The use of extracellular polymeric substances (EPS) stripped from sludge as a supplementary carbon source represents a novel method for enhancing nitrogen removal in low-carbon wastewater. This approach aims to leverage EPS while preserving the sludge metabolic activity. However, the effect of the EPS stripping degree on sludge metabolic activity remains poorly understood. In this study, EPS were progressively removed from activated sludge via ultrasonic centrifugation, and the resulting changes in sludge metabolic activity and settleability were systematically evaluated. The results demonstrate that key metabolic indicators, such as the specific oxygen uptake rate (SOUR), ammonia utilization rate (AUR), nitrogen utilization rate (NUR), and phosphorus release rate (PRR), initially increased and then decreased with increasing EPS removal. Optimal activity rates were observed at a 45% EPS stripping ratio, where SOUR, AUR, NUR, and PRR increased by 24%, 59%, 14%, and 9%, respectively, compared to the original sludge. Notably, the sensitivity to over-stripping varied: PRR inhibition commenced at 67% stripping, while NUR began to decline only after 86% stripping. In contrast, SOUR and AUR were enhanced across all tested stripping levels (up to 100%). This study is the first to establish a biological response relationship between the degree of EPS stripping and overall sludge metabolic activity, identifying 45% stripping as the optimum. It was also found that ultrasonic stripping reduced floc size and increased surface negative charge, thereby impairing sludge settleability. Consequently, practical application requires a balance between metabolic enhancement and sludge settleability.
| [1] |
Ahmed S M , Rind S , Rani K . (2023). Systematic review: External carbon source for biological denitrification for wastewater. Biotechnology and Bioengineering, 120(3): 642–658
|
| [2] |
Chen Y P , Li C , Guo J S , Fang F , Gao X , Zhang P , Li S . (2013). Extraction and characterization of extracellular polymeric substances in biofilm and sludge via completely autotrophic nitrogen removal over nitrite system. Applied Biochemistry and Biotechnology, 169(2): 526–538
|
| [3] |
Cheng Y , Ren H L , Koju R , Li H Y , Hu C Z . (2025). Promotion for deep denitrification and sludge minimization by enhancing sulfur-driven endogenous carbon release and electron transport. Chemical Engineering Journal, 520: 165833
|
| [4] |
D’Abzac P , Bordas F , Van Hullebusch E , Lens P N L , Guibaud G . (2010). Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols. Applied Microbiology and Biotechnology, 85(5): 1589–1599
|
| [5] |
Deng J , Wang H , Huang J H , Hu J , Pan Z Y , Gao R H , Wang L , Xu D . (2025). Synergistic TA/(NH4)2SO4 conditioning for sludge dewatering: mechanisms of EPS redistribution and hydrophobic enhancement. Journal of Environmental Management, 389: 126266
|
| [6] |
Dong J J , Zhang Z M , Yu Z D , Dai X , Xu X Y , Alvarez P J J , Zhu L . (2017). Evolution and functional analysis of extracellular polymeric substances during the granulation of aerobic sludge used to treat p-chloroaniline wastewater. Chemical Engineering Journal, 330: 596–604
|
| [7] |
Feng F , Tang X , Tang C J , Chai L Y . (2024). Start-up and recovery performance of ANAMMOX reactors under long-term starvation. Environmental Science, 45(7): 4074–4081
|
| [8] |
Frølund B , Griebe T , Nielsen P H . (1995). Enzymatic activity in the activated-sludge floc matrix. Applied Microbiology and Biotechnology, 43(4): 755–761
|
| [9] |
Gao J L , Liu Y , Yan Y X , Wan J F , Liu F . (2021). Promotion of sludge process reduction using low-intensity ultrasonic treatment. Journal of Cleaner Production, 325: 129289
|
| [10] |
Gayathri T , Kavitha S , Kumar S A , Kaliappan S , Yeom I T , Banu J R . (2015). Effect of citric acid induced deflocculation on the ultrasonic pretreatment efficiency of dairy waste activated sludge. Ultrasonics Sonochemistry, 22: 333–340
|
| [11] |
Ji J B , Pei J , Ding F H , Zeng C T , Zhou J , Dong W L , Cui Z L , Yan X . (2024). Isolation and characterization of polyester polyurethane-degrading bacterium Bacillus sp. YXP1. Environmental Research, 249: 118468
|
| [12] |
Jin P K , Wang X B , Zhang Q H , Wang X C , Ngo H H , Yang L . (2016). A new activated primary tank developed for recovering carbon source and its application. Bioresource Technology, 200: 722–730
|
| [13] |
Kavitha S , Banu J R , Kumar J V , Rajkumar M . (2016). Improving the biogas production performance of municipal waste activated sludge via disperser induced microwave disintegration. Bioresource Technology, 217: 21–27
|
| [14] |
Kujawska N , Talbierz S , Dębowski M , Kazimierowicz J , Zieliński M . (2021). Effect of the concentration of extracellular polymeric substances (EPS) and aeration intensity on waste glycerol valorization by docosahexaenoic acid (DHA) produced in heterotrophic culture of Schizochytrium sp. Applied Sciences, 11(20): 9573
|
| [15] |
Li H , Jin Y Y , Mahar R B , Wang Z Y , Nie Y F . (2009). Effects of ultrasonic disintegration on sludge microbial activity and dewaterability. Journal of Hazardous Materials, 161(2−3): 1421–1426
|
| [16] |
Li S Q , Zheng M , Wu S , Xue Y , Liu Y C , Wang C W , Huang X . (2019). The impact of ultrasonic treatment on activity of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in activated sludge. Frontiers of Environmental Science & Engineering, 13(6): 82
|
| [17] |
Liao B Q , Allen D G , Droppo I G , Leppard G G , Liss S N . (2001). Surface properties of sludge and their role in bioflocculation and settleability. Water Research, 35(2): 339–350
|
| [18] |
Liu J , Wang Z P , Liu T , Quan X . (2025a). Enhanced resistance to ciprofloxacin stress in integrated floating film activated sludge system filled with surface-modified carriers for simultaneous nitrification and denitrification. Frontiers of Environmental Science & Engineering, 19(4): 52
|
| [19] |
Liu X W , Wu Z Y , Wang S Y , Gu J , Yu R , Ding Z . (2025b). New insights into the effects of Bdellovibrio-and-like organisms for sludge dewatering and reduction: Balancing dual effects of extracellular polymeric substances during sludge biolysis process. Journal of Environmental Chemical Engineering, 13(3): 116355
|
| [20] |
Liu Y , Wang H L , Xu Y X , Fang Y Y , Chen X R . (2017a). Sludge disintegration using a hydrocyclone to improve biological nutrient removal and reduce excess sludge. Separation and Purification Technology, 177: 192–199
|
| [21] |
Liu Y , Wang H L , Xu Y X , Tu Q S , Chen X R . (2017b). Achieving enhanced denitrification via hydrocyclone treatment on mixed liquor recirculation in the anoxic/aerobic process. Chemosphere, 189: 206–212
|
| [22] |
Mahmoud A , Olivier J , Vaxelaire J , Hoadley A F A . (2011). Electro-dewatering of wastewater sludge: influence of the operating conditions and their interactions effects. Water Research, 45(9): 2795–2810
|
| [23] |
Seviour T , Derlon N , Dueholm M S , Flemming H C , Girbal-Neuhauser E , Horn H , Kjelleberg S , van Loosdrecht M C M , Lotti T , Malpei M F . et al. (2019). Extracellular polymeric substances of biofilms: Suffering from an identity crisis. Water Research, 151: 1–7
|
| [24] |
Stamenov D , Đurić S , Jafari T H , Ćirić V , Manojlović M . (2018). Microbiological activity in the soil of various agricultural crops in organic production. Contemporary Agriculture, 67(1): 34–39
|
| [25] |
Sung D W , Song S H , Kim J H , Yoo J Y . (2002). Effects of electron donors on nitrate removal by nitrate and nitrite reductases. Biotechnology and Bioprocess Engineering, 7(2): 112–116
|
| [26] |
Tian S , Zhu Y C , Liu Z W , Zhang G M , Rao J C , Li X . (2022). Optimization of low-intensity ultrasonic irradiation for low-strength sewage treatment in anaerobic baffled reactor. Journal of Environmental Chemical Engineering, 10(3): 108022
|
| [27] |
Wang X B , Chen T T , Gao C Y , Xie Y L , Zhang A L . (2022). Use of extracellular polymeric substances as natural redox mediators to enhance denitrification performance by accelerating electron transfer and carbon source metabolism. Bioresource Technology, 345: 126522
|
| [28] |
Wang X B , Chen T T , Qi X F , Zhang Y D , Gao C Y , Xie Y L , Zhang A L . (2021). Organic matter release from primary sludge by mechanical cutting. Journal of Water Process Engineering, 40: 101896
|
| [29] |
Wang X B , Zhang M T , Zhou Z Q , Qu T T , Ran J R , Zhang J L , Li X , Zhang L X , Zhang A L . (2024a). Effect of extracellular polymeric substances removal and re-addition on anaerobic digestion of waste activated sludge. Journal of Water Process Engineering, 57: 104702
|
| [30] |
Wang X B , Zhang Y D , He D , Ran J R , Zhang M T , Li X , Zhang J L , Zhang L X , Zhang A L . (2024b). Biodegradation of activated sludge extracellular polymeric substances as the electron donors for denitrification. Journal of Environmental Chemical Engineering, 12(2): 112077
|
| [31] |
Wang Y K , Zeng Q Y , Zou S S , Hu C , Chen F , Zhang Y M , Rittmann B E . (2019). Bioavailable electron donors from ultrasound-treated biomass for stimulating denitrification. Journal of Environmental Management, 250: 109533
|
| [32] |
Xu D D , Fan J H , Chen W D , Pan C , Jiang L Y , Kang D , Li W J , Ding S , Zheng P , Hu B L . et al. (2022). Insights into the enhanced effect of Low-Intensity Ultrasound on anammox granular sludge by relieving the embolism. Chemical Engineering Journal, 446: 137470
|
| [33] |
Xu J P , Sun Y X , Liu Y , Yuan W , Dai L , Xu W N , Wang H L . (2019). In-situ sludge settleability improvement and carbon reuse in SBR process coupled with hydrocyclone. Science of the Total Environment, 695: 133825
|
| [34] |
Xu R Y , Shen Q H , Chen L J . (2025). The γ-MnO2/NF mediated peroxymonosulfate activation for expeditious 2,4,6-trichlo-rophenol degradation: performance, pathways, and mechanism. Frontiers of Environmental Science & Engineering, 19(8): 108
|
| [35] |
Xu Y X , Fang Y Y , Wang Z H , Guo D , Liu Y , Huang Y , Fu P B , Jin J H , Wei C W , Wang H L . et al. (2018a). In-situ sludge reduction and carbon reuse in an anoxic/oxic process coupled with hydrocyclone breakage. Water Research, 141: 135–144
|
| [36] |
Xu Y X , Wang H L , Wang Z H , Fang Y Y , Liu Y , Zeng T , Liu Z B , Liu M . (2018b). Hydrocyclone breakage of activated sludge to exploit internal carbon sources and simultaneously enhance microbial activity. Process Safety and Environmental Protection, 117: 651–659
|
| [37] |
Yan Y XLiu FGao J LWan J FDing J YLi T T (2022). Enhancing enzyme activity via low-intensity ultrasound for protein extraction from excess sludge. Chemosphere, 303(Pt 2): 134936
|
| [38] |
Ye J M , Huang L , Zhang Y , Zhong C M . (2025). The impact of low COD/N ratio on denitrification performance and microbial community in an intermittent aeration moving bed membrane bioreactor. Process Biochemistry, 150: 189–201
|
| [39] |
Zhang P , Chen Y P , Guo J S , Shen Y , Yang J X , Fang F , Li C , Gao X , Wang G X . (2014). Adsorption behavior of tightly bound extracellular polymeric substances on model organic surfaces under different pH and cations with surface plasmon resonance. Water Research, 57: 31–39
|
| [40] |
Zhang X Y , Zhao B , An Q , Zhang P . (2023). The influence of different nitrate concentrations on aerobic sludge granulation and the role of extracellular polymeric substances. Journal of Environmental Management, 348: 119226
|
| [41] |
Zheng S Z , Song Y D , Li Y M , Sun L D , Hu B , An M D , Zhou Y X . (2018). Broadening of appropriate demulsifier dosage range for latex-containing wastewater by sulfate addition. Frontiers of Environmental Science & Engineering, 12(6): 4
|
| [42] |
Zubrowska-Sudol M , Walczak J . (2014). Effects of mechanical disintegration of activated sludge on the activity of nitrifying and denitrifying bacteria and phosphorus accumulating organisms. Water Research, 61: 200–209
|
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