Continuous once-through electrooxidation treatment using mixed metal oxide anode for bacterial disinfection

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IntroductionThe rise of hospital-acquired infections (HAIs) like bloodstream infections and antibiotic-resistant bacteria (ARB) is a growing concern, particularly in developing nations1,2,3. Current practices of treating hospital wastewater (HWW) alongside municipal sewage pose a significant risk, as conventional methods like end-of-pipe treatment are ineffective against these resilient microbes2,4. These methods are also impractical for large volumes, costly, and environmentally damaging due to high chemical use and energy consumption4,5. Untreated HWW from sewage treatment plants (STPs) can contaminate nearby water sources and land, potentially escalating public health threats6,7.This study proposes a novel solution: utilizing electrooxidation (EO) as a decentralized on-site treatment system for infectious ward wastewater. This approach would treat wastewater before it mixes with the common effluent, preventing further contamination. Implementing on-site EO has the potential to be a sustainable and effective strategy for combating HAIs and protecting public health.EO is one of the promising technologies for efficiently treating contaminated water. It achieves high oxidation rates by utilizing direct electron transfer (DET) and indirectly mediated oxidation mechanisms through reactive chlorine species (RCS) on the anode surface. This combination makes EO a next-generation solution for wastewater treatment8. In contrast to other electrochemical advanced oxidation processes (EAOP), EO has the advantage of being operable in compact mobile units, consuming low energy, and not necessitating the addition of chemicals9. The EO treatment process produces disinfectant species through the in-situ EO of naturally present ions in the water matrix. This treatment method depends on the anode materials’ properties and the electric current applied to effectively eliminate pathogenic microbes10,11,12.In EO-based water treatment, two primary disinfection mechanisms operate in parallel: indirect oxidation, which involves the generation of RCS or other oxidants in the bulk solution, and direct oxidation, where pollutants and microorganisms are oxidized directly on the anode surface through electron transfer. While indirect oxidation via RCS is often dominant in chloride-containing systems, direct oxidation plays a crucial complementary role, especially on the surface of electrocatalytically active anodes such as mixed metal oxides (MMOs). In direct anodic oxidation, reactive intermediates such as physically adsorbed hydroxyl radicals (•OH) are generated on the anode surface, which can lead to the destruction of bacterial cell membranes or the breakdown of organic contaminants upon direct contact. The surface properties and composition of the MMO anode, particularly the presence of IrO₂ and Pt, promote this mechanism by enabling strong oxidizing potentials and stable hydroxyl radical formation. Thus, both direct and indirect oxidation pathways contribute synergistically to the overall disinfection efficiency, and their combined effect is particularly relevant for the low-energy, continuous EO treatment systems investigated in this study.The potency of the electrochemical disinfection system relies primarily on the type and composition of the anode, which plays a vital role. This is because it significantly affects the generation of oxidants, their properties, and their ability to undergo oxidation. In this context, one of the significant points is the cost of the electrodes being used. Although previous studies have reported using boron-doped diamond (BDD) as an anode13,14, its high cost for treating large volumes of effluents and producing toxic by-products limits its applicability at the ground scale15. In this study, a novel composition of quaternary MMO with oxides of Ti/Ru/Ir/Pt has been used due to its electrochemical stability, affordability, and ability to facilitate the production of a substantial quantity of RCS and reactive oxygen species (ROS) at low current densities16,17. In this study, the notation Ti/Ru/Ir/Pt refers to an MMO coating deposited on a titanium substrate. The actual chemical nature of the coating comprises thermally decomposed oxides, specifically RuO₂, IrO₂, and PtOx, formed via a thermal decomposition route using precursor salts (e.g., RuCl₃, IrCl6, and H₂PtCl₆). During calcination, these precursors are oxidized to their stable oxide forms, resulting in a composite oxide layer primarily consisting of Ru(IV) oxide (RuO₂) and Ir(IV) oxide (IrO₂), with minor contributions from platinum oxides (PtOx). These oxides are well-known for their high oxygen evolution potential, stability in acidic media, and excellent catalytic activity for the EO of contaminants. The titanium substrate is passivated by a thin layer of TiO₂, ensuring corrosion resistance and mechanical stability. The average thickness of the oxide film was ~ 2.5 μm, inter-electrode gapping of 2 cm, and an active surface area of 42 cm2 . The anode composition selected for this particular study (Ti/Ru/Ir/Pt) was chosen to have better removal efficiencies even in extreme conditions. This novel combination of different metal oxides was incorporated on titanium anodes to have an electro-active surface area (active sites) due to the presence of TiO2, better stability due to Ir in acidic solutions as well as a high temperature, good resistance properties of Ru, durable enough at high anodic potentials and electrochemically effective even at lower current densities due to presence of Pt. Moreover, our previous study proves the durability of the MMO anode through cyclic voltammetry analysis, which shows no loss of metal oxides even after 100 recycles. The distinctive properties of this quaternary MMO anode have been discussed in previous research works18.This research explores the application of EO for disinfecting HWW from infectious wards. While EO has demonstrated effectiveness in treating various bacteria found in wastewater19,20, large-scale implementations remain limited. This study presents a novel decentralized EO treatment strategy specifically designed for wastewater generated from infectious wards of hospitals, which are typically rich in pathogenic bacteria yet limited in volume, making them ideal for targeted, on-site treatment. Unlike conventional approaches focusing on treating bulk hospital effluents, this work uniquely emphasizes source-specific treatment to minimize pathogen spread at the origin21. To validate this model, a continuous once-through EO system was applied for the first time on a bacterial consortium representative of actual HWW at low current density and minimal NaCl dose, ensuring both energy and chemical efficiency. This combination of ward-level targeting, operational simplicity, and realistic microbial simulation distinguishes our study from existing research and underlines its practical innovation and scalability22,23.Recent research highlights the significant progress in using electrochemical and photo-assisted technologies to disinfect hospital urine and suppress antibiotic resistance genes (ARGs). A study utilizing a microfluidic flow-through reactor equipped with an MMO anode and a carbon black/PTFE cathode at a current density of 50 A·m⁻² revealed limited ARG and bacterial removal via electro-disinfection alone, but substantial improvements when UV light was added, underscoring the enhanced efficacy of photo-electro-disinfection24. Further, combining ultrasound irradiation with electrochemical disinfection using dimensionally stable anodes (DSA) in real urban wastewater allowed complete microbial inactivation at a minimum current density of 11.46 A·m⁻², demonstrating a synergistic effect that exceeded the efficacy of either technology alone. However, exact treatment times were not always reported; the advances suggest potential for efficient, scalable solutions25,26. Nonetheless, the literature frequently lacks consistency in reporting disinfection times and operational metrics, posing challenges for direct comparison and real-world implementation.This study presents the development of a continuous once-through EO system utilizing an MMO anode for targeted disinfection of a bacterium consortium in simulated wastewater. The primary objective is to offer a decentralized, on-site treatment solution that aligns with the real-time discharge dynamics of infectious ward effluents. Moving beyond traditional batch-mode EO systems, this continuous flow design enables seamless, uninterrupted treatment, making it more suitable for practical field deployment. The novelty of the work lies in its integration of a stable and efficient MMO anode within a simplified, low-maintenance system architecture, addressing the critical need for energy-efficient, scalable, and application-ready disinfection technologies for hospital wastewater management.However, the novelty of the present work lies in the systematic demonstration of a continuous, once-through EO configuration using MMO anodes under low-cost and low-resource conditions specifically tailored for on-site disinfection of hospital infectious ward effluents, an area that remains underexplored. Most prior studies using MMO anodes have employed batch or recirculating systems, which are less practical for decentralized healthcare settings. In contrast, our study demonstrates a simplified, energy-efficient, and durable continuous-flow EO process that requires no holding tanks or complex hydraulic setups. Moreover, the operational conditions were carefully optimized (low current density of 7.14 mA/cm² and NaCl concentration of 0.2 g/L) to mimic realistic hospital scenarios while ensuring high bacterial inactivation efficiency (96% for simulated and 92% for real wastewater). Additionally, the durability testing over 300 cycles, combined with surface characterization (AFM, XPS), and economic analysis (energy consumption of 0.184 kWh/m³ and operational cost of $1.88/m³), provides a comprehensive evaluation of the system’s long-term feasibility, addressing gaps in the existing literature regarding real-world implementation.Materials and methodsChemicals and microorganismsSodium chloride (NaCl) with 99.5% purity, sodium sulfate (Na2SO4), potassium acetate (CH3CO2K), sulfuric acid (H2SO4), and sodium hydroxide (NaOH) were of analytical grade and used as received from Loba Chemicals, Pvt. Ltd., India. Luria Bertani broth for bacterial culturing was procured from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. All the bacterial strains used in this study, Salmonella enterica (MTCC no. 1165), Staphylococcus aureus (MTCC no. 902), Escherichia coli (MTCC no. 448), Acinetobacter calcoacetius (MTCC no. 1948), Bacillus subtilis (MTCC no. 441), Serratia marcescens (MTCC no. 2645), Enterococcus faecalis (MTCC no. 6845), Listeria sp. (MTCC no. 4214), were purchased from IM-Tech, Chandigarh, India. These eight bacterial strains were selected as they are the most common in HWW27. The bacterial inactivation was studied by varying the current density range and NaCl concentration, as reported in our previous study28.Sample preparationA continuous once-through reactor was employed for the EO treatment of simulated wastewater containing a bacterial consortium. This simulated wastewater mimicked human urine composition and was prepared fresh for each experiment. It consisted of 7.7 g potassium acetate, 880 mg sodium sulfate, and 11 mL glacial acetic acid dissolved in 2.2 L of double-distilled water, with sodium chloride concentration adjusted according to the specific reaction. Eight bacterial strains were individually grown in Luria broth for 24 h using a BOD incubator shaker. The concentration of 104 cells/mL for each bacterial strain was taken by confirming the absorbance, which was in the range of 0.8–1.0 a.u., was recorded at a wavelength of 600 nm, and was collectively added to the simulated wastewater for treatment29.Experimental setupA continuous once-through EO process was employed to treat simulated wastewater containing a bacterial consortium in a custom-designed borosilicate glass reactor. The reactor accommodated a sample volume of 2–2.5 L, with a working volume of 2.2 L for the EO experiments. This reactor measured 77.3 mm in internal diameter and 85 mm in external diameter and was positioned on a laboratory jack for stability. A magnetic stirrer ensured uniform electrolyte concentration throughout the reaction by operating at 500–550 rpm. A schematic and actual photograph of the reactor setup are provided in (Fig. 1a&b). Parallel MMO and stainless-steel electrodes (70 mm x 70 mm x 1 mm, 42 cm2 surface area each) were used with a 2 cm inter-electrode spacing. The MMO anode was sourced from Tiaano Pvt. Ltd. (Chennai, India), and the stainless-steel cathode from a local vendor in Mohali, India. A DC power supply (Gayatri Engineers, Maharashtra, India, Model: 0–30 V, 0–2 A) maintained a constant current density throughout the experiment30.Fig. 1Electrooxidation setup in once-through mode at lab-scale. (a) Schematic diagram of the electrooxidation setup under continuous once-through mode for the EO treatment of simulated wastewater containing a bacterial consortium (b) Actual photograph of the electrooxidation setup under continuous once-through mode.Full size imageAnalytical methodsUV-Visible spectroscopyTo assess treatment efficacy, bacterial inactivation was evaluated for both untreated and EO-treated simulated wastewater by measuring the absorbance at 600 nm using a UV-visible spectrophotometer (Analytik Jena, Specord 205). At predetermined intervals, 200 µL samples were withdrawn for analysis. This involved inoculation in 5 mL Luria broth, followed by a 24-hour incubation period. Post-incubation, 2 mL of samples were taken in a quartz cuvette of 1 cm path length and were analyzed at 600 nm for bacterial inactivation31,32,33.Topography & electronic state analysisAtomic Force Microscopy (AFM) (NT-MDT, Russia) was employed to compare the 3D topography of fresh and recycled MMO anodes. X-ray photoelectron spectroscopy (XPS) using a PHI 5000 Versa-Probe III (Physical Electronics) was utilized to analyze the oxidation state of the MMO anodes. The fresh and recycled MMO anodes were washed with 1 N H2SO4 to remove the impurities before analysis. Here, the recycled anode was used for 300 recycle34.Potassium (K+) ion leakage testA potassium ion leakage test (APHA 3500 K) was performed for the bacterial consortium in simulated wastewater to confirm the validation of bacterial inactivation in our study. As the outer membrane of bacteria serves as a barrier for the permeability of the intracellular substances, this test aims to determine the concentration of the potassium ion leaked from the cell after treatment22. The RCS generated during the EO process could damage the cell wall and membrane of the bacterial cell, thus leading to increased seeping of the K+ from the cell. The K+ leakage from the inactivated bacterial cell was studied under different time intervals to analyze the permeability of the bacterial cell membrane through ICP-AES (Inductively Coupled Plasma Atomic Emission Spectroscopy).FE-SEM imaging of bacteriaThe FE-SEM analysis was done to analyze the structural and morphological changes in the E.coli cells in the untreated and EO-treated samples in double-distilled water. In the present study, the microscopic analysis of the EO-treated E.coli cells under optimized conditions (current density = 7.14 mA/cm2, NaCl dose = 0.2 g/L, treatment time = 9 min, flow rate = 40 mL/min) was done. The bacterial consortium simulated wastewater was not used here to avoid interference from other bacterial cells and to get clear images of the damaged E.coli cells. The E.coli cells were washed with PBS (Phosphate buffered solution) and were fixed with 3% glutaraldehyde solution for 3 h at 4 °C. The cells were washed with different ethanol gradients and were kept for air drying. After that, the E.coli cells were analyzed through FE-SEM imaging35.TAC analysis & HOCl detection assayDuring the EO process, the amount of chlorine formed was determined by analyzing the total available chlorine (TAC) in treated and untreated samples using the APHA standard method (4500-Cl B). The chlorine concentration formed during the EO process in the treated samples was analyzed under optimized conditions (current density = 7.14 mA/cm2, NaCl dose = 0.2 g/ L, treatment time = 9 min, flow rate = 40 mL/min). Moreover, TAC is a mixture of reactive intermediates comprising chloramines (NH2Cl) and free chlorine (Cl2, HOCl)36. Among the chloro-oxidant species, HOCl mainly predominates at an acidic pH range of 3–5, leading to the killing of bacteria37. This HOCl then dissociates into a hypochlorite ion depending on the pH of the solution, followed by an acid-base reaction with a pKa value of 7.5515.An RCS probe was used, which binds to the OCl− ion, resulting in the detection of HOCl produced during the EO treatment process. This RCS probe Benz-Thia compound was synthesized via a reaction of –Aminohydroxyl-phenyl-benzothiazole with N, N diethyl-amino salicylaldehyde to form Schiff base Benz-Thia. This compound is designed to function as a ratiometric fluorescent sensor for detecting ClO− ions, utilizing the imine bond as the fluorophore unit. The sensitivity of Benz-Thia for detecting ClO− ions is quantified, revealing the lowest detection limit of 5.5 µM (through fluorescence), indicating its potential for sensitive detection even at low concentrations of ClO− ions. Both the untreated and EO-treated samples were analyzed for HOCl detection through fluorescence studies under the optimized condition (current density = 7.14 mA/cm2, NaCl dose = 0.2 g/ L, treatment time = 9 min, flow rate = 40 mL/min). Around 40 µL of the RCS probe was added to the samples and appropriately mixed, followed by fluorescence analysis at 640 nm38.Experimental procedureThe EO process was conducted under continuous once-through flow with galvanostatic control (constant current) to ensure reproducible results (triplicate experiments). Simulated wastewater pH was adjusted to 3.5 using 1 N HCl and NaOH before the start of the experimental run. In this study, the initial conductivity of the sample solution was approximately 2.6 mS/cm. The NaCl concentration was then varied across different experimental runs to evaluate its effect on the EO treatment performance for simulated wastewater. The conductivity of the sample solution with optimized NaCl concentration of 0.2 g/L was ~ 3.5 mS/cm. Experiments were conducted at room temperature (25 °C ± 2 °C). Current density and NaCl concentration were adjusted based on the specific reaction under study. After each experiment, electrodes were washed with 1 N H2SO4 solution. A 5 L glass tank was the reservoir for continuously circulating the simulated wastewater through the EO reactor using a small water-lifting pump.Parametric optimizationCurrent density is a critical parameter in electro-disinfection, as it significantly influences power consumption and electrode sizing. To evaluate its effect over time, experiments were performed using varying NaCl concentrations (0.2 g/L, 0.3 g/L, and 0.45 g/L) as the supporting electrolyte, with current densities of 7.14, 9.52, and 11.90 mA/cm². The electrolyte concentration notably affected the percentage of bacterial inactivation at different current density levels, primarily due to the generation of RCS. An increase in NaCl concentration led to enhanced inactivation efficiency, highlighting the role of RCS in the disinfection process.Additionally, treatment time and flow rate were investigated as key operational parameters that influence the process’s reaction kinetics and overall cost-efficiency. The effect of treatment duration was assessed at a fixed current density of 7.14 mA/cm² and 0.2 g/L NaCl.Calculation of inactivation efficiency, energy consumption, and operating costThe percentage inactivation efficiency (I) was determined using Eq. (1), which relates the initial concentration of bacteria (C0) to the final concentration of bacteria (C) in the simulated wastewater.$$\:\%\:Inactivation=\:\frac{{C}_{0}-C}{{C}_{0}}*100$$(1)The operating cost of the electrochemical (EC) process is also a crucial factor affecting the feasibility of any wastewater treatment method. It encompasses expenses such as materials (particularly electrodes), electrical energy, maintenance, and other costs. This study specifically considered electrical energy costs and the cost of the electrodes as the operating cost, representing the most significant portion of expenses. The energy consumption is quantified as consumption quantities per cubic meter (m3 of treated wastewater, and its calculation is expressed in Eq. (2).$$\:{E}_{c\:}=\:\frac{\left(V*I*t\right)}{v}$$(2)Ec is the energy consumption (kW-hr/m3), V is the voltage in Volts, I is current in Ampere, t is the treatment time in h, and v is the volume of the treated simulated wastewater in m3.5Results and discussionsElectro-disinfection of simulated bacterial wastewaterThe electro-disinfection of simulated bacterial wastewater containing eight different bacteria using a continuous once-through flow system equipped with MMO anodes was studied regarding operational parameters such as current density, electrolyte concentration, and treatment time. Furthermore, the impact of flow rate on bacterial inactivation was examined at varying rates (200, 130, 80, and 40 mL/min), focusing on the contact time between bacteria and electrodes. Longer contact times are associated with lower flow rates and improved disinfection performance.Effect of current density (j) on % inactivationThis study (Fig. 2a) shows the %inactivation of the bacterial consortium at different current density ranges of 7.14–11.90 mA/cm2.With increasing current density, the % inactivation increases due to an increase in the rate of chloro-oxidant species, as reported in previous studies39,40. The bacterial consortium was ~ 96% inactivated at a minimum current density of 7.14 mA/cm2, depending upon the electrolyte concentration (i.e., 0.2 g/L). It can also be seen that an effective inactivation of ~ 96% was observed when the current density used was 11.90 mA/cm2 in 3 min of treatment time. This 96% inactivation level was possible in 3 min of treatment time due to the formation of HOCl, the dominant chloro-oxidant species at an acidic pH37. However, increased current density resulted in 96% inactivation of the bacterial consortium because the increase in current density accelerates the electron movement, generating more oxidizing species and leading to bacterial cell membrane destruction41. Moreover, an electrical charge corresponding to an energy consumption of 0.19 kWh/m³ was passed through the electrolytic solution at a high current density of 11.90 mA/cm², resulting in faster disinfection rates42. Nevertheless, considering the economic aspect, high current density may not be economically viable due to its high electricity consumption rate. Therefore, 7.14 mA/cm2 was selected as the effective current density with a minimum NaCl concentration of 0.2 g/L at 9 min of treatment time with a 40 mL/min flow rate.Fig. 2Effect of operational parameters. (a) The graph shows the effect of different current densities (7.14-11.90 mA/cm2) on % bacterial inactivation at NaCl concentration of 0.2 g/L. (b) The graph depicts the effect of different electrolytes (0.2–0.45 g/L NaCl) on % bacterial inactivation. (c) The graph shows the effect of treatment time on bacterial inactivation.Full size imageEffect of different electrolyte concentrations (n) on % inactivationThis study’s results show that in (Fig. 2b), it is evident that complete bacterial inactivation was achieved within 3 min at a current density of 11.90 mA/cm² with a NaCl concentration of 0.2 g/L. Increasing the NaCl concentration to 0.3 g/L did not significantly alter the disinfection efficiency, as similar results were obtained under the same treatment time and current density. However, at a higher NaCl concentration of 0.45 g/L, complete bacterial inactivation was still achieved, but only after 5 min of treatment, even at a lower current density of 7.14 mA/cm². Despite lower current density, this delay in inactivation at higher NaCl concentrations can be attributed to the formation of HOCl as the dominant oxidizing species at acidic pH, which promotes indirect oxidation. Although HOCl is effective, the generation and activity of other chloro-oxidants like Cl₂ and ClO⁻ vary depending on chloride concentration, pH, and reaction kinetics. It is also known from earlier studies43 that high NaCl concentrations may lead to fouling and corrosion of electrodes. Therefore, in this study, the NaCl concentration was carefully varied between 0.2 and 0.45 g/L to balance disinfection efficiency with anode stability. The MMO anodes employed here are relatively resistant to fouling due to their combined mediated and direct oxidation capabilities44. Based on these findings, 0.2 g/L NaCl was the optimal dose, providing efficient bacterial inactivation at 7.14 mA/cm² while ensuring long-term anode stability.Effect of electro-disinfection treatment time (t) on % inactivationThis study aimed to confirm the treatment time required for effective inactivation of a bacterial consortium under optimized electrochemical conditions (current density: 7.14 mA/cm²; NaCl concentration: 0.2 g/L). As shown in (Fig. 2c), bacterial inactivation was monitored over a treatment period ranging from 0 to 9 min. A significant increase in inactivation (~ 32%) was observed after 5 min, reaching ~ 96% at 9 min. These results indicate that a minimum residence time of 9 min is essential to achieve effective electro-disinfection (> 90%). Notably, this treatment time is shorter and more cost-effective than similar studies reported in the literature20,21,45. Thus, treatment time is crucial in optimizing EO processes, directly impacting chemical usage, energy consumption, and infrastructure costs46.Effect of flow rate (FR) on % inactivationIn this study, the flow rate of the reactor was also varied from 200 mL/min to 40 mL/min to study its effect on the % inactivation of the bacterial consortium. Figure 3 shows that when the flow rate decreased from 200 mL/min to 40 mL/min, there was an increase in the % inactivation of the bacterial consortium at the current density range of 7.14–11.90 mA/cm2. This could be because as the flow rate decreases, the contact time of bacteria with the electrode surface increases, thus increasing the inactivation efficiency47. From (Fig. 3a), at a minimum current density of 7.14 mA/cm2 and NaCl concentration of 0.2 g/L, 96% bacterial inactivation was obtained at a 40 mL/min flow rate.Whereas, at NaCl concentration of 0.3 g/L ~ 92%, bacterial inactivation was obtained at a minimum current density of 7.14 mA/cm2 but with a flow rate of 80 mL/min, as depicted in (Fig. 3b). At a maximum current density of 11.90 mA/cm2 and NaCl concentration of 0.45 g/L, the bacterial inactivation of 95% was obtained even at a flow rate of 200 mL/min, as shown in (Fig. 3c). Therefore, from an economic point of view, the flow rate of 40 mL/min was preferred as the optimized flow rate with minimum current density of 7.14 mA/cm2 and NaCl concentration of 0.2 g/L for bacterial inactivation. A hydraulic residence time (HRT) of 1 h was selected as the optimal condition for bacterial inactivation under the specified current density and NaCl concentration. Based on this optimized flow rate and a reactor volume of 2.4 L, the HRT of 1 h was calculated using the formula presented in Eq. (3).Fig. 3Effect of different flow rates in continuous once-through mode. The graph shows the effect of different flow rates at the current density range of (7.14-11.90 mA/cm2) and NaCl concentration of (0.22–0.45 g/L). Here, the bacterial inactivation increases with a decrease in the flow rate. (a) At a minimum current density of 7.14 mA/cm2 and NaCl concentration of 0.22 g/L, ~ 96% bacterial inactivation was obtained at a 40 mL/min flow rate. (b) At NaCl concentration of 0.31 g/L ~ 92%, bacterial inactivation was obtained at 7.14 mA/cm2 with a flow rate of 80 mL/min (c) Whereas at maximum current density of 11.90 mA/cm2 and NaCl concentration of 0.45 g/L, ~ 95% bacterial was obtained even at a flow rate of 200 mL/min.Full size image$$\:HRT=\:\raisebox{1ex}{$Volume\:of\:the\:reactor\left(V\right)$}\!\left/\:\!\raisebox{-1ex}{$Flow\:rate\:\left(Q\right)$}\right.$$(3)where V = volume of the reactor in L and Q = Inflow or outflow rate in L/hr.Although ~ 96% bacterial inactivation was achieved within 9 min in a continuous once-through mode under optimized lab-scale conditions, a 1 h HRT was selected to ensure the process’s reliability, robustness, and scalability for practical applications. The 9 min contact time reflects ideal conditions under controlled laboratory settings, where factors such as flow uniformity, bacterial load, and wastewater composition are consistent and tightly regulated. However, such consistency cannot be guaranteed in real-world or pilot-scale systems. Wastewater characteristics such as microbial load, ionic strength, and organic matter vary significantly, which may impact the disinfection efficiency. Therefore, a longer HRT of 1 h was chosen to provide a safety margin that accommodates such variability, ensuring sufficient contact time for effective inactivation even under fluctuating influent conditions. Furthermore, at the selected optimized flow rate of 40 mL/min and reactor volume, the calculated HRT naturally results in ~ 1 h, which supports the system’s hydraulic balance and offers better process control. This extended HRT ensures that the entire volume of wastewater receives adequate electrochemical exposure, improving consistency and treatment reliability in scaled-up applications. Thus, the 1 h HRT ensures effective disinfection under varying operational conditions and supports the transition of the EO process from lab to real-world implementation.The 3D graphs of % inactivation for current density (mA/cm2) & flow rate (mL/min), and NaCl concentration & flow rate (mL/min) are shown in (Fig. 4a & b), respectively. The equation for the surface graph is given in Eq. (4), which depicts the relationship between % inactivation with flow rate, current density, and NaCl concentration.Fig. 4The 3D surface graph of percentage inactivation. (a) The 3D graph shows the effect of different flow rates (40-200 mL/min) on % bacterial inactivation at varying current density range of (7.14-11.90 mA/cm2) at minimum NaCl concentration of 0.2 g/L. (b) The graph shows the effect of different flow rates (40-200 mL/min) on % bacterial inactivation at varying NaCl concentration (0.2–0.45 g/L) at the lowest current density of 7.14 mA/cm2.Full size image$$\:Z=\:{Z}_{0}+\:\underset{0}{\overset{n}{\int\:}}{A}_{i}\:{X}^{i}+\:\underset{0}{\overset{m}{\int\:}}{B}_{i}{Y}^{i}\:$$(4)where z0​: This is the constant term, which represents the percentage of inactivation that occurs even when there is no flow rate, current density, or NaCl concentration.AiXi: These terms represent the linear, quadratic, cubic, quartic, and quintic effects of flow rate on the percentage of inactivation, where i = 0,1,2,3… n.BiYi: These terms represent the linear, quadratic, cubic, quartic, and quintic effects of current density or NaCl concentration on the percentage of inactivation, where i = 0,1,2,3…. m.A and B are the coefficients of a non-linear equation.Further, this equation can be used to find the values of % inactivation (z) at different known values of flow rate (X) and current density or NaCl concentration (Y), where X & Y are the dependent variables, and z is the independent variable.Proposed mechanism for bacterial inactivation through electrooxidationAs stated earlier, the electro-disinfection of the bacterial consortium in this study is primarily attributed to the formation of RCS at the anodic surface during electrolysis. Under acidic pH conditions, HOCl is the dominant RCS, chlorine gas (Cl₂), and hypochlorite ions (OCl-), all contributing to indirect oxidation. These chloro-oxidant species are generated under optimized electrochemical conditions at a current density of 7.14 mA/cm² and a NaCl concentration of 0.2 g/L. The mechanism of RCS generation begins with the oxidation of chloride ions (Cl-) at the anode surface, forming chlorine gas Eq. (5). Chlorine then reacts with water to form HOCl in Eq. (6), which subsequently dissociates to form OCl- in Eq. (7):$$\:2{Cl}^{-\:\:}\to\:{\:Cl}_{2}+2\:{e}^{-}$$(5)$$\:{Cl}_{2\:\:\:\:}+\:{H}_{2\:}O\to\:\:HOCl+\:{H}^{+\:}+{Cl}^{-}\:$$(6)$$\:HOCl\to\:\:{H}^{+}+\:{OCl}^{-}$$(7)These RCS play a crucial role in bacterial inactivation by compromising cell membrane integrity, disrupting enzymatic activity, and causing DNA damage48. Thus, the primary disinfection pathway observed in our study is due to these indirectly acting oxidants. In addition to RCS-mediated inactivation, direct oxidation contributes to electro-disinfection. At low pH, the anodic surface facilitates the adsorption of •OH, which are produced from water oxidation. This direct pathway is described in Eqs. (8)–(10)$$\:{H}_{2}O\to\:\:{\bullet\:}OH+\:{H}^{+\:}+\:{e}^{-}$$(8)$$\:2{\bullet\:}OH\to\:\:{O}_{2}+2{H}^{+}+2{e}^{-}\:$$(9)$$\:2\:{H}_{2}O\to\:\:{O}_{2}+4{H}^{+}+4{e}^{-}$$(10)Although •OH is a potent oxidizing agent with a high redox potential, its contribution to bacterial inactivation is relatively limited in our system. However, its role cannot be completely discounted. Prior studies have also shown a direct relationship between anodic radical activity and microbial inactivation efficiency49,50. In our research, MMO electrodes were used as the active anode material, with an oxygen evolution potential of 1.523 V. This favors the production of RCS over •OH, making indirect oxidation via RCS the dominant disinfection pathway. The •OH generated contributes to inactivation but plays a secondary role. The proposed bacterial inactivation mechanism, combining indirect and direct oxidation routes, is schematically illustrated in (Fig. 5).Fig. 5Proposed inactivation mechanism for bacterial consortium by RCS. The inactivation was due to both direct oxidation through •OH and indirect oxidation mainly due to HOCl species, leading to bacterial DNA damage, cell membrane fragmentation, and protein oxidation at an optimized condition of (current density =7.14 mA/cm2 and NaCl concentration = 0.2 g/L).Full size imageTotal available Chlorine (TAC) analysis and HOCl detection assayThe (Fig. 6a) shows that the TAC level increased from 6 mg/L to 12 mg/L during the EO treatment of simulated wastewater with a bacterial consortium. This was due to the formation of chloro-oxidant species like HOCl, OCl−, and Cl2 at an acidic pH, resulting in the bacteria’s inactivation. Therefore, this suggests that RCS generated during the electrochemical process is responsible for the inactivation of bacteria under optimized conditions. (Fig. 6b) illustrates the spectral response of the Benz-Thia probe upon interaction with chlorinated oxidants. A redshift in the absorption spectrum from 435 nm to 450 nm is observed, along with a ratiometric fluorescence enhancement at ~ 640 nm, indicating the presence of RCS, primarily HOCl51. While the probe is responsive to ClO⁻ ions under neutral to alkaline conditions, it is essential to note that at acidic pH, under which this experiment was conducted, HOCl is the predominant species, and the presence of OCl⁻ is minimal. Therefore, any reference to ClO⁻ formation under acidic conditions has been removed or revised for scientific accuracy.Fig. 6Study of RCS production & membrane disruption test for bacteria. (a) The graph shows the increase in chlorine concentration with an increase in treatment time. At the optimized condition of (current density = 7.14 mA/cm2, NaCl dose = 0.2 g/L, treatment time = 9 min, flow rate = 40 mL/min), the chlorine concentration increased from 0 to 14.2 mg/L within 9 min of treatment time due to the formation of RCS species. (b) The schematic reaction depicts the formation of Benz-Thia through the reaction of Aminohydroxyl-phenyl-benzthiazole with N, N-diethyl-amino salicylaldehyde (c) The graph describes the production of HOCl species at 640 nm at an acidic pH, which increased with an increase in time. (d) The graph depicts the K+ leakage from the bacterial cell, which increased from 5.4 to 7.4 ppm within 8 min of treatment at the same optimized conditions, indicating bacterial inactivation.Full size imageFurthermore, (Fig. 6c) shows the quantified increase in total oxidant concentration (primarily as HOCl) over the treatment period. The oxidant concentration, measured using the Benz-Thia probe, increased from 15 µM at 0 min (no current applied) to 140 µM at 10 min of treatment. Intermediate values were recorded as 56 µM at 2 min, 59 µM at 4 min, 69 µM at 6 min, and 120 µM at 8 min, indicating progressive accumulation of HOCl with increasing electrolysis time.Biological analysisBacterial cell damage analysis through potassium (K+) ion leakage testIt can be seen from (Fig. 6d) that the K+ ion concentration increased from 5.4 to 7.4 ppm after 8 min of treatment time. This concluded that the total K+ concentration was completely released from the bacterial cell, indicating the bacterial consortium’s complete inactivation. This finding can be described as indicative of membrane damage and potential cell viability loss. Moreover, trypan blue staining was done to confirm the complete bacterial inactivation as given in the supplementary file (Fig. S1).FE-SEM imaging of E. coli inactivationThis study (Fig. 7a) shows that the untreated E. coli cells were intact and healthy without any cell damage. However, in (Fig. 7b), it can be observed that there was a cell disruption and morphological change in the treated E.coli cells. The cells were disrupted entirely, mainly due to the production of HOCl, which is a potent RCS. The cell fragments, as seen, were probably due to the bacterial cell lysis leading to the seepage of broken DNA molecules. This concluded the fact that the chloro-oxidant species generated by applying current density and NaCl dose was responsible for the complete inactivation of the bacteria.Fig. 7Confirmatory test of bacterial cell damage & Preliminary test on actual sewage wastewater. FE-SEM images of E.coli under optimized conditions of (current density = 7.14 mA/cm2, NaCl dose = 0.2 g/L, treatment time = 8 min, flow rate = 40 mL/min). (a) E.coli before EO treatment (b) After EO treatment images of E.coli. The arrows show the bacterial cell damage due to RCS production (c) The graph depicts the bacterial inactivation in actual sewage wastewater at the optimized condition (current density = 7.14 mA/cm2, NaCl dose = 0.2 g/L, treatment time = 8 min, flow rate = 40 mL/min). Here, ~ 92% bacterial inactivation can be seen through EO technology within 8 min of treatment time.Full size imageApplication of the electrooxidation process on real wastewaterThe optimized conditions of (current density = 7.14 mA/cm2, NaCl dose = 0.2 g/L, treatment time = 9 min, flow rate = 40 mL/min) obtained from the previous section were applied for the EO treatment of secondary treated sewage wastewater under once through mode at lab-scale at a flow rate of 40 mL/min. It can be seen from (Fig. 7c) that there was ~ 92% bacterial inactivation within a treatment time of 8 min. Thus, this EO treatment can be applied as a tertiary treatment option in the prevailing treatment system. Further pilot-scale studies and comprehensive techno-economic assessments are necessary to evaluate the feasibility of real-world implementation.Characterization of anodesAtomic force microscopy (AFM) imaging of MMOThe topography of the fresh and recycled MMO anodes is shown in (Fig. 8a&b) through 3D AFM images. It can be seen that there was no notable deposition on the surface of the MMO anode after EO treatment, even after 300 recycles. Further, (Fig. 8e&f) shows the surface roughness histogram analysis graph. The average surface roughness (Sa) for fresh and recycled anodes was 61.75 nm and 20.60 nm before and after EO treatment, respectively, after 1 h of EO treatment for 300 recycles. The decrease in the surface roughness for the anode was ~ 66%, possibly due to the wear of materials52. The EO treatment of wastewater may involve many types of direct reactions that take place on the anodic surface through the departure and deposition of electrons53. Further, the cross-sectional analysis of the anode with height profile before and after EO treatment is shown in (Fig. 8c&d).Fig. 83D AFM images & histogram analysis of mixed metal oxide (MMO) anode (a) fresh MMO and (b) recycled MMO. The graph depicts the surface roughness of the anodes through histogram analysis of (c) Fresh MMO and (d) Recycled MMO after EO treatment of simulated wastewater. The average surface roughness (Sa) for fresh MMO was 61.75 nm, and for recycled MMO, it was 20.60 nm. The figure shows the cross-sectional analysis of the mixed metal oxide (MMO) anode along with the height profile using the AFM technique for (e) fresh MMO and (f) recycled MMO after EO treatment of simulated wastewater.Full size imageXPS analysisThe XPS analysis was done to study the metal elements’ surface composition and oxidation state in MMO anodes. The results indicate no drastic change in the peaks of metal elements even after 300 recycles54,55. (Fig. 9a) shows the Ti 2p spectrum in which two peaks corresponding to the binding energies of 458.1 eV and 463.8 eV for Ti 2p3/2 and Ti 2p1/2 oxidation states can be observed56. (Fig. 9b) shows the spectra of O 1s in which one broad peak can be seen at 529.8 eV, corresponding to the formation of Ti-O bonds. Further (Fig. 9c, d,e) shows the spectral signals of Ru 3d, Ir 4f, and Pt 4f, thus indicating their presence in the MMO anode. The Ru spectral signals showing two characteristic peaks were obtained at 280.4 eV and 284.8 eV, corresponding to 3d5/2 and 3d3/2 levels, thus indicating the formation of RuO2 metal oxide. The Ir signal with one prominent peak was obtained at 61.7 eV, corresponding to the Ir 4f7/2 level of Ir3+, thus proposing the formation of IrO2. The two Pt signal spectra were found at 63.5 eV and 75.7 eV, corresponding to the Pt (v) oxidation state at 4f5/2 level due to the formation of Pt-Ti bonds. The results obtained in the present study correspond to the studies reported in previous literature57,58.Fig. 9The XPS spectra of different metals of the MMO anode. The graphs show the spectrum along with their binding energies: (a) Ti 2p spectrum with binding energies 458.1 and 463.8 eV (b) O 1s spectrum with the binding energy of 529.8 eV (c) Ru 3d spectrum with 280.4 and 284.8 eV binding energies (d) Ir 4f with 61.7 eV binding energy and (e) Pt 4f with 63.5 and 75.7 eV binding energies. (f) The durability graph of the MMO anode depicts its stability even after 300 recycles in terms of % bacterial inactivation. There was no significant loss of metal oxides for almost 300 recycles.Full size imageDurability studyThe MMO anode’s stability, durability, and recyclability are critical parameters for evaluating its long-term applicability and economic feasibility. The anode was subjected to 300 treatment cycles using simulated bacterial wastewater to assess these attributes. One “cycle” is defined as a single EO treatment run performed under standardized conditions: a current density of 7.14 mA/cm², NaCl concentration of 0.2 g/L, and treatment duration of 9 min per cycle. After each cycle, the treated effluent was discarded and replaced with fresh simulated wastewater, simulating repeated operational use rather than reusing the same effluent. As shown in Fig. 9f, the MMO anode maintained consistent bacterial inactivation efficiency across all 300 cycles, with negligible performance or structural degradation loss. This suggests a high degree of operational stability. The anode’s exceptional durability can be attributed to IrO₂, known for enhancing the lifespan of MMO electrodes by up to five years. Additionally, incorporating TiO₂, RuO₂, and Pt facilitates the generation of RCS and a smaller fraction of ROS, which promote both direct and indirect oxidation pathways59. The low electrolyte concentration and moderate current density further helped mitigate electrode corrosion, contributing to prolonged anode life. Post-treatment characterization using AFM and XPS, as discussed in Sect. 3.9, confirmed the morphological integrity and chemical stability of the anode surface after prolonged use. These findings reinforce the anode’s suitability for long-term decentralized disinfection applications.Economical cost analysisThe EO disinfection process uses direct current (DC) applied through an MMO anode and a stainless-steel cathode to inactivate bacteria in wastewater. This study’s results demonstrated this approach’s effectiveness, highlighting high bacterial inactivation efficiency, low energy consumption, and cost-effectiveness. The optimized operational parameters were: current density of 7.14 mA/cm², NaCl dose of 0.2 g/L, treatment time of 9 min, and flow rate of 40 mL/min. Under these conditions, sewage wastewater was treated in a continuous once-through mode, achieving approximately 92% bacterial inactivation. The operational cost was calculated based on treatment time, electrode cost, and energy consumption. A detailed cost analysis for bacterial inactivation in sewage wastewater is provided in the supplementary file (Table S1). Based on these parameters, the treatment cost was estimated at 1.8887 $/m³, indicating strong economic viability.Additionally, Table 1 presents the energy consumption and running cost associated with treating a bacterial consortium in simulated wastewater. Table 2 compares the EO method to other disinfection technologies, demonstrating its superior efficiency and affordability. To enhance the context and demonstrate the relevance of our findings, a comparative table in Table 3 summarizes key data from previous studies on EO-based disinfection.Table 1 Cost Estimation and energy consumption at current density = 7.14 mA/cm2, NaCl dose = 0.2 g/ L, 0.3 g/l, 0.45 g/L) for bacterial inactivation in simulated wastewater.Full size tableTable 2 Comparison table with other techniques.Full size tableTable 3 Comparison with other EO based disinfection studies.Full size tableWhile the current findings confirm the technical and economic feasibility of EO disinfection at the lab scale, further considerations are required for scaling up. As treatment volumes increase, operational costs—such as energy demand, electrode degradation, and chemical input—can significantly rise, potentially limiting the technology’s practical implementation. Therefore, strategies for minimizing costs during scale-up are essential. These include optimizing reactor design, improving energy efficiency, extending electrode life, and refining chemical usage. Implementing such measures will enhance the cost-effectiveness of EO, making it suitable for larger-scale applications like on-site hospital wastewater treatment. Ensuring economic sustainability is especially critical in resource-constrained settings, where conventional treatment systems may not be viable. By integrating technical effectiveness with affordability, the EO process holds promise as a scalable, decentralized disinfection solution for real-world wastewater management challenges.ConclusionThis study successfully demonstrated the efficacy of a continuous once-through EO system using MMO anodes for bacterial disinfection in wastewater. The system achieved 96% inactivation in simulated bacterial wastewater at a low current density of 7.14 mA/cm² and NaCl dosage of 0.2 g/L, effectively mimicking hospital infectious ward conditions. A comparable 92% inactivation was also observed in real sewage wastewater under similar conditions, indicating practical applicability. The MMO anode exhibited strong durability, maintaining performance over 300 treatment cycles, with minimal degradation. Energy consumption (0.184 kWh/m³) and operational cost ($1.88/m³) suggest that the process is economically viable for decentralized applications. The continuous, once-through operation eliminates the need for large holding tanks, offering operational simplicity and on-site treatment potential—key for minimizing pathogen transmission and controlling hospital-acquired infections (HAIs). These findings highlight EO technology as a promising decentralized disinfection solution, particularly in healthcare settings. However, caution is needed when projecting to full-scale implementation. Further research is essential to evaluate performance across variable wastewater matrices, pathogen diversity, and long-term operation. Additionally, life-cycle cost analysis, scalability assessments, and integration with other treatment methods will be crucial to fully realize its potential in urban and resource-limited environments fully.Future scopeBuilding on the promising disinfection performance of the continuous EO system, future studies can explore modular reactor designs to support flexible deployment in various healthcare and remote settings. Investigating the electrode’s resilience to complex wastewater matrices, including pharmaceutical residues and antibiotic-resistant bacteria, will provide deeper insight into its practical applicability. Moreover, assessing the real-time system automation, energy optimization strategies, and hybrid integration with renewable energy or complementary treatment technologies could pave the way for a more robust and sustainable decentralized wastewater solution.Data availabilityData is provided within the manuscript or supplementary information files. 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The authors are thankful to the University Grant Commission (UGC), India, for providing an SJSGC fellowship (UGCES-22-GE-PUN-F-SJSGC-13452) to the first author of this article. DC is willing to acknowledge ICMR-Govt of India for extramural funding to the lab (Project File nos. 17 × (3)/Adhoc/63/2022-ITR and 17 × (3)/Adhoc/3/2022-ITR.Author informationAuthors and AffiliationsDepartment of Energy and Environment, Thapar Institute of Engineering and Technology (TIET), Patiala, 147004, Punjab, IndiaPoulomi Chandra & Anoop VermaDepartment of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology (TIET), Patiala, 147004, Punjab, IndiaPoulomi Chandra, Vijay Luxami & Diptiman ChoudhuryCentre of Excellence for Emerging Materials (CEEMS), Thapar Institute of Engineering and Technology, Patiala, 147004, Punjab, IndiaAnoop Verma & Diptiman ChoudhuryUniversity Centre for Research and Development, Chandigarh University, Mohali, 140413, Punjab, IndiaAastha PaltaAuthorsPoulomi ChandraView author publicationsSearch author on:PubMed Google ScholarAnoop VermaView author publicationsSearch author on:PubMed Google ScholarAastha PaltaView author publicationsSearch author on:PubMed Google ScholarVijay LuxamiView author publicationsSearch author on:PubMed Google ScholarDiptiman ChoudhuryView author publicationsSearch author on:PubMed Google ScholarContributions1. Conception and design of the study: D. C., P.C., A.V. 2. Experimentation: P. C. 3. Analysis/Interpretation of data: P. C. 4. Drafting the Manuscript: P. C. 5. Preparation of figures: P. C., D. C. 6. Synthesis of HOCl Sensors: A. P., V. L. 6. Critical reviewing of the manuscript: A. V., D. C. 7. Revising the manuscript critically for important intellectual content: A. V., D. C. 8. Approval of the final manuscript: All.Corresponding authorCorrespondence to Diptiman Choudhury.Ethics declarationsCompeting interestsThe authors declare no competing interests.Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationBelow is the link to the electronic supplementary material.Supplementary Material 1Rights and permissionsOpen Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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