Enhanced nitrogen removal and sludge characteristic in aerobic SBR by simultaneous nitrification and denitrification bacteria
DOI: https://doi.org/10.3846/jeelm.2025.25119Abstract
Four efficient heterotrophic nitrification and aerobic denitrification (HN-AD) strains were applied in sequencing batch reactors (SBRs) via two bioaugmentation strategies to enhance the nitrogen removal and sludge characteristic. Synthetic domestic wastewater with NH4-N concentrations of 30~50 mg L–1, was treated in three SBRs, with DO maintained at 4±0.5 mg L–1. Compared with the control (crude activated sludge), bioaugmentation improved TN removal by 7% averagely, increased nitration rate by 0.54 mg g–1 h–1, and reduced sludge volume index at 30 min (SVI30) by 3.5~14.7 mL g–1. The maximum TN removal efficiency reached 50.37% with effluent TN concentration of 14.64 mg L–1, meeting China’s Class 1A discharge standard (TN ≤ 15 mg L–1). SBR started by bacterial suspension without activated sludge exhibited high adaptability to low carbon/nitrogen ratio, achieving 35% TN removal at C/N = 3 (vs <10% in control), with <5% MLSS fluctuation versus 30% decline in control. Microbial community analysis revealed Saccharibacteria dominance (15.34% vs control’s 8.48%) coupled with 7.6% reduction in filamentous Saprospiraceae (12.78% to 5.18%), collectively explaining the enhanced nitrogen removal and sludge settleability. This study provides the first evidence of granular sludge formation via HN-AD bacterial coaggregation under low C/N conditions, offering a novel strategy for energy-efficient wastewater treatment.
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SBR, bioaugmentation, simultaneous nitrification and denitrification, sludge characteristic, microbial communityHow to Cite
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References
American Public Health Association. (2012). Standard methods for the examination of water and wastewater. Washington DC, USA.
Bassin, J. P., Pronk, M., Kraan, R., Kleerebezem, R., & Loosdrecht, M. C. M. Van. (2011). Ammonium adsorption in aerobic granular sludge, activated sludge and anammox granules. Water Research, 45(16), 5257–5265. https://doi.org/10.1016/j.watres.2011.07.034
Bouchez, T., Patureau, D., Dabert, P., Juretschko, S., Doré, J., Delgenès, P., Moletta, R., & Wagner, M. (2000). Ecological study of a bioaugmentation failure. Environmental Microbiology, 2(2), 179–190. https://doi.org/10.1046/j.1462-2920.2000.00091.x
Bucci, P., Coppotelli, B., Morelli, I., Zaritzky, N., & Caravelli, A. (2022). Micronutrients and COD/N ratio as factors influencing granular size and SND in aerobic granular sequencing batch reactors operated at low organic loading. Journal of Water Process Engineering, 46, Article 102625. https://doi.org/10.1016/j.jwpe.2022.102625
Cao, S., Tao, Y., Fang, J., Du, R., & Peng, Y. (2024). Biological nitrogen removal from nitrate sewage via novel CANDAN process in continuous-flow UASB reactor with municipal wastewater as co-substrate. Chemical Engineering Journal, 488, Article 150847. https://doi.org/10.1016/j.cej.2024.150847
Carucci, A., Cappai, G., Erby, G., & Milia, S. (2019). Aerobic granular sludge formation in a sequencing batch reactor treating agro-industrial digestate. Environmental Technology, 91, 1–22.
Chen, Q., Ni, J., Ma, T., Liu, T., & Zheng, M. (2015). Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR. Bioresource Technology, 183, 25–32. https://doi.org/10.1016/j.biortech.2015.02.022
Chen, W., Lu, Y., Jin, Q., Zhang, M., & Wu, J. (2020). A novel feedforward control strategy for simultaneous nitrification and denitrification (SND) in aerobic granular sludge sequential batch reactor (AGS-SBR). Journal of Environmental Management, 260, Article 110103. https://doi.org/10.1016/j.jenvman.2020.110103
Chi, X., Li, A., Li, M., Ma, L., Tang, Y., Hu, B., & Yang, J. (2018). Influent characteristics affect biodiesel production from waste sludge in biological wastewater treatment systems. International Biodeterioration and Biodegradation, 132, 226–235. https://doi.org/10.1016/j.ibiod.2018.04.010
Choi, D., Cho, K., & Jung, J. (2019). Optimization of nitrogen removal performance in a single-stage SBR based on partial nitritation and ANAMMOX. Water Research, 162, 105–114. https://doi.org/10.1016/j.watres.2019.06.044
Eo, J., & Park, K. C. (2016). Long-term effects of imbalanced fertilization on the composition and diversity of soil bacterial community. Agriculture, Ecosystems & Environment, 231, 176–182. https://doi.org/10.1016/j.agee.2016.06.039
Flemming, H. C., & Wingender, J. (2010). The biofilm matrix. Nature Reviews. Microbiology, 8(9), 623–633. https://doi.org/10.1038/nrmicro2415
Haddaji, C., Chatoui, M., Khattabi Rifi, S., Ettaloui, Z., Digua, K., Pala, A., Anouzla, A., & Souabi, S. (2023). Performance of simultaneous carbon, nitrogen, and phosphorus removal from vegetable oil refining wastewater in an aerobic-anoxic sequencing batch reactor (OA-SBR) system by alternating the cycle times. Environmental Nanotechnology, Monitoring & Management, 20, Article 100827. https://doi.org/10.1016/j.enmm.2023.100827
Hanada, A., Kurogi, T., Giang, N. M., Yamada, T., Kamimoto, Y., Kiso, Y., & Hiraishi, A. (2014). Bacteria of the candidate phylum TM7 are prevalent in acidophilic nitrifying sequencing-batch reactors. Microbes & Environments, 29(4), 353–362. https://doi.org/10.1264/jsme2.ME14052
Hibiya, K., Terada, A., Tsuneda, S., & Hirata, A. (2003). Simultaneous nitrification and denitrification by controlling vertical and horizontal microenvironment in a membrane-aerated biofilm reactor. Journal of Biotechnology, 100, 23–32. https://doi.org/10.1016/S0168-1656(02)00227-4
Hong, P., Sun, X., Yuan, S., Wang, Y., Gong, S., Zhang, Y., Sang, P., Xiao, B., & Shu, Y. (2024). Nitrogen removal intensification of biofilm through bioaugmentation with Methylobacterium gregans DC-1 during wastewater treatment. Chemosphere, 352, Article 141467. https://doi.org/10.1016/j.chemosphere.2024.141467
Hong, P., Wu, X., Shu, Y., Wang, C., Tian, C., Wu, H., & Xiao, B. (2020). Bioaugmentation treatment of nitrogen-rich wastewater with a denitrifier with biofilm-formation and nitrogen-removal capacities in a sequencing batch biofilm reactor. Bioresource Technology, 303(7), Article 122905. https://doi.org/10.1016/j.biortech.2020.122905
Hong, P., Yang, K., Shu, Y., Xiao, B., Wu, H., Xie, Y., Gu, Y., Qian, F., & Wu, X. (2021). Efficacy of auto-aggregating aerobic denitrifiers with coaggregation traits for bioaugmentation performance in biofilm-formation and nitrogen-removal. Bioresource Technology, 337, Article 125391. https://doi.org/10.1016/j.biortech.2021.125391
Hu, D., Liu, L., Liu, W., Yu, L., Dong, J., Han, F., Wang, H., Chen, Z., Ge, H., Jiang, B., Wang, X., Cui, Y., Zhang, W., Zhang, Y., Liu, S., & Zhao, L. (2022). Improvement of sludge characteristics and mitigation of membrane fouling in the treatment of pesticide wastewater by electrochemical anaerobic membrane bioreactor. Water Research, 213, Article 118153. https://doi.org/10.1016/j.watres.2022.118153
Jin, P., Chen, Y., Yao, R., Zheng, Z., & Du, Q. (2019). New insight into the nitrogen metabolism of simultaneous heterotrophic nitrification-aerobic denitrification bacterium in mRNA expression. Journal of Hazardous Materials, 371, 295–303. https://doi.org/10.1016/j.jhazmat.2019.03.023
Jinxiang, Y., Bin, Z., Qiang, A., Yuansheng, H., & Jinsong, G. (2018). Bioaugmentation with A. faecalis strain NR for achieving simultaneous nitrogen and organic carbon removal in a biofilm reactor. Bioresource Technology, 247, 871–880. https://doi.org/10.1016/j.biortech.2017.09.189
Khan, N. A., Singh, S., Ramamurthy, P. C., & Aljundi, I. H. (2024). Exploring nutrient removal mechanisms in column-type SBR with simultaneous nitrification and denitrification. Journal of Environmental Management, 349, Article 119485. https://doi.org/10.1016/j.jenvman.2023.119485
Lang, X., Li, Q., Ji, M., Yan, G., & Guo, S. (2020). Isolation and niche characteristics in simultaneous nitrification and denitrification application of an aerobic denitrifier, Acinetobacter sp. YS2. Bioresource Technology, 302, Article 122799. https://doi.org/10.1016/j.biortech.2020.122799
Li, D., Li, W., Zhang, D., Zhang, K., Lv, L., & Zhang, G. (2023). Performance and mechanism of modified biological nutrient removal process in treating low carbon-to-nitrogen ratio wastewater. Bioresource Technology, 367, Article 128254. https://doi.org/10.1016/j.biortech.2022.128254
Liu, S., Daigger, G. T., Liu, B., Zhao, W., & Liu, J. (2020). Enhanced performance of simultaneous carbon, nitrogen and phosphorus removal from municipal wastewater in an anaerobic-aerobic-anoxic sequencing batch reactor (AOA-SBR) system by alternating the cycle times. Bioresource Technology, 301, Article 122750. https://doi.org/10.1016/j.biortech.2020.122750
Lu, Z., Li, Z., Cheng, X., Xie, J., Li, X., Jiang, X., & Zhu, D. (2023). Treatment of nitrogen-rich wastewater by mixed aeration combined with bioaugmentation in a sequencing batch biofilm reactor: Biofilm formation and nitrogen-removal capacity analysis. Journal of Environmental Chemical Engineering, 11(2), Article 109316. https://doi.org/10.1016/j.jece.2023.109316
Marchant, H. K., Ahmerkamp, S., Lavik, G., Tegetmeyer, H. E., Graf, J., Klatt, J. M., Holtappels, M., Walpersdorf, E., & Kuypers, M. M. M. (2017). Denitrifying community in coastal sediments performs aerobic and anaerobic respiration simultaneously. ISME Journal, 11(8), 1799–1812. https://doi.org/10.1038/ismej.2017.51
Nielsen, P. H. (1996). Adsorption of ammonium to activated sludge. Water Research, 30(3), 762–764. https://doi.org/10.1016/0043-1354(95)00222-7
Ou, C., Shen, J., Zhang, S., Mu, Y., Han, W., Sun, X., Li, J., & Wang, L. (2016). Coupling of iron shavings into the anaerobic system for enhanced 2,4-dinitroanisole reduction in wastewater. Water Research, 101, 457–466. https://doi.org/10.1016/j.watres.2016.06.002
Padhi, S. K., Tripathy, S., Mohanty, S., & Maiti, N. K. (2017). Aerobic and heterotrophic nitrogen removal by Enterobacter cloacae CF-S27 with efficient utilization of hydroxylamine. Bioresource Technology, 232, 285–296. https://doi.org/10.1016/j.biortech.2017.02.049
Remmas, N., Melidis, P., Zerva, I., Kristoffersen, J. B., Nikolaki, S., Tsiamis, G., & Ntougias, S. (2017). Dominance of candidate Saccharibacteria in a membrane bioreactor treating medium age landfill leachate: Effects of organic load on microbial communities, hydrolytic potential and extracellular polymeric substances. Bioresource Technology, 238, 48–56. https://doi.org/10.1016/j.biortech.2017.04.019
Rifi, S. K., Fels, L. E., Driouich, A., Hafidi, M., Ettaloui, Z., & Souabi, S. (2022). Sequencing batch reactor efficiency to reduce pollutant in olive oil mill wastewater mixed with urban wastewater. International Journal of Environmental Science and Technology, 19(11), 11361–11374. https://doi.org/10.1007/s13762-021-03866-2
Shi, P., Zhang, Y., Song, J., Li, P., Wang, Y., Zhang, X., Li, Z., Bi, Z., Zhang, X., Qin, Y., & Zhu, T. (2019). Response of nitrogen pollution in surface water to land use and social-economic factors in the Weihe River watershed, northwest China. Sustainable Cities and Society, 50, Article 101658. https://doi.org/10.1016/j.scs.2019.101658
Wang, H., He, W., Zhang, Z., Liu, X., Yang, Y., Xue, H., Xu, T., Liu, K., Xian, Y., Liu, S., Zhong, Y., & Gao, X. (2024). Spatio-temporal evolution mechanism and dynamic simulation of nitrogen and phosphorus pollution of the Yangtze River economic Belt in China. Environmental Pollution, 357, Article 124402. https://doi.org/10.1016/j.envpol.2024.124402
Wu, H., Shen, J., Jiang, X., Liu, X., Sun, X., Li, J., Han, W., & Wang, L. (2018). Bioaugmentation strategy for the treatment of fungicide wastewater by two triazole-degrading strains. Chemical Engineering Journal, 349, 17–24. https://doi.org/10.1016/j.cej.2018.05.066
Xia, Y., Kong, Y., Thomsen, T. R., & Nielsen, P. H. (2008). Identification and ecophysiological characterization of epiphytic protein-hydrolyzing Saprospiraceae (“Candidatus epiflobacter” spp.) in activated sludge. Applied and Environmental Microbiology, 74(7), 2229–2238. https://doi.org/10.1128/AEM.02502-07
Xiang, Z., Chen, X., Bai, J., Li, B., Li, H., & Huang, X. (2023). Bioaugmentation performance for moving bed biofilm reactor (MBBR) treating mariculture wastewater by an isolated novel halophilic heterotrophic nitrification aerobic denitrification (HNAD) strain (Zobellella B307). Journal of Environmental Management, 325, Article 116566. https://doi.org/10.1016/j.jenvman.2022.116566
Xu, S., Yao, J., Ainiwaer, M., Hong, Y., & Zhang, Y. (2018). Analysis of bacterial community structure of activated sludge from wastewater treatment plants in winter. BioMed Research International, 2018(1), Article 8278970. https://doi.org/10.1155/2018/8278970
Xuesong, D., Bin, Z., Qiang, A., Meng, T., & Jinsong, G. (2019). Role of extracellular polymeric substances in biofilm formation by Pseudomonas stutzeri strain XL-2. Applied Microbiology and Biotechnology, 103(21–22), 9169–9180. https://doi.org/10.1007/s00253-019-10188-4
Zhang, T., Cao, J., Zhang, Y., Fang, F., Feng, Q., & Luo, J. (2020). Achieving efficient nitrite accumulation in glycerol-driven partial denitrification system: Insights of influencing factors, shift of microbial community and metabolic function. Bioresource Technology, 315, Article 123844. https://doi.org/10.1016/j.biortech.2020.123844
Zhang, W., Zhou, S., Sun, J., Meng, X., Luo, J., Zhou, D., & Crittenden, J. (2018). Impact of chloride ions on UV/H2O2 and UV/persulfate advanced oxidation processes. Environmental Science and Technology, 52(13), 7380–7389. https://doi.org/10.1021/acs.est.8b01662
Zhao, B., Cheng, D. Y., Tan, P., An, Q., & Guo, J. S. (2018). Characterization of an aerobic denitrifier Pseudomonas stutzeri strain XL-2 to achieve efficient nitrate removal. Bioresource Technology, 250, 564–573. https://doi.org/10.1016/j.biortech.2017.11.038
Zhao, X., Li, X., Qi, N., Gan, M., Pan, Y., Han, T., & Hu, X. (2017). Massilia neuiana sp. nov., isolated from wet soil. International Journal of Systematic and Evolutionary Microbiology, 67(12), 4943–4947. https://doi.org/10.1099/ijsem.0.002333
Zhou, C., Wu, J., Ma, W., Liu, B., Xing, D., Yang, S., & Cao, G. (2022a). Responses of nitrogen removal under microplastics versus nanoplastics stress in SBR: Toxicity, microbial community and functional genes. Journal of Hazardous Materials, 432, 128715. https://doi.org/10.1016/j.jhazmat.2022.128715
Zhou, L., Al-Dhabi, N. A., Zhang, X., Gao, B., Zhu, Z., Ruth, G., Zhang, X., Tang, W., & Wu, P. (2024). Advanced nitrogen removal from municipal wastewater by autotrophy-heterotrophy coupled anammox system in a novel simultaneous microaerobic/limited-oxygen SBR: Interspecific correlation network. Chemical Engineering Journal, 485, Article 150092. https://doi.org/https://doi.org/10.1016/j.cej.2024.150092
Zhou, Y., Wang, Y., Fu, S., Qiao, W., Zhao, H., & Zhu, L. (2022b). Enhanced nitrogen removal of aquaculture wastewater in the combined biological aerated filter: The effect of GAC location setting. Journal of Chemical Technology & Biotechnology, 97(9), 2519–2527. https://doi.org/10.1002/jctb.7112
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