulfate can be reduced by sulfate-reducing bacteria (SRB) to hydrogen sulfide (H₂S), a compound characterized by foul odor and high toxicity, posing threats to aquatic ecosystems and pipeline infrastructure. To address sulfate-related risks, future engineering strategies will focus on control at both source and end-of-pipe levels: (a) optimizing the dosing ratio of S⁰ to PCL to reduce the absolute yield of sulfate at the source while maintaining denitrification efficiency, by adjusting the proportion of mixotrophic metabolism; (b) coupling this system with a subsequent anaerobic process unit, where the produced sulfate can serve as an electron acceptor for SRB to reduce it to sulfide. This can be followed by a microaerobic process to recover elemental S⁰, achieving a closed-loop cycle and recovery of sulfur, thereby fundamentally resolving the issue of sulfate accumulation [18].

Monitoring of nitrous oxide (N₂O) during denitrification. As a potent greenhouse gas, the emission potential of N₂O represents a core indicator for assessing the environmental sustainability of any biological denitrification process. In subsequent pilot- and large-scale studies, direct measurement of N₂O emissions will constitute an essential research objective. Offline gas chromatography (GC) or online laser gas analyzers will be employed to quantitatively monitor N₂O production rates under different operational conditions—particularly during fluctuations in dissolved oxygen and nitrate loading—and to correlate these emissions with the expression dynamics of functional genes, thereby enabling a comprehensive evaluation of the greenhouse gas footprint of this technology.

The inclusion of biological replicates in biological treatment systems is critical for evaluating the reliability and reproducibility of experimental results. A single-reactor design was adopted for this study due to the following considerations: (a) As a core mechanistic investigation, the primary focus was on deeply resolving the dynamic response of the microbial community and functional genes to conditions using multi-omics technologies, where internal comparisons (across different stages) were prioritized over external replication; (b) To ensure extremely high control and consistency of all conditions (e.g., temperature, pH, DO, and carrier quantity) during the 92-day operation, avoiding potential subtle environmental variations between multiple reactors that could interfere with the sensitive microbial community and gene expression analysis. However, we frankly acknowledge that this precludes statistical quantification of reactor-level variability.

Multiple validations and robustness of the current design: (a) Time-series replicate sampling: Independent biofilm samples were collected at three time points during the stable period of each stage (days 30, 60, and 90) for metagenomic/metatranscriptomic analysis, providing a degree of replicability validation along the temporal dimension; (b) Rigorous technical replicates for chemical analysis: All water quality parameters (NO₃⁻-N, SO₄²⁻, COD) were measured in triplicate, ensuring the precision of the analytical data; (c) Highly consistent performance data: As shown in Figure 1, the system reached a stable performance plateau in each stage with minimal data fluctuation, indicating stable operation and reproducible results. Subsequent pilot-scale studies and optimization experiments targeting specific parameters will employ an experimental design incorporating a minimum of triplicate biological replicates. In particular, when evaluating the optimization of different operational parameters (such as loading and DO), parallel reactors will be established to ensure the statistical significance of the research conclusions and to provide more reliable data support for future technology scaling.

The direct measurement of DO concentration gradients via microsensors or imaging techniques would provide the most intuitive and compelling evidence for the existence of “anoxic microzones.” In the current study, the inference that anoxic microenvironments exist within the sponge carrier relies primarily on indirect evidence from metatranscriptomic data—specifically, the maintained expression of key denitrification genes under high-DO conditions (up to 6.5 mg/L). This indeed represents a limitation of the present work. In subsequent studies, microelectrode DO sensing systems or planar optodes will be employed to accurately map the spatiotemporal distribution of DO within the carrier, thereby providing unambiguous experimental evidence regarding the formation and extent of these anoxic microzones.

In this study, the assessment of metabolic pathway contributions primarily relied on metatranscriptomic data (abundance of functional gene transcripts) and metabolic potential analysis derived from MAGs, combined with indirect inferences based on system performance parameters—such as the stoichiometric relationship between SO₄²⁻ production and COD consumption. Although these multi-layered datasets exhibit strong consistency (e.g., concurrent upregulation of genes related to organic carbon metabolism and sulfur oxidation under high-DO conditions), the evidence remains correlative rather than causative. Therefore, building on the microbial community structure and functional gene expression profiles elucidated in this study, subsequent work will employ direct quantitative techniques such as stable isotope probing (SIP). Using dual labeling with ¹³C and ³⁴S under key conditions like high DO, we aim to precisely quantify the simultaneous carbon and sulfur utilization strategies of mixotrophic microorganisms, thereby providing definitive validation for the conclusions drawn in this work.

5. Conclusions and Outlook

The application of the S⁰-PCL–polyurethane sponge composite carrier in high-oxygen wastewater treatment systems effectively maintained denitrification efficiency under DO stress. The millimeter-scale pores of the carrier created potential anoxic microzones, which ensured sustained high expression of denitrification genes. S⁰ and the carbon released from PCL provide complementary electron fluxes and a mixotrophic metabolic mode, thereby facilitating the denitrification process. The functional microbial community underwent reconfiguration, with sulfur-autotrophic denitrifiers dominating under anoxic conditions. Under high DO, Alicycliphilus (heterotrophic denitrification via PCL hydrolysis), Paracoccus (mixotrophic denitrification coupling sulfur and carbon), and Hyphomonas (aerobic organic matter degradation and DO consumption) formed a resilient consortium to mitigate oxygen inhibition. The novel carrier developed in this study provides a theoretical basis and technical strategies for efficient nitrogen removal in high-DO environments.

The composite carrier technology developed in this research demonstrates clear potential for engineering application. Its most immediate application is as enhanced media in MBBR for treating secondary effluent with high DO, enabling stable advanced nitrogen removal without the need for additional deaeration tanks while effectively controlling effluent COD. This is of great significance for high-standard discharge or water reclamation projects. Furthermore, the carrier can be engineered into modular biofilm units for direct inoculation into nitrate-polluted, oxygen-rich water bodies like rivers and landscape lakes for in situ remediation. This “drop-in” solution avoids the complexity and ecological risks associated with liquid carbon dosing systems, making it particularly suitable for decentralized, non-powered, or operationally constrained environments. To facilitate the translation of this technology from the laboratory to practical application, future work will focus on: (1) Pilot-scale validation under real-world water quality fluctuations to assess long-term carrier durability, biofilm succession, and stability of nitrogen removal performance; (2) A precise evaluation of operational costs and economic benefits, conducting a life-cycle cost comparison with traditional deaeration–heterotrophic denitrification processes; and (3) Optimizing the PCL–sulfur composite ratio and carrier fabrication process to achieve the best economics without compromising performance, laying the groundwork for final commercial scale-up.