The Application of ${\mathbf{S}}_{\mathbf{n}}^{\mathbf{2}\mathbf{-}}$ in Autotrophic Denitrification Process for Advanced Nitrogen Removal in Wastewater Treatment

Abstract

This study presents a cost-effective and feasible technique for the deep denitrification of wastewater, based on sulfur autotrophic denitrification mediated by polysulfides (${\mathrm{S}}_{\mathrm{n}}^{2-}$). Various polysulfides were used as electron donors in an aerobic/anoxic sequencing batch reactor (SBR) to simulate nitrification and denitrification processes. The performance of different polysulfide species and their respective dosages were evaluated to determine the optimal conditions for nitrogen removal. Under optimal nitrogen removal conditions with a dosing of 19.2 mg S/L from Na₂S₃, the system was operated continuously for 38 days, with low sludge production during the process. During stable operation, the system achieved an average removal of 7.3 mg/L of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, corresponding to a removal efficiency of 23.1%. No significant accumulation of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ was observed in the effluent, and the average utilization efficiency of Na₂S₃ reached 83.7%. Continuous dosing of Na₂S₃ promoted the enrichment of sulfur autotrophic denitrification-related microorganisms within the system.

1. Introduction

Nitrogen pollution in aquatic environments continues to pose a serious environmental challenge in China. The excessive accumulation of nitrogen, particularly in the forms of $\mathrm{N}{\mathrm{H}}_4^{+}\text{-}\mathrm{N}$, $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, and $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$, frequently results in eutrophication, algal overgrowth, and water blooms, which severely degrade aquatic ecosystems and water quality, ultimately affecting the habitat of aquatic organisms [1,2]. Therefore, the control of TN concentrations in aquatic environments is critically important. In recent years, China has implemented stricter wastewater discharge regulations, with several regions adopting more stringent TN discharge standards. For instance, the “Standards of water quality for wastewater treatment plant” of Shenzhen [3] mandates that TN concentrations in effluent from wastewater purification plants must be maintained below 10 mg/L (B standard) and 8 mg/L (A standard) [4].

However, conventional wastewater treatment processes often struggle to meet TN standards, especially when the wastewater has low C/N ratios. To enhance the nitrogen removal efficiency, many wastewater treatment plants resort to adding exogenous carbon sources (e.g., sodium acetate, methanol), which significantly increases the operating costs [5]. This challenge underscores the urgent need for low-cost, high-efficiency nitrogen removal technologies that comply with stringent emission standards while reducing operational expenses and minimizing ecological impact.

In recent years, sulfur-based autotrophic denitrification has emerged as a promising solution for low-carbon wastewater treatment due to its advantages, including the absence of additional carbon sources and low sludge production [6,7]. Compared with traditional heterotrophic denitrification, sulfur autotrophic denitrification offers high nitrogen removal efficiency, safe operation, and minimal residual sludge production [8,9,10,11]. Sulfur-based autotrophic denitrification is widely recognized as a cost-effective and sustainable nitrogen removal strategy [12]. A primary reason is the low cost and stable supply of elemental sulfur (S⁰). Sulfur is abundantly available as a byproduct of petroleum refining and natural gas desulfurization. Currently, over 80% of global sulfur production comes from such waste sources. This makes S⁰ an inexpensive commodity, especially when compared with additional carbon sources [13]. Besides the cheap electron donor, sulfur-driven processes produce less sludge because of the lower growth yield of autotrophic microorganism. The biomass yield in sulfur autotrophic denitrifiers is about 0.40–0.57 g VSS/g $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, roughly half of that in heterotrophic systems (0.8–1.2 g VSS/g $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$) [14]. Correspondingly, the cost of sludge disposal is much lower.

Sulfur autotrophic denitrification involves sulfur-oxidizing bacteria that utilize inorganic chemical energy or light energy to convert nitrogen compounds to nitrogen gas under anoxic or anaerobic conditions, using reduced sulfur species (e.g., elemental sulfur (S⁰), sulfide (S²⁻), thiosulfate (${\mathrm{S}}_2{\mathrm{O}}_3^{2-}$), and so on) as electron donors. The reaction principles are described by Equations (1)–(3):

$${\mathrm{S}}^0+1.2\mathrm{N}{\mathrm{O}}_3^{-}+0.4{\mathrm{H}}_2\mathrm{O}\to \mathrm{S}{\mathrm{O}}_4^{2-}+0.6{\mathrm{N}}_2+0.8{\mathrm{H}}^{+}$$

(1)

$${\mathrm{S}}^{2-}+1.6\mathrm{N}{\mathrm{O}}_3^{-}+1.6{\mathrm{H}}^{+}\to \mathrm{S}{\mathrm{O}}_4^{2-}+0.8{\mathrm{N}}_2+0.8{\mathrm{H}}_2\mathrm{O}$$

(2)

$${\mathrm{S}}_2{\mathrm{O}}_3^{2-}+1.6\mathrm{N}{\mathrm{O}}_3^{-}+0.2{\mathrm{H}}_2\mathrm{O}\to 2\mathrm{S}{\mathrm{O}}_4^{2-}+0.8{\mathrm{N}}_2+0.4{\mathrm{H}}^{+}$$

(3)

The low solubility of S⁰ in water limits its application in elemental sulfur autotrophic denitrification (SDAD) technology for wastewater treatment. In SDAD, S²⁻ serves as an electron donor, enabling a fast reaction rate and contributing to the acid-base balance. However, excessively high sulfide concentrations can inhibit microbial activity. In contrast, ${\mathrm{S}}_2{\mathrm{O}}_3^{2-}$ offers high solubility and bioavailability with superior nitrogen removal performance compared to S⁰ and S²⁻. Nevertheless, it consumes substantial alkalinity, increases operational costs, and tends to accumulate nitrite. As a result, its application remains primarily at the experimental stage.

Recent studies have indicated that polysulfides, naturally formed by the abiotic reaction of elemental sulfur with sulfides, exhibit good water solubility. The chemical formula of polysulfides is ${\mathrm{S}}_{\mathrm{n}}^{2-}$, where sulfur atoms form long-chain structures, as illustrated in Figure 1.

${\mathrm{S}}_{\mathrm{n}}^{2-}$ play a crucial role in the sulfur cycle. Under neutral or alkaline conditions, HS⁻ can attack S⁰, causing it to break and form ${\mathrm{S}}_{\mathrm{n}}^{2-}$. These ${\mathrm{S}}_{\mathrm{n}}^{2-}$ can cross cell membranes and undergo intracellular reduction to sulfide by NADPH or oxidation to elemental sulfur, thereby increasing the bioavailability of S⁰ and enhancing sulfur oxidation processes [12,15,16]. ${\mathrm{S}}_{\mathrm{n}}^{2-}$ dominate when sufficient sulfur is present, forming spontaneously when S⁰ coexists with sulfides [17]. Additionally, various enzymes in biological systems facilitate the use of ${\mathrm{S}}_{\mathrm{n}}^{2-}$ as electron donors, promoting autotrophic denitrification and improving wastewater denitrification efficiency. ${\mathrm{S}}_{\mathrm{n}}^{2-}$-based electron donors have been shown to significantly improve autotrophic denitrification performance compared to using S⁰ alone. Xu et al. reported that the addition of a small dose of sulfide (about 20 mg S/L) to sulfur-packed bioreactors increased the specific $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal rate by approximately 50% and reduced the proportion of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removed as $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$, from 1.6% to 0.7%, compared to the sulfide-free control [14]. Similarly, Qiu et al. demonstrated that the sulfur disproportionation-induced ${\mathrm{S}}_{\mathrm{n}}^{2-}$ formation achieved highly efficient autotrophic denitrification, with average denitrification rates of 1.46–1.65 kg/(m³·d) [18]. Bao et al. found that the appropriate addition of Na₂S (0.9 kg/m³/d) to the sulfur-packed system could increase the $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal rate from 33.6% to 97.3%, but excessive addition would lead to the accumulation of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ [19].

However, concerns have been raised regarding the potential toxicity of ${\mathrm{S}}_{\mathrm{n}}^{2-}$, which is primarily attributed to the hydrolysis or oxidation of them to release S²⁻ into the system. S²⁻ is an inhibitor for microbial activity, as it can disrupt membrane integrity, interfere with respiratory enzymes, and generate reactive sulfur species that induce oxidative stress in functional bacteria [20,21]. S⁰, which may also be formed during polysulfide transformation, is essentially non-toxic due to its low solubility and chemical inertness. S⁰ serves as a stable, slow-release electron donor and is well tolerated by sulfur-oxidizing bacteria. Therefore, controlling ${\mathrm{S}}_{\mathrm{n}}^{2-}$ dosage and maintaining favorable sulfur speciation are crucial for mitigating adverse effects and ensuring the stability of sulfur-based autotrophic denitrification systems. Overall, the formation of ${\mathrm{S}}_{\mathrm{n}}^{2-}$ significantly enhances the solubility and bioavailability of S⁰ in water, providing a promising avenue for efficient autotrophic denitrification in nitrogen removal. In addition, the research on polysulfides in denitrification and nitrogen removal remains limited. Further investigation is needed to deepen the understanding in this area. Therefore, this study explores the efficiency of autotrophic denitrification using polysulfides as electron donors.

This study employs an aerobic/anoxic sequencing batch reactor to simulate the nitrification and denitrification processes of the anaerobic/anoxic/oxic (AAO) system. The impact of polysulfides has been investigated by the addition of polysulfides to achieve autotrophic denitrification. The optimization has been identified by monitoring the denitrification performance, sulfur source utilization rate, and variations in nitrogen pollutants under different polysulfide species and dosages. Additionally, this study examines the denitrification efficiency, sludge characteristics, and functional microbial community structure during the stable operation of the polysulfide-driven autotrophic denitrification process to reveal the function of polysulfides in nitrogen removal.

2. Materials and Methods

2.1. Reactor System and Operation

A schematic diagram of the aerobic/anoxic sequencing batch reactor unit is presented in Figure 2. The reactor was constructed from transparent polyvinyl chloride (PVC) tubes with an inner diameter of 100 mm and an effective volume of 3 L. Influent and effluent were introduced and discharged at heights of 40 cm and 18 cm from the bottom of the reactor, respectively, with a drainage ratio of 60%. During the aerobic stage, aeration was provided by an air compressor connected to a diffuser located at the reactor bottom. An electric stirrer was used to ensure a thorough mixing of the sludge and water.

Each operational cycle lasted 8 h, with the time settings for each stage detailed in

Table 1. Aerobic/anoxic sequential batch reaction device time setting (unit/min).

Filling	Aerobic Phase	Anoxic Phase	Aeration Stripping	Settlement	Draining	Cycle
10	240	180	5	40	5	480

. During the aerobic phase, the aeration rate was adjusted to 2 L/min using a flowmeter. During the anoxic phase, an electric stirrer was employed to regulate the speed of 200–300 r/min. Residual dissolved oxygen (DO) may inhibit the activity of denitrifying bacteria, leading to delayed or incomplete $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ reduction. To eliminate residual DO and establish anoxic conditions, a concentrated chemical oxygen demand (COD) solution was added within 5 min of transitioning from the aerobic to the anoxic stage, resulting in a final COD concentration of about 30 mg/L in the 3.0 L reactor volume. The biodegradable organics promoted microbial oxygen uptake, thereby depleting DO to below 0.20 mg/L within 5 min.

2.2. Influent Water Quality

In this study, a simplified synthetic wastewater was used to simulate the nitrogen transformation pathway in the aerobic and anoxic phases of the AAO process, rather than to fully replicate the composition of municipal wastewater. The synthetic feed contained ammonium as the sole nitrogen source, and no organic carbon was added to allow for the evaluation of sulfur-based autotrophic denitrification. The composition of the synthetic wastewater is shown in

Table 2. Influent water quality of aerobic/anoxic sequencing batch reactor.

Components	Concentration (mg/L)	Mean Value (mg/L)	Pharmaceuticals
${\mathrm{NH}}_4^{+}\text{-}\mathrm{N}$	20–25	22.5	NH4Cl
TP	3–5	4.0	KH2PO4
Alkalinity	240–300	270	NaHCO3

.

To enhance the simulation, trace element concentrates were added, consisting of two types with specific compositions and concentrations listed in

Table 3. Trace element concentrate I.

Components	Concentration (g/L)
EDTA-2Na	6.37
FeSO4·7H2O	9.15

and

Table 4. Trace element concentrate II.

Components	Concentration (g/L)
EDTA-2Na	19.11
ZnSO4·7H2O	0.43
CoCl2·6H2O	0.24
MnCl2·4H2O	0.99
CuSO4·5H2O	0.25
NaMo4·2H2O	0.22
NiCl2·6H2O	0.19
NaSeo4·10H2O	0.21
H3BO4	0.014

, respectively. For each liter of synthetic wastewater, 1 mL of Trace Element Concentrate I and 2 mL of Trace Element Concentrate II were added.

2.3. Polysulfide Solutions

Polysulfides are usually synthesized by two methods, sodium hydrosulfide oxidation and sodium sulfide synthesis. The sodium hydrosulfide oxidation method requires harsh preparation conditions, involves a complex process, and is difficult to operate [22,23]. In contrast, the sodium sulfide synthesis method offers simpler preparation conditions and can produce sodium polysulfide at room temperature and atmospheric pressure [24]. Consequently, the sodium sulfide synthesis method was employed in this study to prepare the sodium polysulfide solution. The raw materials for this method include sulfur powder and sodium sulfide, with the preparation process involving the addition of sulfur powder to a sodium sulfide solution. The corresponding chemical reaction is shown in Equation (4):

$$\mathrm{N}{\mathrm{a}}_2\mathrm{S}+(\mathrm{n}-1)\mathrm{S}\to \mathrm{N}{\mathrm{a}}_2{\mathrm{S}}_{\mathrm{n}}$$

(4)

In the equation, n = 1, 2, 3, 4, …

By adjusting the molar ratio of sulfur sulfide to sodium powder, sodium disulfide (Na₂S₂), sodium trisulfide (Na₂S₃), and sodium tetrasulfide (Na₂S₄) solutions were prepared. The specific dosing ratios are presented in

Table 5. Types and preparation solutions of sodium polysulfide.

${\mathbf{S}}_{\mathbf{n}}^{2-}$	Species	Molar Ratio Csodium sulfide:Csulfur powder
n = 1	Na2S	-
n = 2	Na2S2	1:1
n = 3	Na2S3	1:2
n = 4	Na2S4	1:3

.

To ensure complete dissolution of sulfur powder, the sodium polysulfide solution was stirred continuously for 48 to 72 h using a magnetic stirrer. Initially, the sodium sulfide solution is colorless and transparent. However, as the sulfur powder dissolves, the solution color changes from colorless to light yellow and eventually to orange-red.

2.4. Selection of Polysulfide Species

To investigate the autotrophic denitrification performance of different polysulfides as electron donors, appropriate amounts of Na₂S, Na₂S₂, Na₂S₃, and Na₂S₄ solutions were added separately. This study compared pollutant removal efficiency, sulfur source utilization rate, and nitrogen concentration changes throughout the denitrification process. The experiments were conducted in two subseries.

In the first series, the denitrification efficiencies of sodium sulfide and sodium disulfide, when employed as electron donors, were compared. Equal amounts and concentrations of sodium sulfide and sodium disulfide solutions were added to compare their denitrification performance, aiming to determine whether S⁰ in polysulfides was effectively used in the autotrophic denitrification process. The experiment was carried out in two stages. The dosage of sulfur compounds in each stage is shown in

Table 6. The composition of sodium sulfide solution and sodium disulfide solution.

Stage	Species	Concentration (mol/L)	Dosage (mL)	Dosage of S (mg S/L)
I	Na2S	0.1	8.0	8.5
II	Na2S2	0.1	8.0	17.1

.

In the second series, the respective denitrification performance of Na₂S₂, Na₂S₃, and Na₂S₄ when used as autotrophic denitrification electron donors was explored. In this study, the influent S/N ratio (the ratio of sulfur concentration to nitrogen concentration in the influent) was not used as a dosing control parameter. This is because sulfur was added at the transition from the aerobic to the anoxic phase, whereas $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ concentrations at that time varied dynamically across SBR cycles. Therefore, calculating a fixed S/N ratio based on influent $\mathrm{N}{\mathrm{H}}_4^{+}\text{-}\mathrm{N}$ would be inappropriate and unrepresentative of actual electron demand during denitrification. Additionally, due to variations in S⁰ and S²⁻ content among the systems, the theoretical dosages required for the removal of each gram of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ differed accordingly. To compare the nitrogen removal efficiencies of different polysulfide types, their required dosages were calculated based on the theoretical removal of 10 mg/L of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$. The experiment was carried out in three stages. The dosage of polysulfides in each stage is shown in in

Table 7. Polysulfide types and corresponding dosages at operational stages.

Stage	Species	Dosage of S (mg S/L)	$\mathbf{Theoretically}\mathbf{Removable}\mathbf{N}{\mathbf{O}}_3^{-}\text{-}\mathbf{N}$Content (mg/L)
I	Na2S2	21.4	10.0
II	Na2S3	22.5	10.0
III	Na2S4	23.1	10.0

.

To quantitatively evaluate the roles of S⁰ and S²⁻ in $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, the contribution and utilization rates in various polysulfide-based autotrophic denitrification systems were calculated by Equations (5) to (8).

$${\mathrm{R}}_{{\mathrm{S}}^0}={\displaystyle \frac{\mathrm{M}-{\mathrm{T}}_{{\mathrm{S}}^{2-}}}{{\mathrm{T}}_{{\mathrm{S}}^0}-{\mathrm{T}}_{{\mathrm{S}}^{2-}}}}$$

(5)

$${\mathrm{R}}_{{\mathrm{S}}^{2-}}={\displaystyle \frac{{\mathrm{T}}_{{\mathrm{S}}^0}-\mathrm{M}}{{\mathrm{T}}_{{\mathrm{S}}^0}-{\mathrm{T}}_{{\mathrm{S}}^{2-}}}}$$

(6)

In the equations,

• M—actual generation of $\mathrm{S}{\mathrm{O}}_4^{2-}$, in mg/L;
• ${\mathrm{T}}_{{\mathrm{S}}^{2-}}$—theoretical production of $\mathrm{S}{\mathrm{O}}_4^{2-}$ when actual removal of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ entirely due to S²⁻, in mg/L;
• ${\mathrm{T}}_{{\mathrm{S}}^0}$—theoretical production of $\mathrm{S}{\mathrm{O}}_4^{2-}$ when actual removal of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ entirely due to S⁰, in mg/L;
• ${\mathrm{R}}_{{\mathrm{S}}^{2-}}$—proportion of the contribution of S²⁻ to the actual removal of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, in %;
• ${\mathrm{R}}_{{\mathrm{S}}^0}$—proportion of the contribution of S⁰ to the actual removal of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, in %.

$${\mathsf{\eta }}_{{\mathrm{S}}^0}={\displaystyle \frac{{\mathrm{R}}_{{\mathrm{S}}^0}\times {\mathrm{m}}_{\mathrm{N}}}{{\mathrm{M}}_{\mathrm{N}}\times {\mathrm{r}}_{{\mathrm{S}}^0}}}$$

(7)

$${\mathsf{\eta }}_{{\mathrm{S}}^{2-}}={\displaystyle \frac{{\mathrm{R}}_{{\mathrm{S}}^{2-}}\times {\mathrm{m}}_{\mathrm{N}}}{{\mathrm{M}}_{\mathrm{N}}\times {\mathrm{r}}_{{\mathrm{S}}^{2-}}}}$$

(8)

In the equations,

• ${\mathrm{M}}_{\mathrm{N}}$—theoretical generation of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, in mg/L;
• ${\mathrm{m}}_{\mathrm{N}}$—actual generation of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, in mg/L;
• ${\mathrm{r}}_{{\mathrm{S}}^{2-}}$—proportion of the contribution of S²⁻ to the theoretical removal of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, in %;
• ${\mathrm{r}}_{{\mathrm{S}}^0}$—proportion of the contribution of S⁰ to the theoretical removal of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$, in %;
• ${\mathsf{\eta }}_{{\mathrm{S}}^{2-}}$—relative utilization rate of S²⁻ to $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, in %;
• ${\mathsf{\eta }}_{{\mathrm{S}}^0}$—relative utilization rate of S⁰ to $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, in %.

2.5. Optimization of Polysulfide Dosage

The previous study identified the optimal polysulfide electron donor among Na₂S, Na₂S₂, Na₂S₃, and Na₂S₄. In this section, the influence of different doses of optimal polysulfides on the nitrogen removal efficiency of autotrophic denitrification was examined. The optimal polysulfide dosage was selected based on a comprehensive assessment of denitrification performance, including $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal efficiency, $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ accumulation, sulfur utilization efficiency, and potential signs of process inhibition. The experiments in this section were conducted into six operational stages, with the corresponding polysulfide dosages outlined in

Table 8. The dosage of polysulfides.

Stage	Species	$\mathbf{Concentration}\mathbf{of}{\mathbf{S}}_{\mathbf{n}}^{2-}$ Solution (mol/L)	$\mathbf{Volume}\mathbf{of}{\mathbf{S}}_{\mathbf{n}}^{2-}$ Solution (mg/L)	$\mathbf{Dosage}\mathbf{of}{\mathbf{S}}_{\mathbf{n}}^{2-}$ (mg S/L)	$\mathbf{Theoretically}\mathbf{Removable}\mathbf{N}{\mathbf{O}}_3^{-}\text{-}\mathbf{N}$Content (mg/L)
I	Na2S3	0.1	4.0	12.8	5.7
II	Na2S3	0.1	6.0	19.2	8.5
III	Na2S3	0.1	8.0	25.6	11.4
IV	Na2S3	0.1	12.0	38.4	17.1
V	Na2S3	0.1	16.0	51.2	22.8
VI	Na2S3	0.1	20.0	64.0	28.5

.

2.6. Research on the Stable Operation of the Polysulfide Autotrophic Denitrification Process

Building upon prior research, the selected optimal electron donor and its dosage were used to operate the system stably for 38 days. Active sludge discharge was not carried out during operation. Denitrification performance was evaluated by measuring the concentrations of nitrogen pollutants in the initial anoxic phase and effluent and assessing sulfur source utilization. Sludge samples were collected regularly to analyze changes in sludge concentration and settling performance at different operating stages. The sampling times of sludge samples are shown in

Table 9. Sampling schedule of sludge sample.

Groups	Sampling Period	Sulfur Concentration in System mg S/L
I	Day 1 of operation	19.2
II	Day 12 of operation	19.2
III	Day 22 of operation	19.2
IV	Day 38 of operation	19.2

. Additionally, microbial community structure succession at the genus level was examined to understand its role in sulfur autotrophic denitrification.

3. Results and Discussion

3.1. Influence of Polysulfide Species on the Effectiveness of Autotrophic Denitrification for Nitrogen Removal

3.1.1. Comparison of Nitrogen Removal Performance Among Different Polysulfide Species

A comparative analysis was conducted to evaluate the denitrification efficiency of Na₂S and Na₂S₂. $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal was primarily expressed as the absolute concentration (mg/L) reduced. This approach reflects the practical challenge in AAO systems, where residual $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ concentrations of several mg/L must be further removed to meet TN discharge standards. The concentration changes in the initial anoxic phase and effluent are presented in Figure 3. After the addition of sodium sulfide solution during Stage I, the average concentrations of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$in the initial anoxic phase and effluent were 31.6 mg/L and 26.4 mg/L, respectively, resulting in the removal amount of 5.2 mg/L (removal efficiency of 16.5%). Simultaneously, 2.4 mg/L of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ was generated, corresponding to an accumulation rate of 46.3%. $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ accumulation was expressed as the percentage of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removed that was transformed into $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$, calculated by dividing the net increase in $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ by the net decrease in $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ during the anoxic phase. Previous studies have indicated that a lower S/N ratio in sulfide autotrophic denitrification systems promotes nitrite accumulation [25,26,27]. Therefore, it was hypothesized that the primary cause of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$accumulation in the experimental effluent is the low S/N ratio in the system.

During Stage II, the average concentrations of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ in the initial anoxic phase and effluent were 31.8 mg/L and 25.9 mg/L, respectively, corresponding to a removal amount of 5.9 mg/L (removal efficiency of 18.6%). No accumulation of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ was detected in the effluent. These results demonstrated that compared to sodium sulfide, an equivalent dose of sodium disulfide achieved an additional 0.7 mg/L of nitrogen removal without leading to $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ accumulation.

At present, study on the autotrophic denitrification of polysulfides remains limited. The prevailing perspective suggests that polysulfides are more readily utilized by microorganisms as electron shuttles. Within microbial cells, polysulfides are converted through a series of enzymatic reactions into S⁰ and S²⁻ (with S²⁻ representing the total dissolved sulfide in the system), which are subsequently utilized. In this study, the analysis of polysulfide utilization in denitrification systems was primarily based to the perspective corresponding to the S²⁻. During the evaluation of S⁰ utilization in the sodium disulfide system, it was initially assumed that all S²⁻ contributed to autotrophic denitrification, theoretically supporting a $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal amount of up to 4.6 mg/L. However, the observed average removal amount of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ was 5.9 mg/L, indicating that S⁰ in sodium disulfide also contributed to denitrification. The specific utilization rate of S⁰ requires further analysis in conjunction with the generation of sulfate ($\mathrm{S}{\mathrm{O}}_4^{2-}$). This aspect will be investigated in detail in Section 3.1.2.

Previous studies have demonstrated the considerable potential of polysulfides as electron donors in autotrophic denitrification. Several types of polysulfides, namely Na₂S₂, Na₂S₃, and Na₂S₄, have been identified. In this study, the denitrification performance of each type of polysulfide as an electron donor was further investigated.

The variations in initial and effluent concentrations of$\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$and $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ across the three operational stages are shown in Figure 4. In Stage I, the average initial and effluent concentrations of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ were 32.1 mg/L and 24.6 mg/L, respectively, resulting in an average $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal amount of 7.5 mg/L (removal efficiency of 23.4%). In Stage II, the average initial and effluent concentrations $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ were 31.6 mg/L and 24.5 mg/L, respectively, resulting in an average $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal amount of 7.1 mg/L (removal efficiency of 22.5%). In Stage III, the average initial and effluent concentrations of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ were 31.7 mg/L and 26.2 mg/L, resulting in an average $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal of 5.5 mg/L (removal efficiency of 17.4%). Notably, $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ accumulation in the effluent was negligible throughout all operational stages. In terms of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, both Na₂S₂ and Na₂S₃ demonstrated better performance than Na₂S₄.

3.1.2. Sulfur Utilization Efficiency Among Different Polysulfide Species

The utilization of Na₂S and Na₂S₂ in the autotrophic denitrification process was analyzed initially. The autotrophic denitrification processes involving S⁰ and Na₂S are illustrated in Equations (9) and (10) [28,29]:

$$\begin{gathered} {1.1\mathrm{S}}^0+\mathrm{N}{\mathrm{O}}_3^{-}+0.76{\mathrm{H}}_2\mathrm{O}+0.08\mathrm{N}{\mathrm{H}}_4^{+}+0.4\mathrm{C}{\mathrm{O}}_2 \\ \to 0.08{\mathrm{C}}_5{\mathrm{H}}_7{\mathrm{O}}_2\mathrm{N}+0.5{\mathrm{N}}_2+1.1\mathrm{S}{\mathrm{O}}_4^{2-}+{1.28\mathrm{H}}^{+} \end{gathered}$$

(9)

$$\begin{gathered} \mathrm{H}{\mathrm{S}}^{-}+1.228\mathrm{N}{\mathrm{O}}_3^{-}+0.438\mathrm{HC}{\mathrm{O}}_3^{-}+{0.573\mathrm{H}}^{+}+0.093\mathrm{N}{\mathrm{H}}_4^{+}+0.027\mathrm{C}{\mathrm{O}}_2 \\ \to 0.093{\mathrm{C}}_5{\mathrm{H}}_7{\mathrm{O}}_2\mathrm{N}+0.614{\mathrm{N}}_2+0.866{\mathrm{H}}_2\mathrm{O}+\mathrm{S}{\mathrm{O}}_4^{2-} \end{gathered}$$

(10)

The utilization efficiency of the sulfur source was analyzed based on the theoretical and actual removal of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$. Theoretically, the added Na₂S is capable of removing 4.59 mg/L of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$. However, in practice, the autotrophic denitrification process using Na₂S led to the accumulation of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$. Therefore, when calculating the utilization efficiency, the actual removal of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ and the accumulation of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ were uniformly converted into the number of electrons utilized. Additionally, the added Na₂S₂ is theoretically capable of removing 8.0 mg/L of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$. By calculation, the average utilization efficiencies of Na₂S and Na₂S₂ were 86.6% and 76.0%, respectively, as shown in Figure 5.

Further analysis of S⁰ utilization in Na₂S₂ system was conducted performed under the assumption that S²⁻ utilization was identical in both Na₂S and polysulfide systems. Based on the actual utilization efficiency of 86.6% measured in the Na₂S system, an average S⁰ utilization efficiency of 65.4% in Na₂S₂ was calculated. It is suggested that S⁰ dissolved from polysulfides can serve as an electron donor in autotrophic denitrification.

The utilization efficiencies of Na₂S₂, Na₂S₃, and Na₂S₄ as electron donors in autotrophic denitrification were compared based on the amount of $\mathrm{S}{\mathrm{O}}_4^{2-}$ generated. According to the reaction illustrated in Equations (5) and (6), the amount of $\mathrm{S}{\mathrm{O}}_4^{2-}$ generated is related to the type of electron donor used in autotrophic denitrification. When S⁰ and S²⁻ were employed as electron donors in autotrophic denitrification, the corresponding $\mathrm{S}{\mathrm{O}}_4^{2-}$ yields were 7.5 mg/L and 5.6 mg/L per mg of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removed, respectively.

To investigate the utilization of different polysulfide species, it was initially assumed that S⁰ or S²⁻ were the sole electron donors involved in the autotrophic denitrification process. Theoretical $\mathrm{S}{\mathrm{O}}_4^{2-}$ production was calculated based on the actual $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, assuming either S⁰ or S²⁻ served as the sole electron donor. By comparing actual and theoretical production of $\mathrm{S}{\mathrm{O}}_4^{2-}$, the utilization efficiencies of S⁰ and S²⁻ in the autotrophic denitrification of polysulfides were determined. The actual $\mathrm{S}{\mathrm{O}}_4^{2-}$ production values were 46.3 mg/L, 47.0 mg/L, and 36.3 mg/L when different polysulfides were employed as electron donors, as shown in Figure 6.

When Na₂S₂ was added, the actual $\mathrm{S}{\mathrm{O}}_4^{2-}$ production values closely matched the theoretical production values for autotrophic denitrification using S²⁻ as the sole electron donor. When Na₂S₃ was added, the actual $\mathrm{S}{\mathrm{O}}_4^{2-}$ production values fell between the theoretical production values from S⁰ and S²⁻ systems. When Na₂S₄ was added, the actual $\mathrm{S}{\mathrm{O}}_4^{2-}$ production values were more consistent with the theoretical values for autotrophic denitrification using S⁰ alone. These results suggest that the utilization efficiencies of S⁰ and S²⁻ differ among polysulfide species, and that microorganisms may exhibit selectivity in their using S⁰ and S²⁻ during autotrophic denitrification.

The respective contributions of S⁰ and S²⁻ from polysulfides during autotrophic denitrification were further explored. The individual contributions of S⁰ and S²⁻ to nitrogen removal are shown in Figure 7. The contribution rates of S⁰ in Na₂S₂, Na₂S₃, and Na₂S₄ were 31.9%, 52.7%, and 55.4%, respectively. With an increasing proportion of S⁰ in the polysulfide compounds, its contribution to denitrification also increased.

In addition, due to the differences in $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal among various types of polysulfides, it is necessary to compare the overall utilization efficiency of each polysulfide with the individual utilization efficiencies of S⁰ and S²⁻. As shown in Figure 8a, the average utilization efficiencies of Na₂S₂, Na₂S₃, and Na₂S₄ were 75.1%, 71.2%, and 58.7%, respectively. The overall utilization efficiencies of Na₂S₂ and Na₂S₃ were similar, while the utilization efficiency of Na₂S₄ was significantly lower, indicating that approximately 40% of the sulfur source remained unutilized in the Na₂S₄ autotrophic denitrification system.

The utilization efficiencies of S⁰ and S²⁻ in polysulfides during autotrophic denitrification are shown in Figure 8b. It is indicated that the utilization efficiency of S²⁻ was higher than that of S⁰ when different types of polysulfides were used as electron donors. Specifically, the utilization efficiency of S²⁻ approached 90% in both the Na₂S₂ and Na₂S₃ systems, whereas it was only 80.8% in the Na₂S₄ system. Additionally, S⁰ utilization efficiency remained relatively low across all polysulfide systems, with corresponding values of 55.2%, 62.3%, and 42.9%. These findings suggest that microorganisms preferred to utilize S²⁻ over S⁰ as an electron donor for autotrophic denitrification.

In the Na₂S₄ system, although S⁰ accounted for over half (55.4%) of the $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, its actual utilization rate remained relatively low. This phenomenon may be attributed to the accumulation of underutilized S²⁻, which could inhibit microbial activity and further limited the efficiency of S⁰ utilization. As a result, the actual contribution of S⁰ to $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal in the Na₂S₄ system was only 3.0 mg/L, compared to 3.7 mg/L in the Na₂S₂ system, despite a lower percentage (52.7%) in the latter. This comparison suggests that the higher S⁰ contribution in the Na₂S₄ system did not reflect an improvement in S⁰ utilization efficiency but rather resulted from the relatively higher S⁰ content in Na₂S₄.

The comparative analysis of the autotrophic denitrification performance between Na₂S₂ and Na₂S₃ showed that although Na₂S₂ achieved 0.4 mg/L more $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, Na₂S₃ exhibited a 6.3% higher S⁰ utilization rate. Furthermore, the higher proportion of S⁰ in Na₂S₃ compared to Na₂S₂ resulted in a 30% reduction in the sulfur dosage required to achieve equivalent $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal.

S⁰ is less expensive and more readily available on the market. Na₂S₃ presented greater potential for $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal and was therefore considered a more suitable electron donor for autotrophic denitrification in terms of both efficiency and economic cost. Accordingly, in subsequent studies, Na₂S₃ was selected as the electron donor for polysulfide-based autotrophic denitrification, with all subsequent references to polysulfides specifically referring to Na₂S₃.

3.1.3. Time-Dependent Changes in Nitrogen Pollutants Among Different Polysulfide Species

As can be seen from Figure 9, during the process of autotrophic denitrification of polysulfides, the majority of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal was achieved within the first 40 min of the reaction. At 20 min of the reaction, the $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal rates of each system peaked at 10.0 mg/(L·h), 8.9 mg/(L·h), and 7.9 mg/(L·h), respectively. Subsequently, the $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal rates gradually declined. It can be seen that the $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal rate of the Na₂S₂ system was higher than that of the Na₂S₃ and Na₂S₄ systems. This may be attributed to the fact that S²⁻ has a higher denitrification rate than S⁰ under equivalent conditions. Moreover, some studies have pointed that S²⁻ can provide alkalinity supplementation for sulfur autotrophic denitrification to a certain extent, thereby enhancing the denitrification rate [30,31,32]. As the reaction progressed, the concentration of S²⁻ in the system decreased, and the promotion effect of S²⁻ on S⁰ denitrification gradually disappeared, so the removal rate gradually declined. The Na₂S₂ system had a relatively higher S²⁻ concentration compared to the Na₂S₃ and Na₂S₄ systems, which contributed to its higher overall denitrification rate.

At 60 min of the reaction, the $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ concentrations in all systems peaked at 0.6 mg/L, 0.6 mg/L, and 0.5 mg/L, indicating that during the first 60 min, the rate of reduction of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ to $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ was higher than the rate of conversion of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ to N₂. After 60 min of reaction, $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ concentrations began to decrease, and no $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ accumulation was observed at the end of the reaction.

3.2. Influence of Polysulfide Dosage on the Effectiveness of Autotrophic Denitrification for Nitrogen Removal

3.2.1. Comparison of Nitrogen Removal Effect at Different Na₂S₃ Dosages

The nitrogen removal performance under varying polysulfide dosages is presented in Figure 10. Increasing the polysulfide dosage led to a gradual decrease in the $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ concentration in the effluent. Although occasional fluctuations were observed in the effluent, the $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ concentration generally remained below 2.0 mg/L, with no significant accumulation of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$. The respective, average $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal amount at each stage was 4.9 mg/L, 7.4 mg/L, 8.6 mg/L, 10.9 mg/L, 12.5 mg/L, and 13.7 mg/L.

In stages V and VI, the sulfur concentration increased from 51.2 mg S/L to 64.0 mg S/L, theoretically improving $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal amount by 5.7 mg/L. However, the actual $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal amount increased by only 1.2 mg/L. This discrepancy may be attributed to the fact that when the polysulfide dosage increases, the rising S²⁻ concentration could inhibit the biological activity of autotrophic denitrifying bacteria, consequently affecting the bioavailability of S⁰ and reducing autotrophic denitrification efficiency [9,33,34,35]. Therefore, although increasing polysulfide dosage improved $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, it simultaneously reduced the polysulfide utilization efficiency.

3.2.2. Sulfur Utilization Efficiency at Different Na₂S₃ Dosages

The production of $\mathrm{S}{\mathrm{O}}_4^{2-}$ under varying polysulfide dosages is presented in Figure 11. As shown in Figure 11a, $\mathrm{S}{\mathrm{O}}_4^{2-}$ production exhibited an increasing trend with increasing polysulfide dosages. The actual $\mathrm{S}{\mathrm{O}}_4^{2-}$ production consistently fell between the theoretical values obtained when autotrophic denitrification was carried out using S⁰ or S²⁻ as the sole electron donor. Figure 11b shows that the contribution of S²⁻ from polysulfides to denitrification, initially increased and then decreased as the polysulfide dosage rose. It is suggested that denitrifying bacteria tend to increasingly prefer S⁰ over S²⁻ as an electron donor at higher polysulfide concentrations.

The utilization efficiencies of polysulfides under different dosages is shown in Figure 12. As the polysulfide dosage increased, the overall utilization efficiency exhibited a declining trend. At dosages of 12.8 mg S/L and 19.2 mg S/L, the sulfur utilization efficiencies were 85.7% and 85.5%, respectively. However, when the dosage reached 64.0 mg S/L, sulfur utilization declined to 47.2%, indicating that more than half of the polysulfides remained unused during the autotrophic denitrification process for $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal. Additionally, the utilization efficiencies of S²⁻ and S⁰ also showed a declining trend. This reduction may be attributed to the increased S²⁻ concentration in the system, which likely inhibited the biological activity of denitrifying bacteria, and subsequently decreased the overall sulfur utilization efficiency.

3.2.3. Time-Dependent Changes in Nitrogen Pollutants at Different Na₂S₃ Dosages

To examine the effect of polysulfide dosing on time-dependent concentration changes in nitrogen pollutants during autotrophic denitrification, the changes in $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ and $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ concentrations and reaction rates in the anoxic stage were analyzed. As shown in Figure 13, the $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal rate increased with increasing polysulfide dosages. At polysulfide dosages of 38.4–64.0 mg S/L, the average $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal rates remained high during the first 40 min of the reaction. After 60 min, the rate declined to 3.2–3.8 mg/(L·h) and continued to decline thereafter. It is indicated that increasing the sulfur concentration enhances the initial $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal rate, but the rate declines significantly in the later stages due to sulfur depletion. After 120 min, the concentrations of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ and $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ stabilized, suggesting the completion of the nitrogen removal process.

The $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ concentrations showed a rise-and-fall trend, peaking at 0.5, 0.6, 0.7, 0.8, 1.3, and 1.6 mg/L at 60 min in the respective systems. It is suggested that during the first 60 min, the reduction of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ to $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ outpaced the subsequent conversion of $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ to N₂. At 80 min, the $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ removal rates reached peaks across all systems. In systems with polysulfide dosages of 38.4–64.0 mg S/L, the reaction rates remained high between 80 and 100 min, ranging from 1.0 to 2.5 mg/(L·h). After 100 min, $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ concentration decreased to 0 mg/L in all systems.

3.3. Study on Stable Operation of Polysulfide Autotrophic Denitrification Process

Previous studies have demonstrated that polysulfides possess strong potential for nitrogen removal. When the polysulfide dosage ranged from 12.9 to 19.2 mg S/L, the autotrophic denitrification process using polysulfides exhibited both high nitrogen removal efficiency and sulfur utilization efficiency. Therefore, in this section, a polysulfide (Na₂S₃) dosage of 19.2 mg S/L was selected to investigate the performance of the polysulfide-based autotrophic denitrification process.

3.3.1. Effect of Nitrogen Removal

The system was operated stably for 38 days, and the experimental results are shown in Figure 14. The concentration of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$in the effluent gradually decreased with the continuous operation. After 9 days of operation, the $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ concentrations in the initial anoxic phase and effluent of the system were 31.6 mg/L and 24.3 mg/L, respectively, with an average $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal of 7.3 mg/L. There was no $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ accumulation in the effluent. The utilization efficiency of the polysulfide was 83.7% during stable operation.

3.3.2. Changes in Sludge Characteristics

The changes in sludge concentration and the settling performance of the system during stable operation are shown in Figure 15.

Sludge concentrations exhibited minor variations with the absence of active sludge discharging. During the operation period, the MLSS level was maintained at 2400–2800 mg/L. It is indicated that the sludge production during the polysulfide-based autotrophic denitrification process was relatively low. MLVSS can reflect the microbial content in the sludge. The trend of it indicates that some microorganisms were initially inhibited by polysulfide exposure, resulting in a decrease in MLVSS during the initial dosing stage. At the same time, those microorganisms that could not adapt to polysulfides were eliminated and discharged out of the reactor [35]. With the continuous dosing of polysulfides, those microorganisms had better environmental adaptability gradually increased, which was reflected in the rebound of MLVSS concentration. However, after 38 days of operation, the SV₅ and SVI₅ values of the sludge increased, implying that the settling performance of the sludge deteriorated. This may be attributed to the toxic effects of polysulfides on floc-forming bacteria, placing them at a competitive disadvantage against filamentous bacteria, thereby resulting in a deteriorated sludge-settling performance [36].

3.3.3. Changes in Microbial Community Structure

The microbial community structure of sludge before and after the operation of polysulfide-based autotrophic denitrification process was analyzed, with emphasis on genus-level changes among sulfur-autotrophic denitrifying microorganisms. As shown in Figure 16, the relative abundances of four key genera, Thiobacillus, Thauera, Pseudomonas, and Thiothrix, increased significantly after 38 days of operation, with fold increases of 124.3, 1.5, 0.6 and 1.0, respectively.

Among them, Thiobacillus was the dominant genus in the system with a relative abundance of 13.3%. Thiobacillus is recognized as one of the most common sulfur autotrophic denitrifying bacteria, and the significant enrichment indicated its key role in the process of sulfur autotrophic denitrification of polysulfides [37,38]. Members of Thauera are common heterotrophic denitrifying bacteria capable of utilizing organic substrates as electron donors. Recent studies have reported that some denitrifiers in sulfur-based systems have also been classified under the genus Thauera, with demonstrated metabolic capacity for sulfide utilization in autotrophic growth and denitrification [39]. Members of Pseudomonas have been reported in several studies to be able to participate in the sulfur cycle. They can utilize S⁰ and S^2- as electron donors for sulfur autotrophic denitrification [40]. In addition, Others include the genera such as Sulfuricella, Sulfuriferula, Sulfurimicrobium, Desulfuromonas, Sulfuritortu, and Sulfurivermis, which were closely associated with sulfur cycling. Although not directly involved in the autotrophic denitrification process, these genera are capable of utilizing sulfur sources such as sulfide and elemental sulfur as electron donors for autotrophic growth [41].

With the addition of polysulfides, the relative abundances of genera related to sulfur autotrophic denitrification and sulfur oxidation processes have increased. It is suggested that these microorganisms become progressively enriched within the polysulfide-based autotrophic denitrification system and play essential roles in sustaining the sulfur autotrophic denitrification process.

3.4. Cost Analysis

During the stable operation of the polysulfide-based autotrophic denitrification process, a $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal of 7.3 mg/L was achieved at a polysulfide dosing concentration of 19.2 mg S/L. Na₂S₃ was synthesized using the sodium sulfide method, with a molar ratio of sodium sulfide to sulfur powder of 1:2. The market price of sodium sulfide is approximately CNY 2300/ton (CNY 2.3/kg), while sulfur powder is relatively inexpensive, typically ranging from CNY 1500 to 2500/ton (CNY 1.5 to 2.5/kg). Cost values for sodium sulfide and sulfur powder were based on typical market prices in China, obtained from industrial procurement references and supplier quotation. Consequently, the estimated cost of producing Na₂S₃ is CNY 1.9~2.4/kg. Then the cost-effectiveness of the electron donor can be evaluated as CNY 0.036~0.046/m³, and the cost-effectiveness of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal can be evaluated as CNY 4.9~6.3/kg·$\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$.

4. Conclusions

In this study, the feasibility of polysulfide-based autotrophic denitrification for nitrogen removal was evaluated using an aerobic/anoxic sequencing batch reactor. The type and dosage of polysulfides were optimized. Polysulfides synthesized chemically from sodium sulfide and sulfur powder could serve as electron donors to facilitate autotrophic denitrification. Among them, Na₂S₃ demonstrated better denitrification efficiency, lower preparation cost, and greater application potential. Under the dosage for the same theoretical $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removal, the Na₂S₃ system removed $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ of 7.1 mg/L, achieved a sulfur utilization efficiency of 71.2%, and generated $\mathrm{S}{\mathrm{O}}_4^{2-}$ of 47.0 mg/L. When the dosages of Na₂S₃ were 12.8 mg S/L and 19.2 mg S/L, the sulfur utilization efficiencies were 85.7% and 85.5%, respectively, with 4.9 mg/L and 7.4 mg/L of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ removed. Further increasing the polysulfide dosage resulted in decreased sulfur utilization. During stable operation of the polysulfide-based autotrophic denitrification process, the system removed 7.3 mg/L of $\mathrm{N}{\mathrm{O}}_3^{-}\text{-}\mathrm{N}$ with no $\mathrm{N}{\mathrm{O}}_2^{-}\text{-}\mathrm{N}$ accumulation, achieving a sulfur utilization efficiency of 83.7% at an Na₂S₃ dosage of 19.2 mg S/L. The process generated less sludge. However, sludge settling performance deteriorated after 38 days of continuous operation. Moreover, the addition of polysulfides promoted the enrichment of sulfur autotrophic denitrification-related microorganisms. Among them, Thiobacillus was the dominant genus, with a relative abundance of 13.3%, representing a 124-fold increase compared to conditions without the addition of polysulfides.