Removal of pathogenic indicator microorganisms during partial nitrification: the role of free nitrous acid

Highlights

● 83% ± 13% E. coli and 59% ± 27% Enteroco ccus were removed by partial nitrification.

● FNA exposure leads to surface collapse of E. coli and Enterococcus .

● Bacteria inactivation was due to the breakdown of cell walls and cell membranes.

● Enterococcus was more resistant to FNA treatment than E. coli .

Abstract

Digested wastewater contains pathogenic microorganisms and high ammonia concentrations, which can pose a potential risk to public health. Effective removal of pathogens and nitrogen is crucial for the post-treatment of digested wastewater. Partial nitrification-anammox is an energy-saving nitrogen removal process. Free nitrous acid (FNA), an intermediate product of partial nitrification, has the potential to inactivate microorganisms. However, the efficiency and mechanisms of FNA-related inactivation in pathogens during partial nitrification remains unclear. In this study, Enterococcus and Escherichia coli (E. coli) were selected to investigate the efficiency and mechanisms of FNA-related inactivation in partial nitrification process. The results revealed that 83% ± 13% and 59% ± 27% of E. coli and Enterococcus were removed, respectively, in partial nitrification process at FNA concentrations of 0.023−0.028 mg/L. When the concentration of FNA increased from 0 to 0.5 mg/L, the inactivation efficiencies of E. coli and Enterococcus increased from 0 to 99.9% and 89.9%, respectively. Enterococcus exhibited a higher resistance to FNA attack compared to E. coli. 3D-laser scanning microscopy (3D-LSM) and scanning electron microscopy (SEM) revealed that FNA exposure caused the surface collapse of E. coli and Enterococcus, as well as visible pore formation on the surface of E. coli cells. 4',6-Diamidino-2-phenylindole dihydrochloride n-hydrate (DAPI)/propidium iodide (PI) and biomolecule leakage confirmed that inactivation of E. coli and Enterococcus occurred due to breakdown of cell walls and cell membranes. These findings indicate that partial nitrification process can be used for the removal of residual pathogenic microorganisms.

Graphical abstract

Keywords

Partial nitrification / Free nitrous acid / Pathogenic indicator microorganism / Inactivation / Cell structure

References

[1] Abbew A W, Amadu A A, Qiu S, Champagne P, Adebayo I, Anifowose P O, Ge S. (2022). Understanding the influence of free nitrous acid on microalgal-bacterial consortium in wastewater treatment: a critical review. Bioresource Technology, 363: 127916

[2] Akhiar A, Battimelli A, Torrijos M, Carrere H. (2017). Comprehensive characterization of the liquid fraction of digestates from full-scale anaerobic co-digestion. Waste Management, 59: 118–128

[3] Alegbeleye O O, Sant’Ana A S. (2020). Manure-borne pathogens as an important source of water contamination: an update on the dynamics of pathogen survival/transport as well as practical risk mitigation strategies. International Journal of Hygiene and Environmental Health, 227: 113524

[4] Amiri M, Crawford G A, Earthman J C. (2021). Quantitative percussion diagnostics for evaluating porosity and surface roughness of cold sprayed and laser deposited materials. Journal of Materials Research and Technology, 14: 312–323

[5] APHA (2005). Standard Methods for the Examination for Water and Wastewater, 21st ed. Washington, DC: APHA, AWWA and WEF

[6] Bai X, Schenk P M, Yuan Z, Lant P A, Pratt S. (2015). Enhanced triacylglyceride extraction from microalgae using free nitrous acid pre-treatment. Applied Energy, 154: 183–189

[7] Borowski S, Domanski J, Weatherley L. (2014). Anaerobic co-digestion of swine and poultry manure with municipal sewage sludge. Waste Management, 34(2): 513–521

[8] Chen H, Zhang M. (2013). Effects of advanced treatment systems on the removal of antibiotic resistance genes in wastewater treatment plants from Hangzhou, China. Environmental Science & Technology, 47(15): 8157–8163

[9] Chislett M, Guo J, Bond P L, Jones A, Yuan Z. (2020). Structural changes in cell-wall and cell-membrane organic materials following exposure to free nitrous acid. Environmental Science & Technology, 54(16): 10301–10312

[10] Chislett M, Guo J, Bond P L, Wang Y, Donose B C, Yuan Z. (2022). Reactive nitrogen species from free nitrous acid (FNA) cause cell lysis. Water Research, 217: 118401

[11] Chislett M, Guo J, Bond P L, Yuan Z. (2021). Structural changes in model compounds of sludge extracellular polymeric substances caused by exposure to free nitrous acid. Water Research, 188: 116553

[12] Cho M, Kim J, Kim J Y, Yoon J, Kim J H. (2010). Mechanisms of Escherichia coli inactivation by several disinfectants. Water Research, 44(11): 3410–3418

[13] Dennehy C, Lawlor P G, Gardiner G E, Jiang Y, Cormican P, McCabe M S, Zhan X. (2017). Process stability and microbial community composition in pig manure and food waste anaerobic co-digesters operated at low HRTs. Frontiers of Environmental Science & Engineering, 11(3): 4

[14] Duan H, Gao S, Li X, Ab Hamid N H, Jiang G, Zheng M, Bai X, Bond P L, Lu X, Chislett M M, Hu S, Ye L, Yuan Z. (2020). Improving wastewater management using free nitrous acid (FNA). Water Research, 171: 115382

[15] Gao S H, Fan L, Yuan Z, Bond P L. (2015). The concentration-determined and population-specific antimicrobial effects of free nitrous acid on Pseudomonas aeruginosa PAO1. Applied Microbiology and Biotechnology, 99(5): 2305–2312

[16] Guo Y, Luo Z, Shen J, Li Y Y. (2022). The main anammox-based processes, the involved microbes and the novel process concept from the application perspective. Frontiers of Environmental Science & Engineering, 16(7): 84

[17] Han X, Zhang S, Yang S, Zhang L, Peng Y. (2020). Full-scale partial nitritation/anammox (PN/A) process for treating sludge dewatering liquor from anaerobic digestion after thermal hydrolysis. Bioresource Technology, 297: 122380

[18] Huffman D. (2000). Inactivation of bacteria, virus and Cryptosporidium by a point-of-use device using pulsed broad spectrum white light. Water Research, 34(9): 2491–2498

[19] Jiang C, Xu S, Wang R, Zhou S, Wu S, Zeng X, Bai Z, Zhuang G, Zhuang X. (2018). Comprehensive assessment of free nitrous acid-based technology to establish partial nitrification. Environmental Science. Water Research & Technology, 4(12): 2113–2124

[20] Jiang G, Gutierrez O, Yuan Z. (2011). The strong biocidal effect of free nitrous acid on anaerobic sewer biofilms. Water Research, 45(12): 3735–3743

[21] Jiang G, Keating A, Corrie S, O’Halloran K, Nguyen L, Yuan Z. (2013). Dosing free nitrous acid for sulfide control in sewers: results of field trials in Australia. Water Research, 47(13): 4331–4339

[22] JiangG, Yuan Z (2013). Synergistic inactivation of anaerobic wastewater biofilm by free nitrous acid and hydrogen peroxide. Journal of Hazardous Materials, 250–251: 91–98

[23] Li M, Song G, Liu R, Huang X, Liu H. (2022). Inactivation and risk control of pathogenic microorganisms in municipal sludge treatment: a review. Frontiers of Environmental Science & Engineering, 16(6): 70

[24] Li Y, Liu H, Li G, Luo W, Sun Y. (2018). Manure digestate storage under different conditions: chemical characteristics and contaminant residuals. Science of the Total Environment, 639: 19–25

[25] Li Y, Wang D, Yang G, Yuan X, Xu Q, Yang Q, Liu Y, Wang Q, Ni B J, Tang W, Jiang L. (2020). Enhanced dewaterability of anaerobically digested sludge by in-situ free nitrous acid treatment. Water Research, 169: 115264

[26] LiangY, Ma R, NghiemA, XuJ, TangL, WeiW, Prommer H, GanY (2022). Sources of ammonium enriched in groundwater in the central Yangtze River Basin: anthropogenic or geogenic? Environmental Pollution, 306: 119463

[27] Lin M, Wang A, Ren L, Qiao W, Wandera S M, Dong R. (2022). Challenges of pathogen inactivation in animal manure through anaerobic digestion: a short review. Bioengineered, 13(1): 1149–1161

[28] Ma B, Yang L, Wang Q, Yuan Z, Wang Y, Peng Y. (2017). Inactivation and adaptation of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria when exposed to free nitrous acid. Bioresource Technology, 245: 1266–1270

[29] Manero A, Blanch A R. (1999). Identification of Enterococcus spp. with a biochemical key. Applied and Environmental Microbiology, 65(10): 4425–4430

[30] Maniakova G, Salmeron I, Nahim-Granados S, Malato S, Oller I, Rizzo L, Polo-Lopez M I. (2021). Sunlight advanced oxidation processes vs ozonation for wastewater disinfection and safe reclamation. Science of the Total Environment, 787: 147531

[31] Mao G, Wang D, Bai Y, Qu J. (2023). Mitigating microbiological risks of potential pathogens carrying antibiotic resistance genes and virulence factors in receiving rivers: benefits of wastewater treatment plant upgrade. Frontiers of Environmental Science & Engineering, 17(7): 82

[32] Ofori I, Maddila S, Lin J, Jonnalagadda S B. (2018). Chlorine dioxide inactivation of Pseudomonas aeruginosa and Staphylococcus aureus in water: the kinetics and mechanism. Journal of Water Process Engineering, 26: 46–54

[33] Oguma K, Katayama H, Mitani H, Morita S, Hirata T, Ohgaki S. (2001). Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair. Applied and Environmental Microbiology, 67(10): 4630–4637

[34] Qiu S, Li Z, Hu Y, Yang Q, Chen L, Liu R, Zhan X. (2021). N₂O generation via nitritation at different volumetric oxygen transfer levels in partial nitritation-anammox process. Journal of Cleaner Production, 293: 126104

[35] Schwarzenbach R P, Escher B I, Fenner K, Hofstetter T B, Johnson C A, von Gunten U, Wehrli B. (2006). The challenge of micropollutants in aquatic systems. Science, 313(5790): 1072–1077

[36] Vadivelu V M, Yuan Z, Fux C, Keller J. (2006). The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched nitrobacter culture. Environmental Science & Technology, 40(14): 4442–4448

[37] Vergara-Castiblanco C A, Freire-Santos F, Oteiza-Lopez A M, Ares-Mazas M E. (2000). Viability and infectivity of two Cryptosporidium parvum bovine isolates from different geographical location. Veterinary Parasitology, 89(4): 261–267

[38] Wang H, Wang J, Zhou M, Wang W, Liu C, Wang Y. (2022). A versatile control strategy based on organic carbon flow analysis for effective treatment of incineration leachate using an anammox-based process. Water Research, 215: 118261

[39] Wang Q, Ye L, Jiang G, Jensen P D, Batstone D J, Yuan Z. (2013). Free nitrous acid (FNA)-based pretreatment enhances methane production from waste activated sludge. Environmental Science & Technology, 47(20): 11897–11904

[40] Wen G, Liang Z, Xu X, Cao R, Wan Q, Ji G, Lin W, Wang J, Yang J, Huang T. (2020). Inactivation of fungal spores in water using ozone: kinetics, influencing factors and mechanisms. Water Research, 185: 116218

[41] WuJ, YangQ, LuoW, SunJ, XuQ, ChenF, ZhaoJ, Yi K, WangX, WangD, et al. (2018). Role of free nitrous acid in the pretreatment of waste activated sludge: extracellular polymeric substances disruption or cells lysis? Chemical Engineering Journal, 336: 28–37

[42] Zhan X, Xiao L. (2017). Livestock Waste 2016−International Conference on Recent Advances in Pollution Control and Resource Recovery for the Livestock Sector. Frontiers of Environmental Science & Engineering, 11(3): 16

[43] Zhang T, Wang Q, Khan J, Yuan Z. (2015). Free nitrous acid breaks down extracellular polymeric substances in waste activated sludge. RSC Advances, 5(54): 43312–43318

[44] Zhang Y, Jiang Y, Wang S, Wang Z, Liu Y, Hu Z, Zhan X. (2021). Environmental sustainability assessment of pig manure mono- and co-digestion and dynamic land application of the digestate. Renewable & Sustainable Energy Reviews, 137: 110476

[45] Zhao Q, Ma J, Zeb I, Yu L, Chen S, Zheng Y, Frear C. (2015). Ammonia recovery from anaerobic digester effluent through direct aeration. Chemical Engineering Journal, 279: 31–37

[46] Zheng M, Wang Z, Meng J, Hu Z, Liu Y, Yuan Z, Hu S. (2021). Inactivation kinetics of nitrite-oxidizing bacteria by free nitrous acid. Science of the Total Environment, 752: 141876

[47] Zhou B W, Shin S G, Hwang K, Ahn J H, Hwang S. (2010). Effect of microwave irradiation on cellular disintegration of Gram positive and negative cells. Environmental Science & Technology, 87(2): 765–770

Acknowledgements

This research was supported by the Natural Science Foundation of Anhui Province (China) (No. 2208085ME145), the Key Project of Science and Technology in Anhui Province (China) (No. 1801041130), and the National Key R&D Program of China (No. 2019YFC0408502).

Conflict of Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11783-024-1793-6 and is accessible for authorized users.

RIGHTS & PERMISSIONS

2024 Higher Education Press 2024