Insights into seasonal temperature shifts on a robust composite-substrate autotrophic denitrification reactor for treating municipal secondary effluent

Abstract

Sulfur autotrophic denitrification (SAD) is a promising biological nitrogen removal technology without CO₂ emissions. However, the impact of seasonal temperature variations on SAD performance, especially in the treatment of actual municipal secondary effluent, remains unclear. To address this issue, a composite substrate SAD reactor (i.e., SPSAD), where element S⁰ and pyrite (v:v/1:3) were uniformly mixed in hollow plastic balls that served as the filler, was developed in this paper. The performance of the SPSAD reactor was comprehensively evaluated under 190 d of seasonal variation. The results indicated that the nitrate removal loading (NRL), nitrate removal efficiency (NRE), and PO₄³⁻-P removal rate decreased from 0.060 kg NO₃⁻-N /(m³·d), 93.2% and 67.9% to 0.032 kg NO₃⁻-N /(m³·d), 49.9% and 30.2%, respectively, when the temperature decreased from 35 °C to 9 °C. The SPSAD reactor was effective at performing denitrification under temperature variations. Additionally, the ratio of ΔSO₄²⁻ to ΔNO₃⁻-N gradually decreased from 6.48 to 5.34 as the temperature decreased, revealing a shift in the predominant electron donor for denitrification from S⁰ to pyrite. Microbial analysis revealed that the average abundance of Proteobacteria was 53.11%, making it the dominant phylum in the reactor. Thiobacillus was significantly enriched as the predominant genus responsible for denitrification, with its abundance decreasing from 33.2% at Stage I (25–35 °C) to 26.4% at Stage III (9–11 °C). The feasibility and advantages of NO₃⁻-N and PO₄³⁻-P removal via the SPSAD reactor were discussed and demonstrated in practical applications. This study provides stakeholders with scientific support for the deep treatment of municipal secondary effluent in cold areas.

Graphical abstract

Keywords

Sulfur Autotrophic / NO ₃ ⁻-N / PO ₄ ³⁻-P / Seasonal Temperature / Secondary effluent

Highlight

	● A robust reactor resistance to low temperature was developed for N and P removal.
	● NRL of 0.032 kg NO3−-N /(m3·d) and NRE of 30.2% were achieved at 10.4 °C.
	● Thiobacillus was enriched and the predominant genera serving for denitrification.
	● Feasibility and merit of SPSAD were demonstrated in municipal secondary effluent.

References

[1] Chen D, Wang H Y, Yang K, Ma F. (2018). Performance and microbial communities in a combined bioelectrochemical and sulfur autotrophic denitrification system at low temperature. Chemosphere, 193: 337–342

[2] Chen X Y, Yang L, Chen F, Song Q N, Feng C P, Liu X, Li M. (2022). High efficient bio-denitrification of nitrate contaminated water with low ammonium and sulfate production by a sulfur/pyrite-based bioreactor. Bioresource Technology, 346: 126669

[3] Chen Z Q, Pang C, Wen Q X. (2023). Coupled pyrite and sulfur autotrophic denitrification for simultaneous removal of nitrogen and phosphorus from secondary effluent: feasibility, performance and mechanisms. Water Research, 243: 120422

[4] Cui P, Wan N H, Li C Y, Zou L, Ma M, Du J, Jiang Y. (2024). Comparative analysis of sulfur-driven autotrophic denitrification for pilot-scale application: pollutant removal performance and metagenomic function. Bioresource Technology, 413: 131433

[5] Di Capua F, Ahoranta S H, Papirio S, Lens P N L, Esposito G. (2016). Impacts of sulfur source and temperature on sulfur-driven denitrification by pure and mixed cultures of Thiobacillus. Process Biochemistry, 51(10): 1576–1584

[6] Demir Ö, Atasoy A D, Çalış B, Çakmak Y, Capua F D, Sahinkaya E, Uçar D. Impact of temperature and biomass augmentation on biosulfur-driven autotrophic denitrification in membrane bioreactors treating real nitrate-contaminated groundwater. Science of the Total Environment, 853: 158470

[7] Fajardo C, Mora M, Fernández I, Mosquera-Corral A, Campos J L, Méndez R. (2014). Cross effect of temperature, pH and free ammonia on autotrophic denitrification process with sulphide as electron donor. Chemosphere, 97: 10–15

[8] Feng L J, Sun X R, Wang J Q, Xie T N, Wu Z L, Xu J K, Wang Z Y, Yang G F. (2024). Performance and microbial mechanism in sulfide-driven autotrophic denitrification by different inoculation sources in face of various sulfide and sulfate stress. Bioresource Technology, 413: 131443

[9] GengY W (2021). Application of a Novel Sulfur Autotrophic Denitrification Reactor for Advanced Denitrification. Dissertation for the Doctoral Degree. Suzhou: Suzhou University of Science and Technology (in Chinese)

[10] Hu H D, Shi Y J, Liao K W, Ma H J, Xu K, Ren H Q. (2019). Effect of temperature on the characterization of soluble microbial products in activated sludge system with special emphasis on dissolved organic nitrogen. Water Research, 162: 87–94

[11] JiX Y (2022). Study on Preparation of Anhydrous Calcium Hydrogen Phosphate by Micro-Impinging Stream Reactors and its Application Properties. Thesis for the Masteral Degree. Beijing: Beijing University of Chemical Technology (in Chinese)

[12] Kim J H, Guo X J, Park H S. (2008). Comparison study of the effects of temperature and free ammonia concentration on nitrification and nitrite accumulation. Process Biochemistry, 43(2): 154–160

[13] Kong Z, Feng C P, Chen N, Tong S, Zhang B G, Hao C B, Chen K. (2014). A soil infiltration system incorporated with sulfur-utilizing autotrophic denitrification (SISSAD) for domestic wastewater treatment. Bioresource Technology, 159: 272–279

[14] Li H B, Li Y F, Guo J B, Song Y Y, Hou Y N, Lu C C, Han Y, Shen X F, Liu B W. (2021). Effect of calcinated pyrite on simultaneous ammonia, nitrate and phosphorus removal in the BAF system and the Fe²⁺ regulatory mechanisms: electron transfer and biofilm properties. Environmental Research, 194: 110708

[15] Li H Y, Xu Y H, Dong H, Min J, Xu H Y, Sun D Z, Liu X Y, Dang Y, Qiu B, Mennella T. . (2024). Evidence of autotrophic direct electron transfer denitrification (DETD) by Thiobacillus species enriched on biocathodes during deep polishing of effluent from a municipal wastewater treatment plant. Chemical Engineering Journal, 495: 153389

[16] Li R H, Morrison L, Collins G, Li A M, Zhan X M. (2016). Simultaneous nitrate and phosphate removal from wastewater lacking organic matter through microbial oxidation of pyrrhotite coupled to nitrate reduction. Water Research, 96: 32–41

[17] Li R H, Wei D Y, Wang W, Zhang Y W. (2020a). Pyrrhotite-sulfur autotrophic denitrification for deep and efficient nitrate and phosphate removal: synergistic effects, secondary minerals and microbial community shifts. Bioresource Technology, 308: 123302

[18] Li T, Gao Y L, Tang Y Y, Xu Y J, Ren H Q, Huang H. (2022). A new LDH based sustained-release carbon source filter media to achieve advanced denitrogenation of low C/N wastewater at low temperature. Science of the Total Environment, 838: 156488

[19] Li Y F, Guo J B, Li H B, Song Y Y, Chen Z, Lu C C, Han Y, Hou Y N. (2020b). Effect of dissolved oxygen on simultaneous removal of ammonia, nitrate and phosphorus via biological aerated filter with sulfur and pyrite as composite fillers. Bioresource Technology, 296: 122340

[20] Li Y Y, Wang Y L, Wan D J, Li B, Zhang P Y, Wang H J. (2020c). Pilot-scale application of sulfur-limestone autotrophic denitrification biofilter for municipal tailwater treatment: performance and microbial community structure. Bioresource Technology, 300(C): 122682

[21] Lin Q, De Vrieze J, Li J B, Li X Z. (2016). Temperature affects microbial abundance, activity and interactions in anaerobic digestion. Bioresource Technology, 209: 228–236

[22] Liu H, Zeng W, Fan Z W, Li J M, Zhan M J, Peng Y Z. (2021). Effect of iron on enhanced nitrogen removal from wastewater by sulfur autotrophic denitrification coupled to heterotrophic denitrification under different substrate ratios. Chemical Engineering Journal, 421: 129828

[23] Liu X Z, Zhao C S, Xu T T, Liu W, Chen Q F, Li L Z, Tan Y, Wang X K, Dong Y N. (2023). Pyrite and sulfur-coupled autotrophic denitrification system for efficient nitrate and phosphate removal. Bioresource Technology, 384: 129363

[24] LuN N (2020). Performance of Nitrogen and Collaborative Phosphorus Removal via Composite Sulfur Substrate Autotrophic Denitrification. Thesis for the Masteral Degree. Xuzhou: China University of Mining and Technology (in Chinese)

[25] Lu S P, Gischkat S, Reiche M, Akob D M, Hallberg K B, Küsel K. (2010). Ecophysiology of Fe-cycling bacteria in acidic sediments. Applied and Environmental Microbiology, 76(24): 8174–8183

[26] Lv X, Zhang W X, Deng J S, Feng S Y, Zhan H Z. (2024). Pyrite and humus soil-coupled mixotrophic denitrification system for efficient nitrate and phosphate removal. Environmental Research, 247: 118105

[27] Ma W J, Zhang H M, Ma Z S, You X J, Wei X Y, Li Y, Tian Y. (2024). Meta-analyzing the mechanism of pyrogenic biochar strengthens nitrogen removal performance in sulfur-driven autotrophic denitrification system: evidence from metatranscriptomics. Water Research, 253: 121296

[28] Meynet P, Joss A, Davenport R J, Fenner K. (2024). Impact of long-term temperature shifts on activated sludge microbiome dynamics and micropollutant removal. Water Research, 258: 121790

[29] MiuB, JiangY, LiuP P, Wang D L, HaoW, LiangP, HuangX (2019). Effect of low temperature on sulfur autotrophic denitrification and its improvement. China Water & Wastewater,35(5): 105–109109

[30] Panswad T, Doungchai A, Anotai J. (2003). Temperature effect on microbial community of enhanced biological phosphorus removal system. Water Research, 37(2): 409–415

[31] Sahinkaya E, Dursun N, Kilic A, Demirel S, Uyanik S, Cinar O. (2011). Simultaneous heterotrophic and sulfur-oxidizing autotrophic denitrification process for drinking water treatment: control of sulfate production. Water Research, 45(20): 6661–6667

[32] Shi M, Li X, Dang P Z, Xu Q, Huang T Y, Yuan Y, Huang Y, Zhou C. (2024). Effects of O₂ on accumulation of nitrous and elemental sulfur and microbial community structure in double short-cut sulfur autotrophic denitrification system. Bioresource Technology, 409: 131243

[33] Wang S Q, Yuan Y, Liu F, Liu R D, Zhang X Z, Jiang Y B. (2025). Coupling thiosulfate-driven denitrification and anammox to remove nitrogen from actual wastewater. Bioresource Technology, 417: 131840

[34] Wang T, Li X, Wang H, Xue G, Zhou M D, Ran X C, Wang Y Y. (2023). Sulfur autotrophic denitrification as an efficient nitrogen removals method for wastewater treatment towards lower organic requirement: a review. Water Research, 245: 120569

[35] Wang T, Wang H, Ran X C, Wang Y Y. (2024). Salt stimulates sulfide-driven autotrophic denitrification: microbial network and metagenomics analyses. Water Research, 257: 121742

[36] Wang W, Wei D Y, Li F C, Zhang Y W, Li R H. (2019). Sulfur-siderite autotrophic denitrification system for simultaneous nitrate and phosphate removal: from feasibility to pilot experiments. Water Research, 160: 52–59

[37] Wang X H, Hu M, Xia Y, Wen X H, Ding K. (2012). Pyrosequencing analysis of bacterial diversity in 14 wastewater treatment systems in China. Applied and Environmental Microbiology, 78(19): 7042–7047

[38] Weng Z S, Ma H, Ma J, Kong Z, Shao Z, Yuan Y, Xu Y, Ni Q, Chai H. (2022). Corncob-pyrite bioretention system for enhanced dissolved nutrient treatment: carbon source release and mixotrophic denitrification. Chemosphere, 306: 135534

[39] Xu B K, Shi L S, Zhong H, Wang K. (2019). The performance of pyrite-based autotrophic denitrification column for permeable reactive barrier under natural environment. Bioresource Technology, 290: 121763

[40] Xu Z S, Li Y N, Zhou P P, Song X S, Wang Y H. (2022). New insights on simultaneous nitrate and phosphorus removal in pyrite-involved mixotrophic denitrification biofilter for a long-term operation: performance change and its underlying mechanism. Science of the Total Environment, 845: 157403

[41] Yang D X, Zhang C Y, Ge S J, Xie Y Q, Yuan L M. (2024). Performance of single PN/A reactor under wide fluctuation of nitrogen load. Environmental Science and Pollution Research International, 31: 58083–58092

[42] Yang Y, Chen T H, Morrison L, Gerrity S, Collins G, Porca E, Li R H, Zhan X M. (2017). Nanostructured pyrrhotite supports autotrophic denitrification for simultaneous nitrogen and phosphorus removal from secondary effluents. Chemical Engineering Journal, 328: 511–518

[43] Yang Y, Chen T H, Zhang X, Qing C S, Wang J, Yue Z B, Liu H B, Yang Z. (2018). Simultaneous removal of nitrate and phosphate from wastewater by siderite based autotrophic denitrification. Chemosphere, 199: 130–137

[44] ZhangX X, Hu X L, ZhangL, WangH Y (2022). Effect of HRT on operation performance of sulfur/siderite autotrophic denitrification system. Industrial of Water Treatment, 42(11): 177–183183

[45] ZhangY W (2018). Study of the Behavior and Mechanism of Nitrogen and Phosphorus Removal in Wastewater by Pyrite. Dissertation for the Doctoral Degree. Nanjing: Nanjing University (in Chinese)

[46] Zhao L F, Xue L Y, Wang L, Liu C, Li Y. (2022). Simultaneous heterotrophic and FeS₂-based ferrous autotrophic denitrification process for low-C/N ratio wastewater treatment: nitrate removal performance and microbial community analysis. Science of the Total Environment, 829: 154682

[47] ZhaoN N (2016). Study on Synthesis Process Optimization and Performance of FePO₄. Dissertation for the Doctoral Degree. Tianjin: Hebei University of Technology (in Chinese)

[48] Zhou J M, Ding L, Cui C Z, Lindeboom R E F. (2024). High nitrite accumulation in hydrogenotrophic denitrification at low temperature: transcriptional regulation and microbial community succession. Water Research, 263: 122144

[49] Zhou W L, Sun Y J, Wu B T, Zhang Y, Huang M, Miyanaga T, Zhang Z J. (2011). Autotrophic denitrification for nitrate and nitrite removal using sulfur-limestone. Journal of Environmental Sciences, 23(11): 1761–1769

[50] Zhu S M, Chen S L. (2002). The impact of temperature on nitrification rate in fixed film biofilters. Aquacultural Engineering, 26(4): 221–237

[51] Zuo F M, Sui Q W, Yu D W, Zhang J Y, Gui S L, Wang Y Y, He Y W, Wei Y S. (2024). A temperature-resilient anammox process for efficient treatment of rare earth element tailings wastewater via synergistic nitrite supply of partial nitritation and partial denitrification. Bioresource Technology, 407: 131111

RIGHTS & PERMISSIONS

Higher Education Press 2025