Application Potential of Sulfur-Based Autotrophic Denitrification in Low Carbon Wastewater Treatment: Efficiency, Cost and Greenhouse Gas Emission Reduction

Abstract

With the continuous improvement of wastewater treatment standards, advanced nitrogen removal from municipal wastewater treatment plant effluents faces severe challenges. This paper systematically analyzes the application potential of sulfur-based autotrophic denitrification (SAD) technology in advanced wastewater treatment, focusing on its denitrification efficiency, operational costs, and carbon reduction benefits. Compared to conventional heterotrophic denitrification (HD), SAD technology demonstrates significant advantages, including high denitrification efficiency, low operational costs, low sludge production, and low CO₂ emission, through the reduction of external organic carbon source addition and energy consumption. Among the autotrophic denitrification processes, SAD has the highest denitrification rate with low cost and low safety risk. Through sulfur source selection and process optimization, the denitrification rate could reach 1.2 kg N/m³·d, and the accumulation of byproducts can be effectively controlled. As calculated, SAD can reduce over 55% sludge production, reduce 50–80% operational costs, and reduce over 80% greenhouse gas (GHG) emissions. Despite challenges such as long start-up periods, SAD technology shows promising application prospects for advanced treatment of low C/N ratio wastewater. Future research should focus on process optimization and scale-up engineering applications to promote the large-scale implementation of this technology.

1. Introduction

In China, wastewater treatment contributes about 1–2% of the country’s overall carbon emissions [1]. Driven by the goal of carbon neutrality, the limit of total nitrogen in effluent is becoming more and more strict in the field of urban sewage treatment [2]. Meanwhile, the need for effective nitrogen removal from wastewater with a low carbon-to-nitrogen ratio has grown more pressing, which has led to an important change in the denitrification strategy in the process of biological nitrogen removal [3]. Under the guidance of low-carbon development, the sewage treatment industry is facing the dual challenges of synergistic regulation of nitrogen removal efficiency and carbon emissions.

At present, conventional biological nitrogen removal (BNR) technologies, which rely on microbial metabolism, are still widely applied, but their performance and sustainability are limited by many factors [4]. During denitrification, a large amount of oxygen for aeration is required, along with additional carbon sources to compensate for alkalinity consumption [5]. Nevertheless, the accumulation of external carbon may lower the denitrification rate and lead to nitrite accumulation. As a result, traditional heterotrophic denitrification (HD) is often associated with several limitations, including high energy demand, high operational costs, excessive sludge production, and dependence on external carbon sources and potential secondary pollution [6,7,8]. These drawbacks contradict the principles of low-carbon development, thereby necessitating the investigation and implementation of novel BNR strategies that align with environmental protection policies, ecological balance, and energy-saving objectives [9].

In traditional HD, external carbon sources are often added to provide electron donors; that is, external carbon sources are used to assist biological denitrification. Methanol, ethanol, and acetic acid/acetate are the most commonly used carbon sources. However, their higher market prices have led to increased operating costs, and there are also flammable and explosive risks for methanol and ethanol [10]. Although the solid carbon source is usually cheaper, the carbon release rate is usually unstable, which affects the controllability of the denitrification process. In addition, although some newly developed carbon sources have alleviated the above problems to some extent, they still face limitations such as high cost, temperature sensitivity, component complexity, and possible environmental risks caused by by-products [11]. Therefore, there are still multiple challenges such as economy, safety and stability in the practical application of external carbon sources, and it is urgent to develop more sustainable alternative carbon sources and optimize dosing strategies.

In this context, autotrophic denitrification has received increasing attention as a low-carbon alternative, which can significantly reduce the risk of organic pollution and excess sludge generation. Hence, this approach is regarded as an effective, appropriate, and eco-friendly biological method for nitrogen removal [12]. Autotrophic denitrification relies on inorganic substances such as hydrogen (H₂), reduced sulfur compounds, and ferrous ions as electron donors [13]. Among them, sulfur-based autotrophic denitrification (SAD) demonstrates notable benefits in achieving advanced nitrogen removal from municipal wastewater. This process obtains energy through the oxidation of reduced sulfides and uses nitrate as the terminal electron acceptor [14]. SAD not only has low operating costs and no direct carbon dioxide emissions, but also has extremely low biomass and sludge yield due to its autotrophic characteristics [15]. Given these advantages, the SAD process has gained wide attention as a promising option for lowering nitrate concentrations in both water and wastewater treatment (Figure 1). The pilot-scale fixed-bed and packed-bed systems have achieved efficient denitrification in a short hydraulic retention time in the treatment of secondary effluent [12]. The key functional microorganisms in SAD are mainly Thiobacillus and Thiomonas, which carry complete denitrification gene clusters [16]. A successful biological nitrogen removal process usually depends on a stable and balanced microbial community, and various functional floras ensure system stability and treatment performance through synergy [17].

In general, the pilot and comprehensive studies on municipal wastewater show that SAD is a practical, environmentally friendly and economically feasible deep nitrogen removal technology [18]. The purpose of this paper is to systematically compare the differences in nitrogen removal efficiency between SAD and traditional HD and other autotrophic denitrification processes, explore its cost-effectiveness improvement strategy, and analyze its operating costs compared with traditional technologies. In addition, this review also evaluates the potential of SAD in carbon emission reduction and broader environmental benefits and, finally, clarifies the application prospect of this technology in urban sewage treatment, pointing out the key direction of future process optimization.

2. Metabolic Mechanisms and Efficiency of SAD

2.1. Electron Donor Metabolic Mechanisms and Key Microbial Differences of SAD

Among various autotrophic denitrification and HD pathways, SAD has attracted significant attention due to its unique electron donors and metabolic mechanisms, as is shown in Figure 2. In this process, CO₂, HCO₃⁻, and CO₃²⁻ serve as inorganic carbon sources, while reduced sulfur compounds or iron sulfides act as electron donors. Biofilms formed on the donor surface function as reduction sites for nitrate, and the energy released from chemolithoautotrophic oxidation drives the denitrification reaction [19]. To elucidate the coupling between reduced sulfur oxidation and nitrate reduction, the metabolic pathways and electron transfer mechanisms of SAD are illustrated below (Figure 3).

SAD both supplies electrons from reduced sulfides and shapes microbial communities [24]. Within SAD systems, Thiobacillus and Thiomonas are the most critical genera [25]. Although their growth rates are relatively slow and their abundance is typically 30–40%, both harbor complete sulfur-oxidation and denitrification gene sets, and their complementary metabolic behaviors enable complete nitrate reduction [12]. Meta-transcriptomic and meta-genomic annotations revealed that sox clusters, sqr, and classical denitrification-encoding genes (narG/napA, nirS/nirK, norB, nosZ) were all significantly detected in SAD reactors, exhibiting pronounced expression changes upon supplementation with different electron donors or exposure to thermal shock, indicating that the dynamic gene expression of microbial communities is a key mechanism explaining operational performance fluctuations [26]. When elemental sulfur is used as the electron donor, the microbial metabolism is more complex compared with soluble reduced sulfur compounds, leading to the highest microbial diversity in SAD systems [27].

In contrast, HD generally relies on methanol, acetate, or other soluble organic carbon sources as electron donors, degrading organic matter to obtain energy. Although the process is relatively simple and its functional microorganisms—primarily Pseudomonas and Ferribacterium—exhibit rapid growth rates and high denitrification activity [28], the electron donor utilization efficiency and nitrate reduction potential are often lower than those of SAD [29]. This highlights the advantage of SAD in terms of electron donor efficiency and nitrogen removal performance, especially in low C/N conditions.

Other autotrophic denitrification pathways provide additional options for nitrate removal. Hydrogen autotrophic denitrification (HAD) uses H₂ as a clean electron donor. Due to its low ionization energy, electrons can be readily released without intermediate reactions, granting a clear kinetic advantage and allowing “zero organic carbon addition.” However, its performance is highly constrained by gas–liquid mass transfer and hydrogen supply strategies [30]. The dominant microbial genera in HAD systems include Paracoccus, Rhodobacter, Acinetobacter, and Pseudomonas [31], indicating a partial overlap with both SAD and autotrophic denitrification microbial communities.

Research indicates that multivalent metal cycling can significantly regulate electron flow and reactive species formation [32]. Iron-driven and pyrite/pyrrhotite-based autotrophic denitrification (IAD) represents another important pathway. In the Fenton process, the Fe³⁺/Fe²⁺ cycle achieves pollutant degradation through electron transfer, whereas in IAD, Fe(II)/Fe(0) acts as an electron donor to drive nitrate reduction [33].In Fe(II)-based liquid systems, core microbial consortia typically consist of Gallionellaceae, Acidovorax, and Dechloromonas, which together form the “iron oxidation–denitrification” mechanism [34]. In contrast, Fe(0)-based systems are enriched with classical denitrifiers such as Pseudomonas and Paracoccus, accompanied by iron-cycling microorganisms [35]. For FeS₂/FeS mineral systems, sulfur-oxidizing genera including Thiobacillus, Sulfurimonas, Sulfuricurvum, and Sulfuricella play a dominant role in driving denitrification [36]. By comparison (

Table 1. Comparative Analysis of Typical Functional Microorganisms in AD and HD Pathways.

Type	Representative Functional Genera	Dominant Phyla	Environmental Preferences/Sensitivities	Refs.
HD	Pseudomonas, Paracoccus, Acinetobacter, Bacillus	Proteobacteria, Firmicutes	Fast growth, requires sufficient organic carbon, highly sensitive to DO and temperature changes	[28]
IAD	Gallionella (Gallionellaceae), Acidovorax, Dechloromonas, Thiobacillus	Proteobacteria, Nitrospirae	Kinetics generally slower; strongly affected by mineral dissolution/passivation, pH, alkalinity; requires longer HRT	[34]
HAD	Hydrogenophaga, Paracoccus, Thauera, Pseudomonas	Proteobacteria, Bacteroidetes	Sensitive to gas–liquid mass transfer; temperature-sensitive; prone to NO2− accumulation at low temperature	[37]
SAD	Thiobacillus, Sulfurimonas, Sulfuricurvum, Sulfuricella	Proteobacteria	Tolerant to salinity and sulfate stress; diverse sulfur-oxidizing pathways; performs well under low C/N conditions	[38]

), the differences in the primary microbial populations of SAD and the predominant bacterial genera of HD, HAD, and IAD, as well as their environmental preference sensitivity, were identified.

Through the above comparative analysis, different denitrification pathways exhibit distinct characteristics in terms of electron donor selection, microbial community structure, and reaction kinetics. HD benefits from high microbial growth rates and well-established engineering applications, but it is strongly dependent on external carbon sources and often associated with higher sludge production and secondary pollution risks. HAD shown emerging potential, yet its performance remains constrained by gas–liquid mass transfer limitations, temperature sensitivity, and hydrogen supply strategies. IAD generally proceeds at slower kinetics and is strongly affected by mineral dissolution and passivation, as well as pH and alkalinity variations [39]. In contrast, SAD demonstrates relatively high electron donor utilization efficiency while maintaining diverse and stable microbial communities [40].

2.2. Efficiency of SAD Compared to Conventional HD Processes

Compared with HD, SAD showed significant characteristics and potential in nitrate removal efficiency, volumetric loading capacity and adaptability to low C/N ratio wastewater [22]. X. Li et al. [41] demonstrated that HD can achieve denitrification rates as high as 1.4 kg N/(m³·d) during the start-up phase, with total nitrogen removal rates reaching 90–99%. However, the effluent total nitrogen concentration struggles to be further reduced to below 5 mg/L. The work of S.-S. Wang et al. [42] further indicated that under COD loading conditions below 90 mg/L, the denitrification efficiency of HD filters is similar to that observed in SAD filters; However, as COD concentration increases (C/N ratio rises), HD exhibits higher denitrification efficiency but is accompanied by a greater risk of COD exceedance.

Regarding electron donor types, common carbon sources for HD include methanol/ethanol, acetate/volatile fatty acids (VFAs), glucose, and starch/solid-phase slow-release carbon sources (SRC). Among these, acetic acid and VFAs have garnered increasing attention in recent years due to their kinetic advantages and potential for on-site preparation via anaerobic fermentation [10,43]. Methanol/ethanol, favored for its mature process and readily available supply, remains widely adopted in engineering applications despite posing certain safety concerns and risks of elevated effluent COD [44]. Glucose-based carbon sources, while readily assimilated, may increase N₂O emissions and alter microbial community structures under specific operating conditions, as indicated by some studies [45]. Solid-phase starch and other SRC are suitable for decentralized or deep polishing applications, reducing the need for continuous dosing. However, they exhibit lower peak rates, and their release behavior is significantly influenced by material properties and operational conditions [46].

In contrast, SAD demonstrates superior advantages in specific scenarios due to its excellent adaptability to low C/N ratios and high-salinity water bodies (e.g., seawater, aquaculture effluent, saline groundwater) [12]. Sun et al. [47] noted that due to the longer generation time of autotrophic communities, SAD system startup is generally slower than in heterotrophic systems. However, composite media and anti-clogging measures can effectively shorten the startup period while maintaining high flux. Concurrently, pilot-scale studies have demonstrated that S⁰-fixed-bed filters can achieve nitrate loading rates of 1158 mg/(L·d) and reaction rates of 164 g NO₃⁻-N/(m³·h) during long-term operation [12], while Li [48] reported TN removal rates of 96.4% in constructed wetland systems under 24 h HRT conditions.

In summary,

Table 2. Comparison of Denitrification Efficiency Between SAD and HD Under Different Carbon Source Conditions.

Type	Carbon Source	Applicable Scenarios	Representative Removal Efficiency	Representative Volumetric Rate/Load	Operational Considerations/By-Products	Refs.
HD	Methanol	Municipal plant post-denitrification, media bed/biological filter	Typically, 90–99% removal under proper dosing	Pilot reports show instantaneous rates from 0.5 up to 1.4 kg N·m−3·d−1 in optimized systems or startup peaks	Note the flammability risks and safety requirements; over-injection may cause COD levels in effluent to exceed standards and pose N2O risks	[11,44]
	Acetate/VFAs	Tertiary/post denitrification, SBRs, packed beds	Very high >90% NO3− removal; faster kinetics and higher specific denitrification rates than methanol	Higher specific denitrification rates reported vs. methanol; Volumetric rates depend on HRT/biomass	Fast kinetics, can be prepared on-site, requires fermentation equipment/raw materials, ingredient fluctuations, over-dosage may leave residual COD	[10,49]
	Glucose	Primarily used for laboratory mechanism research and certain pilot projects	Achieves high nitrate removal ≈85–95% (under appropriate C/N ratio and HRT)	Initial/instantaneous rates are high (easily assimilated by microorganisms), but long-term rates depend on community succession	High instantaneous rates require long-term monitoring. Nitrite/N2O issues are prone to occur in high-load/short HRT systems	[50,51]
	Starch/SRC	Decentralized/remote wastewater treatment stations, aquaculture water polishing	Long-term removal efficiency typically ranges from 70–95%	Instantaneous volumetric flow rates are generally lower than for soluble small-molecule carbon, exhibiting lower peaks and long-term stability	Release rates depend on material, temperature, and microbes; show initial lag; need replacement; risk clogging, local anaerobia, and short COD spikes	[52,53]
SAD	-	Low C/N municipal secondary effluents for advanced nitrogen removal; saline/brackish groundwater	In most studies, NO3−-N removal >90%; Constructed wetlands and filters treating real/simulated tailwater reach 96%	Fixed bed/packed filter: peak 164 g NO3−-N·m−3·h−1 (≈3.9 kg N ·m−3·d−1); long-term maximum load ≈ 1158 mg·L−1·d−1(≈1.158 kg N·m−3·d−1); stable even at short HRT	Manage sulfate formation and alkalinity consumption; anti-clogging and backwashing to maintain hydraulics; suppress H2S accumulation	[12]

summarizes the denitrification efficiency under different carbon source conditions for HD and compares it with SAD. The results indicate that HD offers higher rates and established process advantages but is constrained by carbon source availability, COD risks, and N₂O emissions. In contrast, SAD demonstrates superior stability and stress tolerance under low C/N ratios and saline conditions. Therefore, carbon source selection and process adaptation require comprehensive trade-offs between rate, operational convenience, safety, greenhouse gas (GHG) control, and cost.

2.3. Efficiency of SAD Compared to Other Autotrophic Denitrification Processes

The AD process utilizing inorganic sulfur (elemental sulfur, sulfides, or sulfide minerals such as pyrite/chalcopyrite powder) as an electron donor requires no external organic carbon input. Consequently, it is particularly suitable for polishing/deep denitrification of secondary effluent, agricultural runoff, aquaculture wastewater, and certain industrial effluents characterized by organic carbon scarcity. This process represents an engineering-wise highly attractive low-carbon/low-chemical-consumption alternative technology [54].

Han et al. [55] developed an electrochemical–membrane composite hydrogen supply HAD process, which maintained stable operation and achieved a 97.8% nitrate removal efficiency when applied to real groundwater, with effluent NO₃⁻-N levels around 2.2 ± 1.0 mg/L. Thant et al. [56] reported the coupling process of sulfur autotrophic denitrification bed and vertical flow constructed wetland (SADN-VFCW), which achieved high nitrate removal under short HRT and was suitable for deep nitrogen removal and reuse of sewage. Although both SAD and HAD are feasible in low C/N wastewater treatment, their limiting mechanisms are different: HAD is temperature-sensitive and prone to nitrite (NO₂⁻) accumulation, while SAD is more tolerant to temperature changes [57]. Compared with IAD, SAD is more stable and has a higher volumetric rate under short HRT and high load Ntagia and Lens [58]. Research indicates that vertical flow constructed wetlands (IC-CW) based on zero-valent iron–carbon micro-electrolysis (IC-ME) technology have been developed for advanced treatment of reclaimed water [59]. Although IAD can still achieve high removal rate in low C/N groundwater or drinking water treatment, it usually requires longer contact time. For example, the denitrification rate is 134.7 ± 14.1 mg NO₃⁻-N/(L·d) at HRT of about 18 h, and the removal efficiency is 81.8 ± 6.0%, reflecting its relatively slow reaction kinetics. Research has found that electron donation from Fe⁰-dependent redox cycles, in synergy with carbon carriers, can significantly enhance the rate and stability of IAD [60].

Table 3. Comparative Analysis of the Denitrification Efficiency of SAD and Other Autotrophic Denitrification Technologies.

Type	Applicable Scenarios	Representative Removal Efficiency	Representative Volumetric Rate/Load	Achievable Effluent NO3−–N NO3−-N	Operational Considerations	Refs.
SAD	Low C/N municipal secondary effluents for advanced nitrogen removal; reclaimed water; saline wastewater	Typically 92 ± 5%, with reported ranges of 80–98% under various operational conditions.	Peak: 164 g NO3−-N·m−3·h−1 (≈3.9 kg N·m−3·d−1); maximum load: 1158 mg·L−1·d−1 (≈1.158 kg N·m−3·d−1) (fixed-bed/packed filter)	Typically, 2–10 mg·L−1; <5 mg·L−1 achievable in advanced processes (municipal effluents)	Sulfate generation and alkalinity consumption; risk of clogging, requires backwashing to maintain hydraulic performance	[12]
HAD	Groundwater/drinking water denitrification; post-treatment of reclaimed water	90–99%; 97.8% in real groundwater	On the order of 135 g N·m−3·d−1 (≈0.135 kg N·m−3·d−1) under optimized MBfR conditions; strongly affected by H2 supply and mass transfer	2.2 ± 1.0 mg·L−1 (real groundwater, continuous operation)	H+ production causing pH decrease; requires alkalinity or CO2 dosing; sensitive to temperature and mass transfer	[61]
IAD	Groundwater/drinking water (low C/N); mineral-driven systems	Typically, 85 ± 7%, with reported ranges of 70–95% under comparable hydraulic and nitrogen loading conditions; ≈94% at HRT of 24 h	Continuous-flow P-FBR: 168 mg NO3−·L−1·d−1 (≈0.168 N kg·m−3·d−1) at HRT of 24 h; efficiency drops significantly at 12 h, with NO2− accumulation	10 mg NO3−·L−1 (as NO3−; at HRT of 24 h)	Limited by mineral dissolution/passivation; influenced by pH/alkalinity and co-release of metals/sulfate; sensitive to organic co-substrates	[58]

summarizes the performance comparison between SAD and other autotrophic denitrification processes. Compared with other autotrophic denitrification processes, SAD has a more stable nitrate removal capacity and a higher reaction rate under low C/N conditions, which is more suitable for actual urban sewage purification scenarios.

SAD has demonstrated clear engineering feasibility and cost advantages at field/engineering scales (particularly for organic-poor effluents and sites with readily available low-cost sulfur sources). However, its long-term stable operation relies on engineered control of backwash strategies, pH/alkalinity compensation, and sulfate byproduct loading. Concurrently, routine monitoring of N₂O and sulfate impacts on downstream environments must be incorporated during scaled-up applications [12].

2.4. Denitrification Performance and Kinetics

To quantify the rate advantage of SAD, kinetic parameters of SAD and other denitrification systems (heterotrophic, hydrogen autotrophic, and iron autotrophic) were compiled and compared under comparable operating conditions (25–30 °C, influent nitrate 50–200 mg L⁻¹) from literature published over the past five years.

The reaction of SAD generally follows quasi-zero-order or pseudo-first-order kinetic models, depending on the particle size of the sulfur source and substrate concentration. Reported values for the “zero-order rate constant (r₀)” range from 20 to 350 mg NO₃⁻-N L⁻¹ h⁻¹, while the “first-order rate constant (k₁)” ranges from 0.05 to 0.35 h⁻¹ [62]. Under pilot-scale fixed-bed conditions, SAD achieved a maximum volumetric removal rate of 164 g NO₃⁻-N m⁻³ h⁻¹, indicating high reactivity at the S⁰–biofilm interface [63].

Traditional heterotrophic denitrification typically exhibits the highest apparent kinetics when soluble carbon sources are abundant. In common literature, its instantaneous or initial volume removal rates and empirical specific rates significantly exceed those of most autotrophic pathways. From the perspective of kinetic constants, the specific growth rate μmax of heterotrophic communities and the corresponding first-order or zero-order apparent constants generally fall within higher ranges. This enables HD to rapidly achieve high removal rates under conditions of short HRT and high loading [64]. HAD exhibits a kinetic profile characterized by “high potential rates but engineering limitations”: under membrane-based hydrogen feed (MBfR) or high-efficiency gas–liquid mass transfer conditions, HAD exhibits exceptionally high instantaneous surface flux and volumetric rates (with apparent first-order constants or initial rates approaching and, under certain conditions, exceeding reported engineering rates for SAD when hydrogen supply and mass transfer are sufficient). However, in conventional liquid-phase reactors, the estimated first-order apparent constants decrease significantly due to low H₂ solubility and mass transfer limitations, exhibiting greater temperature sensitivity [61]. IAD typically exhibits the slowest kinetic response: its rate is constrained by mineral dissolution rates, surface passivation, and accompanying chemical reactions (pH, alkalinity, dissolved metals). Consequently, when given similar HRT, IAD often demonstrates lower apparent first-order constants or requires longer contact times to achieve comparable removal rates [58].

Overall, SAD exhibits a favorable balance between rate and stability. Its volumetric rate exceeded heterotrophic systems while offering lower energy consumption, reduced sludge production, and significantly decreased greenhouse gas emissions. Its near-zero-order reaction characteristic indicates that the overall rate is controlled by electron donor (S⁰) diffusion rather than enzyme limitation. Future studies can further obtain intrinsic rate constants (kint) and apparent activation energies through standardized kinetic experiments with uniform HRT, packing ratio, and biomass conditions, enabling comparability across different systems.

3. Costs and Strategies to Improve the Economic Efficiency of SAD

3.1. Comparison with the Cost of the Traditional HD Process

SAD operating costs primarily include expenses for chemical reagents (electron donors and alkalinity-adjusting agents) and energy consumption. A comparison of operating costs between HD and SAD systems from various studies is presented in Table 4. Compared to the HD process, SAD is an economical biological denitrification technology that can reduce operating costs by approximately 80%. Firstly, the price of reduced inorganic sulfur compounds (RISCs) (such as S⁰, S²⁻, S₂O₃²⁻, or SO₃²⁻: 0.13–0.25 $/kg) [12] is much lower than that of conventional organic carbon sources (such as methanol or acetate: 0.4–1.6 $/kg) [65]; Secondly, compared to HD, the SAD process reduces sludge production, thereby lowering sludge treatment costs. Furthermore, under equivalent denitrification efficiency, electron donor consumption in SAD is 19–59% lower than in HD [66].

In the pilot-scale SAD bioreactor for actual wastewater treatment, the total operating cost can be as low as USD 0.016 per ton, of which the chemical reagent cost is about USD 0.012 per ton [42]. Under the same denitrification rate (nitrate removal rate of 15 mg N/L) and hydraulic retention time (1.4 h), the operating cost of the HD process with sodium acetate as carbon source was about USD 0.037 per ton, indicating that SAD could save more than 50% of the operating cost, showing better economy and industrial application potential. Another study based on enrichment of autotrophic denitrifying bacteria (using elemental sulfur) showed that the total treatment cost was about 0.38 euros per cubic meter of wastewater, providing a reference for laboratory-scale autotrophic denitrification cost estimation [67]. Liu et al. [16] further pointed out that the pyrite-assisted autotrophic denitrification (PSAD) biofilter can save about 17% of the cost compared with traditional SAD. The operating cost of the HD process is about 4.98 times that of SAD, while PSAD can reduce 83.33% compared with HD.

Table 4. Comparison of operating costs of HD and SAD systems based on comparable treatment efficiency and hydraulic retention time.

Process/System Type	Electron Donor/Carbon Source	Reported Operating Cost (USD/m3)	Notes	Unit Denitrification Cost (USD/m3)	Refs.
SAD filter	Elemental sulfur	0.016	The cost based on the consumables of HD was 2.23 times higher than SAD	0.16	[42]
HD filter	Acetic acid byproduct	0.037		0.37
SAD (lab)	Elemental sulfur	0.41	Real wastewater tested	4.1	[16]
Pyrite-assisted SAD (PSAD) biofilter	Pyrite	0.34	Lower cost due to pyrite price & reduced chemicals	3.4
HD biofilter	CH3COONa	2.04	Typical in municipal WWTPs	20.4
SAD filter	Elemental sulfur	0.009		0.09	[68]
HD filter	Sodium acetate	0.057		0.57
SAD filter	Autotrophic denitrifying filter media	0.029	SAD filters save approximately 37.9% in operating costs compared to HD filters	0.29	[69]

Table 4. Comparison of operating costs of HD and SAD systems based on comparable treatment efficiency and hydraulic retention time.

Process/System Type	Electron Donor/Carbon Source	Reported Operating Cost (USD/m3)	Notes	Unit Denitrification Cost (USD/m3)	Refs.
SAD filter	Elemental sulfur	0.016	The cost based on the consumables of HD was 2.23 times higher than SAD	0.16	[42]
HD filter	Acetic acid byproduct	0.037		0.37
SAD (lab)	Elemental sulfur	0.41	Real wastewater tested	4.1	[16]
Pyrite-assisted SAD (PSAD) biofilter	Pyrite	0.34	Lower cost due to pyrite price & reduced chemicals	3.4
HD biofilter	CH3COONa	2.04	Typical in municipal WWTPs	20.4
SAD filter	Elemental sulfur	0.009		0.09	[68]
HD filter	Sodium acetate	0.057		0.57
SAD filter	Autotrophic denitrifying filter media	0.029	SAD filters save approximately 37.9% in operating costs compared to HD filters	0.29	[69]

In summary, the operating cost of SAD is generally low when treating low carbon nitrogen ratio (C/N) wastewater, and its economic advantages are particularly prominent in terms of electron donor and energy costs. The actual cost varies with system scale, sulfur source type (such as elemental sulfur, pyrite, etc.), and process combination, but SAD still shows significant cost competitiveness under most configurations.

3.2. Strategies to Improve the Economic Efficiency of SAD

By optimizing the source of electron donors, improving the reactor design and developing the coupling process, the economic performance of SAD can be further improved (Figure 4).

First, replacing high-purity sulfur with low-cost or waste-source sulfur materials (such as pyrite or industrial desulfurization by-products) can effectively reduce material costs without affecting denitrification efficiency [70,71,72]. For example, Wang et al. [19] used natural pyrite as an electron donor to construct a pyrite autotrophic denitrification (PAD) system (Figure 4a). Yunfu pyrite (B-YPy38 and B-YPy48) exhibited outstanding performance, achieving a NO₃⁻-N removal efficiency of 88.6 ± 0.6% while maintaining relatively low costs.

In addition, the engineering carrier or composite filter material can improve the efficiency of sulfur utilization, prolong the service life of the material, and reduce the demand for electron donors required for unit denitrification. Tong et al. [73] prepared immobilized electron donors by loading elemental sulfur on polyurethane foam (PFSF) (Figure 4b). The denitrification efficiency was 97.3%, which was higher than that of sodium sulfite (91.1%), and the unit nitrate removal cost was only $0.9/kg NO₃⁻-N, which was much lower than the $8.0/kg NO₃⁻-N of the sodium sulfite system. A corncob-sulfur mixed nutrient filler was developed by Ma et al. [74]. In a packed-bed reactor, the removal rate of NO₃⁻-N was 98.62%, and the effluent concentration was as low as 0.31 mg/L, with low raw material cost and high treatment efficiency.

Secondly, from the standpoint of energy conservation and reduced consumption, integrating SAD with HD and anaerobic ammonia oxidation is anticipated to represent a key pathway for achieving high-efficiency, low-energy denitrification in the future [13,75]. Such coupled processes not only enhance nitrogen removal efficiency and lower energy use, but also enable the simultaneous removal of pollutants like chromate and suppress sulfate generation, making them particularly advantageous for treating industrial wastewater with high ammonia nitrogen levels [76]. The maximum denitrification rate of the pyrite-PHBV mixed nutrient denitrification system constructed by Zhou et al. [77] reached 0.65 mg NO₃⁻-N/(L·h), the sulfate production was less than 5 mg/L, the denitrification rate reached 96%, and the cost was significantly reduced (Figure 4c). Zeng et al. [78] used S⁰-driven autotrophic denitrification (S⁰AD) to treat leachate after partial nitrification/anammox pretreatment (Figure 4d), and its operating costs were 6 times and 8 times lower than those of thiosulfate and sulfide systems, respectively.

Figure 4. Strategies to improve economic efficiency of SAD (a) using natural pyrite as an electron donor [19]; (b) loading elemental sulfur on the surface of polyurethane foam to develop an immobilized electron donor [73]; (c) introducing PHBV and controlling the pyrite sizes achieved pyrite-based autotrophic–heterotrophic denitrification systems [77]; (d) elemental sulfur-driven autotrophic denitrification coupled partial nitritation and anaerobic ammonium oxidation (anammox) (PN/A) pretreatment [78].

Figure 4. Strategies to improve economic efficiency of SAD (a) using natural pyrite as an electron donor [19]; (b) loading elemental sulfur on the surface of polyurethane foam to develop an immobilized electron donor [73]; (c) introducing PHBV and controlling the pyrite sizes achieved pyrite-based autotrophic–heterotrophic denitrification systems [77]; (d) elemental sulfur-driven autotrophic denitrification coupled partial nitritation and anaerobic ammonium oxidation (anammox) (PN/A) pretreatment [78].

In summary, by selecting economic sulfur sources, improving sulfur utilization efficiency, and promoting process coupling, the operating cost of SAD can be further reduced, so that it has significant economic and environmental benefits in low C/N ratio wastewater treatment. In practical application, the above strategies should be flexibly selected and optimized according to the factors such as the price of regional sulfur resources, wastewater quality, and emission standards, so as to maximize the cost-effectiveness.

3.3. Integrated Testing and Optimization Strategies for Process Enhancement and Scale-Up of SAD

SAD has been widely recognized as an efficient and sustainable denitrification method in low-carbon wastewater treatment systems. Its advantages, including the absence of external organic carbon sources and low sludge production, have made it a focal point of research. However, the SAD process generates acid and consumes alkalinity, leading to pH decline and increased sulfate concentrations as byproducts, which limit its widespread application [12]. To address these challenges, the comprehensive testing framework originally developed for evaluating anaerobic biodegradability of industrial wastewater [79] can be adapted for systematic assessment and optimization of sulfur-driven denitrification processes. This approach bridges the gap between laboratory research and full-scale implementation.

Laboratory-scale testing serves as the initial step in assessing SAD feasibility, focusing on electron donor screening, pH and alkalinity optimization, and microbial community analysis. Research indicates that the type of sulfur source significantly influences reaction conditions and microbial community structure [80]. When sulfide or thiosulfate serves as the electron donor, denitrification can proceed across a broader pH range (pH 4–8); however, when elemental sulfur (S⁰) is used, maintaining pH above 6 is typically required to ensure activity. More critically, the acid-producing nature of the SAD process necessitates optimized pH buffering strategies. Static batch experiments elucidate denitrification kinetics and sulfur oxidation efficiency under varying S/N stoichiometric ratios, pH levels, and temperatures. These tests provide critical insights for optimizing nutrient balance and trace element supplementation, which is vital for maintaining electron transfer efficiency and promoting complete nitrate reduction.

During the transition to pilot-scale testing, integrated strategies should incorporate actual wastewater matrices to evaluate the robustness of sulfur-driven systems. Multi-stage reactors enable simultaneous assessment of hydraulic retention time, sulfur utilization efficiency, and denitrification performance. Studies indicate that under optimal operating conditions, SAD systems can shorten hydraulic retention time while maintaining high denitrification rates. For instance, Wang et al. [63] established a pilot-scale sulfur-based autotrophic denitrification system achieving an optimal hydraulic retention time of 0.21 h, a maximum denitrification load of 1158 mg/(L·d), and a denitrification rate of 164 gNO₃⁻-N/(m³·h)). Effective backwashing is fundamental to long-term stable and efficient denitrification performance, restoring normal denitrification capabilities within 0.5 h at an operational cost of only $0.013/t. Additionally, pilot-scale trials should focus on process coupling and hybridization, such as integrating SAD with other process systems to achieve energy-efficient total nitrogen removal. Zhang et al. [81] developed an integrated autotrophic denitrification process based on sulfur and zero-valent iron within an anoxic fluidized bed membrane bioreactor (AnFB-MBR). At hydraulic retention times of 1.0–5.0 h, the AnFB-MBR achieved nitrate removal rates of 1.22 g NO₃⁻-N L⁻¹d⁻¹ with NO₃⁻-N concentrations ranging from 40 to 200 mg L⁻¹. The autotrophic denitrification process using zero-valent iron exhibits an alkalinity-producing characteristic, perfectly compensating for the alkalinity consumption of the SAD process. This integrated system enables internal pH self-regulation, significantly reducing reliance on external alkalinity sources. It also substantially decreases sulfate production due to shared denitrification loads, achieving reductions of 29.3–70.3% and 31.2–50.9%.

At the full-scale application level, it is crucial to confirm the integrated system’s stable operation under actual water quality and temperature variations, validate optimal operational parameters such as HRT, and calculate its operating costs. Dynamic feedback control is achieved by integrating automated sensors for redox potential, pH, nitrate, and sulfide concentrations into a supervisory control and data acquisition system. Combined with process modeling tools like activated sludge or sulfur–nitrogen coupling models, this system enables predictive optimization of sulfur dosage, aeration strategies, and effluent quality.

Overall, by integrating laboratory inhibition screening, kinetic modeling, pilot-scale validation, and intelligent process control, this framework guides the design of robust, economical, and environmentally sustainable sulfur-driven denitrification technologies for next-generation low-carbon wastewater treatment plants.

4. GHG Emission Reduction Effects of SAD

4.1. GHG Reduction Effects

In recent years, the emergence of the circular economy and carbon neutrality concepts has shifted the focus of denitrification processes beyond mere pollutant removal. Increasing emphasis is now placed on resource and energy recovery, as well as carbon emission reduction [82]. There are significant differences in the composition of GHG emissions between SAD and HD (Table 5). During the HD process, the metabolism of organic carbon sources inevitably produces carbon dioxide (CO₂). Under operating conditions such as C/N ratio, dissolved oxygen (DO), carbon source type, pH and temperature, incomplete denitrification can also lead to the release of N₂O. The carbon emissions of the HD system include CO₂ and N₂O, and its greenhouse effect is about 298 times that of CO₂ [83].

In contrast, SAD uses reduced sulfur as an electron donor, does not rely on organic carbon sources, and does not produce CO₂ during metabolism [12]. Its GHG emission mainly comes from N₂O, which is usually produced under conditions of high nitrate concentration or insufficient electron donors [70]. The formation of N₂O can be effectively inhibited by maintaining a low NO₃⁻-N concentration and sufficient electron donors. Elemental sulfur (S⁰) or sulfides serve as stable, low-energy electron donors. Recent studies indicate that under comparable nitrate loading conditions, SAD systems exhibit significantly lower N₂O emissions than heterotrophic systems. N₂O emissions in the SAD system usually account for 0.01–0.8% of the NO₃⁻-N load, which is much lower than that in the HD process (N₂O emissions account for 2.3–20% of the NO₃⁻-N load) [12] The emission reduction mechanism can be explained through two distinct pathways: thermodynamic characteristics of electron donors and differences in biochemical enzyme pathways. Their slow rate of electron release upon oxidation maintains the system at a relatively stable reduction potential (Eh ≈ +200–350 mV), facilitating the complete reduction sequence of denitrification: NO₃⁻ → NO₂⁻ → NO → N₂O → N₂. Thermodynamically, the standard free energy of the coupled oxidation of S⁰ and reduction of nitrate is approximately −743 kJ mol⁻¹ N. This stable energy release prevents the accumulation of intermediate products in heterotrophic systems caused by fluctuations in organic carbon [84]. The dominant bacterial genera in the SAD system all carry complete narG–nirS/nirK–norB–nosZ gene clusters, with significantly enhanced N₂O reductase activity encoded by nosZ. Metagenomic and transcriptomic results indicate that the abundance of the nosZ gene is approximately 1.5–3 times higher than in heterotrophic systems, thereby strengthening the N₂O → N₂ reduction process [26]. In addition, the iron–sulfur autotrophic denitrification (ISAD) system usually has an emission level below the detection limit due to the lack of compounds that induce N₂O production, which is significantly lower than that of pure sulfur or iron-based denitrification systems [70]. For example, Feng et al. [85] evaluated three types of vertical flow constructed wetlands (VFCWs) filled with quartz sand (C-CW), elemental sulfur (S-CW), and a sulfur–siderite mixture (SS-CW). The results indicated that SS-CW attained the highest total nitrogen removal efficiency (91.6 ± 2.2%) while simultaneously producing the lowest CH₄ and N₂O emissions.

Table 5. Comparison of GHG emissions between HD and SAD.

Process Type	Electron Donor	N2O Emission (% of Nitrate Load)	Carbon Emission (g/m3)	Refs.
HD	Methanol	-	35.48	[86]
SAD	S0	-	−14.19
	S2O32−	-	−12.31
	FeS2	-	−40.16
HD	Methanol	3.01	-	[87,88,89]
	Acetate	2.3–19.91	-
SAD	Sulfur	0.01−0.8	-

Notes: Carbon emission is calculated for the anaerobic stage for the denitrification of 50 mg/L of NO₃⁻ to N₂.

Table 5. Comparison of GHG emissions between HD and SAD.

Process Type	Electron Donor	N2O Emission (% of Nitrate Load)	Carbon Emission (g/m3)	Refs.
HD	Methanol	-	35.48	[86]
SAD	S0	-	−14.19
	S2O32−	-	−12.31
	FeS2	-	−40.16
HD	Methanol	3.01	-	[87,88,89]
	Acetate	2.3–19.91	-
SAD	Sulfur	0.01−0.8	-

Notes: Carbon emission is calculated for the anaerobic stage for the denitrification of 50 mg/L of NO₃⁻ to N₂.

In summary, from the perspective of the composition and formation mechanism of GHG emission, SAD effectively alleviates the dependence on organic carbon, thereby reducing carbon emissions during denitrification and the carbon footprint of biological nitrogen removal [90]. However, although SAD does not directly produce CO₂, it has potential N₂O release problems, and attention should still be paid to the accumulation and release of N₂O that may be caused by operating conditions (such as low pH) [91].

4.2. Environmental Benefits Assessment of SAD

SAD utilizes inorganic sulfides, such as S₂O₃²⁻, S⁰, S²⁻, and FeS_2, as electron donors without the need for additional organic carbon sources. This approach not only saves the economic cost of purchasing and adding carbon sources but also avoids the secondary pollution risk associated with effluent caused by organic carbon addition [92]. This feature makes SAD suitable for treating wastewater with a low carbon-to-nitrogen ratio (C/N) [93]. In addition, the microbial yield of SAD is low, and the excess sludge production is usually 55% less than that of HD [12], which significantly reduces the environmental burden of sludge treatment, transportation and final disposal.

However, SAD inevitably produces sulfate (SO₄²⁻), and excessive concentrations of SO₄²⁻ in water can cause harm to aquatic ecosystems. Excessive human intake can also lead to diarrhea, dehydration and gastrointestinal diseases. SAD can efficiently remove nitrate nitrogen without added organic carbon, but it is accompanied by sulfate formation (S⁰ → SO₄²⁻). Based on the stoichiometry of elemental sulfur (5S⁰ + 6NO₃⁻ + 2H₂O → 5SO₄²⁻ + 3N₂ + 4H⁺), theoretically, approximately 5.72 mg SO₄²⁻ is produced for every 1 mg NO₃–N NO₃⁻-N removed (calculated in this study). However, measured ratios in pilot and engineering studies often deviate significantly from this value (reported ranges in the literature: ~0.86–7.54 mg SO₄²⁻/mg NO₃–N NO₃⁻-N). This indicates that processes such as incomplete oxidation, sulfur reduction, precipitation, or dilution occur in actual systems, resulting in measured sulfate levels below theoretical values [40]. At present, China’s drinking water standard stipulates a SO₄²⁻ concentration limit of 250 mg/L [87]. If not effectively controlled, SO₄²⁻ may be converted into sulfides by sulfate-reducing bacteria (SRB), especially in reducing environments such as sediments and soils [94]. Therefore, effective sulfate control strategies must be developed.

The emission reduction strategy mainly includes two aspects: source control and end treatment. On the source side, sulfate production can be effectively reduced through mixed nutrient denitrification [95], precise control of sulfur conversion pathways (S²⁻ to S⁰), and the development of new S⁰ recovery technologies. For example, the foamed FeSO₄ modified limestone sulfur concrete (FFLSC) prepared by Chen et al. [96] did not detect H₂S at HRT of 1 h, mainly due to the formation of FeS inhibiting the release of H₂S. In terms of end treatment, reverse osmosis, reverse electrodialysis, chemical technology and biological treatment can be used to reduce the concentration of SO₄²⁻ in the effluent to the allowable range of regulations, but these technologies may bring higher operating costs [70,87]. Wang et al. [97] coupled SAD with microbial electrochemical denitrification system, which reduced the demand for sulfur while achieving deep denitrification, and provided a new idea for nitrate pollution control under the limited electron donor scenario.

4.3. Sustainability Discussions from a Life Cycle Perspective

Building upon economic and GHG emission assessments, this study further examines the environmental sustainability of SAD process from a life cycle assessment (LCA) perspective [98].

Compared to conventional heterotrophic denitrification processes, SAD exhibits significantly reduced carbon source requirements. This enables the reduction of energy consumption and indirect carbon emissions associated with the production and transportation of external organic carbon sources (such as methanol, acetic acid, etc.) within the system boundaries [63]. According to recent LCA studies, sulfur-based denitrification can reduce energy consumption and carbon footprint per unit of nitrogen removal by approximately 30–60% and 40–70%, respectively [30]. SAD-generated fluoride- or sulfur-containing sludge can be stabilized for use in construction materials or metal recovery. Concurrently, some studies indicate that the sulfate solution produced during the reaction can undergo chemical conversion (e.g., recovery of calcium sulfate and sodium sulfate) for secondary resource utilization. Compared to conventional denitrification, this pathway holds potential value for “byproduct resource recovery” [26].

From the perspective of comprehensive lifecycle benefits, sulfur autotrophic denitrification demonstrates higher environmental sustainability in terms of energy consumption, carbon emissions, and resource recovery potential. However, its actual environmental performance remains contingent upon operational management (alkali replenishment, backwash frequency), sulfur source acquisition methods (natural/byproduct sulfur), and downstream byproduct disposal pathways. Therefore, future full-scale validation studies should employ dynamic LCA or system scaling methods to quantify “net emission reduction benefits,” while conducting synergistic assessments with socioeconomic indicators (cost, resource efficiency) to establish a multidimensional sustainability framework.

5. Conclusions and Prospects

SAD demonstrates significant benefits for treating wastewater with a low C/N ratio. It provides high nitrogen removal efficiency, produces minimal sludge, adapts well to temperature variations, and maintains strong performance under high volumetric loading and long-term operation. Although the system has a long start-up time, the operation performance can be effectively improved by optimizing the carrier and the control strategy. In terms of economy, the operating cost of SAD is significantly lower than that of traditional HD. The cost of electron donor is low, the sludge production is low, and the cost can be saved by 50–80%. If the low-cost sulfur source and composite carrier are further used, the cost-effectiveness can be greatly improved. From the perspective of environmental sustainability, SAD avoids CO₂ emission from organic carbon metabolism, and the emission of N₂O is much lower than that of HD, which effectively reduces the carbon footprint. This process also lowers reliance on external organic carbon sources and minimizes the risk of excess organic matter in the treated effluent, but sulfate as a by-product, still needs to be effectively managed through source control or end treatment. In general, SAD represents a highly efficient, cost-effective, and eco-friendly approach to biological nitrogen removal, which is especially suitable for advanced treatment of urban sewage, low C/N ratio industrial wastewater and high salt wastewater and has important engineering application value.

Whereas, in the actual promotion and system optimization, SAD still faces a series of challenges, and future research needs to be promoted from both theoretical innovation and engineering application.

(1)

Future research should further explore the metabolic mechanism and community synergy of sulfur-oxidizing functional microorganisms (such as Thiobacillus and Thiomonas) and optimize the bacterial community structure through microbial ecological engineering methods to improve system stability and nitrogen removal efficiency.

(2)

To address issues such as low mass transfer efficiency and sulfur media clogging, there is an urgent need to develop composite sulfur carriers with high specific surface area, controlled-release properties, and excellent mass transfer performance. This will enhance electron transfer processes and effectively suppress sulfate accumulation.

(3)

To resolve alkalinity imbalance and microbial community instability, promote deep integration of SAD with low-carbon-consumption processes like anaerobic ammonium oxidation (ANAMMOX), establishing SAD–ANAMMOX synergistic systems to achieve self-balancing alkalinity and microbial ecological regulation, thereby enhancing system resilience to disturbances.

(4)

Pilot-scale and large-scale engineering studies should be intensified to establish intelligent control strategies based on real-time water quality and operational characteristics. This will drive the SAD process toward modularization, standardization, and intelligent upgrades, providing reliable support for its widespread application.