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Article

Comparison of Denitrification Performance and Regulation Strategies of Corncob/PHBV and Sulfur in Circulation Packed-Bed Reactor

by
Yumeng Gao
1,2,
Yongyou Hu
1,2,*,
Donghui Liang
1,3,*,
Jianhua Cheng
1,2,
Xiaoqiang Zhu
4,
Guobin Wang
4 and
Jieyun Xie
4
1
School of Environment and Energy, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China
2
The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China
3
College of Urban and Rural Construction, Zhongkai University of Agriculture and Engineering, Zhongkai Road, Haizhu District, Guangzhou 510225, China
4
Guangzhou Pengkai Environment Technology Co., Ltd., Guangzhou 511493, China
*
Authors to whom correspondence should be addressed.
Sustainability 2026, 18(10), 4836; https://doi.org/10.3390/su18104836
Submission received: 24 March 2026 / Revised: 5 May 2026 / Accepted: 11 May 2026 / Published: 12 May 2026
(This article belongs to the Special Issue Advanced Technologies and Innovative Process Control Strategies Used for Sustainable Wastewater Treatment and Reuse)
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Abstract

To enhance nitrogen removal in low-carbon sewage through heterotrophic and sulfur autotrophic denitrification, a corncob-sulfur circulating packed-bed reactor (CS-CPBR) and a PHBV-sulfur circulating packed-bed reactor (PS-CPBR) were constructed. The results showed that the total nitrogen (TN) and NO3−-N removal efficiencies of both reactors improved with increasing hydraulic retention time(HRT) and NaHCO3 dosage. With a HRT of 2 h, a NaHCO3 dosage of 0.4 g/L, and an influent NO3−-N concentration of 30 mg/L, the TN removal efficiencies were 87.9% and 94.0%, respectively. The PS-CPBR exhibited a higher nitrogen removal rate and efficiency. Mechanistic studies indicated that the lower bioavailability of corncob restricted microbial activity in the CS-CPBR, leading to the difference in denitrification efficiency between the two reactors. Metagenomic analysis revealed that, relative to the CS-CPBR, the PS-CPBR significantly enriched denitrifying bacterial genera such as Sinirhodobacter, Rhodobacter, and Brachymonas. The PS-CPBR also harbored higher abundances of denitrification genes (nirS, norC, nosZ), thereby strengthening the complete denitrification pathway. This study provides experimental data to guide carbon source selection and process control for solid carbon-sulfur denitrification in advanced nitrogen removal.

1. Introduction

In China, the discharge standard for municipal wastewater treatment plants sets a TN limit of 15 mg/L for secondary treatment effluent. However, more stringent TN discharge limits have been established in coastal areas and ecologically sensitive regions [1]. Since the bioavailable chemical oxygen demand (COD) in secondary treatment effluent is largely depleted [2], heterotrophic denitrification (HD) is insufficient to achieve efficient advanced nitrogen removal. In this context, autotrophic denitrification has emerged as a promising alternative [3]. Among these methods, sulfur-based autotrophic denitrification (SAD) is the most representative technology. It has been reported that sulfur-packed bed reactors can achieve TN removal efficiencies of 95–100%, with volumetric nitrogen loading rates ranging from 0.5 to 2.5 kg NO3−-N/(m3·d) [4]. Nevertheless, SAD is inherently constrained by slow reaction kinetics and high alkalinity consumption, with approximately 4.57 g of alkalinity (as CaCO3) being consumed per gram of NO3−-N reduced [5]. Therefore, exogenous alkalinity supplementation is required to maintain the stable operation of the system.
Recent studies have demonstrated that coupling HD with SAD can effectively overcome these limitations. Lee et al. [6] employed an upflow sulfur-packed filter bed, supplemented with methanol or sodium acetate as additional carbon sources to treat nitrate-laden leachate with an influent NO3−-N concentration of 700–900 mg/L. The results showed that the contribution of HD to nitrogen removal reached 60% and 44%, respectively. Complete denitrification was achieved without external alkalinity supplementation, while the system attained a peak volumetric nitrate-nitrogen loading rate of 5.05 kg NO3−-N/(m3·d) [6]. Despite these advantages, the dosage of liquid carbon sources is difficult to control precisely in practice [7], often leading to overdosing and suppressed SAD metabolism [8]. In contrast, solid carbon sources, characterized by slow carbon release and serving as biofilm carriers, offer a viable alternative for the stable and controllable operation of a coupled HD-SAD system.
Although the nitrogen removal performance of the solid carbon source-sulfur coupled denitrification system exceeds that of the SAD system [9], the co-existing heterotrophic and autotrophic denitrifying bacteria compete for the electron acceptor and ecological niches. Under carbon-abundant conditions, heterotrophic bacteria establish competitive dominance because of their faster growth rates, leading to the competitive displacement and metabolic inhibition of sulfur autotrophic bacteria [8]. Therefore, the selection and dosage of the solid carbon source are key factors in determining the microbial community structure and nitrogen removal efficiency in coupled sulfur-based denitrification systems.
Solid carbon sources are broadly classified into natural and synthetic types, which differ in the released carbon compounds, electron donation efficiency, and engineering cost. Natural carbon sources release macromolecular organic matter composed primarily of cellulose, hemicellulose, lignin, proteins, and phospholipids [10]. Their release period is relatively short, making it difficult to sustain long-term system operation [11]. Among natural carbon sources, corncob is regarded as a particularly favorable material due to its high cellulose content, wide availability, and low cost, with carbon release rates ranging from 29.06 to 794.6 mg/g [12]. Synthetic carbon sources, such as polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate-co-valerate (PHBV), and polybutylene succinate (PBS), hydrolyze to release low-molecular-weight organic compounds, primarily acetic and butyric acid [13]. These components are readily utilized by denitrifiers, and synthetic carbon sources offer a long-lasting carbon release cycle. In particular, the abundance of hydrophilic groups in PHBV enhances its biodegradability and microbial utilization, yielding a nitrogen removal rate of 32.01 mg N/(L·h) [14]. In the context of advanced nitrogen removal from municipal secondary treatment effluent, an alkali-modified corncob-sulfur coupled system achieved a NO3−-N removal efficiency of 98.62% and a TN removal loading rate of 13.61 mg TN/(L·h) [15], whereas a PHBV-sulfur coupled system attained a TN removal loading of 14.86 mg TN/(L·h) [16]. Although both corncob-sulfur and PHBV-sulfur systems exhibit promising nitrogen removal performance, direct comparisons of their long-term operational stability are still needed. The carbon release characteristics of each solid carbon source type directly influence the community structure and the symbiotic interactions between heterotrophic and autotrophic bacteria, which in turn govern the nitrogen removal mechanism and overall system performance. Currently, most studies on corncob-sulfur and PHBV-sulfur denitrification systems have been conducted under single operating conditions. Consequently, the long-term behavior under gradients of key operating parameters and the biological mechanisms underlying the performance differences between the two systems have yet to be fully elucidated.
To this end, a corncob-sulfur external circulating packed bed reactor (CS-CPBR) and a PHBV-sulfur external circulating packed bed reactor (PS-CPBR) were constructed. This study aimed to (1) compare their nitrogen removal performance under varying operating conditions and identify the optimal parameters; (2) elucidate the intrinsic mechanisms underlying the performance differences, with an emphasis on carbon release characteristics, microbial community assembly, and functional gene expression; and (3) explore the regulatory role of NaHCO3 beyond its function in alkalinity supplementation. The results are expected to provide theoretical support for advanced nitrogen removal from municipal secondary treatment effluent using solid carbon-sulfur denitrification.

2. Materials and Methods

2.1. Materials

All reagents are listed in Text S1 of Supplementary Material. Corncob, elemental sulfur, and PHBV were sieved to 3–5 mm particles, rinsed with ultrapure water to remove impurities, and vacuum-dried before use.
The activated sludge used as inoculum was collected from the anaerobic tank of a municipal wastewater treatment plant in Guangzhou, China. The sludge was separately acclimated in a heterotrophic denitrification medium and a sulfur autotrophic denitrification medium [17] (detailed in Table S1). The two acclimated sludges were then mixed at a volume ratio of 1:1, with a mixed liquor suspended solids (MLSS) concentration of 5.98 g/L and a mixed liquor volatile suspended solids (MLVSS) concentration of 3.50 g/L. The mixed sludge was washed three times with sterile phosphate-buffered saline (PBS) before inoculation.

2.2. Reactors Construction and Startup

The sequencing batch external circulating packed-bed reactors were made of organic glass, with a diameter of 70 mm and a reactor height of 600 mm. The reactors adopted a double-layer packing structure. The lower layer was packed with 600 g of elemental sulfur, and the upper layer was packed with 300 g of the solid carbon source. A carbon-to-sulfur mass ratio of 0.5 was selected based on preliminary optimization experiments, which indicated that this ratio enabled complete NO3−-N removal without inhibiting SAD. The detailed optimization procedure and corresponding data are provided in the Supplementary Materials (Figure S1).
Two reactors, designated as CS-CPBR and PS-CPBR, were operated in parallel, with corncob and PHBV serving as the respective solid carbon sources. The volume ratio, and the packing height ratio of carbon source to sulfur, were 0.64 in the CS-CPBR and 1.28 in the PS-CPBR. At setup, each reactor was inoculated with 0.6 L of activated sludge and 0.5 L of simulated wastewater, while the external water tank was filled with 3 L of the same synthetic wastewater (composition details in Table S2). Wastewater was pumped from the external tank into the bottom of the reactors and returned from the top effluent to establish an external circulation loop, with a constant influent flow rate of 160 mL/min selected to ensure stable operation. The 3 L of simulated wastewater in the external water tank was replaced daily. Reactor startup was considered successful when NO3−-N removal efficiencies stabilized at approximately 90% and biofilm formation was observed on the packing surface (Figure S2). Following startup, the suspended activated sludge was drained from the reactors, and both biofilm reactors were operated in sequencing batch mode, treating 3 L of wastewater per cycle.

2.3. Reacters Operation

To investigate the effects of the HRT, NaHCO3 dosage, and influent NO3−-N concentration on pollutant removal performance, nine experimental periods were conducted under the conditions listed in Table 1. The reactors were operated in a batch recirculation mode. 3 L of synthetic wastewater was filled in the external tank, recirculated for the designated reaction time, and drained before the next batch. All synthetic wastewater was freshly prepared before use. Nitrogen gas was employed to strip dissolved oxygen. At the end of each period, backwashing was performed at a flow rate of 240 mL/min for 10 min to remove excess biomass and prevent packing clogging. Water samples were collected from the external tank for the determination of TN, NO3−-N, NO2−-N,NH4+-N, SO42−, COD, pH and alkalinity. Only data obtained from the steady state were used for analysis.

2.4. Kinetic Experiments

During the stable operation of the reactors in periods 4–9, kinetic batch tests were performed to evaluate the nitrogen removal kinetics of the CS-CPBR and the PS-CPBR. Water samples were collected from the external tank at predetermined time intervals (0, 0.25, 0.5, 1, 1.5, and 2 h), and the TN concentrations were measured. The TN removal profiles were subsequently fitted to a kinetic model.

2.5. Analysis of Functional Genes and Microbial Communities

To evaluate the effects of the NaHCO3 dosage on the microbial community and nitrogen removal genes in the biofilm, and to assess the differences between the CS-CPBR and the PS-CPBR, biofilm samples were collected from both reactors during stable operation (periods 4–6) and sent to LC-Bio Technology Co., Ltd. (Hangzhou, China) for metagenomic analysis. The samples were designated CS-0.05, CS-0.2, CS-0.4, PS-0.05, PS-0.2, and PS-0.4, respectively. Metagenome libraries were sequenced on an Illumina NovaSeq 6000 platform using the PE150 strategy. Raw reads were quality-filtered using the Fastp software (v0.23.4) to remove adapter contamination, low-quality bases, and undetermined bases. The clean reads were subjected to de novo assembly for each sample using MEGAHIT (v1.2.9) and used for functional and taxonomic annotation. Differentially abundant taxa were identified based on predefined statistical criteria.

2.6. Analytical Testing Methods

Conventional water quality parameters and alkalinity were analyzed according to the methods described in [18]. The surface morphologies of the corncob, PHBV, and elemental sulfur samples collected before and after reactor startup were characterized using scanning electron microscopy (SEM; ZEISS Sigma 300, Oberkochen, Germany). To track changes in dissolved organic matter (DOM), water samples were collected from the reactors during periods 4–6 and were analyzed using three-dimensional excitation-emission matrix (3D-EEM; Hitachi F-7000, Tokyo, Japan).

2.7. Methods of Statistical Analysis of Data

Data were processed and plotted using Excel and Origin 2021. The 3D-EEM fluorescence spectral data were analyzed with MATLAB R2022b. The nitrogen removal process was fitted with a quasiprimary kinetic model [19].

3. Results and Discussion

3.1. Nitrogen Removal Performance of CS-CPBR and PS-CPBR

3.1.1. Effects of HRT

During periods 1–3, the TN and NO3−-N removal efficiencies of both the CS-CPBR and the PS-CPBR declined with decreasing HRT (Figure 1a,b). At an HRT of 4 h, both reactors achieved TN and NO3−-N removal efficiencies exceeding 95% (Figure 1a,b), while effluent NO3−-N and NO2−-N concentrations remained below 0.93 mg/L (Figure 1b,c). When the HRT was shortened to 1–2 h during periods 2 and 3, the nitrogen removal performance of both reactors deteriorated, though the extent of the decline differed between the two systems. In the CS-CPBR, TN and NO3−-N removal efficiencies decreased by 25.4–47.2% and 14.5–39.7%, respectively, which were higher than the decreases observed in the PS-CPBR. These results indicate that sufficient reaction time is a critical factor for efficient biological nitrogen removal [20].
The effluent alkalinity in both reactors ranged from 161 to 182 mg/L as CaCO3, exceeding the influent alkalinity by 36–57 mg/L (Figure 1d). The pH remained between 6.95 and 7.49 (Figure 1d), which falls within the optimal range for denitrifying bacteria [21]. During the initial phase of reactor operation, the rapid leaching of soluble small molecules led to markedly higher effluent COD concentrations in the CS-CPBR than in the PS-CPBR (Figure 1c). Subsequently, effluent COD gradually stabilized in the CS-CPBR as hemicellulose was hydrolyzed and refractory cellulose was degraded by microorganisms [2]. In the PS-CPBR, the controlled slow hydrolysis of PHBV maintained consistently low effluent COD levels. The low-molecule-weight organic acids released from PHBV were readily utilized by heterotrophic denitrifiers, thereby sustaining efficient nitrogen removal even at a limited reaction time. In contrast, the macromolecular organics derived from corncob exhibited poor bioavailability, which weakened denitrification in the CS-CPBR. When the HRT was shortened to 1 h, the TN removal efficiency of CS-CPBR dropped sharply. In the effluent, NO3−-N accounted for 77.4% of the TN, and NO2−-N accounted for 19.4%. Compared with the HRT of 2 h, the residual NO3−-N concentration increased by 7.5 mg/L, whereas effluent pH and COD showed no significant changes. Correspondingly, the contribution of HD decreased by 7.8% (Figure 2a). This deterioration in TN removal at a reduced HRT in the CS-CPBR was primarily attributed to the diminished removal of NO3−-N through the HD pathway. As the HRT decreased from 4 h to 1 h, the contribution of SAD in the PS-CPBR declined from 60.7% to 48.1% (Figure 2b). Since SAD proceeds at a slower reaction rate than HD, denitrifiers prefer to utilize the PHBV hydrolysis products when the reaction time is limited. This explains why the SAD contribution in the PS-CPBR was more sensitive to HRT variation.

3.1.2. Effects of NaHCO3 Dosage

As the influent NaHCO3 dosage increased from 0.05 to 0.4 g/L, the influent pH rose from 7.3 to 7.8, and the influent alkalinity increased from 48 to 308 mg/L as CaCO3. The TN and NO3−-N removal efficiencies of the CS-CPBR and the PS-CPBR increased from 45% to 88% and from 56% to 94%, respectively. At a NaHCO3 dosage of 0.4 g/L (period 6), NO2−-N was nearly undetectable in both reactors (Figure 1c), indicating that NaHCO3 supplementation enhanced microbial nitrite reduction. The PS-CPBR consistently outperformed the CS-CPBR in nitrogen removal across all tested NaHCO3 dosages, which is primarily attributable to the dominance of HD in the system (Figure 2b). When the NaHCO3 dosage was in the range of 0.2–0.4 g/L, the two reactors exhibited different effluent characteristics. From period 5 to period 6, the effluent alkalinity of the PS-CPBR increased to 300 and 430 mg/L as CaCO3 (Figure 1d), while the contribution of SAD declined to 34.9% and 34.2% (Figure 2b). In contrast, this trend was absent in the CS-CPBR. These differences likely stem from the distinct carbon release kinetics and organic carbon bioavailability between PHBV and corncob. The highly bioavailable carbon released from PHBV sustained HD as the dominant pathway and SAD as a subordinate process in the PS-CPBR, whereas HD and SAD remained balanced in the CS-CPBR.
Figure S3 illustrates the differences in effluent DOM between the two reactors under varying NaHCO3 dosages [22]. The fluorescence intensity in Region IV (microbial metabolites) was significantly higher for the CS-CPBR than for the PS-CPBR, and detectable signals were also observed in Regions I, II and III. This indicates that the DOM leached from the CS-CPBR triggered unsteady-state explosive microbial metabolism, leading to substantial carbon consumption by non-denitrifying bacteria. Increasing the NaHCO3 dosages enhanced microbial metabolic activity and promoted the secretion of extracellular polymeric substances (EPS) in the reactors (Figure S4), which supported the long-term stable operation of the systems. High electronic conductivity is fundamental for efficient electron transfer [23], and EPS are known to facilitate electron transfer between microorganisms and substrates. Under elevated NaHCO3 conditions, the PS-CPBR exhibited a grater increase in EPS secretion (134.3% vs. 82.1%) and a higher protein-to-polysaccharide (PN/PS) ratio (0.93 vs. 0.49) compared with the CS-CPBR, indicating superior colonization capacity and system stability.

3.1.3. Effects of Influent NO3−-N Concentration

With the HRT fixed at 2 h and the NaHCO3 dosage maintained at 0.4 g/L, the effect of the influent NO3−-N concentration on the denitrification performance of the CS-CPBR and the PS-CPBR was evaluated in periods 7–9. As the influent NO3−-N concentration increased from 10 to 30 mg/L, both reactors showed a decline in nitrogen removal efficiency and an increase in effluent NO2−-N accumulation (Figure 1c). At 30 mg/L, the TN removal efficiency of the PS-CPBR decreased to 84%, while its NO3−-N removal efficiency remained at 90% (Figure 1a). Under the same conditions, the TN and NO3−-N removal efficiencies of the CS-CPBR dropped to 71% and 84%, respectively (Figure 1a,b). These results indicate that the PS-CPBR possessed greater tolerance to elevated nitrogen loading than the CS-CPBR. Concurrently, the contribution of SAD decreased by 4% in the PS-CPBR (Figure 2b) and by 21.6% in the CS-CPBR (Figure 2a). High nitrate loading intensifies electron competition between nitrate reductase and nitrite reductase, thereby inhibiting nitrite reduction [24,25]. Accumulated nitrite can suppress the activity of sulfur autotrophic denitrifiers, leading to the attenuation of SAD. Throughout the operational period, the maximum effluent sulfate concentrations of the CS-CPBR and the PS-CPBR were 108.1 mg/L and 120.6 mg/L, respectively. Values for both reactors were lower than the typical effluent sulfate concentration of 273.5 mg/L reported for sulfur-based autotrophic denitrification processes [26]. This demonstrates that the solid carbon source-sulfur denitrification process achieves superior control of sulfate by-product generation.

3.1.4. Kinetic Analysis of Nitrogen Removal

The kinetic fitting analysis was conducted based on the reaction processes in the CS-CPBR and the PS-CPBR. The results demonstrated that TN removal adhered to a pseudo-first-order kinetic model (Figure 3). The fitting parameters including k and R2 are presented in Table S3. As the NaHCO3 dosage increased from 0.05 to 0.4 g/L, the TN removal rate constant of the PS-CPBR rose from 0.1725 h−1 to 0.5258 h−1 (Figure 3b). In contrast, the rate constant of the CS-CPBR peaked at 0.3992 h−1 at 0.2 g/L NaHCO3 and then declined to 0.2830 h−1 at 0.4 g/L (Figure 3a). This is presumably ascribed to the inhibition of enzymatic activity caused by high osmotic pressure under excessive NaHCO3 [27]. When the influent NO3−-N concentration was raised from 10 to 30 mg/L, the TN removal rate constant decreased from 1.0395 h−1 to 0.1695 h−1 for the CS-CPBR (Figure 3c) and from 1.7577 h−1 to 0.1679 h−1 for the PS-CPBR (Figure 3d). These results demonstrate that the influent nitrate loading exerted a pronounced effect on TN removal kinetics, indicating slower overall nitrogen removal under high-nitrate conditions. In summary, the nitrogen removal rate in these systems can be sustained by appropriately increasing the NaHCO3 dosage.

3.2. Nitrogen Removal Mechanism of CS-CPBR and PS-CPBR

3.2.1. Nitrogen Balance and Alkalinity Balance

The nitrogen balance was calculated for the CS-CPBR and the PS-CPBR in period 6 (Figure 4). In the reactors, nitrogen loss accounted for 81.7% and 90.9% of the influent total nitrogen, while residual NO3−-N constituted 10.2% and 5.2%, respectively, indicating more complete denitrification in PS-CPBR. This superior denitrification performance can be partly attributed to the greater bioavailability of PHBV. By comparison, the lignocellulosic structure of corncob limited the rate and extent of organic carbon release in the CS-CPBR. Hydrothermal pretreatment to disrupt the lignocellulosic matrix could enhance the carbon release capacity of corncob [28] and thereby improve the overall denitrification efficiency. Based on the proportion of NH4+-N, the dissimilatory nitrate reduction to ammonium (DNRA) pathway contributed 0.78% and 1.16% to nitrate reduction in the CS-CPBR and the PS-CPBR, respectively [29], indicating that DNRA is a minor competing pathway in both reactors. Notably, the proportion of biomass nitrogen in the CS-CPBR (5.2%) was significantly higher than that in the PS-CPBR (1.4%), reflecting a greater contribution of nitrogen assimilation in the CS-CPBR.
Alkalinity is a critical factor for maintaining the stable operation of heterotrophic and sulfur-autotrophic denitrification systems. Theoretically, HD generates 3.57 g CaCO3 per gram of NO3−-N removed, whereas SAD consumes 4.57 g CaCO3 per gram of NO3−-N removed [30]. The measured alkalinity changes per unit of nitrate removed are presented in Table 2. The CS-CPBR showed smaller fluctuations in alkalinity, which can be attributed to the sustained alkalinity release ability of corncob [31]. During periods 1–9, the alkalinity changes per unit of nitrate removed in the PS-CPBR remained positive, confirming that alkalinity did not act as a limiting factor for SAD. Notably, in periods 5 and 6, the measured alkalinity changes in the PS-CPBR reached 3.8 and 4.1 g CaCO3/g NO3−-N, which exceeded the theoretical alkalinity of HD. The excess alkalinity is likely attributable to additional alkalinity-generating metabolic pathways such as incomplete sulfide oxidation [32] and heterotrophic ammonification [33].

3.2.2. Microbial Community Composition

Figure 5 illustrates the differences in the microbial community structure of the biofilms from the CS-CPBR and the PS-CPBR under varying influent NaHCO3 dosages. At the phylum level (Figure 5a), Pseudomonadota, Bacteroidota, Bacillota, and Campylobacterota were the dominant phyla in the CS-CPBR and the PS-CPBR, together representing approximately 85% of the total community. Pseudomonadota, a phylum that encompasses diverse anoxic denitrifying bacteria [34,35], showed absolute dominance in both reactors (66–77%). The majority of Pseudomonadota lack a complete canonical sulfur-oxidation pathway but are capable of reducing sulfur compounds [36]. Bacteroidota, Bacillota, and Campylobacterota are also anoxic denitrifiers [37,38]. Members of the Bacteroidota phylum harbor heterotrophic denitrifiers equipped with sulfur-oxidation capacity, which are relevant to the coupled denitrification system [39].
The microbial community composition at the genus level is shown in Figure 5b). Sinirhodobacter and Rhodobacter were the dominant genera in the CS-CPBR and the PS-CPBR, together accounting for 25–41%. Rhodobacter has been reported to function as a heterotrophic denitrifier [40], whereas Sinirhodobacter has been reported to be an autotrophic nitrate-reducing genus [41]. The combined relative abundance of these two genera was 3.5–13.2% higher in the PS-CPBR than in the CS-CPBR. In addition, the PS-CPBR was further enriched in the heterotrophic denitrifier Brachymonas (1–7.2%) [40]. The CS-CPBR exhibited a greater enrichment of Ottowia, a facultative anaerobic denitrifier capable of mixotrophic metabolism [42]. Overall, the PS-CPBR harbored a higher relative abundance of highly efficient denitrifying genera, which contributed to its superior nitrogen removal efficiency. When the NaHCO3 dosage was increased from 0.05 to 0.2 g/L, the relative abundance of the sulfur-reducing genus Desulfurella decreased in the PS-CPBR but increased in the CS-CPBR. Desulfurella can supply additional electron donors through sulfur metabolism. However, these excess electron donors may stimulate DNRA and divert electrons away from the denitrification pathway [29].
Cluster heatmap analysis (Figure 5c) revealed distinct enrichment patterns of functional genera between the CS-CPBR and the PS-CPBR. Sulfurimicrobium [43] and Thiomonas [44] were identified as the signature genera associated with sulfur-oxidation and nitrogen metabolism in the CS-CPBR, whereas Thioclava [45] dominated in the PS-CPBR. The functional genera involved in nitrogen and sulfur cycling in the two reactors responded divergently to an increasing NaHCO3 dosage. In the CS-CPBR, the sulfur-oxidizing Desulfurella, together with carbon-hydrolyzing Chryseobacterium [46] and Sphingopyxis [47], were enriched. In PS-CPBR, the carbon-hydrolyzing genera Cloacibacterium [48], Paludibacter [49], and Thauera [50] were enriched. NaHCO3 exerted a stronger regulatory effect on the carbon-hydrolyzing bacteria in the PS-CPBR, thereby supplying more available substrates for denitrifiers.

3.2.3. Denitrification Functional Genes

The expression levels of functional genes involved in the nitrogen cycle in the biofilms of the CS-CPBR and the PS-CPBR under different NaHCO3 dosages were normalized using the Transcripts Per Million method [51] (Figure 6). The genes narG, narH, narI, narJ, and narZ encode membrane-bound nitrate reductase; napA and napC encode periplasmic nitrate reductase; nirK and nirS encode nitrite reductase; norB and norC encode nitric oxide reductase; and nosZ encodes nitrous oxide reductase [52,53,54]. As the NaHCO3 dosage increaed from 0.05 to 0.4 g/L, the expression of asnB was upregulated in both reactors, indicating that NaHCO3 stimulated microbial growth and metabolic function through the upregulation of asnB expression [55]. Enhancing the global metabolic activity of microorganisms is an effective strategy for improving the operational performance of biological wastewater treatment systems [56]. However, the two reactors exhibited differences in the expression of nitrogen removal functional genes. The expression abundances of core denitrification genes, including nirS, norC, and nosZ, were significantly higher in the PS-CPBR than in the CS-CPBR. This suggests that the sequential reduction pathway from nitrite to nitric oxide, nitrous oxide, and dinitrogen gas proceeded more smoothly in the PS-CPBR. Electron transfer along this denitrification cascade can drive a higher nitrogen removal rate. In contrast, the CS-CPBR showed elevated expression of nrfC, which mediates the DNRA process [57], thereby diverting electrons away from canonical denitrification and inhibiting the complete reduction of nitrite to dinitrogen gas [58]. Thus, the type of solid carbon source critically governs the allocation of electron flow in denitrification systems. As demonstrated by Wang et al., the inhibition of denitrifying enzyme activities within the electron transport chain can alter the nitrate reduction pathway [59]. In this study, the slow carbon release kinetics of corncob led to unstable performance in the CS-CPBR by shunting a fraction of electrons toward the competing DNRA pathway. Conversely, the sustained and stable carbon supply in PS-CPBR was conducive to activating the tricarboxylic acid (TCA) cycle [60], which facilitated the sequential transfer of electrons along the complete denitrification chain. These findings indicate that optimizing the carbon release characteristics of solid carbon sources to direct electron flow preferentially toward the complete denitrification pathway is a key strategy for enhancing the nitrogen removal efficiency of sulfur-based mixotrophic denitrification systems.

3.2.4. Nitrogen Removal Mechanisms of the CS-CPBR and PS-CPBR

During stable reactor operation, the dominant heterotrophic denitrifiers Sinirhodobacter and Rhodobacter utilized the carbon released from corncob or PHBV to upregulate key denitrification genes (narZ, napC, nirK, norB, nosZ). The elevated expression of these genes drove the sequential reduction steps of complete denitrification, thereby enabling efficient nitrate removal. The better nitrogen removal performance of the PS-CPBR over the CS-CPBR was primarily attributable to the higher bioavailability of PHBV, which enhanced the dominance of HD. The PS-CPBR exhibited a greater enrichment of denitrifying genera (Sinirhodobacter, Rhodobacter, Brachymonas), and elevated expression of denitrification genes (nirS, norC, nosZ), thereby reinforcing the complete denitrification pathway. In contrast, the lower bioavailability of corncob restricted the overall nitrogen removal efficiency of the CS-CPBR.

4. Conclusions

(1)
Under optimal conditions (HRT = 2 h, NaHCO3 = 0.4 g/L, influent NO3−-N = 30 mg/L), the total nitrogen removal efficiencies of the CS-CPBR and the PS-CPBR reached 87.9% and 94.0%, respectively. To sustain efficient nitrogen removal, the following control strategies are recommended: For the CS-CPBR, the NaHCO3 dosage should be maintained within 0.05–0.2 g/L to maintain alkalinity balance, while a prolonged HRT is advisable under high nitrate loading to preserve TN removal performance. For PS-CPBR, the NaHCO3 dosage should be kept at 0.2–0.4 g/L to balance alkalinity. Maintaining the HRT between 2 h and 4 h under high nitrate nitrogen loading can effectively suppress nitrite accumulation and secure stable system operation.
(2)
The carbon source type governs its bioavailability, which in turn dictates the denitrification performance of the solid carbon source-sulfur system. Due to the higher bioavailability of PHBV, HD functioned as the dominant nitrogen removal pathway in the PS-CPBR. The PS-CPBR exhibited an enrichment of denitrifying genera (e.g., Sinirhodobacter, Rhodobacter, Brachymonas, etc.) and an upregulation of denitrification functional genes (nirS, norC, nosZ), resulting in better nitrogen removal capacity than the CS-CPBR.
(3)
NaHCO3 increased the relative abundance of carbon-hydrolyzing and sulfur-oxidizing bacteria to supply more electron donors for denitrification. NaHCO3 also promoted the secretion of EPS and enhanced microbial activity, which strengthened system stability and nitrogen removal efficiency.
(4)
Further studies using actual municipal secondary effluent and extended operational periods are warranted to validate the long-term performance of the CS-CPBR and the PS-CPBR and to assess their engineering feasibility in practical applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su18104836/s1, Text S1. Chemicals; Table S1. Components of domestication medium; Table S2. Composition of laboratory water; Table S3. Kinetic fitting parameters for TN removal under different operating conditions; Figure S1. Nitrogen removal experiments of corncob/sulfur, PHBV/sulfur under different investment ratio:(a) NO3−-N, (b) NO2−-N, (c) COD and (d) HD/SAD; Figure S2. Electron microscope (SEM) photographs of corncob, PHBV and sulfur before (a–c) and after (d–f) biofilm coverage; Figure S3. The 3D-EEM spectra of dissolved organic matter in effluents from CS-CPBR (a–c) and PS-CPBR (d–f) under different NaHCO3 dosages; Figure S4. Protein and polysaccharide contents of EPS under NaHCO3 dosages.

Author Contributions

Conceptualization, Y.H.; methodology, Y.G.; software, Y.G., and D.L.; validation, D.L.; formal analysis, Y.G. and D.L.; investigation, Y.G.; resources, G.W.; writing—original draft, Y.G.; writing—review and editing, Y.H.; supervision, Y.H.; project administration, X.Z., G.W. and J.X.; funding acquisition, Y.H. and J.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, grant number (21477039) and Guangzhou Pengkai Environment Technology Co., Ltd.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are not publicly available due to privacy issues.

Conflicts of Interest

The authors employed by Guangzhou Pengkai Environment Technology Co., Ltd. are Xiaoqiang Zhu, Guobin Wang, and Jieyun Xie. The company only provided funding for the research and had no role in study design, data collection, analysis, interpretation, manuscript writing, or the decision to submit for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CS-CPBRCorncob-sulfur circulation packed-bed reactor
PS-CPBRPHBV-sulfur circulation packed-bed reactor
TNTotal nitrogen
HRTHydraulic retention time
CODChemical oxygen demand
HDHeterotrophic denitrification
SADSulfur-based autotrophic denitrification
PLAPolylactic acid
PCLPolycaprolactone
PHBVPolyhydroxybutyrate-co-valerate
PBSPolybutylene succinate
MLSSMixed liquor suspended solids
MLVSSMixed liquor volatile suspended solids
SEMScanning electron microscopy
3D-EEMThree-dimensional excitation-emission matrix
DOMDissolved organic matter
EPSExtracellular polymeric substances
PNProtein
PSPolysaccharide
EETExtracellular electron transfer
DNRADissimilatory nitrate reduction to ammonium

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Figure 1. Concentration and removal rate of TN (a) and NO3−-N (b), concentration of NO2−-N, COD (c) and pH, alkalinity (d) in CS-CPBR and PS-CPBR.
Figure 1. Concentration and removal rate of TN (a) and NO3−-N (b), concentration of NO2−-N, COD (c) and pH, alkalinity (d) in CS-CPBR and PS-CPBR.
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Figure 2. The proportion of SAD/HD in CS-CPBR (a) and PS-CPBR (b).
Figure 2. The proportion of SAD/HD in CS-CPBR (a) and PS-CPBR (b).
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Figure 3. Kinetics of TN removal in CS-CPBR and PS-CPBR at different NaHCO3 dosages (a,b) and influent NO3−-N concentrations (c,d).
Figure 3. Kinetics of TN removal in CS-CPBR and PS-CPBR at different NaHCO3 dosages (a,b) and influent NO3−-N concentrations (c,d).
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Figure 4. Nitrogen balance in the CS-CPBR (a) and PS-CPBR (b).
Figure 4. Nitrogen balance in the CS-CPBR (a) and PS-CPBR (b).
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Figure 5. Community composition at phylum (a), genus (b) level, and the clustering heatmap of genus-level taxa in CS-CPBR and PS-CPBR (c) under different NaHCO3 dosages.
Figure 5. Community composition at phylum (a), genus (b) level, and the clustering heatmap of genus-level taxa in CS-CPBR and PS-CPBR (c) under different NaHCO3 dosages.
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Figure 6. Contribution ratio of denitrification functional genes in CS-CPBR and PS-CPBR under different NaHCO3 dosages.
Figure 6. Contribution ratio of denitrification functional genes in CS-CPBR and PS-CPBR under different NaHCO3 dosages.
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Table 1. The operating parameters of reactors at different periods.
Table 1. The operating parameters of reactors at different periods.
Period123456789
Cycle1–1011–2021–3031–4041–5051–6061–7071–8081–90
HRT (h)421222222
NaHCO3 (g/L)0.10.10.10.050.20.40.40.40.4
NO3−-N (mg/L)303030303030102030
Table 2. The alkalinity changes per unit nitrate removed in CS-CPBR and PS-CPBR (g CaCO3/g NO3−-N).
Table 2. The alkalinity changes per unit nitrate removed in CS-CPBR and PS-CPBR (g CaCO3/g NO3−-N).
Period123456789
CS-CPBR1.721.141.50−0.111.300.78−0.580.700.48
PS-CPBR1.281.442.481.413.804.101.161.901.78
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MDPI and ACS Style

Gao, Y.; Hu, Y.; Liang, D.; Cheng, J.; Zhu, X.; Wang, G.; Xie, J. Comparison of Denitrification Performance and Regulation Strategies of Corncob/PHBV and Sulfur in Circulation Packed-Bed Reactor. Sustainability 2026, 18, 4836. https://doi.org/10.3390/su18104836

AMA Style

Gao Y, Hu Y, Liang D, Cheng J, Zhu X, Wang G, Xie J. Comparison of Denitrification Performance and Regulation Strategies of Corncob/PHBV and Sulfur in Circulation Packed-Bed Reactor. Sustainability. 2026; 18(10):4836. https://doi.org/10.3390/su18104836

Chicago/Turabian Style

Gao, Yumeng, Yongyou Hu, Donghui Liang, Jianhua Cheng, Xiaoqiang Zhu, Guobin Wang, and Jieyun Xie. 2026. "Comparison of Denitrification Performance and Regulation Strategies of Corncob/PHBV and Sulfur in Circulation Packed-Bed Reactor" Sustainability 18, no. 10: 4836. https://doi.org/10.3390/su18104836

APA Style

Gao, Y., Hu, Y., Liang, D., Cheng, J., Zhu, X., Wang, G., & Xie, J. (2026). Comparison of Denitrification Performance and Regulation Strategies of Corncob/PHBV and Sulfur in Circulation Packed-Bed Reactor. Sustainability, 18(10), 4836. https://doi.org/10.3390/su18104836

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