Enhanced resistance to ciprofloxacin stress in integrated floating film activated sludge system filled with surface-modified carriers for simultaneous nitrification and denitrification

Highlights

	● Modified carriers promoted SND resistance to CIP stress with IC50 of 36.54 mg/L.
	● Modified carriers induced more EPS to trap CIP to mitigate the toxicity.
	● Activities of AMO, NXR, NAR, and NIR were less suppressed on modified carriers.
	● Target preventing and efflux pumping were the main mechanisms to resist CIP.

Abstract

While the effectiveness of integrated floating film activated sludge (IFFAS) system filled with surface-modified carriers has been demonstrated in promoting simultaneous nitrification and denitrification (SND) performance, the effect of antibiotic stress on this system remains unexplored. Herein, this study investigated the stress response of SND in IFFAS under short- and long-term exposure to ciprofloxacin (CIP), a commonly used antibiotic. Results indicated the significantly higher semi-inhibitory concentration of CIP in the IFFAS system with modified carriers (36.54 mg/L) than that in IFFAS with conventional high density polyethylene (HDPE) carriers (28.77 mg/L). IFFAS system with modified carriers exhibited improved resistance to CIP toxicity compared to IFFAS using HDPE carriers or conventional activated sludge under long-term exposure to CIP concentrations from 50 to 3000 μg/L. The surface-modified carriers demonstrated a multifaceted strategy to mitigate the inhibitory effects of CIP, such as enhancing production of extracellular polymeric substances (EPS) to serve as a potential protective barrier against CIP toxicity, the less suppression of key enzyme activities involved in nitrogen removal, as well as inducing the upregulation of antibiotic resistance genes (ARGs) (qepA, qnrB/C) and the integrase gene (intI1) to enhance target prevention and efflux pumping mechanisms for resistance to CIP. These findings collectively underscored the efficacy of modified carriers in attenuating CIP toxicity within the SND system.

Graphical abstract

Keywords

Simultaneous nitrification and denitrification (SND) / Ciprofloxacin / Modified carrier / Resistance / Extracellular polymeric substances (EPS)

References

[1] Amorim C L, Maia A S, Mesquita R B, Rangel A O, Van Loosdrecht M C, Tiritan M E, Castro P M. (2014). Performance of aerobic granular sludge in a sequencing batch bioreactor exposed to ofloxacin, norfloxacin and ciprofloxacin. Water Research, 50: 101–113

[2] Arakawa Y. (2020). Systematic research to overcome newly emerged multidrug-resistant bacteria. Microbiology and Immunology, 64(4): 231–251

[3] Biswal B K, Wang B, Tang C J, Chen G H, Wu D. (2020). Elucidating the effect of mixing technologies on dynamics of microbial communities and their correlations with granular sludge properties in a high-rate sulfidogenic anaerobic bioreactor for saline wastewater treatment. Bioresource Technology, 297: 122397

[4] Bush N G, Diez-Santos I, Abbott L R, Maxwell A. (2020). Quinolones: mechanism, lethality and their contributions to antibiotic resistance. Molecules, 25(23): 5662

[5] Cai Y, Yan Z, Ou Y, Peng B, Zhang L, Shao J, Lin Y, Zhang J. (2022). Effects of different carbon sources on the removal of ciprofloxacin and pollutants by activated sludge: mechanism and biodegradation. Journal of Environmental Sciences, 111: 240–248

[6] Chen Y, Dong K, Zhang Y, Zheng J, Jiang M, Wang D, Zhang X, Huang X, Zhou L, Li H. (2024). Enhancing biofilm formation in the hydrogen-based membrane biofilm reactor through bacterial Acyl-homoserine lactones. Frontiers of Environmental Science & Engineering, 18(11): 142

[7] Flemming H C, Wingender J, Szewzyk U, Steinberg P, Rice S A, Kjelleberg S. (2016). Biofilms: an emergent form of bacterial life. Nature Reviews. Microbiology, 14(9): 563–575

[8] Gu C, Gao P, Yang F, An D, Munir M, Jia H, Xue G, Ma C. (2017). Characterization of extracellular polymeric substances in biofilms under long-term exposure to ciprofloxacin antibiotic using fluorescence excitation-emission matrix and parallel factor analysis. Environmental Science and Pollution Research, 24(15): 13536–13545

[9] Guo X, Yan Z, Zhang Y, Kong X, Kong D, Shan Z, Wang N. (2017). Removal mechanisms for extremely high-level fluoroquinolone antibiotics in pharmaceutical wastewater treatment plants. Environmental Science and Pollution Research, 24(9): 8769–8777

[10] Jia A, Wan Y, Xiao Y, Hu J. (2012). Occurrence and fate of quinolone and fluoroquinolone antibiotics in a municipal sewage treatment plant. Water Research, 46(2): 387–394

[11] Jing A S, Liu T, Quan X, Chen S, Zhang Y B. (2019). Enhanced nitrification in integrated floating fixed-film activated sludge (IFFAS) system using novel clinoptilolite composite carrier. Frontiers of Environmental Science & Engineering, 13(5): 69

[12] Khan M M T, Chapman T, Cochran K, Schuler A J. (2013). Attachment surface energy effects on nitrification and estrogen removal rates by biofilms for improved wastewater treatment. Water Research, 47(7): 2190–2198

[13] Kim D, Nguyen L N, Oh S. (2020). Ecological impact of the antibiotic ciprofloxacin on microbial community of aerobic activated sludge. Environmental Geochemistry and Health, 42(6): 1531–1541

[14] Kim J K, Park K J, Cho K S, Nam S W, Park T J, Bajpai R. (2005). Aerobic nitrification-denitrification by heterotrophic Bacillus strains. Bioresource Technology, 96(17): 1897–1906

[15] Li J L, Li J W, Peng Y Z, Wang S Y, Zhang L, Yang S H, Li S. (2020). Insight into the impacts of organics on anammox and their potential linking to system performance of sewage partial nitrification-anammox (PN/A): a critical review. Bioresource Technology, 300: 122655

[16] Li W H, Sheng G P, Liu X W, Yu H Q. (2008). Characterizing the extracellular and intracellular fluorescent products of activated sludge in a sequencing batch reactor. Water Research, 42(12): 3173–3181

[17] Li Z L, Cheng R, Chen F, Lin X Q, Yao X J, Liang B, Huang C, Sun K, Wang A J. (2021). Selective stress of antibiotics on microbial denitrification: Inhibitory effects, dynamics of microbial community structure and function. Journal of Hazardous Materials, 405: 124366

[18] Lindberg R, Jarnheimer P A, Olsen B, Johansson M, Tysklind M. (2004). Determination of antibiotic substances in hospital sewage water using solid phase extraction and liquid chromatography/mass spectrometry and group analogue internal standards. Chemosphere, 57(10): 1479–1488

[19] Liu K, Sun M, Ye M, Chao H, Zhao Y, Xia B, Jiao W, Feng Y, Zheng X, Liu M. . (2019). Coexistence and association between heavy metals, tetracycline and corresponding resistance genes in vermicomposts originating from different substrates. Environmental Pollution, 244: 28–37

[20] Liu T, Jia G, Quan X. (2018). Accelerated start-up and microbial community structures of simultaneous nitrification and denitrification using novel suspended carriers. Journal of Chemical Technology and Biotechnology, 93(2): 577–584

[21] Liu T, Xu J, Tian R, Quan X. (2021). Enhanced simultaneous nitrification and denitrification via adding N-acyl-homoserine lactones (AHLs) in integrated floating fixed-film activated sludge process. Biochemical Engineering Journal, 166: 107884

[22] Mahendran B, Lishman L, Liss S N. (2012). Structural, physicochemical and microbial properties of flocs and biofilms in integrated fixed-film activated sludge (IFFAS) systems. Water Research, 46(16): 5085–5101

[23] Martínez J L. (2008). Antibiotics and antibiotic resistance genes in natural environments. Science, 321(5887): 365–367

[24] Michael I, Rizzo L, Mcardell C S, Manaia C M, Merlin C, Schwartz T, Dagot C, Fatta-Kassinos D. (2013). Urban wastewater treatment plants as hotspots for the release of antibiotics in the environment: a review. Water Research, 47(3): 957–995

[25] Nguyen T T, Bui X T, Luu V P, Nguyen P D, Guo W, Ngo H H. (2017). Removal of antibiotics in sponge membrane bioreactors treating hospital wastewater: comparison between hollow fiber and flat sheet membrane systems. Bioresource Technology, 240: 42–49

[26] Nguyen V, Karunakaran E, Collins G, Biggs C A. (2016). Physicochemical analysis of initial adhesion and biofilm formation of Methanosarcina barkeri on polymer support material. Colloids and Surfaces. B, Biointerfaces, 143: 518–525

[27] Porras J, Bedoya C, Silva-Agredo J, Santamaria A, Fernandez J J, Torres-Palma R A. (2016). Role of humic substances in the degradation pathways and residual antibacterial activity during the photodecomposition of the antibiotic Ciprofloxacin in water. Water Research, 94: 1–9

[28] Su Z, Chen L, Wen D. (2024). Impact of wastewater treatment plant effluent discharge on the antibiotic resistome in downstream aquatic environments: a mini review. Frontiers of Environmental Science & Engineering, 18(3): 36

[29] Tang M, Zhou S, Huang J, Sun L, Lu H. (2022). Stress responses of sulfate-reducing bacteria sludge upon exposure to polyethylene microplastics. Water Research, 220: 118646

[30] Tran N H, Reinhard M, Gin K Y. (2018). Occurrence and fate of emerging contaminants in municipal wastewater treatment plants from different geographical regions: a review. Water Research, 133: 182–207

[31] Xu J, Sheng G P, Ma Y, Wang L F, Yu H Q. (2013). Roles of extracellular polymeric substances (EPS) in the migration and removal of sulfamethazine in activated sludge system. Water Research, 47(14): 5298–5306

[32] YiK, WangD, YangQ, Li X, ChenH, SunJ, AnH, WangL, Deng Y, LiuJ, ZengG (2017). Effect of ciprofloxacin on biological nitrogen and phosphorus removal from wastewater. Science of the Total Environment, 605–605: 368–375

[33] Zhang L, Song Z, Dong T, Fan X, Peng Y, Yang J. (2023). Mitigating mechanism of nZVI-C on the inhibition of anammox consortia under long-term tetracycline hydrochloride stress: Extracellular polymeric substance properties and microbial community evolution. Journal of Hazardous Materials, 452: 131035

[34] Zhang Q Q, Ying G G, Pan C G, Liu Y S, Zhao J L. (2015). Comprehensive evaluation of antibiotics emission and fate in the river basins of China: source analysis, multimedia modeling, and linkage to bacterial resistance. Environmental Science & Technology, 49(11): 6772–6782

[35] Zhao T, Liu X, Huai L, Feng R, Yan T, Xu W, Zhao Y. (2024). Fabrication of the TiO₂/Ti₃C₂ loaded ceramic membrane targeting for photocatalytic degradation of PPCPs: ciprofloxacin, tetracycline, and ibuprofen. Frontiers of Environmental Science & Engineering, 18(10): 123

[36] Zhou H, Li X, Chu Z, Zhang J. (2016). Effect of temperature downshifts on a bench-scale hybrid A/O system: process performance and microbial community dynamics. Chemosphere, 153: 500–507

Conflict of Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by the Natural Science Foundation of Liaoning Province of China (No. 2023-MS-099).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11783-025-1972-0 and is accessible for authorized users.

RIGHTS & PERMISSIONS

2025 Higher Education Press 2025