Challenges in Operating a Microbial Electrolysis Cell (MEC): Translating Biofilm Activity to Electron Flow and Hydrogen

Abstract

Microbial electrolysis cells (MECs) are bioreactors that utilize electroactive microorganisms to catalyze the oxidation of organic substrates in wastewater, generating electron flow for hydrogen production. Despite the concept, a persistent performance gap exists where metabolically active anodic biofilms frequently fail to achieve expected current densities by the flow of electrons to produce hydrogen. This review examines the multiple causes that lead to the disconnect between robust biofilm development, electron transfer, and hydrogen production. Factors affecting biofilm generation (formation, substrate selection, thickness, conductivity, and heterogeneity) are discussed. Moreover, factors affecting electron transfer (electrode configuration, mass transfer constraints, key electroactive species, and metabolic pathways) are discussed. Also, substrate diffusion limitations, proton accumulation causing inhibitory pH gradients in stratified biofilms, elevated internal resistance, electron diversion to competing processes like hydrogenotrophic methanogenesis consuming H₂, and detrimental biofilm aging, impacting hydrogen production, are studied. The critical roles of electrode materials, reactor configuration, and biofilm electroactivity are analyzed, emphasizing advanced electrochemical (CV, EIS, LSV), imaging (CLSM, SEM, AFM), and omics (metagenomics, transcriptomics, proteomics) techniques essential for diagnosing bottlenecks. Strategies to enhance extracellular electron transfer (EET) (advanced nanomaterials, redox mediators, conductive polymers, bioaugmentation, and pulsed electrical operation) are evaluated for bridging this performance gap and improving energy recovery. The review presents an integrated framework connecting biofilm electroactivity, EET kinetics, and hydrogen evolution efficiency. It highlights that conventional biofilm metrics may not reflect actual electron flow. Combining electrochemical, microelectrode, and omics insights allows precise evaluation of EET efficiency and supports sustainable MEC optimization for enhanced hydrogen generation.

1. Introduction

Bioelectrochemical systems (BESs) use microorganisms as biocatalysts for the production of energy and wastewater treatment [1]. Electrons are either received from the cathode for reduction processes or transferred from the oxidized substrate to the anode in these systems’ microbially catalyzed electrode reactions [2]. Many microorganisms have recently been found to have electrochemical activity, which allows them to survive while exchanging electrons with an electrode. BESs are now positioned as a promising sustainable energy and chemical production technology as a result of this breakthrough, which has generated a lot of research interest [3].

Microbial fuel cells (MFCs), which produce electricity, and MECs, which consume electrons and produce energy, are the two main types of BESs. Even though the oxidation reactions in the anode are similar for both systems, the cathode reactions are different. While MECs need an external energy input to drive the reaction and produce desired products, MFCs generate electrical energy through thermodynamically favorable reactions [4,5]. MECs are a cutting-edge technology that can treat wastewater and produce clean hydrogen. In contrast to conventional techniques, MECs use bacteria to break down organic waste and produce high-purity hydrogen gas from it, including sewage, agricultural residues, and industrial byproducts [6]. As organic material is being broken down by specialized bacteria inside the anode chamber, protons, electrons, and carbon dioxide are released as byproducts. While the protons move through the system in the direction of the cathode, the released electrons move through an external circuit [7]. MECs need a small external voltage input, usually at least 0.2 volts, to enable hydrogen generation at the cathode, in contrast to MFCs, which generate electricity naturally. Direct current (DC) can be used to provide this required power input [5].

The anodic biofilm serves as the fundamental biocatalytic engine within MECs. This structured community of microorganisms adheres to the electrode surface, forming a conductive matrix embedded within extracellular polymeric substances (EPSs) [8]. Its primary role is the oxidation of organic substrates, liberating electrons and protons. Crucially, the biofilm facilitates the transfer of these liberated electrons to the anode via mechanisms such as direct contact through outer-membrane cytochromes, conductive microbial nanowires, or soluble redox shuttles [9]. The efficiency of this EET process, governed by the biofilm’s composition, structure, thickness, and electroactivity, directly dictates the overall current generation and, consequently, the hydrogen production efficiency at the cathode [10]. Therefore, understanding the formation, function, and limitations of anodic biofilms is paramount for optimizing MEC performance, particularly when addressing the observed disconnect between robust biofilm growth and suboptimal electrochemical output.

MECs demonstrate versatility in utilizing a wide range of organic substrates from wastewater and biomass [11]. These include simple molecules like glucose, lactate, and ethanol, as well as complex organics such as cellulose, proteins, lipids, and even real wastewater matrices like domestic sewage or food processing effluent [12]. However, acetate (CH₃COO⁻) has emerged as the predominant and most extensively studied substrate in fundamental MEC research [13]. Its preference stems from several key advantages: it serves as a direct electron donor for well-characterized exoelectrogenic bacteria, especially those belonging to the genus Geobacter, undergoes straightforward oxidation without requiring complex fermentation steps, offers high reproducibility for mechanistic studies, and is a prevalent intermediate in anaerobic digestion and many wastewaters [14]. Consequently, acetate functions as a model substrate, simplifying the investigation of core electron transfer mechanisms and performance bottlenecks, which is the central focus of this review examining the disconnect between biofilm activity and electron flow to the cathode for hydrogen production [15].

The observed decoupling between high biofilm activity and low current or hydrogen output is one of the most puzzling and frequent problems in the functioning of MECs [16]. Robust biofilm formation on the anode surface initially indicates ideal conditions for EET and system performance. This is frequently verified by dense microbial colonization, high cellular viability, and active metabolic indicators [17]. The actual electrochemical outputs, such as current density and hydrogen production, however, are far below theoretical or expected values, according to several studies, even when the biofilm appears to be healthy and electroactive. Quantitative analyses from previous MEC studies clearly highlight this disparity. Laboratory-scale systems have reported current densities of only 0.5 to 0.8 A m⁻² despite active and well-developed anodic biofilms, compared with theoretical expectations exceeding 1.5 to 2.0 A m⁻². Corresponding hydrogen recoveries were typically limited to 50–65%, indicating that even metabolically active biofilms often fail to achieve efficient electron transfer, underscoring the magnitude of the biofilm–hydrogen performance gap discussed throughout this review [5,18]. The disconnect is due to the combination of physical, chemical, and biological elements. For instance, when a biofilm becomes too thick or layered, it can create redox gradients that leave the inner layers inactive from an electrochemical standpoint. As a result, there are fewer actively metabolizing cells available to transfer electrons to the anode [19]. Also, the restrictions in mass transfer within the biofilm and at the electrode interface can create localized regions where there is not enough substrate and where protons accumulate. This leads to pH gradients that inhibit performance, which can finally raise the internal resistance of the system [20]. Moreover, localized substrate depletion zones and proton accumulation may result from mass transfer restrictions in the biofilm and at the electrode interface, producing inhibitory pH gradients and raising the system’s internal resistance [21]. To make matters worse, the hydrogen generated at the cathode may be lost through diffusion or eaten by hydrogenotrophic methanogens, which would lower net hydrogen recovery [22]. These elements highlight the fact that MEC performance cannot be accurately predicted by microbial activity alone, which is frequently assessed using biomass density or enzymatic markers. Biologically active biofilms are necessary for a high-performing MEC system, but so are precisely calibrated electrochemical interfaces, ideal electrode arrangements, and low transport resistances. Since improving biofilm growth without addressing related inefficiencies may paradoxically reduce rather than increase energy recovery, the conundrum thus emphasizes the urgent need for more integrative and system-level approaches to optimize MECs. The advancement of MECs supports sustainable development goals (SDGs), particularly SDG 6 (Clean Water and Sanitation) and SDG 7 (Affordable and Clean Energy), by combining wastewater treatment with renewable hydrogen generation [11].

This review examines the causes underlying the frequent discrepancy in MECs, where active biofilms exhibit robust growth but generate suboptimal current density and hydrogen yield, using acetate as a model substrate. It critically analyzes key factors contributing to this performance gap, including proton and substrate transport dynamics, electron-transfer mechanisms within biofilms, and the influence of electrode materials and system architecture on overall performance. Despite extensive progress in biofilm cultivation and electrode engineering, most existing reviews treat biofilm electroactivity and hydrogen generation as parallel phenomena rather than interdependent processes. Several reviews have examined biofilm behavior, electron-transfer mechanisms, and hydrogen production in MECs, but most address these elements separately rather than as coupled system phenomena. Sleutels et al. (2012) discussed biofilm activity and hydrogen generation independently, without analyzing their mechanistic correlation [1]. Lapinsonnière et al. (2012) compared enzymatic and microbial biocatalysts but did not quantitatively link biofilm electron transfer to hydrogen yield [2]. Logan et al. (2019) synthesized advances in electroactive microorganisms but did not connect biofilm redox behavior to hydrogen production [3]. Swaminathan et al. (2024) reviewed MECs for wastewater treatment and hydrogen recovery, yet treated biofilm electroactivity and hydrogen output as distinct performance indicators [11]. Call et al. (2009) reported both biofilm activity and hydrogen yield, but did not integrate these data into explaining observed discrepancies [18].

The apparent disconnect between strong biofilm activity and poor electrochemical performance arises from unquantified inefficiencies in electron transfer per unit of metabolic activity. To systematically interpret this relationship, a coupled electrochemical–biological diagnostic framework is introduced that integrates cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) signatures with microelectrode-derived redox/pH gradients and multi-omics indicators of microbial metabolism. By correlating these datasets with hydrogen output, the framework provides a quantitative measure of biofilm-specific EET efficiency, transforming observational discrepancies into diagnosable system bottlenecks. This review thereby establishes an analytical perspective and serves as a comprehensive resource for advancing MEC efficiency toward practical applications in wastewater treatment and sustainable hydrogen production [3,23].

2. Working Principle of MEC

MECs use a closely coordinated series of microbial and electrochemical processes to convert biodegradable organics into hydrogen and other products with added value. It is essential to base the discussion on the fundamental workings of MECs because this review centers on the perplexing discrepancy between high biofilm activity and low electrical output. Therefore, this section (i) describes the half-cell reactions that propel proton and electron flows, (ii) explains the energetic and thermodynamic framework that sets MECs apart from traditional water electrolysis.

2.1. Anode and Cathode Reactions in MECs

The core aspect of MECs is the two separate half-reactions. In the anode chamber, electroactive microorganisms break down the substrate provided and utilize it to generate energy, but also result in the release of protons and electrons in the process:

$${\mathrm{CH}}_3\mathrm{CO}{\mathrm{O}}^{-} + 4{\mathrm{H}}_2\mathrm{O} \to 2\mathrm{HC}{\mathrm{O}}_{3 }^{-}+ 9{\mathrm{H}}^{+} + 8{\mathrm{e}}^{-}$$

(1)

In a biofilm, the transfer of electrons generated by oxidation occurs in several ways, including direct contact, conductive pili, cytochromes, and redox mediators. As shown in Figure 1, electrons travel from the biofilm to the anode, then to the cathode through an external circuit. To keep everything balanced, the protons, along with other cations, move across the proton-exchange membrane or electrolyte. When a small external voltage manages to overcome kinetic barriers, the electrons and protons come together at the cathode, resulting in the production of hydrogen gas:

$$8{\mathrm{H}}^{+} + 8{\mathrm{e}}^{-} \to { 4\mathrm{H}}_2$$

(2)

Net overall reaction:

$${\mathrm{CH}}_3\mathrm{CO}{\mathrm{O}}^{-} + 4{\mathrm{H}}_2\mathrm{O} \to 2\mathrm{HC}{\mathrm{O}}_{3 }^{-}+ 4{\mathrm{H}}_2$$

(3)

The cell is powered by electron flow, and ohmic losses are determined by the internal ionic circuit, which is made up of protons and other ions. These losses can be increased by excessive biofilm thickness or electrolyte resistance, which also decouples microbial activity from detectable current [24].

2.2. Thermodynamics and Energy Inputs

The oxidation of acetate (substrate) is strongly exergonic (ΔG°′ ≈ −847 kJ mol⁻¹) from a thermodynamic perspective, giving microbes plenty of metabolic energy. To move reaction (2) forward, an external bias (usually 0.1–1.0 V) must be applied because proton reduction to H₂ is endergonic at biological pH (E₀′ ≈ −0.41 V versus standard hydrogen electrode (SHE)) [25]. Since this bias is significantly less than the 1.23 V needed for water electrolysis, MECs are inherently more energy-efficient [26]. However, in reality, the real energy input is increased by concentration polarization within thick biofilms, ohmic resistance across the electrolyte and membrane, and activation overpotentials at both electrodes [27]. It is crucial to accurately analyze these voltage losses to comprehend why a metabolically active biofilm can still produce low electrical yields.

3. Factors Contributing to the Disconnect Between Biofilm, Electron Transfer Mechanism, and Hydrogen Production

The factors that contribute to ineffective electron transfer from anode to cathode and production of hydrogen at the cathode, even though there is an active biofilm formation at the anode, are examined in this section. Although biofilm activity is necessary, system-level performance and effective EET are not always ensured by it. The three interconnected factors are discussed, which depend on biofilm, electron transfer mechanisms, and hydrogen production.

3.1. Factors Affecting Biofilm Development

Biofilm development at the anode is the first critical step governing microbial attachment, substrate utilization, and overall current generation in MECs.

3.1.1. Stages of Biofilm Formation

The development of biofilms on conductive surfaces follows a predictable sequence of events: (i) initial adhesion, in which planktonic cells attach using pili and van der Waals forces [28]; (ii) microcolony formation, which is characterized by rapid cell division and the onset of EPS secretion [29]; (iii) mutation, in which a three-dimensional architecture rich in EPS and water channels emerges [30]; and (iv) late-stage consolidation or dispersal, where shear stress or nutrient limitations cause cell death, sloughing, or phenotypic changes [31]. Each stage in MECs influences the community’s electrical connectivity; mature biofilms provide longer conduction paths but higher ohmic losses, while early monolayers maximize direct anode contact. Figure 2 highlights this progression visually, showing how the initially porous and metabolically active biofilm transitions into a denser structure with restricted water-channel networks, which corresponds to the reduced EET efficiency and increased internal resistance observed in mature and aging biofilms. The selection of suitable microbial species plays a pivotal role in ensuring successful biofilm formation and electrochemical performance in MECs. Electroactive bacteria used for anodic colonization must possess several essential characteristics, including strong adhesion to conductive anodes, high EET capability via direct or mediated pathways, tolerance to anaerobic conditions, and metabolic flexibility toward substrates. Among the most studied exoelectrogenic bacteria, Geobacter sulfurreducens and Desulfuromonas acetoxidans form dense, conductive biofilms and perform DET using outer-membrane c-type cytochromes (OmcZ, OmcS) and conductive pili (nanowires) [32,33]. These species achieve high coulombic efficiencies of greater than 90% and current densities exceeding 1.5 A·m⁻² when acetate is used as the electron donor [21]. Shewanella oneidensis also exhibits EET ability through MET using self-secreted flavins and quinones, enabling biofilm growth under variable redox conditions [3,34].

Non-exoelectrogenic but robust biofilm formers such as Pseudomonas aeruginosa contribute to biofilm stability via abundant EPS production, which improves adhesion and mechanical resilience [35,36]. Mixed culture inocula from wastewater environments often outperform pure cultures by combining fermentative, syntrophic, and exoelectrogenic species such as Geobacter, Shewanella, and Pseudomonas, providing a greater substrate utilization range and enhanced system stability [37,38].

3.1.2. Selection of Substrate in MEC

Substrate selection is a critical factor shaping biofilm development, community structure, and EET efficiency in MECs. Using acetate as a simple, readily oxidizable substrate, the researchers observed the formation of dense, homogeneous biofilms dominated by specialist exoelectrogens such as Geobacter sulfurreducens. These biofilms exhibited high current densities and efficient EET, primarily through direct electron transfer (DET) mechanisms involving outer-membrane cytochromes and conductive nanowires [32]. In contrast, complex substrates, such as sucrose or wastewater, lead to the development of stratified biofilms composed of syntrophic consortia, including fermenters and anaerobes. This stratification results in heterogeneous metabolic zones within the biofilm, increasing mass transfer resistance, creating redox gradients, and intensifying competition for electrons. Consequently, more energy is diverted toward fermentation processes rather than direct anode respiration, which can reduce overall MEC efficiency [39]. Therefore, substrate type directly impacts biofilm architecture, electroactivity, and the hydrogen yield.

3.1.3. Biofilm Thickness, Conductivity, and Heterogeneity

Internal diffusion limitations arise when biofilms thicken beyond 50 to 100 µm: protons produced at the anode must escape, while substrate must pass through EPS matrices [40]. The percentage of cells that contribute electrons can be reduced by creating an electrochemically inert inner core through steep substrate and pH gradients [41]. In addition, the density and alignment of cytochromes determine the biofilm’s bulk conductivity; a heterogeneous distribution of these structures results in spatially varying resistances [33]. Excess EPS, metal precipitates, or dead biomass further hinder charge transport [42]. Recent redox mapping studies confirmed that internal potential and pH gradients strongly control biofilm conductivity and electron flux [43]. Therefore, there is an ideal biofilm thickness where conductive path length and electron-generation capacity are balanced; going over this threshold results in decreasing current even with high microbial activity. However, a 50 to 100 µm threshold is not universal and can vary with substrate type, hydrodynamic regime, and anode potential. Thicker biofilms (up to 150 µm) can remain active under high-flow or complex-substrate conditions, whereas thinner layers (50 µm) perform better under diffusion-limited or high-potential environments [40,44]. Quantitative studies have shown that biofilm architecture has a substantial impact on mass transfer and electron conduction. Zhang et al. (1994) reported that biofilm porosity can decrease substantially with depth, from 0.84 to 0.93 in upper layers to 0.58 to 0.67 in deeper, compacted regions, which correspondingly lowers the effective diffusivity ratio from 68–81% to 38–45%, representing more than a 50% reduction in diffusivity in denser zones [45]. Torresi et al. (2017) [46] further demonstrated that thin, less porous biofilms (porosity 75%) exhibit diffusivity factors (f) of less than 0.1. In contrast, thicker and more porous biofilms (porosity > 90%) achieve f-values of 0.2 to 0.8, indicating improvements in mass-transfer capacity [46]. Such reductions in porosity and water-channel openness restrict proton and substrate mobility, increase electron-transfer resistance, and can decrease bulk biofilm conductivity by up to an order of magnitude. The activity decline illustrated in Figure 2 mirrors the quantitative trends, where increased thickness and compaction reduce diffusivity and electron-transfer pathways.

Therefore, controlling biofilm structure and composition is essential, as excessive growth or poor conductivity can initiate the redox and mass-transfer bottlenecks that lower MEC efficiency.

3.2. Factors Affecting Electron Transfer Mechanisms

Even with well-developed biofilms, inefficient electron transfer between microorganisms and the anode remains a major cause of low current and hydrogen output.

3.2.1. Electrode Material and Configuration

The density of biofilm colonization, the strength of microbial adhesion, and the effectiveness of electron transfer are all significantly influenced by the anode’s material and surface architecture [47]. Because of their high conductivity, stability, and biocompatibility, carbon-based materials such as graphite rods, carbon cloth, and carbon felt are used extensively [48]. However, these materials’ specific surface area, porosity, and roughness can differ greatly, which affects the electroactivity and morphology of biofilms [49]. Carbon nanotube (CNT) sponges and graphene-coated foams are examples of high-surface-area structures that offer more colonization sites and allow for three-dimensional growth, which may increase current output [50]. As summarized in Table 1, materials with higher surface area and conductivity, such as carbon felt, carbon cloth, and CNT/graphene composites, consistently produce higher normalized current densities, reinforcing the correlation between electrode microstructure and EET performance. However, if these intricate structures are not appropriately optimized, they can also trap dead biomass and produce mass transfer dead zones [51].

Table 1. Comparison of commonly used carbon-based anode materials in MECs with respect to surface area, conductivity, and biofilm compatibility.

Anode Material	Surface Area (m2g−1)	Conductivity (S cm−1)	Biofilm Compatibility	Reference
Graphite Rod	0.5–1	10–100	Good; supports mature biofilms	[52]
Carbon Felt	10–20	10–100	Excellent; promotes dense, uniform biofilms	[52,53]
Carbon Cloth	5–10	10–100	Very good; rapid biofilm formation and high current generation	[52,54]
Carbon Paper	5–10	10–100	Good; higher current density than graphite rod	[52]
CNT/Graphene	100–2042.5	1.32–204	Excellent; enhances electron transfer and microbial attachment	[55,56]

Table 1. Comparison of commonly used carbon-based anode materials in MECs with respect to surface area, conductivity, and biofilm compatibility.

Anode Material	Surface Area (m2g−1)	Conductivity (S cm−1)	Biofilm Compatibility	Reference
Graphite Rod	0.5–1	10–100	Good; supports mature biofilms	[52]
Carbon Felt	10–20	10–100	Excellent; promotes dense, uniform biofilms	[52,53]
Carbon Cloth	5–10	10–100	Very good; rapid biofilm formation and high current generation	[52,54]
Carbon Paper	5–10	10–100	Good; higher current density than graphite rod	[52]
CNT/Graphene	100–2042.5	1.32–204	Excellent; enhances electron transfer and microbial attachment	[55,56]

3.2.2. Mass Transfer Limitations Through the Connections and the Membrane

Mass transfer limitations in the movement of substrates like acetate and products such as protons and hydrogen through the biofilm matrix and electrolyte can affect MEC efficiency [57]. As biofilms thicken, the transport of acetate to deeper layers slows and becomes the limiting factor [15]. At the same time, protons generated during acetate oxidation accumulate in the biofilm, causing steep pH gradients [58]. A lower pH in the inner biofilm can hinder redox enzyme activity and microbial metabolism, leading to reduced electron transfer and current density even when outer layers function well [34]. Beyond the challenges within the biofilm, poor fluid mixing near the electrode surface creates stagnant diffusion boundary layers and decreases mass transport [59]. The situation is intense in MECs using cation exchange membranes; slow proton transfer leads to counter-ion accumulation and increased internal resistance [60].

3.2.3. Key Electroactive Species

Usually, obligate anaerobes that are well-known for their EET dominate MEC anodes [61]. DET is a speciality of Geobacter sulfurreducens, which can achieve high coulombic efficiencies under optimal conditions through dense networks of conductive pili and c-type cytochromes [62,63]. Although it can withstand slightly higher salinity, Desulfuromonas acetoxidans plays a similar role [64]. Although facultative anaerobes like Shewanella oneidensis can colonize anodes, they typically play a supporting role when a particular substrate is the only carbon source because they depend on soluble flavins for mediated electron transfer (MET) [65]. Hydrogenotrophic methanogens that compete for electrons or fermenters that cross-feed substrate can also be found in mixed-culture systems [66]. A recent review also highlights how electrode potential influences EET gene regulation and microbial community structure [67].

In mixed-culture MEC biofilms, the balance between direct electron transfer DET and MET varies with substrate composition, anode potential, and microbial community structure [68,69]. DET typically dominates in acetate-fed systems enriched with Geobacter species, where cytochromes and conductive pili support efficient electron conduction with minimal diffusion losses [70,71]. In contrast, complex substrates promote the activity of fermenters and syntrophic partners that increase secretion of soluble redox mediators, elevating the contribution of MET. But it reduces the overall electron transfer efficiency due to mediator diffusion, instability, and redox cycling losses [10,72]. Anode potential also modulates pathway dominance, with more positive potentials promoting cytochrome expression and DET, whereas lower potentials increase mediator release and MET activity [73]. Each pathway introduces characteristic efficiency losses where DET is limited by redox cofactor density and increased internal resistance in thick or heterogeneous biofilms. Moreover, MET suffers from diffusion constraints, mediator instability, and lower coulombic recovery [71]. These interacting factors explain why mixed-culture biofilms can exhibit high metabolic activity yet inadequate electron delivery to the anode. Overall, biofilm conductivity and electron sink competition are determined by the relative abundance of these guilds.

3.2.4. Metabolic Pathways of Substrate-Oxidizing Exoelectrogens

Specialized exoelectrogens with the capacity to transfer electrons straight to an electrode are the main agents responsible for oxidation in MECs. The most well-known of these are the species of Desulfuromonas and Geobacter [70]. These microorganisms use the tricarboxylic acid cycle (TCA) to metabolize acetate, a model substrate. Acetyl-CoA (Acetyl coenzyme A) is initially produced from acetate, then it enters the TCA cycle and is oxidized to CO₂. As a result, reduced cofactors such as NADH (Nicotinamide adenine dinucleotide) and FADH₂ (Flavin adenine dinucleotide) are produced, which are subsequently utilized to propel electron transfer via an electron transport chain that is membrane-bound (Figure 3a) [74]. Figure 3a visually summarizes this pathway by mapping acetate oxidation, NADH/FADH₂ generation, and subsequent electron transfer through membrane-bound carriers, clarifying the sequence of biochemical reactions. The dominance of DET or MET directly shapes the net electron flux entering the anode, especially in mixed-culture systems where fermenters, methanogens, and exoelectrogens compete for electrons under different substrate and potential regimes. Hari et al. (2016) [75] reported that more positive anode potentials and acetate-rich environments favor DET, resulting in higher current outputs. In contrast, fermentable substrates and lower anode potentials promote methanogenic competition and increase reliance on MET, leading to greater electron diversion to methane and reduced coulombic efficiency [75]. Geobacter sulfurreducens can transfer electrons through outer-membrane c-type cytochromes or conductive pili, also referred to as nanowires [62]. DET is made possible by these structures, which physically join the bacteria to the anode surface [76]. High-resolution cryo-electron microscopy (cryo-EM) and spectroscopic studies show that Geobacter forms multi-heme cytochrome filaments (OmcS, OmcE, OmcZ) with stacked hemes that create conductive electron-tunneling paths [77,78]. Recent reviews on exoelectrogenic communities emphasize how multi-omics and imaging tools clarify these EET networks [79]. These coexist with type IV pili (PilA), which anchor cells and align redox proteins for efficient contact [80]. The balance between cytochrome-based conduction and pili-assisted interfaces depends on anode potential and biofilm conditions. Pili provide structural linkage, while OmcZ or OmcS filaments act as high-conductance pathways, especially at the biofilm–electrode boundary. This cooperation enhances long-range EET but can lead to transport bottlenecks where internal redox gradients limit electron flow despite high microbial activity [81].

In thick biofilms, electron flow becomes limited by redox and mass-transfer constraints rather than by microbial metabolic potential alone. Across biofilms ranging from tens to hundreds of micrometers, redox and pH gradients can create zones with unfavorable potentials for outer-membrane cytochromes, thereby lowering effective EET per cell. Microelectrode and CLSM mapping at varying anode potentials demonstrate spatial stratification of electroactivity and potential-dependent enrichment of specific anodic populations, clarifying why high biomass does not always correspond to proportional current output [82]. Furthermore, some exoelectrogens generate soluble redox mediators that transfer electrons to the electrode [83].

The overall efficiency of these molecular-scale EET pathways ultimately governs the electrochemical performance of the entire MEC system. In thick biofilms, spatially uneven cytochrome- and pili-mediated electron transfers lead to the formation of localized redox and pH gradients that generate potential drops across the biofilm matrix. Such gradients can increase anode internal resistance by approximately 20 to 40%, resulting in noticeable reductions in current density and hydrogen yield. Consequently, even metabolically active biofilms may underperform electrochemically because heterogeneity in redox conditions and limited conductivity restrict effective electron flow. Previous studies have confirmed that uneven redox potentials and proton accumulation diminish biofilm conductivity and constrain EET efficiency [84,85]. This coupling between molecular-level activity and system-scale behavior is illustrated in Figure 3b, which complements Figure 3a by linking cytochrome-driven electron transfer and redox stratification to overall reactor-level hydrogen generation.

Understanding these electron transfer limitations is crucial for developing targeted electrode modifications and microbial strategies to enhance EET performance.

3.3. Factors Affecting the Production of Hydrogen at the Cathode

Hydrogen generation efficiency at the cathode depends not only on electron availability but also on redox balance, mass transport, and competing biological reactions.

3.3.1. Redox Imbalance and Internal Resistance

Efficient EET relies on a delicately balanced redox environment. When the anode potential is overly positive compared to the redox potential of microbial electron carriers, like c-type cytochromes, EET becomes thermodynamically challenged [85]. This redox mismatch from factors like biofilm overgrowth, surface fouling, or voltage fluctuations can trap electrons or divert them through less efficient pathways, reducing MEC performance [86]. The overall efficiency in MECs is significantly compromised by internal resistance, including ohmic resistance from the electrolyte and separator, activation overpotential at the electrode interface, and concentration overpotential from local substrate depletion [87]. These resistive losses in MECs absorb part of the external voltage, leaving less energy available for current generation and hydrogen evolution [88].

3.3.2. Hydrogen Losses and Methanogenesis

Even when electrons successfully reach the cathode, hydrogen yield is not guaranteed. Hydrogen generated at the cathode can diffuse back or cross over to the anode, where it may be consumed by hydrogenotrophic microorganisms, reducing the hydrogen yield [89]. Among these, methanogens, particularly hydrogenotrophic types, convert H₂ into CH₄ in MECs, thereby reducing both coulombic and hydrogen recovery efficiencies [89,90]. This biological short-circuiting is more pronounced in mixed-culture MECs and during extended operation without selective microbial enrichment [18]. Additionally, some hydrogen is physically lost by leakage or solubilization into the electrolyte [91].

Cui et al. (2019) reported that, under uninhibited mixed-culture conditions, hydrogen content in the biogas decreased from 85 to 90% to nearly 0%, while methane increased to greater than 94%, demonstrating almost complete diversion of H₂ to methanogenesis [92]. Hou et al. (2014) similarly showed that hydrogen yield declined from 3.78 to 0.03 mol H₂ mol⁻¹ acetate when methanogenesis was active, corresponding to 99% hydrogen loss [93]. He et al. (2022) summarized that methanogenesis typically accounts for 40 to 60% of total hydrogen losses in MECs, where H₂ purity dropped from 83% to 25% and from 93% to 6%, accompanied by CH₄ increases from 9% to 66% and 5.5% to 94%, respectively [94]. Beyond biological consumption, physical diffusion and crossover of H₂ through membranes, tubing, and imperfect seals can further reduce apparent recovery. Bibliometric and content analysis by Zhao et al. (2020) highlighted that decreasing internal resistance and H₂ diffusion is crucial for improving MEC hydrogen yields, particularly in single-chamber and poorly sealed systems [95]. Collectively, these studies confirm that methanogenic hydrogen consumption represents the primary cause of H₂ loss in mixed-culture MECs, frequently exceeding losses from physical diffusion.

3.3.3. Biofilm Stratification and Aging

Internal stratification in thickened anodic biofilms leads to substrate starvation and metabolic byproduct accumulation in inner layers, with only outer layers remaining metabolically active [96]. Spatially differentiated activity in biofilms leads to a decline in the fraction of electroactive cells contributing to current, even though total biomass may continue to increase [39]. Furthermore, the buildup of EPS and dead cells in deeper biofilm layers forms a dense, poorly conductive matrix that hinders mass transport and increases resistance [97]. As a result of these aging effects, which are summarized in Table 2, microbial activity of the biofilm gradually becomes decoupled from functional outputs such as hydrogen evolution or current density [98].

Table 2. Changes in biofilm viability, EPS concentration, and conductivity with biofilm age.

Biofilm Age	Viability (Live/Dead Ratio)	EPS Concentration (mgg−1 Biofilm)	Conductivity (Sm−1)	Reference
Young (early)	High(>80% live)	Moderate	High	[84,97,99]
Mature	Moderate (~60–70% live)	Increased (protective response)	Moderate	[84,97,99]
Aged (late)	Low(<40% live, dead core)	High (thick EPS layer)	Lower (Dead layer may remain conductive)	[84,97,99]

Table 2. Changes in biofilm viability, EPS concentration, and conductivity with biofilm age.

Biofilm Age	Viability (Live/Dead Ratio)	EPS Concentration (mgg−1 Biofilm)	Conductivity (Sm−1)	Reference
Young (early)	High(>80% live)	Moderate	High	[84,97,99]
Mature	Moderate (~60–70% live)	Increased (protective response)	Moderate	[84,97,99]
Aged (late)	Low(<40% live, dead core)	High (thick EPS layer)	Lower (Dead layer may remain conductive)	[84,97,99]

Hence, improving cathodic reactions requires simultaneous control of biofilm activity, internal resistance, and hydrogen losses to competing microbial pathways. Overall, the observed gap between biofilm activity and hydrogen yield arises from these interacting factors, providing a clear foundation for the diagnostic and optimization strategies discussed in the following sections.

4. Monitoring and Characterization Techniques of Biofilm Performance

To identify performance bottlenecks in MECs and direct focused enhancements, accurate evaluation of biofilm function is crucial. Modern characterization approaches cover three complementary domains: electrochemical probes that quantify real-time electron transfer; imaging techniques that reveal biofilm architecture; and high-throughput omics that decode community composition and metabolic potential. A comprehensive understanding of how structural, electrochemical, and genetic factors interact to affect current and hydrogen yields is made possible by combining these tools.

4.1. Electrochemical Techniques: CV, EIS, and LSV

Electrochemical methods offer rapid, non-destructive insights into the redox behavior and resistive losses of living biofilms. Cyclic voltammetry (CV) scans the anode potential to identify redox-active peaks associated with outer-membrane cytochromes and soluble mediators, allowing evaluation of electron-transfer kinetics. The shape and height of CV peaks can indicate biofilm health. Sharp, well-defined peaks suggest efficient redox coupling, whereas flattened curves often signal fouling or redox imbalance [100]. Electrochemical impedance spectroscopy (EIS) applies small-amplitude AC perturbations over a frequency range to resolve charge-transfer resistance, double-layer capacitance, and mass-transfer diffusion elements to monitor biofilm development and aging [101]. Linear sweep voltammetry (LSV) is used to map anode polarization behavior and determine onset potentials [102]. Together, CV, EIS, and LSV build a quantitative fingerprint of biofilm electroactivity that can be tracked over time or compared across electrode materials.

Several emerging in situ techniques now provide direct insight into electron-transfer processes within MEC biofilms. Plasmonic imaging enables label-free, real-time visualization of cytochrome redox activity at the single-cell level, revealing spatial heterogeneity in EET [103]. In situ Raman spectroscopy can monitor the redox state of outer-membrane cytochromes within intact, hydrated biofilms during current generation [104]. Oblique Incident Reflectivity Difference (OIRD) offers spatially resolved, real-time mapping of EET flux at the biofilm–electrode interface [105]. Cyclic voltammetry coupled with microsensors has also been applied to semi-quantitatively track electron-transfer activity and hydrogen involvement directly within living biofilms [106].

In addition, recent in situ studies tracking biofilm behavior under different anode potentials clarify how start-up voltage and operating regime shape electroactivity and stability in MEC systems [107,108].

4.2. Microscopic and Imaging Methods: CLSM, SEM, and AFM

When analyzing electrochemical data, it is crucial to visualize the morphology of biofilms. Confocal laser scanning microscopy (CLSM) enables non-destructive, three-dimensional visualization and quantification of biofilm thickness, porosity, and structure in hydrated biofilms [109]. Scanning electron microscopy (SEM) provides high-resolution images of conductive pili networks, mineral precipitates, and cell-to-electrode contact points in dehydrated biofilm samples [63]. Atomic force microscopy (AFM) measures biofilm stiffness, adhesion forces, and surface topography in liquid environments relevant to MECs. By combining CLSM volumetrics with AFM nanomechanics and SEM ultrastructure, scientists can link spatial differences in electron-transfer efficiency to physical heterogeneity.

4.3. Omics Tools: Metagenomics, Transcriptomics, and Proteomics

In addition to what can be inferred from electrochemistry and imaging alone, high-throughput omics can reveal the genetic and functional blueprint of biofilms in MECs [110]. Metagenomics can identify key microbial groups and gene abundances related to hydrogenotrophs, fermenters, exoelectrogenic taxa, and important pathways like pilin assembly, hydrogenase expression, and c-type cytochrome production [111]. Transcriptomic (RNA-sequencing) analysis can identify gene expression responses to oxidative stress and cell membrane changes under different electrical and redox conditions [112]. Proteomics provides the strongest correlation with phenotype by confirming the presence and activity of metabolic enzymes and redox proteins [113]. Combining these layers or multi-omics approaches (Figure 4) in MECs can identify key microbial groups, gene abundances, gene expression changes, and active proteins, revealing functional bottlenecks such as increased methanogenic hydrogenases and inhibited cytochrome synthesis [114].

Collectively, these electrochemical, imaging, and omics techniques provide the diagnostic basis for identifying electron-transfer inefficiencies and guiding process improvements.

5. Techniques for Enhancing the Electron Transfer Mechanism and Hydrogen Production

Recent studies have been dedicated to addressing the limitations associated with MECs by improving the efficiency of EET between electroactive microbes and electrodes. While the activity of microorganisms is crucial, the overall performance of the system relies on how effectively electrons are transferred from microbial metabolism to the external circuit. This section discusses several promising approaches that have emerged to enhance EET, including the development of advanced anode materials, the use of redox mediators and conductive polymers, the strategic modification of microbial communities through bioaugmentation, and the implementation of novel electrical inputs employing pulsed or alternating voltage methods.

5.1. Acetate as Model Substrate

Acetate is a preferred substrate in MEC research due to the consistent electrochemical behavior and abundance in wastewater. For exoelectrogenic bacteria, especially those belonging to the genus Geobacter, which are renowned for their effective EET mechanisms, acetate, a short-chain volatile fatty acid, is the perfect electron donor [115]. In MECs, acetate is oxidized, which releases electrons that are transported to the anode via the biofilm. Hence, acetate is highly suitable for researching the direct relationship between electrochemical outputs like current density and hydrogen production and microbial metabolic activity [116]. Additionally, unlike longer-chain or aromatic organics, acetate does not require complex enzymatic degradation, which reduces confounding variables in experimental designs [117]. The widespread use of acetate in wastewater treatment streams and anaerobic fermentation processes gives its application in MECs an additional practical significance [118]. Four useful benefits account for acetate’s dominance as a research substrate. (i) Simple metabolism: because it is a two-carbon molecule, it can undergo direct oxidation through the TCA without going through the hydrolytic or fermentative processes [119]. (ii) Compatibility: canonical exoelectrogens with well-characterized extracellular electron-transfer (EET) pathways, like Desulfuromonas acetoxidans and Geobacter sulfurreducens, grow robustly on acetate [70]. (iii) reproducibility: standardizing performance metrics and facilitating mechanistic comparisons across laboratories using the same, simple substrate [120]. (iv) Significance: Since acetate is widely present in municipal wastewaters and fermentation effluents, laboratory results are consistent with actual feedstocks [121]. Because of these benefits, acetate is perfect for examining basic EET issues and locating systemic bottlenecks that more complex organics might obscure. In addition to making system operation simpler, the direct use of acetate eliminates the need for fermentation intermediates and offers a more controlled platform for researching how electrode materials, system architecture, and environmental factors affect MEC performance.

Regardless of the benefits, the use of acetate has some other difficulties, including inadequate scalability, biofilm stratification, and disparities between hydrogen yield and microbial activity [122]. These problems show that although acetate makes the biological environment simpler, it also reveals more profound system-level inefficiencies pertaining to internal resistances, mass transport constraints, and electron transfer kinetics [123]. Therefore, when transferring bench-scale insights to practical applications, acetate’s limitations must be carefully considered, even though it is still a crucial model substrate for basic MEC research [124]. Also, acetate-based MECs often fail to replicate the complexity of real wastewater systems, where mixed substrates and microbial interactions can cause lower reproducibility. Thus, while acetate serves as an excellent model substrate for mechanistic studies, its scalability to real environmental applications remains limited [70].

5.2. Advanced Electrode Materials

Microbial attachment and biofilm integrity can be enhanced by surface functionalisation, such as the addition of carboxylic, hydroxyl, or amine groups to the anode [125]. Advanced carbon-based nanomaterials with their remarkable properties, such as high surface area, excellent conductivity, and customizable surface chemistry, enhance the performance of MECs [126]. A study illustrated that the use of graphene, known for its unique two-dimensional honeycomb structure, stands out due to its ultra-low electron resistance and large surface area, which promote superior and consistent microbial colonization [127]. Interfacial charge-transfer resistance can also be decreased by covering the anode with metal oxides [128,129]. Indirect effects of electrode geometry and spacing on ion transport can also affect the efficiency of electron recovery [130]. Graphene-based composites with metal oxides or polymers enable tunable surface charge and wettability, supporting superior microbial colonization and electrochemical performance [131]. On the other hand, CNTs provide a highly conductive 3D scaffold that enhances microbial adhesion and shortens charge transfer distances [132]. A study reported that electrodes modified with CNTs or graphene oxide increased current density from 7.5 A m⁻² to 32.5 A m⁻² (≈ 4.33-fold) compared with unmodified electrodes [50]. Recent advances in surface-functionalized nanocarbons and transition-metal composites further enhance electron transport and microbial adhesion [23]. Figure 5 compiles the current densities reported for various nanostructured electrodes, including CNT/GO composites, N-MWCNT/graphene aerogels, PANI/CNT/rGO hybrids, rGO–FeMoO₄/MnO₂ composites, NiCo₂O₄-coated graphite rods, N-doped graphene, PPy–Pd nanohybrids, MnO/CNF@graphene membranes, and Pt–Ni/graphene nanoplatelets. These data illustrate the wide performance range achievable through nanomaterial-based electrode engineering. It shows that CNT and graphene-based hybrid electrodes exhibit the highest current densities, while PPy–Pd nanohybrids achieve the largest relative improvement despite moderate absolute performance. In contrast, Pt–Ni/graphene nanoplatelets provide the highest absolute current density, reflecting their superior catalytic activity but smaller fold-increase over baseline. Collectively, these trends illustrate how nanomaterial composition, catalytic functionality, and structural design jointly determine the magnitude of MEC performance enhancement. The combination of high conductivity and optimal biofilm interaction positions these materials as exceptional candidates for enhancing electron harvesting.

Despite their excellent performance, many advanced materials face cost and scalability barriers. Long-term stability, potential leaching of nanoparticles, and challenges in large-scale synthesis may restrict their industrial use. Therefore, optimization must balance performance gains with environmental and economic sustainability [23].

5.3. Improving Reactor Mixing, Biofilm Architecture, and Electrode Design

To improve electron transfer efficiency in MECs, addressing mass transfer limitations is crucial. Enhancing reactor mixing through mechanical stirring, gas sparging, or flow recirculation can reduce stagnant boundary layers. Moreover, it can increase the turbulence near the electrode surface, which improves substrate and proton transport throughout the system. These interventions improve substrate and proton transport throughout the system, supporting higher current densities and more stable MEC operation. Real-time, in situ biofilm monitoring was used to identify optimal biofilm thickness (100–150 μm) for maximum current generation [140]. Better mixing also facilitates the uniform distribution of microbial communities and prevents massive accumulation or deposition of inhibitory byproducts. A study demonstrates that, under flow conditions, an increased diversity within the biofilm community and higher levels of EPSs contributed to greater microbial resilience against heavy metal stress. This enhanced mixing mitigated the negative effects of toxic byproducts, such as copper ions [141]. Developing controlled, thinner biofilms through periodic shear stress, hydraulic control, or quorum-sensing inhibitors can ensure better penetration of the substrate (acetate) into the inner layers, minimize pH gradients, and maintain optimal conditions for microbial metabolism and redox enzyme activity. Thinner biofilms also allow faster diffusion of protons away from the anode, preventing acidic microenvironments that hinder electron transfer [142].

Additionally, integrating engineered microchannel electrodes or 3D-structured materials not only increases surface area for biofilm growth but also enhances convective flow and nutrient access at the micro-scale level. Such architectures shorten diffusion distances within the electrode-biofilm interface and improve electron collection efficiency [143]. Researchers investigated the use of 3D-printed anode electrodes with precisely defined geometries, including microchannels in the hundreds of micrometer range. These electrodes were fabricated using advanced additive manufacturing techniques with conductive copper-based Electrifi filament, and sometimes they were further enhanced with surface modifications to improve electrochemical activity. The research demonstrated that spiral-shaped 3D-printed electrodes significantly improved mass transport and biofilm colonization compared to traditional designs, leading to up to 2.6 times higher current density and a fivefold increase in hydrogen production rate. The one-cycled spiral electrode exhibited a maximum current density of 13 A m⁻², compared with 3.8 A m⁻² for the rod electrode, and achieved a hydrogen generation rate of approximately 0.71 L H₂ m⁻² h⁻¹, exceeding the rod electrode’s 0.14 L H₂ m⁻² h⁻¹, confirming its superior electrochemical and hydrogen-evolution performance. The enhanced performance was attributed to the increased surface area, improved contact between the electrode and electrolyte, and more efficient charge transfer enabled by the engineered microchannel structures [144]. Advanced designs may incorporate hydrophilic coatings, such as poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate (PEDOT/PSS), to three-dimensional carbon felt anodes, significantly enhancing both mass transfer and microbial attachment. The super-hydrophilic surface created by the coating improves water penetration and gas release, which increases the effective surface area available for biofilm growth and accelerates hydrogen production [53]. These strategies collectively aim to reduce internal resistance, stabilize the electrochemical environment, maintain uniform pH, and ensure consistent electron delivery from microbes to the anode.

Excessive mixing or shear stress may detach biofilms and disrupt electron pathways, reducing system stability. Reactor designs must therefore be optimized to enhance mass transfer without compromising biofilm integrity or increasing energy input [145].

5.4. Use of Redox Mediators and Conductive Polymers

Researchers have investigated redox mediators and conductive polymers as they can enhance the connection between microbial electron donors and electrode surfaces [146]. Redox mediators like neutral red, flavins, and humic acids facilitate indirect electron transfer by shuttling electrons from microbial cells to the anode and are beneficial in situations with thick or poorly connected biofilms [147,148,149]. However, concerns about stability and toxicity arise with the use of synthetic mediators [147]. Conversely, conductive polymers such as polyaniline (PANI) and polypyrrole (PPy) create biocompatible, electroactive films on electrode surfaces, enhancing microbial adherence and electron flow [150,151]. These polymer coatings can drastically lower activation overpotentials and help maintain long-term biofilm stability [152].

The use of synthetic mediators introduces toxicity and cost concerns, while conductive polymer coatings may degrade under prolonged operation. Selecting stable, biocompatible materials or exploring naturally secreted mediators can minimize these drawbacks [153].

5.5. Use of Hydrophobic Membrane, Methanogen Inhibitors, and Selective Electrocatalysts

To minimize hydrogen losses caused by methanogenesis and diffusion, several targeted strategies have been developed. One effective approach is the use of hydrophobic membranes, which act as selective barriers. A study demonstrated that using gas-permeable hydrophobic membranes enables rapid and selective extraction of hydrogen gas. Also, it effectively prevents the hydrogen loss to methanogenesis and back-diffusion. By actively harvesting hydrogen through gas-permeable hydrophobic membranes under vacuum, the system maintained a high hydrogen partial pressure at the cathode and completely suppressed methane formation, achieving hydrogen yields 3.2-fold higher (0.74 to 2.35 L H₂ m⁻² h⁻¹) than those obtained by traditional spontaneous gas release. Switching to active membrane-based harvesting immediately halted ongoing methanogenesis, increased current density by 36% (from 4.46 to 6.07 A m⁻²), and enhanced overall energy efficiency, rendering the process energy-positive. It allows hydrogen gas to be collected more efficiently while limiting its crossover to the anode, where it could otherwise be consumed by hydrogenotrophic methanogens [154].

The use of methanogen inhibitors like 2-bromoethanesulfonate (2-BES) is a suitable method to selectively suppress methanogenic archaea, thereby minimizing hydrogen losses without significantly affecting exoelectrogenic bacteria. A recent study demonstrated that adding 2-bromoethanesulfonate (2-BES) to MECs effectively inhibits methanogenesis, resulting in a 79.5% reduction in methane production and a 145.5% increase in hydrogen yield compared to controls. Importantly, the use of 2-BES did not significantly alter the exoelectrogenic bacterial community, indicating that it selectively suppresses methanogenic archaea without harming the bacteria responsible for electron transfer and hydrogen production. The study also found that even low concentrations of 2-BES (500 μM) were sufficient to achieve these effects, allowing for rapid and targeted control of electron flow toward hydrogen rather than methane [155]. In addition to chemical inhibition, periodic cathode sterilization or selective biofilm removal techniques, such as in situ magnetic scraping, can effectively prevent the establishment and persistence of methanogens on the cathode surface during long-term operation. For example, a study demonstrated that in situ biofilm removal from cathodes using magnets significantly reduced biofilm thickness and improved power output, indicating that regular biofilm management can help maintain optimal microbial communities and system performance [156].

Additionally, cathode materials modified with selective electrocatalysts, like cobalt phosphide on nickel foam (CoP-NF), have been shown to favor the hydrogen evolution reaction (HER) while resisting microbial colonization. A study demonstrated that the CoP-NF catalyst improved hydrogen production rates to three times that of bare nickel foam, increased energy efficiency, and maintained superior stability during long-term operation, making it highly effective for boosting coulombic efficiency and overall hydrogen recovery [157]. Similarly, biocathode-based MECs have achieved high hydrogen yields with reduced energy input by coupling electrocatalysis and biofilm activity [158]. Other commonly used materials for hydrogen evolution catalysts include Pt/C, Ni- and Co-based catalysts, and a range of non-precious metal electrocatalysts such as transition-metal phosphides (e.g., CoP, Ni₂P), and carbides (e.g., Mo₂C, WC), all of which offer favorable activity under neutral and alkaline MEC conditions [159,160,161,162]. Cobalt phosphide nanoparticles grown on pristine graphene have demonstrated outstanding HER activity, with onset potentials as low as 7 mV, overpotentials of 91 mV at 10 mA cm⁻², and excellent durability over 2000 CV cycles [163]. Similarly, mixed Co–W phosphide nanoparticles self-assembled on graphene have achieved exceptionally low overpotentials and high stability in both acidic and alkaline conditions, outperforming many non-noble-metal HER catalysts [164]. Such catalysts offer high intrinsic activity, good cycling stability, and compatibility with low-cost supports, making them promising candidates for improving cathodic hydrogen evolution in MECs. Collectively, these strategies contribute to reducing biological hydrogen losses and improving the energy output and reliability of MECs.

While these methods improve hydrogen recovery, chemical inhibitors such as 2-BES raise operational costs and may harm beneficial microbial populations. Similarly, membrane maintenance and catalyst deactivation present long-term challenges that must be addressed before field implementation [165].

5.6. Bioaugmentation Strategies

To enhance the EET capabilities of the electroactive ecosystem of native communities, high-performing electroactive microorganisms are added through a method called bioaugmentation [166]. Strains like Shewanella oneidensis and Geobacter sulfurreducens have been effectively integrated into mixed-culture systems, resulting in improved startup times and increased current generation [37]. Furthermore, creating syntrophic co-cultures, such as pairing Geobacter with hydrogen-producing fermenters, can enable a consistent flow of electrons [167]. Bioaugmentation also allows for adaptation to challenging conditions such as high salinity and the presence of toxic compounds, improving both pollutant removal and energy production [168].

Nevertheless, challenges remain in ensuring the long-term survival and integration of these introduced species, especially in open systems where ecological competition and drift occur. Successful application requires maintaining balance within the native community to prevent ecological instability [145].

5.7. Pulsed or Alternate Voltage Strategies

Electrically modulating the anode potential alters microbial community composition and activity, thereby stimulating microbial electroactivity and influencing biofilm dynamics [86]. Pulsed voltage, where voltage is periodically applied on and off or values are alternated, disrupts EPS buildup, enhances mass transport, and stimulates electrogenic gene expression by increasing the electroactivity of biofilm proteins [169]. It also prevents biofilm overgrowth and improves substrate penetration by maintaining a more homogeneous and active biofilm layer [99]. Pulsed-electric-field treatment enhanced anodic performance, increasing the maximum current density from 1.3 to 3.1 A m⁻², corresponding to approximately a 2.4-fold improvement in electrochemical activity [170]. Alternate current overlays or low-frequency square waves can also promote redox cycling of microbial electron carriers such as cytochromes, ferredoxin, quinones, and flavins [171]. However, tuning pulse duration, frequency, and amplitude is always critical to avoid electrochemical damage or microbial inhibition [172]. Electrical modulation improves biofilm activity but may also cause electrode corrosion or stress responses in microorganisms if not properly tuned. Optimizing pulse frequency and intensity is therefore crucial to avoid detrimental side effects [11]. Combining electrical modulation with adaptive control systems may offer an effective way to maintain optimal performance in real time.

5.8. MEC Optimization in Complex Wastewater

While MECs are promising for integrating wastewater treatment with hydrogen production, the effectiveness of optimization strategies in real, complex wastewaters containing heavy metals, high salinity, and refractory organics has been only partially demonstrated. Elevated concentrations of Cu²⁺, Ni²⁺, and Zn²⁺ ions can deform microbial cells, disrupt membrane potential, and suppress enzymatic electron transfer, leading to reduced biofilm electroactivity and lower hydrogen yields. However, enhanced hydrodynamic mixing and enrichment of metal-tolerant microbial consortia can mitigate these effects. Under flow conditions, MECs maintained approximately 47% performance under high Cu²⁺ stress, compared to only 14% under static conditions, due to increased EPS production and microbial diversity that improved heavy-metal resistance [141,173,174]. Salinity fluctuations can similarly alter microbial community structure and decrease charge-transfer efficiency, yet selective enrichment of halotolerant exoelectrogens has restored stable performance. Salinity-adapted MECs achieved up to 91% organic removal efficiency and a 1.57–1.70 increase in hydrogen production compared with non-adapted systems [175,176]. Industrial wastewaters containing recalcitrant compounds, such as etching terminal wastewater (ETW), have also been successfully treated using MECs, achieving simultaneous heavy-metal removal and mineralization of complex organics with mineralization rates of 42.2 mg L⁻¹ h⁻¹ and hydrogen generation rates of 0.1138 m³m⁻²d⁻¹ [174,177,178]. These findings collectively indicate that optimization strategies, such as enhanced mixing, advanced electrode materials, and microbial adaptation, are adaptable to challenging wastewater matrices, though further long-term pilot studies are required to validate their performance under industrial-scale conditions.

These integrated material, biological, and operational strategies address the root causes of poor EET and hydrogen loss, forming the basis for next-generation MEC optimization. Although nanomaterials and advanced reactor architectures significantly enhance EET efficiency and hydrogen generation, these performance gains often come with notable scalability constraints. Nanostructured electrodes, such as CNT-, graphene-, or metal-oxide-modified surfaces, offer high conductivity and large surface areas, but their large-scale production remains cost-intensive and energy-demanding, with additional concerns about nanoparticle leaching and environmental persistence [11,23]. Complex 3D-printed or multilayer reactor designs improve mass transfer and biofilm distribution but introduce higher fabrication complexity, increased material consumption, and challenges in long-term hydraulic stability and fouling management [144]. Another study further emphasizes that nanomaterial-coated electrodes may suffer performance deterioration under extended operation due to mechanical detachment, surface oxidation, or loss of catalytic activity [179]. These findings underscore that while advanced materials and engineered architectures deliver measurable performance benefits in controlled laboratory settings, practical implementation requires careful consideration of cost, durability, environmental compatibility, and manufacturability to ensure scalable and sustainable MEC deployment.

6. Future Research Directions

Future research should concentrate on combining cutting-edge diagnostics, creative bioengineering techniques, and extensive system-level enhancements to address persistent issues in microbial electrochemical cells (MECs). This will aid in bridging the gap between the overall efficacy of the system and biofilm performance.

6.1. Better In Situ Biofilm Characterization

Current diagnostic tools may hinder the delicate structure of living biofilms or provide only limited data. There are microsensors, such as microelectrodes for redox, pH, and H2 gradients, that provide continuous, minimally invasive insight [180]. Combining these sensors with optical coherence tomography (OCT) or digital holographic microscopy (DHM) may enable the acquisition of real-time 3D maps of biofilm morphology and internal mass transfer [181]. Concatenating sensor data with computational fluid dynamics (CFD) models can enable predictive control of biofilm architecture and activity [180]. Emerging research on integrated microbial–electrode architectures and 3D printing approaches is redefining future MEC scalability [182].

6.2. Real-Time Metabolic Activity Monitoring

Tracking metabolic fluxes dynamically is essential for understanding them; structural data alone is not enough. Surface-enhanced Raman spectroscopy (SERS), especially when combined with machine learning, offers a fast and sensitive way to identify microbes and track their metabolic activity in real time [183]. Recent studies also show some potential of combining SERS with other technologies such as mass spectrometry and microfluidics. These combined techniques can help in understanding metabolism at the single-cell level [184]. Machine-learning-based models have recently been employed to predict current density and hydrogen production in MECs from multi-parameter datasets, enabling data-driven optimization of system performance [185]. These approaches allow researchers to track metabolic activity more dynamically than structural data alone.

6.3. Engineering of Synthetic Microbial Consortia

With each microorganism playing its unique role, whether it is producing hydrogen, generating electricity, or altering extracellular polymer substances (EPS), synthetic biology is opening exciting possibilities for crafting specialized microbial communities [10]. The design of such synthetic consortia typically involves metabolic modeling to predict cooperative interactions, genetic circuit engineering to regulate redox enzyme or cytochrome expression, and adaptive co-culture optimization to maintain community stability. Recent studies have employed CRISPR-based gene editing and quorum-sensing control to fine-tune microbial interactions and enhance extracellular electron transfer [186,187]. Advances in synthetic biology now enable the rational design of genetic circuits that can activate EPS-degrading enzymes when conductivity decreases or upregulate cytochrome expression in Geobacter at specific anode potentials [188]. This level of engineering enables the development of tailored solutions that could have a significant impact on environmental and energy challenges. Moreover, electron transfer can be boosted by pairing electrogenic microbes with fermentative partners that produce electron shuttles, such as flavins. It is important to create metabolic dependencies or use quorum-sensing kill switches to keep the group balanced, ensuring that these microbial communities remain stable and effective [16]. New perspectives suggest that co-culturing electrogens and methanogens under controlled redox regimes can enhance EET balance and system stability [189]. Furthermore, deep-learning-based control frameworks have been proposed to automatically adjust voltage and environmental conditions in MECs, improving hydrogen yield and operational stability [190].

6.4. Integrative System-Level Diagnostic Framework for Bridging the Biofilm–Electron–Hydrogen Gap

To unify electrochemical, biological, and reactor-scale observations, an integrated diagnostic model linking microbial metabolic activity to real hydrogen productivity can be proposed. This model couples the following:

• Electrochemical metrics: CV/EIS parameters reflecting redox kinetics and internal resistance [23].
• Physicochemical gradients: microelectrode measurements of redox potential, pH, and dissolved H₂ across biofilm depth [82,84].
• Omics-based activity indicators: expression of c-type cytochromes, conductive pili genes (e.g., omcZ, pilA), and hydrogenase enzymes identified via metagenomic or proteomic analysis [85].

By normalizing current generation and hydrogen yield to these biological and electrochemical variables, the framework enables calculation of a Biofilm-Specific EET Efficiency (BSEE), a metric representing electrons successfully transferred to the anode per unit of microbial metabolic activity. Implementing this quantitative approach can allow predictive modeling of MEC performance, early identification of redox bottlenecks, and adaptive control of biofilm thickness and potential regime for maximal hydrogen evolution. These future research directions emphasize predictive control, synthetic consortia, and sustainable system integration, directly supporting the problem–cause–solution framework of this review. Future MEC work should assess sustainability indicators such as life-cycle energy use, carbon reduction, and material recyclability. Integrating MECs with wastewater and bioenergy infrastructures can accelerate their contribution to clean-energy and circular-economy goals. Emerging advances in electrochemical reactor engineering and catalyst design, such as recent developments in CO₂-conversion electrocatalysis and high-pressure electrolysis systems, provide useful conceptual parallels that may inspire future MEC scale-up strategies [191,192].

7. Conclusions

The persistent disconnect between robust biofilm activity and suboptimal hydrogen output in MECs stems from multiple factors that disrupt electron flow from anode to cathode. Biofilm architecture, particularly thickness more than 50–100 µm, induces severe mass transfer constraints, where substrate starvation and proton accumulation create an acidic environment, which leads to the inactivation of inner layers. Concurrently, electron transfer efficiency and hydrogen production efficiency are compromised by redox imbalances at the anode interface, ohmic losses from electrolyte resistance, and the diversion of electrons toward methanogens rather than hydrogen evolution. These factors collectively decouple metabolic activity from measurable current, revealing that biofilm viability alone cannot guarantee system performance. Advanced monitoring and characterization techniques are pivotal in diagnosing these issues. The techniques include electrochemical methods such as CV, EIS, and LSV, which quantify electron transfer kinetics and resistive losses; imaging tools such as CLSM, SEM, and AFM used to visualize stratified biofilms and dead zones; and multi-omics approaches, which can decode genetic imbalances like upregulated methanogenic hydrogenases. These diagnostics transform observational anomalies into actionable targets, revealing that biofilm viability alone cannot predict system performance.

Strategies to bridge this gap can make the entire system efficient. Advanced electrode materials like graphene-CNT hybrids enhance microbial adhesion and shorten electron transfer paths, boosting current densities by 2.6–4.3-fold. Optimizing the process, including pulsed voltage regimes, reactor mixing, and 3D-printed microchannel anodes, can mitigate mass transfer barriers and maintain electroactive biofilms. Biological interventions, such as selective methanogen suppression (e.g., 2-BES inhibitors increasing H₂ yield by 145%) and bioaugmentation with Geobacter, further redirect electrons toward hydrogen production.

Future advancements demand integrated approaches. Future systems need built-in sensors to track biofilm behavior and nutrient flow. Real-time diagnostics combining microsensors, OCT, and machine learning could dynamically map biofilm metabolism and transport limitations, enabling predictive control. Engineered synthetic consortia, programmed via genetic circuits to optimize EET pathways or EPS management, may sustain electroactivity under industrial conditions. Ultimately, translating MECs into viable energy solutions requires the co-design of materials, microbiology, and reactor engineering, prioritizing not only peak performance but also economic viability and long-term resilience for the conversion of organic substrates to hydrogen valorization. Collectively, this review reframes the MEC performance problem through a system-level diagnostic lens. Rather than viewing biofilm growth, electron transfer, and hydrogen evolution as separate elements, it connects them via a measurable BSEE metric. Integrating electrochemical (CV, EIS), microelectrode (pH/redox profiling), and omics (gene/protein expression) data provides the basis for real-time mapping of energy losses and redox imbalances. This conceptual framework offers a path to predictive control of MEC operation, where dynamic feedback between microbial metabolism and electrochemical signals can optimize hydrogen yield while minimizing internal resistance. In summary, MECs bridge environmental protection and renewable energy generation, aligning with SDGs 6 and 7. Their dual role in wastewater treatment and hydrogen production underscores their relevance for sustainable development.