Operando investigation of the synergistic effect of electric field treatment and copper for bacteria inactivation

The overuse of chemical disinfectants like chlorine and antibiotics raises concerns about disinfection byproducts (DBPs), carcinogenicity, and antimicrobial resistance (AMR). The WHO estimates AMR will cause 20 million deaths by 2050. This necessitates safer, sustainable alternatives. Electric field treatment (EFT) is an emerging technology that inactivates pathogens via electroporation, a process distinct from electrochemical methods. However, high voltage and energy requirements limit its widespread use. Locally enhanced electric field treatment (LEEFT), using nanowire-modified electrodes, addresses these limitations, achieving significant inactivation at low voltages and energy consumption. While LEEFT shows promise, the nanowires' long-term robustness needs improvement. EFT disrupts cell membranes through electroporation, making it synergistic with chemical disinfectants that also target cell membranes. Studies have shown successful synergistic inactivation using EFT with chlorine and ozone, significantly reducing chemical concentrations and DBP formation. Copper (Cu) is a known biocide damaging cell membranes and intracellular components. While Cu is used in various applications, its use in drinking water disinfection is limited due to potential health risks at high concentrations. Previous research demonstrated enhanced microbial inactivation using a combined EFT-copper ionization cell system with low effluent copper concentrations, suggesting potential for point-of-use applications. However, the synergistic mechanism at the cellular level requires further investigation. A lab-on-a-chip (LOAC) device was used to study the EFT-Cu system *in situ* at the microscale, offering advantages over bulk electroporator-based experiments, enabling detailed observation and quantification of the synergistic effect with varying parameters (pulse width, electric field strength, time, and Cu concentration).

The literature review section highlights the drawbacks of traditional chemical disinfection methods, emphasizing the growing concerns regarding DBP formation from chlorine and the emergence of AMR to antibiotics. The review introduces EFT as a promising alternative, detailing its mechanism of action and the challenges associated with its implementation at scale. Existing research on LEEFT and synergistic combinations of EFT with other disinfectants (chlorine and ozone) is summarized, showcasing the potential for reduced chemical usage and DBP formation. The known biocidal properties of copper and its limitations due to potential toxicity at higher concentrations are discussed, along with previous findings on the combined EFT-Cu system. The review concludes by justifying the use of a LOAC device for in-depth mechanistic studies at the single-cell level.

A LOAC device was fabricated using photolithography and lift-off methods to deposit gold electrodes onto a glass substrate. The electrodes' curved edge design creates a linear electric field strength gradient. *Staphylococcus epidermidis* was used as a model bacterium. Cells were immobilized on the chip using positively charged poly-L-lysine. Copper ion solutions (0-2 mg/L) were prepared with sodium sulfate to maintain consistent conductivity. Square-wave pulsed voltage shocks (50-80 V) with varying parameters (pulse width: 500 ns, 1 µs, 2 µs; period; pulse number; effective treatment time: 20ms; duty cycle: 0.1%) were applied using a high-speed pulse generator. Propidium iodide (PI) staining was used to quantify inactivation. Microscopy images (differential interference contrast and fluorescence) were analyzed using MATLAB to determine inactivation percentages. Time-series experiments were conducted (Cu-only, EFT-only, EFT-Cu) over 180 minutes to observe inactivation rates. Single-cell analysis was performed to observe membrane permeability changes in each condition.

Experiments with Cu-only showed that 1 mg/L Cu induced ~15% inactivation, while 2 mg/L induced ~30%. EFT-Cu combination experiments showed a clear synergistic effect. Increasing Cu concentration increased inactivation across all pulse widths. A threshold electric field strength was needed for electroporation, after which inactivation increased rapidly. The lethal electroporation threshold (LET, 50% inactivation) decreased with increasing Cu concentration. Longer pulse widths led to higher inactivation at lower electric field strengths. Analysis of slope values (inactivation per mg/L Cu) revealed a synergistic dose response, which increased with electric field strength. The synergistic dose response also increased with pulse width, enabling the same inactivation at lower electric field strengths. Time-series experiments showed a rapid, non-residual effect of EFT-only, a slow increase in inactivation for Cu-only, and a combined effect in EFT-Cu, showcasing the residual effect of Cu and faster overall inactivation. At higher electric field strengths, EFT-Cu's inactivation rate was up to five times faster than the theoretical additive. Single-cell analysis showed that Cu-only staining took ~44s for complete saturation, EFT-only was much faster (~3.3 s), and EFT-Cu showed an intermediate rate (~21s). This indicates increased cell permeability due to EFT, enhancing Cu uptake and accelerating inactivation.

The findings demonstrate a clear synergistic effect between EFT and Cu in bacterial inactivation. The increased cell permeability caused by EFT facilitates faster Cu ion uptake, leading to more rapid and efficient inactivation compared to either method alone. The faster inactivation rate observed with EFT-Cu suggests that this approach could reduce treatment time or Cu concentration while maintaining high disinfection efficiency. These results support the development of a combined EFT-Cu approach for water disinfection, potentially offering a more sustainable and effective alternative to traditional chemical methods. The observed synergistic effect at the microscale provides valuable insights into the underlying mechanisms and opens avenues for further optimization and scale-up.

This study successfully demonstrated the synergistic effect of EFT and Cu for bacterial inactivation using a LOAC device. EFT enhances cell permeability, increasing Cu uptake and accelerating inactivation. This approach has potential for efficient water disinfection, offering a sustainable alternative to chemical methods. Future research should investigate a wider range of parameters (pulse widths, treatment times, water quality factors, other disinfectants) and explore scaling up the technology for practical applications.

The study's limitations include the focus on specific parameters and the generation of reactive oxygen species (ROS) at higher electric field strengths. Further research is needed to investigate ROS effects and a wider range of parameters. The microscale study provides valuable insights but further investigations are needed to fully understand the synergy and optimize for different applications. The study used a single bacterial strain, and further research should investigate the generalizability of the findings to other microorganisms.