Electric Field Effects on Microbial Cell Properties: Implications for Detection and Control in Wastewater Systems

Abstract

Electric fields (EFs) have emerged as effective, non-chemical tools for modulating microbial populations in complex matrices such as wastewater. This review consolidates current advances on EF-induced alterations in microbial structures and functions, focusing on both vegetative cells and spores. Key parameters affected include membrane thickness, transmembrane potential, electrical conductivity, and dielectric permittivity, with downstream impacts on ion homeostasis, metabolic activity, and viability. Such bioelectrical modifications underpin EF-based detection methods—particularly impedance spectroscopy and dielectrophoresis—which enable rapid, label-free, in situ microbial monitoring. Beyond detection, EFs can induce sublethal or lethal effects, enabling selective inactivation without chemical input. This review addresses the influence of field type (DC, AC, pulsed), intensity, and exposure duration, alongside limitations such as species-specific variability, heterogeneous environmental conditions, and challenges in achieving uniform field distribution. Emerging research highlights the integration of EF-based platforms with biosensors, machine learning, and real-time analytics for enhanced environmental surveillance. By linking microbiological mechanisms with engineering solutions, EF technologies present significant potential for sustainable water quality management. Their multidisciplinary applicability positions them as promising components of next-generation wastewater monitoring and treatment systems, supporting global efforts toward efficient, adaptive, and environmentally benign microbial control strategies.

1. Introduction

Microbial contamination in wastewater poses a major challenge for both environmental quality and public health. Untreated effluents contain diverse microorganisms—including bacteria, viruses, protozoa, archaea, and fungi—that can disseminate pathogens, trigger disease outbreaks, and contribute to antimicrobial resistance.

Monitoring microbial content in wastewater is essential for protecting public health, evaluating treatment performance, and safeguarding natural ecosystems [1]. Wastewater contains a wide range of microorganisms—including pathogenic bacteria, viruses, protozoa, and fungal spores—that signal fecal contamination or potential disease outbreaks. Common bacterial species detected in untreated municipal wastewater include Escherichia coli (up to 10⁸ CFU/mL), Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella spp., and spore-forming Clostridium perfringens [2]. In addition to bacteria, viruses, and protozoa, wastewater also harbors significant populations of archaea, yeasts, and fungi. Archaea, particularly methanogens, are involved in anaerobic digestion and biogas production, while yeasts and filamentous fungi contribute to organic matter degradation, biofilm formation, and in some cases opportunistic pathogenicity. Their presence highlights the need for detection and control strategies that address the full microbial diversity of wastewater ecosystems [2,3,4]. Viral agents such as norovirus and adenovirus, as well as protozoan parasites like Giardia lamblia and Cryptosporidium parvum, are also commonly reported in wastewater samples [5]. The persistence of these microorganisms poses risks to downstream environments, especially when effluents reach surface or groundwater bodies. Monitoring typically relies on indicators such as E. coli and Enterococcus [6], but insufficient control or ineffective inactivation can promote the spread of antibiotic-resistant bacteria—such as multidrug-resistant P. aeruginosa—and other pathogens, threatening human health and ecological balance. Consequently, agencies including the WHO and EPA have established microbial quality standards for wastewater discharges and reclaimed water reuse (e.g., ≤1000 CFU/100 mL fecal coliforms in treated effluents for unrestricted irrigation, per WHO guidelines) [7,8]. Against this background, Figure 1 contrasts conventional microbiological approaches with emerging electric field-based strategies, highlighting the shift toward faster, label-free, and in situ detection. Traditional methods such as culture-based assays, staining, and microscopy remain widely used for cultivable microorganisms [9], while novel techniques including electric field exposure, impedance or capacitance spectroscopy, and biosensor platforms offer real-time, label-free, and more targeted microbial monitoring [10]. Conventional approaches for microbial detection and inactivation in wastewater, although widely used, face several important limitations. Culture-based methods are slow and restricted to cultivable organisms, while molecular techniques may overestimate viable counts. Disinfection strategies such as chlorination, UV irradiation, or ozonation reduce overall microbial loads but are less effective in turbid or complex matrices and may generate undesirable byproducts. These gaps highlight the need for alternative solutions that can provide faster, more selective, and reliable results in real-world wastewater environments. Figure 1 summarizes this transition, focusing on representative technologies rather than an exhaustive survey.

Traditional microbiological methods such as culture-based assays and microscopy remain valuable, yet they are time-consuming, labor-intensive, and restricted to cultivable organisms. Notably, only 1–10% of environmental microorganisms are estimated to grow under standard laboratory conditions. To overcome these limitations, electric field (EF)-based methods have emerged as promising alternatives for microbial monitoring and control. Unlike chemical or thermal approaches, EF techniques interact directly with the structural and functional properties of microbial cells, enabling rapid, label-free, and potentially in situ analyses. Methods such as electrical impedance spectroscopy (EIS), dielectrophoresis (DEP), and pulsed electric fields (PEFs) exploit intrinsic microbial electrical properties for detection, discrimination, or inactivation. These approaches provide faster response times, higher selectivity, and greater adaptability to fluctuating wastewater conditions, making them attractive candidates for next-generation environmental monitoring systems. In contrast, emerging electric field (EF)-based technologies provide faster, more selective, and potentially real-time alternatives for microbial detection, classification, and even inactivation in situ [11,12]. Enhancing microbial assessment strategies that exploit electrical and dielectric properties (e.g., membrane potential, conductivity) is therefore critical for adaptive and resilient wastewater management [13].

Conventional detection approaches—including culture-based assays, staining, and molecular methods such as qPCR—each present important limitation. Culture-based techniques detect only viable and cultivable organisms, failing to capture viable but non-culturable (VBNC) cells and thus underestimating microbial diversity [14,15]. For example, Clostridium perfringens spores, frequently found in sewage, can persist undetected in cultures but later resurface under favorable conditions. Indeed, plate cultures recover only ~10% of microbial taxa in wastewater, while metagenomic analyses reveal hundreds of uncultivable species per milliliter [3,4]. These methods are also time-intensive, often requiring 24–72 h for results. Molecular approaches such as qPCR offer faster and more sensitive detection (2–4 h), but detect total DNA, including from dead cells, potentially overestimating viable load by up to 30–50% in effluents, especially after chlorination [16].

In contrast, inactivation strategies—including chlorination, UV disinfection, and ozonation—are designed to reduce microbial loads but also exhibit drawbacks [17]. Chlorination may generate carcinogenic byproducts (e.g., trihalomethanes), and its efficacy declines in turbid or organic-rich water. UV treatment is ineffective against highly resistant spores (Bacillus subtilis, Cryptosporidium) unless applied at high doses, with turbidity > 10 NTU reducing penetration by more than 50%. Ozonation is highly effective but energy-intensive, requiring advanced infrastructure that is less feasible in decentralized contexts [18,19]. Many of these disinfection approaches depend on centralized facilities, multiple treatment steps, and skilled personnel, limiting their applicability in low-resource or small-scale water reuse systems [20].

These limitations underscore the need for alternative approaches that combine rapid detection with effective microbial control. EF-based methods are attractive because they act selectively on microbial structures such as the membrane, influencing potential, conductivity, and permeability, while enabling real-time, in situ analysis without extensive sample preparation or chemical reagents [21]. Unlike traditional chemical or thermal strategies, EFs provide a non-invasive physical mechanism to probe and manipulate microbial behavior [22,23,24]. Exposure to electric fields has been shown to induce diverse changes—including alterations of membrane potential, thickness, conductivity, and dielectric properties—whose outcomes depend on the intensity, duration, and frequency of the applied field, and may be reversible or permanent [25].

In the case of Escherichia coli, for instance, irreversible electroporation generally occurs at field strengths around 10 kV/cm, while Saccharomyces cerevisiae tends to require slightly lower intensities—approximately 7.5 kV/cm—when subjected to pulses lasting from microseconds to milliseconds [26]. These differences are directly linked to structural and compositional features of the cell envelope. Gram-negative bacteria such as E. coli possess relatively thin membranes (4–6 nm) and a porin-rich outer membrane, which facilitates charge redistribution and lowers the electroporation threshold [27,28,29]. In contrast, yeast cells like S. cerevisiae have thicker and more rigid walls composed mainly of glucans and mannans, providing mechanical resistance but still allowing reversible permeabilization under strong pulsed fields [26,30]. Such structural variability explains the species-dependent sensitivity to electric fields. A notable study by Kunlasubpreedee et al. explored the influence of high-frequency, low-voltage alternating electric fields (AEFs) on membrane permeability and biofilm formation in E. coli and P. aeruginosa. Their results showed that applying an AEF of 3.05 V/cm at 20 MHz significantly limited bacterial adhesion during early biofilm stages. For E. coli, cell length increased from 2.04 µm in control conditions to 3.42 µm upon AEF treatment, while P. aeruginosa exhibited a shift from 1.51 µm to 2.22 µm, pointing to a comparable stress response. These findings suggest that even low-intensity AEFs can provoke notable alterations in cell structure and function, affecting membrane integrity, morphology, and adhesion, without relying on heating or electrochemical pathways [31].

Pulsed Electric Fields (PEFs) have also been extensively explored, especially for their role in electroporation, enhancing membrane permeability, facilitating DNA uptake, or leading to microbial inactivation [32]. Research has further shown that applying high specific energy through PEF can result in microbial reductions of up to 5 log units, particularly in liquid food systems with high electrical conductivity [33].

Beyond inactivation, electric fields open new avenues for non-destructive microbial detection. Techniques like electrical impedance spectroscopy (EIS) and dielectrophoresis (DEP) take advantage of the cells’ intrinsic electrical properties to achieve label-free, real-time analysis [34,35]. For instance, impedance-based sensors have successfully detected E. coli in aqueous media at concentrations as low as 10³ CFU/mL, highlighting the sensitivity and applicability of these approaches for rapid, reagent-free microbial monitoring [36].

Moreover, electric field-based strategies can be fine-tuned to selectively affect specific microbial populations—for example, targeting spores differently from vegetative cells—or to improve the performance of biosensors. A notable advantage of these methods is their reduced sensitivity to turbidity or interfering compounds, which makes them highly suitable for use in decentralized or real-time monitoring systems [10]. Given these benefits, electric fields are being increasingly incorporated into bioanalytical tools and environmental surveillance platforms. Their versatility offers valuable opportunities not only for advancing fundamental research but also for practical microbiological applications, particularly in areas such as water reuse, biosecurity, and the surveillance of antimicrobial resistance. In this review, particular attention is given to dielectrophoresis as a promising technique for microbial capture and discrimination, which will be discussed in detail in the dedicated section. Beyond DEP, this review provides a broader integration of electric field-based strategies for microbial detection and control in wastewater. The unique contribution of this work lies in combining both detection and inactivation approaches within a single framework, linking fundamental biophysical mechanisms with engineering applications such as biosensors and treatment processes. By highlighting strengths, limitations, and research opportunities, the review aims to support both scientific progress and practical implementation in environmental biotechnology.

Research Trends and Bibliometric Insights

To gain deeper insight into how scientific interest has developed regarding the use of electric fields for microbial control in wastewater, a bibliometric analysis was carried out using data from the Web of Science Core Collection covering the period 2015–2024. The findings reveal a consistent upward trend in the number of publications addressing this topic, with significant peaks observed in 2022 and 2023—each accounting for 19 articles—and 18 publications already indexed in 2024, reflecting continued momentum in the field. This trend (Figure 2) suggests a growing awareness of the potential of electric fields as non-chemical tools for microbial manipulation and detection in environmental systems.

The bibliometric component of this review was based on data retrieved from the Web of Science Core Collection (2015–2024) using the query: (“electric field” OR “pulsed electric field”) AND (“microorganism” OR “bacteria” OR “spores”) AND (“wastewater” OR “waste water”). A total of 126 articles were selected. The records were exported as plain text and processed using VOSviewer v1.6.19 to generate keyword co-occurrence maps and thematic clusters.

To complement this quantitative analysis, a co-occurrence network of keywords was generated using VOSviewer [37]. The map revealed distinct thematic clusters (Figure 3).

These clusters highlight the interdisciplinary character of the field, combining microbiology, bioelectrochemistry, and environmental engineering. The bibliometric findings confirm that electric field-based strategies are emerging as a robust and innovative direction for future research and practical implementation in wastewater monitoring and treatment. The central positioning of keywords such as “electric field”, “microbial community”, and “membrane fouling” reflects their foundational role across multiple research directions. The green cluster emphasizes biological processes and microbial activity, while the blue cluster is more focused on electrochemical systems and electron transfer mechanisms. The occurrence of distinct yet interconnected terms such as disinfection, photocatalysis, and ultrasound points toward emerging or specialized applications that are likely to gain increased significance soon. At the same time, the intersection of environmental-related keywords with electrochemical terminology reflects a growing integration between conventional wastewater engineering and more refined, bioelectrical approaches.

In parallel with the bibliometric analysis, a narrative synthesis was conducted. Articles were selected based on their relevance to the topic (electric field interactions with microbial systems in wastewater), experimental depth, and thematic coverage. Priority was given to recent publications (2015–2024), papers published in Q1/Q2 journals in relevant fields (Environmental Sciences, Microbiology, Biotechnology), and/or with significant citation impact. Seminal or mechanistic studies were included even when citation counts were low.

This review sets out to consolidate existing knowledge on how electric fields influence microbial cells, focusing on responses at the membrane and cytoplasmic levels. Additionally, it explores how these effects can be harnessed for practical purposes, especially in microbial detection and control strategies within wastewater treatment systems.

To build a solid conceptual basis, the next section introduces the main types of electric fields employed in microbiological and wastewater-related studies, detailing the associated parameters and mechanisms through which they act on microbial structures.

2. Basics of Electric Fields and Microbial Systems

2.1. Types of Electric Fields

In microbiological applications, electric fields are typically categorized according to specific attributes such as waveform, intensity, frequency, and pulse duration [38]. Among these, three main types have garnered the most attention in scientific literature:

(a)

Direct Current (DC) Electric Fields

Direct current fields maintain a constant voltage over time and can directly influence microbial physiology by affecting cell orientation, membrane polarization, and electrochemical gradients. These effects make DC fields particularly relevant in bioelectrochemical systems such as microbial fuel cells and microbial electrolysis cells, where they support electrogenic activity and cell migration (electrotaxis) [39].

Applications of DC stimulation have demonstrated enhanced biofilm growth and improved electron transfer efficiency. For example, the use of an encapsulated alginate bioanode in a microbial electrolysis cell significantly increased biofilm formation, boosting current generation up to 4.14 A·m⁻² while enriching Geobacter spp. to nearly 80% of the community. At the same time, chemical oxygen demand (COD) removal reached 62–88%, showing the dual benefit of energy recovery and wastewater treatment [40].

Overall, these results show that DC fields can steer microbial organization and enhance bioelectrochemical performance.

(b)

Alternating Current (AC) Electric Fields

Alternating current fields periodically switch polarity across a wide frequency spectrum, from Hz to MHz, and their biological effects are strongly frequency dependent. They are particularly important for analytical applications such as impedance spectroscopy and dielectrophoresis, where microbial dielectric responses provide the basis for label-free discrimination [41].

Studies consistently show that microorganisms exhibit species-specific dielectric signatures under AC exposure. Asami et al. measured E. coli suspensions across 10 kHz–100 MHz, identifying distinct dispersions near 1 MHz and quantifying parameters such as membrane capacitance, cytoplasmic conductivity, and wall permittivity [42]. These features enable real-time classification of mixed microbial populations. At higher frequencies, alternating fields around 20 MHz (≈3 V/cm) significantly inhibited the early adhesion of E. coli and P. aeruginosa, reducing cell attachment during biofilm initiation [31]. Conversely, very low-strength fields (<1 V/cm) primarily caused reversible membrane polarization without damaging cellular structures, supporting their use in continuous monitoring. Taken together, AC fields offer dual advantages: they can generate detailed dielectric fingerprints for microbial detection and, under specific conditions, serve as non-chemical strategies for controlling biofilm formation in water and wastewater systems.

(c)

Pulsed Electric Fields (PEFs)

Pulsed electric fields consist of short, high-voltage pulses in the microsecond to millisecond range and are widely applied to induce either reversible or irreversible electroporation depending on field strength and pulse duration [43]. Beyond their established role in food and medical applications, PEFs are emerging in wastewater and sludge valorization, where pretreatment can release intracellular compounds from microbial or algal cells, enhancing substrate accessibility for bioconversion and improving methane generation or nutrient recovery [44]. Recent studies highlight both inactivation and stimulation effects. For example, Rosenzweig et al. combined nanosecond PEF (600 ns, 21 kV/cm) with low doses of hydrogen peroxide, achieving > 5 log reductions in E. coli, Listeria, and Salmonella—a result not reached by either treatment alone [29]. Similarly, Tongdonyod et al. reported > 6 log reductions in E. coli in coconut water at 20 kV/cm without altering substrate properties, confirming the effectiveness of PEFs in low-conductivity matrices [45]. At the same time, low-intensity PEF can reversibly enhance microbial function: in Pseudomonas putida, stimulation accelerated growth and denitrification, coupled with upregulation of transport and respiration genes [46]. Electroporation has also been used as a delivery tool—for instance, to load gold nanoparticles into vesicles derived from P. aeruginosa—demonstrating PEF’s potential in nano-enabled biotechnologies [47].

Collectively, these studies underline the versatility of PEFs for both microbial inactivation and stimulation, depending on operating conditions. As shown in Figure 4, electric fields relevant to wastewater microbiology can be categorized into Direct Current, Alternating Current, and Pulsed Electric Fields, each associated with characteristic parameters (yellow) and representative applications (blue).

2.2. Microbial Structures Relevant to Electrical Interaction

The interaction between electric fields and microorganisms is largely governed by the structural organization of microbial cells. Several cellular components act as key interfaces for field-induced responses, each contributing differently to the cell’s overall electrical behavior [48].

2.2.1. Cell Membrane and Cell Wall

The structural organization of the microbial envelope plays a decisive role in how cells respond to electric fields. The lipid bilayer, reinforced in many organisms by a rigid cell wall (peptidoglycan in bacteria, chitin in fungi), introduces both mechanical resistance and dielectric heterogeneity [49,50,51,52]. Gram-negative bacteria such as E. coli and P. aeruginosa possess relatively thin envelopes (4–6 nm), which makes them more easily polarized and prone to reversible electroporation at moderate field strengths (~10 kV/cm) [27,28,29]. By contrast, Gram-positive species like B. subtilis and E. faecalis develop thick, multilayered peptidoglycan walls (20–30 nm), which increase the threshold for electroporation and provide greater resilience to electrical stress [53,54]. Experimental evidence confirms these principles: in microfluidic systems, millisecond pulses of 10 kV/cm were sufficient to permanently disrupt E. coli, while S. cerevisiae, with its thicker wall, required slightly higher intensities (~7.5 kV/cm) to achieve lysis and metabolite release [26,30]. Such results demonstrate how envelope composition not only dictates susceptibility to inactivation but also enables practical applications like high-resolution metabolomics and real-time monitoring. Overall, dielectric properties such as membrane capacitance (10⁻⁶–10⁻⁴ S/m) directly govern whether electrical exposure leads to reversible poration, irreversible breakdown, or controlled extraction of intracellular content [55,56].

2.2.2. Cytoplasm

The cytoplasm functions as a conductive medium where ions, metabolites, and macromolecules interact to define the cell’s dielectric behavior. In E. coli, for example, the total inorganic ion concentration is about 300 mM, with potassium (K⁺) as the dominant species, although levels fluctuate with osmotic conditions [57,58]. Such ionic content explains why cytoplasmic conductivity typically falls between 0.1 and 1 S/m, depending on metabolic activity [59]. In wastewater isolates like Pseudomonas and Aeromonas, higher conductivity has been linked to stronger electrochemical responsiveness, directly affecting how intracellular gradients shift when exposed to electric fields [60,61]. Structural components also modulate these properties. The outer membrane of P. aeruginosa, enriched in proteins such as β-barrel porins, creates selective diffusion pathways that regulate nutrient flow while contributing to its dielectric signature [62]. On a molecular scale, ions align water molecules and reduce the dielectric constant of cytosolic water compared to pure water, while metabolites such as amino acids can increase modestly [59]. Together, these factors show that the dielectric behavior of the cytoplasm is not static but shaped by ionic balance, metabolic state, and membrane architecture. This interplay is critical for interpreting impedance measurements, where subtle intracellular changes can manifest as detectable shifts in bulk dielectric signals.

2.2.3. Spores and Dormant Forms

Bacterial spores, particularly those produced by Bacillus and Clostridium species, represent some of the most resilient microbial structures. Their multilayered envelopes and dehydrated cores (<30% water) confer both mechanical resistance and markedly reduced dielectric permittivity, setting them apart from vegetative cells [63]. These traits explain why electroporation thresholds for spores often exceed 30 kV/cm, a value consistently reported across experimental and applied studies [64,65]. The physical basis of this resistance lies in their dense architecture and limited water content, which not only hinders inactivation but also provides distinctive dielectric signatures detectable by techniques such as dielectrophoresis or time-domain dielectric spectroscopy [66].

Evidence across multiple models supports this principle. For instance, Clostridium sporogenes spores typically require 30–50 kV/cm to achieve effective inactivation [67], while C. botulinum spores withstand even advanced processes such as high-pressure thermal treatments (300–1200 MPa at 30–75 °C) [68]. These findings emphasize that spore inactivation demands either extreme field intensities or combined approaches, such as coupling PEF with heat or pressure. At the same time, their unique dielectric properties make spores more distinguishable than vegetative cells, enabling label-free detection and selective separation in complex microbial systems.

Overall, the remarkable durability of spores arises from the same characteristics that allow their electrical discrimination. This duality—resistance to inactivation yet amenability to detection—highlights both the challenges and opportunities of targeting dormant forms in wastewater environments.

2.2.4. Surface Structures (e.g., Pili, Flagella, Biofilms)

Surface appendages such as pili and flagella are not only structural features but also key determinants of how bacteria interact with electric fields. By altering local field distribution, these structures influence adhesion, motility, and aggregation. In Pseudomonas aeruginosa, for instance, the retraction of electrically stimulated type IV pili drives electrotaxis—directed movement along electric field gradients—which in turn promotes biofilm development on electrode surfaces [69]. Beyond locomotion, these pili act as mechanosensors: when they contact a surface, their retraction triggers intracellular signaling cascades that elevate cyclic di-GMP, a messenger central to biofilm formation [70,71].

Electric fields themselves can modulate the conformation and function of pili and flagella. Studies have shown that exposure may alter their structure and dynamics, thereby change adhesion capacity or promote aggregation under certain conditions [72]. Once biofilms are established, they evolve into intricate three-dimensional networks that complicate field penetration. In mixed-species wastewater biofilms, the EPS matrix and layered architecture generate strong dielectric heterogeneity and capacitive behavior that can screen the applied field, cause non-uniform penetration and limit selective action on inner layers. As a result, EF responses often reflect interfacial effects and detachment rather than uniform inactivation throughout the biofilm [73,74].

Beyond these shielding effects, biofilms can also display significant electrochemical activity. For example, Geobacter sulfurreducens biofilms can reach current densities of ~2 A/m² under controlled conditions, highlighting their role in electron transfer. Biofilm thickness, which may range from a few micrometers in early stages to over 50 µm in mature consortia, further influences diffusion processes and the efficacy of antimicrobial treatments. Advanced techniques such as cyclic voltammetry and confocal microscopy have confirmed that electrode-associated biofilms modify hydrogen adsorption and oxygen reduction, underscoring the complexity of their electrical behavior. Low-frequency AC fields (0.1–2 Hz) and direct currents (50–250 µA) have been used to disrupt biofilm integrity and increase susceptibility to antimicrobials like gentamicin, illustrating the potential of electric stimulation for control applications [74].

Together, these findings emphasize that microbial appendages and biofilm architectures are critical modulators of field–microbe interactions. Incorporating such structural and bioelectrical insights into predictive models can greatly improve the specificity and efficiency of electric field-based detection and inactivation strategies, particularly under the complex conditions typical of real wastewater environments. The structural characteristics of selected microorganisms relevant to their interaction with electric fields are summarized in

Table 1. Structural characteristics of selected microorganisms relevant to their interaction with electric fields.

Microorganism/Structure	Relevant Structural Characteristics	Electrical Parameters/Response to EF	Refs.
Gram-negative bacteria (E. coli, P. aeruginosa)	Membrane thickness 4–6 nm; thin peptidoglycan layer; P. aeruginosa has asymmetric outer membrane with LPS; porins for ionic exchange	Reversible electroporation at ~10 kV/cm; membrane capacitance 10−6–10−4 S/m; cytoplasmic conductivity 0.1–1 S/m; detectable via EIS and DEP	[26,27,28,29,30,53,59]
Gram-positive bacteria (B. subtilis, E. faecalis)	Thick peptidoglycan wall (20–30 nm); multilayered envelope	Higher electroporation threshold than Gram-negatives; lower membrane conductivity (~0.5–1 × 10−6 S/m)	[53,54]
Yeast (S. cerevisiae)	Thick wall with glucans and mannans	Electroporation at ~7.5 kV/cm; used for intracellular extraction	[26,30]
Spores (Bacillus, Clostridium spp.)	Multilayered structure; low water content (<30%); highly resistant wall	Activation/inactivation threshold >30 kV/cm; low dielectric permittivity; high PEF resistance	[63,64,65,66,67,68]
Surface structures (pili, flagella, biofilm)	Type IV pili in P. aeruginosa responsive to EF; biofilm thickness 1–50 μm, high mechanical resistance	EF can induce electrotaxis, modify adhesion, detach biofilm (AC 0.1–2 Hz, DC 50–250 μA); affects local field distribution	[69,70,71,72,73,74]
Cytoplasmic properties	Main ions: K+, Na+; cytoplasmic conductivity ~0.1–1 S/m; dielectric constant ~25–30 (hydrated)	EF influences ion distribution and polarization at mid–high frequencies; measurable via impedance	[57,58,59]

.

To illustrate these multi-level interactions in a unified view, Figure 5 presents a schematic representation of the main electric field effects on a model bacterium.

3. Membrane-Level Bioelectrical and Dielectric Properties

3.1. Membrane Thickness

Membrane thickness is one of the most decisive structural traits governing microbial interactions with electric fields. Thin envelopes, such as those of Gram-negative bacteria, typically measure ~15 nm in total, with a 4 nm inner bilayer and a minimal peptidoglycan layer, as in E. coli [75]. By contrast, Gram-positive species like B. subtilis or E. faecalis develop peptidoglycan layers of 25–40 nm, interlaced with teichoic acids, producing robust walls that alter their dielectric response [76,77]. These differences mean that thinner membranes polarize more quickly under applied fields, while thicker ones provide greater resistance.

This principle is well captured by the Schwan equation, which shows the inverse relationship between membrane thickness and induced transmembrane potential [78]. In practice, this explains why Gram-negative bacteria reach electroporation thresholds at lower field strengths, whereas Gram-positive bacteria require higher intensities. Spores represent an extreme case: their multilayered envelopes confer strong mechanical resistance, but also create localized hotspots where electric stress can concentrate and induce dielectric breakdown [79].

Recognizing these structural distinctions is essential for predictive modeling. By linking membrane thickness to polarization dynamics, electroporation thresholds, and stress distribution, researchers can better tailor electric field parameters to achieve controlled inactivation or analytical precision, particularly in complex systems such as wastewater.

3.2. Membrane Potential (ΔΨ)

The membrane potential is a fundamental electrochemical property that arises from the uneven distribution of ions across the microbial membrane. In most bacteria, ΔΨ typically ranges from −100 to −200 mV, depending on species, metabolic state, and external conditions [80]. This gradient is sustained by proton pumps and ATP-driven ion transporters, which also maintain pH homeostasis [81,82]. Beyond its role in energy transduction, ΔΨ contributes to active transport [83], motility regulation via flagellar rotation [84], and even antibiotic resistance, as shown by classic studies linking membrane polarization to aminoglycoside susceptibility [85]. These foundational works, although dated, remain essential for understanding bacterial physiology.

In actively respiring cells, ΔΨ is dynamic. For example, E. coli exhibits values around −220 mV during early exponential growth and about −140 mV in late exponential phase, reflecting shifts in metabolic activity and ion balance [86]. This potential is directly affected by external electric fields, which induce asymmetric polarization: depolarization at the anode-facing side and hyperpolarization at the cathode-facing side. Such field-driven effects have been demonstrated in both E. coli and Bacillus subtilis and highlight how proliferative capacity influences polarization responses [87].

Theoretical models such as the Schwan equation [78] predict that the induced transmembrane potential is proportional to both applied field strength and cell size. For a bacterium ~1 µm in diameter, exposure to 10 kV/cm can generate ΔΨ values sufficient to trigger electroporation, a phenomenon confirmed by uptake of propidium iodide in experimental studies [87,88]. When ΔΨ exceeds a critical threshold of ~1 V, nanoscale pores form in the bilayer [89]. These can reseal after short exposures (reversible electroporation) or remain open after strong pulses, leading to irreversible disruption and cell death.

Beyond viability, field-induced polarization also alters intracellular ion fluxes, promotes reactive oxygen species generation, and disrupts metabolism—mechanisms now being explored for antimicrobial and biosensing applications [90]. As emphasized by Pucihar et al., controlling ΔΨ dynamics under EF exposure is central for microbial inactivation, permeability tuning, and real-time monitoring in wastewater treatment [91]. Overall, understanding the relationship between ΔΨ and electric field strength provides the foundation for designing effective EF-based technologies across diverse microbial species.

3.3. Membrane Electrical Conductivity

Membrane electrical conductivity is a critical parameter that reflects the ability of microbial cells to support ionic current flow under an applied electric field. This property depends on membrane composition, lipid phase state, the presence of embedded proteins, and overall ionic permeability. Biological membranes are composed of a dynamic lipid bilayer interspersed with proteins that carry out most of the membrane’s specialized functions—including ion transport, which plays a key role in determining membrane conductivity. As described by Alberts et al. [49], these membrane proteins regulate the selective movement of ions across the bilayer, thereby sustaining ionic gradients essential for cellular function.

In addition to protein activity, the phase state of the lipid bilayer significantly influences membrane permeability and conductivity. Transitions between liquid-disordered and more ordered or gel-like states can alter the membrane’s permeability to small molecules, as shown by Frallicciardi et al. [92]. Such changes directly impact the flow of ions and, thus, the membrane’s electrical properties. Ion channels and transporters embedded in the bilayer contribute further to this conductivity by enabling controlled ionic movement across the membrane [93].

Taken together, membrane electrical conductivity arises from the combined influence of lipid composition, bilayer phase behavior, protein function, and ionic permeability. Understanding this complex interplay is essential for refining electric-field-based techniques aimed at microbial inactivation or bioelectrical diagnostics.

Membrane conductivity reflects the ability of a microbial cell to allow ionic current to pass under the influence of an external electric field. This property is highly dependent on structural and compositional factors, including the nature of the lipids, the presence of membrane proteins, and environmental conditions such as temperature and ionic strength [94].

In Escherichia coli, reported membrane conductivity values typically range between 1 × 10⁻⁶ and 3 × 10⁻⁶ S/m, with variations attributed to environmental influences. This relatively high conductivity is largely due to the abundance of porin proteins in the outer membrane, which facilitate passive ion transport and differentiate E. coli from Gram-positive bacteria [95,96]. In Pseudomonas aeruginosa, conductivity values are slightly higher—around 5 × 10⁻⁶ S/m—reflecting its metabolically active membrane and the presence of anion-selective channels such as protein P, which enhance ionic permeability [97].

By contrast, Bacillus subtilis, a Gram-positive bacterium with a thicker peptidoglycan wall and fewer porin-like channels, exhibits significantly lower membrane conductivity. Values typically range from 0.5 × 10⁻⁶ to 1 × 10⁻⁶ S/m, a difference primarily linked to its dense cell wall structure and limited ion-permeable protein content [28]. Understanding these differences in membrane conductivity among microbial species is fundamental for optimizing electric-field-based applications such as electroporation, antimicrobial treatments, and biosensing technologies. These differences affect how microbial cells respond to applied electric fields. Higher membrane conductivity enables more efficient charge redistribution and faster polarization but also leads to a lower threshold for field-induced membrane destabilization.

Exposure to electric fields can transiently or permanently alter membrane conductivity. Reversible electroporation typically increases membrane conductance by 10–100×, facilitating molecule transport (e.g., dyes, antibiotics, DNA). In E. coli, conductance can increase from 2 × 10⁻⁶ S/m to ~10⁻⁴ S/m immediately after exposure to 10–15 kV/cm pulses. In wastewater disinfection contexts, this enhanced conductivity is both a marker of electroporation and a tool for impedance-based microbial detection. Electrical impedance spectroscopy (EIS) systems use these shifts to differentiate between viable, damaged, and lysed cells [98,99]. Monitoring conductivity shifts in microbial membranes enables real-time feedback in disinfection systems. Moreover, integrating conductivity data with field strength allows predictive modeling of cell viability [100]. In one configuration, a continuous electric field of just 2 kV/cm was sufficient to achieve a >4.5-log reduction in E. faecalis, highlighting the strong susceptibility of this Gram-positive bacterium even at moderate field strengths [101]. In mixed microbial communities from wastewater, average membrane conductivity measurements can inform system-wide susceptibility to electric treatments, guiding pulse design and frequency selection in PEF reactors. Understanding membrane conductivity profiles across diverse species is thus essential for optimizing electroporation protocols, improving sensor design, and enhancing microbial inactivation strategies in complex aqueous environments [102].

3.4. Dielectric Permeability of the Membrane

Dielectric permittivity (ε) is a key parameter describing how a material polarizes in response to an external electric field. In microbial membranes, it reflects the ability of lipid and protein components to store electrical energy. This property affects how the cell responds to high-frequency alternating fields and determines the cell’s behavior in techniques like electrical impedance spectroscopy and dielectrophoresis. Microbial membranes typically exhibit relative dielectric permittivity values (ε_r) in the range of 5–12, depending on composition and hydration state [103,104]. E. coli membranes show dielectric permittivity around 5–9, consistent with fluid-phase lipid bilayers [105]. No direct measurements of the full-cell membrane dielectric permittivity of P. aeruginosa were found. However, typical values for lipid bilayer interiors lie between 2 and 4 ε_r, and dielectric behavior of bacterial suspensions may exhibit much higher apparent values due to volumetric polarization effects [105,106]. Direct measurements of the full-cell dielectric permittivity for Bacillus subtilis are not available. However, dielectric spectroscopy at 15–16 GHz reveals that B. subtilis exhibits lower dielectric constant responses compared to E. coli, suggesting a lower ε_r overall [107]. Dormant spores exhibit even lower permittivity values (<4), owing to their dehydrated state and multilamellar protective structures. This is supported by dielectric measurements showing extremely low conductivity and minimal mobile ion content in dormant Bacillus cereus spores at 50 MHz, indicative of very low dielectric response [108]. Dielectric permittivity is frequency dependent. At low frequencies (kHz range), the whole cell polarizes and behaves like a particle in a medium, dominated by membrane properties. At higher frequencies (MHz–GHz), the field penetrates the membrane and interacts with the cytoplasm, where ionic content drives internal polarization [109,110]. In practice, this means at 100 kHz, viable E. coli cells exhibit strong interfacial (β-) polarization, while spores—or cells rendered non-viable—lose this response, evidencing minimal dielectric activity at that frequency range [111]. At frequency above approximately 10 MHz, the cell membrane behaves almost like a short circuit, allowing the electrical current to penetrate the cytoplasm. As a result, intracellular conductivity becomes the dominant contributor to the overall impedance, although membrane permittivity still subtly influences the impedance spectrum at these high frequencies [112]. Dielectric permittivity differences are exploited in DEP-based separation, where cells are sorted according to their dielectric signature—such as spores versus vegetative bacteria—through frequency-selective polarization in non-uniform electric fields [113]. EIS leverages changes in membrane permittivity and conductivity following electroporation or cellular stress as a sensitive, label-free method to assess cell viability in real time [114]. In electroporated E. coli populations, membrane integrity is compromised, and impedance-based measurements—such as those carried out using a capacitive Wheatstone bridge—can detect real-time changes in dielectric properties. While precise quantitative reductions in effective permittivity are not consistently reported, such approaches clearly reveal diminishing dielectric signatures correlating with viability loss confirmed via plating assays [115]. A clear understanding of microbial dielectric permittivity is critical for advancing electric field-based detection systems, fine-tuning operational frequency ranges, and enhancing the sensitivity of in situ biosensors in complex wastewater environments [116]. Following the discussion on structural features,

Table 2. Membrane-level bioelectrical and dielectric properties of selected microorganisms.

Property	Microorganism/Example	Reported Values/Characteristics	Notes on Electric Field Interaction	Refs.
Membrane Thickness	E. coli (Gram-negative)	Inner membrane ~4 nm; peptidoglycan ~3 nm; total ~15 nm	Thin membranes polarize faster; lower electroporation threshold (~10 kV/cm)	[75,78]
	B. subtilis (Gram-positive)	Peptidoglycan layer 25–40 nm	Thicker wall increases mechanical resistance; higher electroporation threshold	[76,79]
	E. faecalis (Gram-positive)	Cell wall ~40 nm with teichoic acids	High structural robustness under EF	[77]
Membrane Potential	General bacteria	Resting potential −100 to −200 mV; E. coli: −220 mV (early exponential) to −140 mV (late exponential)	Drives ion transport, motility; EF causes depolarization/hyperpolarization depending on orientation	[80,81,82,83,84,85,86,87]
Membrane Conductivity	E. coli	1 × 10−6 to 3 × 10−6 S/m; increases to ~10−4 S/m after electroporation	Higher conductivity → faster polarization, lower EF threshold for breakdown	[95,98]
	P. aeruginosa	~5 × 10−6 S/m	Linked to fluid membrane and protein P channels	[97]
	B. subtilis	0.5 × 10−6 to 1 × 10−6 S/m	Thick wall, fewer ion channels → lower conductivity	[28]
Dielectric Permittivity	E. coli	εr ≈ 5–9	Fluid-phase lipid bilayer; strong β-polarization at kHz	[103,111]
	B. subtilis	Lower εr than E. coli (exact value NA)	Reduced dielectric response at GHz	[107]
	Spores (Bacillus, Clostridium)	<4 (dehydrated core)	Low water content → high EF resistance (>30 kV/cm); distinct DEP separation	[108,113]

focuses on membrane-level parameters—such as electrical conductivity and dielectric behavior—that govern microbial sensitivity to electric stimulation.

4. Cytoplasmic Bioelectrical and Dielectric Properties

4.1. Cytoplasmic Conductivity

Cytoplasmic conductivity plays a central role in shaping the electrical response of microbial cells to external fields. It governs internal charge redistribution and contributes significantly to the complex impedance of cells, especially at intermediate to high frequencies (10 kHz–10 MHz) [117]. This parameter depends on intracellular ion concentrations, metabolic activity, pH, and the macromolecular composition of the cytosol. When an electric field is applied, the cytoplasm behaves as a conductive medium, allowing charge transfer between internal and external environments through electroporated or naturally permeable membranes. In dielectric models, cytoplasmic conductivity affects the reactive (imaginary) component of the cell’s impedance, modulating phase shift and relaxation frequencies observed in impedance spectroscopy [117]. For example, viable E. coli cells with intact membranes typically exhibit cytoplasmic conductivity around 0.22 S/m and a relative permittivity near 100, producing a pronounced β-dispersion in the ~1–10 MHz range [117,118]. Following pulsed electric field exposure, conductivity can decrease markedly due to ion leakage and metabolic arrest, changes that are detectable by electrical impedance spectroscopy or time-domain reflectometry and correlate with viability loss confirmed by plate counts [32]. Cytoplasmic conductivity is highly sensitive to the internal ionic environment and pH. Elevated concentrations of K⁺, Na⁺, Cl⁻, and organic acids—such as in metabolically active P. aeruginosa—are associated with higher conductivities compared to nutrient-deprived cells [119,120]. Experimental work has shown that lowering intracellular pH from ~7.2 to ~6.5 can reduce conductivity by up to 20% through altered protein protonation and reduced buffering capacity [121]. In wastewater environments, microbes are frequently exposed to fluctuations in ionic strength and pH during aerobic–anaerobic cycling or after chemical treatment, which alters their impedance signatures. Real-time monitoring systems can exploit these variations to assess microbial viability and stress state [98]. Spore-forming organisms such as Clostridium spp. have very low internal conductivity (<0.05 S/m) due to dehydration and ionic shielding within their core, making them less responsive in impedance-based assays but still distinguishable through dielectric contrast methods [122]. Understanding the dynamic nature of cytoplasmic conductivity is therefore essential for evaluating microbial viability, electrosensitivity, and stress responses—parameters that are increasingly used in wastewater treatment for both detection and selective inactivation.

4.2. Dielectric Permeability of the Cytoplasm

Dielectric permittivity defines how well the cytoplasmic medium becomes polarized under an external electric field. It is vital for understanding microbial responses to electric stimuli, especially at high frequencies where the field penetrates beyond the cellular envelope. Cytoplasm is a richly structured environment, full of water, ions, proteins, nucleic acids, and various inclusions like polyphosphate granules. Its dielectric permittivity determines how effectively the cell’s interior can polarize when subjected to oscillating fields—particularly prominent above ~1 MHz [123]. Studies using a refined multi-shell modeling approach reported that E. coli cells possess a cytoplasm relative permittivity of about 100, paired with a cytoplasmic conductivity near 0.22 S/m. This high permittivity supports substantial polarizability, manifesting as notable β-dispersion around 1–10 MHz [115,118]. Cellular stress—such as electroporation or thermal damage—impairs membrane integrity and ionic balance, lowering polarization capacity. While exact figures are scarce, single-cell impedance studies have shown discernible shifts in the dielectric signature corresponding to compromised viability (e.g., plasma-activated water exposure) [124]. Though bacteria lack membrane-bound organelles, they do accumulate inclusions like glycogen, PHB, or polyphosphate granules. These dense bodies shift cytoplasmic dielectric properties. Moreover, when modeling “dry” bacterial cores (proteins and nucleic acids), cytoplasmic permittivity estimates range from 5 to 6.5. Under ambient (hydrated) conditions, values rise significantly—between 25 and 30 for E. coli—reflecting strong hydration effects and biological complexity [125]. Spore cores, such as those in Clostridium spp., feature dehydrated, densely packed dipicolinic acid–calcium complexes. This structure dramatically limits polarizability—their effective dielectric permittivity remains very low, often under 30, distinguishing them clearly from vegetative cells via dielectric techniques [126]. The cytoplasm inside microbial cells—busy with water, ions, big molecules, and storage inclusions—behaves differently depending on how hydrated or stressed it is. For E. coli, that permittivity hits around 100 when healthy, which helps it polarize sharply under high-frequency fields. Throw in stress or damage, and that ability drops noticeably. Even more interesting: “dry” spores barely polarize, while hydration and internal granules push permittivity way up. That dielectric fingerprint is exactly what tools like high-frequency impedance or dielectrorotation sensors use to tell live cells from stressed or dormant ones. Given that electric fields not only affect microbial membranes but also penetrate the cytoplasm,

Table 3. Cytoplasmic Bioelectrical and Dielectric Properties.

Property	Microorganism/State	Value/Range	Notes	Refs.
Cytoplasmic Conductivity	E. coli (viable)	0.22 S/m	Typical intact membrane; pronounced β-dispersion at 1–10 MHz	[117,118]
	E. coli (post-PEF)	↓ significantly	Reduction due to ion leakage and metabolic arrest	[32]
	P. aeruginosa (active)	Higher than E. coli	Linked to high intracellular K+, Na+, Cl−, and organic acids	[119,120]
	Low pH (~6.5)	↓ ~20%	Reduced protein protonation and buffering capacity	[121]
	Clostridium spores	<0.05 S/m	Dehydration and ionic shielding; low impedance response	[122]
Dielectric Permittivity	E. coli (hydrated)	εr ~100	High polarizability; β-dispersion around 1–10 MHz	[115,118]
	E. coli (dry core model)	5–6.5	Represents proteins and nucleic acids without hydration	[125]
	E. coli (ambient hydrated)	25–30	Strong hydration effects	[125]
	Clostridium spores	<30	Dense dipicolinic acid–calcium complexes; low polarizability	[126]

Notes: “↓” indicates a decrease compared to the control or initial value.

highlights cytoplasmic bioelectrical and dielectric properties that contribute to cellular polarization, electromechanical stress distribution, and inactivation thresholds.

Building upon the cellular-level insights discussed in Section 3 and Section 4, the following section explores how these electrical and dielectric properties are exploited in practice—specifically in the development of detection, monitoring, and disinfection technologies for wastewater systems.

5. Electric Field Effects on Microbial Physiology

Electric fields exert powerful, often species-dependent effects on microorganisms, ranging from reversible physiological changes to complete inactivation. The outcome depends on field intensity, pulse duration and waveform, as well as structural resilience and metabolic state of the target cells.

5.1. Vegetative Cells

In vegetative bacteria such as Escherichia coli and Pseudomonas aeruginosa, EF exposure first causes membrane depolarization followed by pore formation, a process known as electroporation. At moderate field strengths (~10 kV/cm) and short pulses, electroporation is reversible, enabling transient permeability increases useful for biosensing and molecular uptake [127,128,129]. When field intensity exceeds ~15 kV/cm or pulses are prolonged, irreversible poration occurs, leading to membrane rupture, cytoplasmic leakage, and cell death, as confirmed by CFU counts and luminescence assays [130,131]. Sublethal exposure can also alter ATP synthesis, enzyme activity, surface charge, and motility (e.g., reduced swarming in P. aeruginosa) [132], serving as biomarkers of stress [133,134,135].

Overall, vegetative cells are highly responsive to EF exposure, with outcomes ranging from mild physiological stress to complete inactivation, depending on applied parameters.

5.2. Spores

Bacterial spores such as those of Bacillus subtilis and Clostridium perfringens are more resilient due to multilayered, dehydrated structures. PEF in the range of 25–35 kV/cm may trigger germination by releasing dipicolinic acid–calcium complexes or, under suboptimal conditions, inhibit germination and induce membrane reorganization detectable via dielectric relaxation [136,137]. When combined with mild heat (40–50 °C) or high-pressure pretreatments, PEF can synergistically activate spores, offering potential for advanced disinfection strategies, though studies remain limited [138].

Thus, spores require higher EF thresholds than vegetative cells, but their responses open opportunities for combined treatment approaches.

5.3. Broader Metabolic Effects

Beyond direct membrane permeabilization, EFs can disrupt key physiological processes. They collapse the proton motive force (PMF) and membrane potential, induce reactive oxygen species (ROS), impair glucose uptake, and alter NADH activity [139,140]. EF exposure also modulates stress-response gene expression (e.g., soxS, katG), influencing microbial adaptation. In wastewater, baseline stresses from nutrient limitation and redox fluctuations may amplify these effects, sometimes promoting biofilm formation in Pseudomonas spp. or inducing sporulation in B. subtilis under repeated sublethal pulses [141,142]. These metabolic impacts highlight the dual potential of EFs to either damage microbes or trigger adaptive survival responses, depending on context.

5.4. Electrical Impedance Spectroscopy (EIS)

EIS is a label-free method that monitors microbial viability through frequency-dependent impedance patterns. Using low- to mid-frequency AC (1 kHz–10 MHz), it detects changes in membrane integrity, cytoplasmic conductivity, and cell concentration. Viable E. coli typically shows β-dispersion around 100 kHz–1 MHz, whereas inactivated cells display reduced polarization capacity, with capacitance drops of up to 60%. Portable EIS systems achieve sensitivities down to 10³ CFU/mL in real effluent without labeling or filtration [143,144,145]. EIS therefore provides a rapid, non-invasive diagnostic tool for real-time monitoring of microbial dynamics in wastewater.

5.5. Dielectrophoresis

DEP exploits the motion of polarizable cells in non-uniform AC fields, enabling separation by size, morphology, or dielectric properties [143,146,147]. It distinguishes viable from non-viable cells and spores from vegetative forms, outperforming filtration or culture-based enrichment. DEP is increasingly used in microelectrode and microfluidic platforms for microbial capture, improving the detection limits of impedance-based biosensors and supporting real-time, label-free analysis [148,149]. However, high ionic strength in wastewater can dampen efficiency, biofilms may mask signatures, and scaling to treatment plants requires optimization of electrode design and flow conditions [150,151,152].

Despite these challenges, DEP offers powerful capabilities for selective microbial detection and discrimination.

5.6. Field-Assisted Biosensors

Biosensors integrated with EF techniques show enhanced sensitivity through improved analyte transport, surface capture, and modulation of microbial activity. Electrochemical impedance sensors with EF-assistance have reached detection limits as low as 2 CFU/mL [153]. In wastewater contexts, DEP-assisted immunosensors combine microbial concentration with amperometric detection, significantly accelerating binding rates and reducing detection times. Field-assisted biosensors thus represent a promising frontier for online pathogen monitoring in complex effluent streams.

Compared to conventional sterilization approaches, such as chlorination, UV or ozonation, which present drawbacks like carcinogenic by-product formation, reduced efficiency in turbid waters, or high energy demand [17,18,19], electric field-based treatments provide a non-thermal alternative. Pulsed electric fields, for instance, can achieve significant microbial reductions while maintaining the physicochemical integrity of the treated substrate, thus avoiding the quality losses typically associated with thermal sterilization [33].

6. Challenges and Research Opportunities

Exploring electric field–based technologies in wastewater treatment has revealed both promise and complexity. These approaches hold great potential for real-time microbial monitoring, selective control, and sustainable disinfection, but their transition from laboratory proof-of-concept to large-scale implementation requires careful consideration of technical and environmental challenges.

Complex mixed-species biofilms introduce additional limitations: EPS-mediated shielding and steep micro-gradients (O₂, pH, ionic strength) distort local field distribution, create hot-spots, and confound impedance signatures; detachment may release viable aggregates; deeper layers can remain unaffected unless energy input increases—raising Joule-heating and corrosion risks. These factors complicate reproducibility and scale-up, and call for anti-fouling electrodes, controlled shear/flow, and combined strategies (e.g., EF with mild chemical or enzymatic cleaning) [73,74,148,149,150,151,152]. At the same time, pilot-scale demonstrations have shown that EF-based methods can be successfully applied for real-time microbial monitoring and selective inactivation in wastewater, supporting their transition from proof-of-concept studies to practical implementation. Although impedance biosensors are promising—miniaturized and label-free—they still face reliability constraints in harsh wastewater environments (e.g., biofouling, pH shifts, chemical interferences) [150].

A key practical limitation is the difficulty of achieving uniform electric field distribution in complex matrices such as wastewater. High ionic conductivity, turbidity, and suspended solids can generate shielding effects, local hotspots, and heterogeneous microbial exposure, ultimately reducing reproducibility and scalability [33,44,146,147]. Beyond physical complexity, sensor reliability in harsh wastewater environments remains a critical issue. Impedance biosensors, although promising as miniaturized and label-free tools, often suffer from biofouling, chemical interferences, and pH-driven drift [145]. This highlights the need for standardized protocols, durable electrode materials, and hybrid detection platforms that integrate electrical readouts with complementary molecular or omics-based analyses [149,154].

Promising opportunities are emerging to address these limitations. Advanced signal deconvolution methods, such as Distribution of Relaxation Times (DRT), and machine-learning-based pattern recognition can help extract microbial signals from complex backgrounds [148,149]. Anti-fouling electrode coatings, self-powered sensor designs based on microbial fuel cells (MFCs), and multi-modal detection platforms that couple EIS with optoelectronic or omics modules are under development to enhance both robustness and specificity [149,154]. These innovations, together with pilot-scale demonstrations in real facilities, could provide the basis for reliable, decentralized, and energy-efficient wastewater monitoring and control systems.

7. Outlook: Electric Field-Based Wastewater Monitoring and Control

As global demand for resilient, adaptive, and sustainable wastewater treatment continues to grow, electric field (EF)–based technologies are emerging as transformative tools for microbial monitoring and targeted disinfection. Their compatibility with real-time data acquisition, selective physical interaction with microorganisms, and non-chemical mode of operation position them as strong candidates for integration into next-generation smart water systems.

7.1. Smart Biosensing: Integrating AI with EF-Based Readouts

The convergence of artificial intelligence (AI) and EF-based biosensing is set to redefine microbial diagnostics. AI-driven algorithms can enhance pattern recognition in impedance spectra, predict microbial behavior from dielectric signatures, and optimize EF parameters under dynamic conditions [155,156]. Machine learning models trained on EIS or DEP datasets have been shown to classify microbial species, detect anomalies, and estimate microbial loads with minimal human intervention. Such systems could be embedded in autonomous water quality stations, enabling continuous, intelligent surveillance of microbial risks in wastewater [157,158].

7.2. Targeted Microbial Control in Decentralized Wastewater Systems

EF-based solutions offer unique advantages in decentralized or low-infrastructure settings, including portable PEF devices, field-assisted biosensors, and microfluidic DEP platforms. These can be adapted for on-site treatment facilities, agricultural runoff management, or emergency sanitation deployments [159].

Such systems hold potential for targeted inactivation of pathogens, including antimicrobial-resistant strains, while minimizing disruption of beneficial microbial communities in reuse or bioreactor applications. The low chemical footprint and modular design of EF-based platforms align well with circular economy principles and decentralized water reuse strategies [160].

7.3. Hybrid Biotechnological–Electrical Approaches

Merging EF methods with biotechnological tools opens new opportunities for microbial manipulation and process optimization. Potential applications include electro-fermentation, steering microbial metabolism with low-voltage fields [161]; electro-biostimulation, enhancing biofilm growth for pollutant removal or energy recovery [162]; electroporation-assisted gene transfer, enabling rapid adaptation or functionalization of engineered strains in situ [163]. These hybrid approaches pave the way for programmable microbial system engineered communities designed to respond predictably to electric cues, blending synthetic biology with physical field control. Recent electrochemical studies further expand the applicability of EF-based systems by demonstrating their potential not only for microbial detection but also for the monitoring of heavy metal contaminants in wastewater [164,165]. This broader scope strengthens the case for EF methods as versatile environmental tools. Such practical applications underline that EF-based technologies are not limited to experimental proof-of-concept studies but hold real potential for integration into operational wastewater monitoring, decentralized reuse, and smart treatment systems.

8. Conclusions

Electric field technologies represent a promising and versatile approach for investigating and manipulating microorganisms, with potential in the monitoring and treatment of wastewater. While current results highlight their ability to interact directly with structural and functional cellular components—such as membranes, cytoplasmic content, and dielectric properties—their broader applicability still requires validation at pilot and full scale.

Evidence from the literature shows that exposure to electric fields can modulate membrane potential and integrity, cytoplasmic permittivity and ion fluxes, as well as overall cell viability and metabolic activity. These effects are not inherently destructive; under controlled conditions, they can be tuned or reversed, enabling the selective detection, stimulation, or inactivation of microbial populations.

A major advantage of these technologies lies in their capacity to characterize microbial communities in real time, without labels or reagents, which is particularly valuable in complex matrices such as wastewater. Techniques including impedance spectroscopy, dielectrophoresis, and pulsed electric field inactivation are already being adapted into modular and scalable platforms for environmental biotechnology.

Nevertheless, challenges remain in translating laboratory-scale findings into robust, field-ready solutions. Further investigations are necessary to better understand the relationships between specific electric field parameters and microbial structural or functional responses across diverse species and environmental contexts. Advancing model precision, enhancing sensor integration, and improving the scalability of these systems will be key steps toward enabling their broader application in real-world settings. Overall, electric field-based technologies represent a promising frontier in microbial analysis and control, with the potential to complement and enhance conventional water quality strategies. Their multidisciplinary nature encourages continued exploration at the intersection of microbiology, physics, engineering, and environmental science, paving the way for intelligent and adaptive water management systems.