Optimizing Hydrogen Production Through Efficient Organic Matter Oxidation Performed by Microbial Electrolysis Cells

Abstract

Microbial electrolysis cells (MECs) represent a pioneering technology for sustainable hydrogen production by leveraging bioelectrochemical processes. This study investigates the performance of a single-chamber cathodic MEC, where a cation exchange membrane separates the electrically active bioanode from the cathode. The system was constantly fed with a synthetic carbonaceous solution, employing a working potential of +0.3 V vs. SHE and an organic loading rate of 2 gCOD/Ld with a hydraulic retention time of 0.3 d. Notably, no methanogenic activity was detected, likely due to the establishment of an alkaline pH in the cathodic chamber. Under these conditions, the system exhibited good performance, achieving a current density of approximately 115 A/m³ and a hydrogen production rate of 1.28 m³/m³d. The corresponding energy consumption for hydrogen production resulted in 6.32 kWh/Nm³ H₂, resulting in a slightly higher energetic cost compared to conventional electrolysis; moreover, an average energy efficiency of 85% was reached during the steady-state condition. These results demonstrate the potential of MECs as an effective and sustainable approach for biohydrogen production by helping the development of greener energy solutions.

1. Introduction

The urgency to address climate change has never been more pronounced. Across the globe, rising temperatures, variable weather patterns, and the increasing frequency of extreme events serve as stark reminders of the impact of greenhouse gases (GHGs) on our planet [1,2]. Predominantly, human activities—ranging from industrial processes to transportation—have significantly contributed to the accumulation of these gases in the atmosphere, leading to adverse environmental and socio-economic effects. In response, international communities, and notably the European Union, have set ambitious targets to mitigate GHG emissions and transition toward a more sustainable and low-carbon future [3]. In detail, European policymakers have recognized the importance of transitioning to cleaner energy sources. The EU’s roadmap for a sustainable future is characterized by a commitment to reducing CO₂ emissions, enhancing energy efficiency, and fostering innovation in renewable energy technologies [4]. These objectives not only support global climate goals but also drive economic competitiveness and technological leadership within Europe. As a result, the escalating challenges posed by greenhouse gas emissions call for immediate and innovative responses.

Hydrogen began to be used as an encouraging energy vector capable of decarbonizing sectors traditionally subject to fossil fuels [5]. However, the environmental benefits of hydrogen are highly dependent on its production method. Traditional hydrogen production, predominantly through steam methane reforming (SMR), is heavily reliant on natural gas. Although SMR is currently the most cost-effective method, it, unfortunately, releases significant amounts of carbon dioxide (CO₂) into the atmosphere, thus perpetuating the cycle of greenhouse gas emissions and contributing to climate change [6].

On the contrary, green hydrogen constitutes a cleaner alternative, generated via water electrolysis driven by renewable energy sources such as hydroelectric, wind, or solar power [7,8]. This process results in virtually zero emissions, aligning with the stringent environmental objectives of regions like the European Union. The EU’s commitment to reducing GHG emissions is encapsulated in policies and frameworks such as the European Green Deal, which seeks to achieve climate neutrality by 2050 [3]. These initiatives not only aim to drastically reduce emissions but also stimulate technological innovation and create new opportunities for sustainable growth in the energy sector.

Among these transformative strategies, innovative technologies are emerging that promise to improve the productivity and sustainability of green hydrogen production.

Some examples include using mixed photosynthetic cultures, such as purple bacteria (PPB), which are capable of balancing redox potential through hydrogen production via the enzyme nitroreductase, and also exploring the potential of wastewater or by-products as substrates for clean energy production [9,10]. Dark fermentation produces hydrogen anaerobically from organic substrates, generating volatile fatty acids and CO₂, making it useful for waste valorization [11]. In contrast, bio-photolysis relies on photosynthetic microbes, such as algae, to split water using solar energy, though its efficiency is limited by oxygen sensitivity. Integrating these processes can enhance hydrogen yields, as fermentation by-products serve as substrates for photoheterotrophic hydrogen production [12].

Among these new technologies, one such breakthrough is the application of microbial electrolysis cells (MECs). MECs represent a novel approach that harnesses the natural metabolic processes of microorganisms to drive hydrogen production at relatively low energy costs [13]. Unlike conventional electrolysis systems, which require high energy input and operate at elevated temperatures, MECs operate under mild conditions, making them a potentially transformative technology in the renewable energy field.

The principle behind MECs is rooted in bioelectrochemistry. In these systems, bacteria serve as biological catalysts that break down organic substrates and facilitate the transfer of electrons to an electrode. Furthermore, one of the key rewards of MECs is their high degree of optimization potential, as their performance can be enhanced by adjusting operational parameters, selecting efficient catalytic materials [14,15,16], or improving the system design by integrating advanced membrane technology, preventing its aging to improve ion transport and system stability in a long-term period [17,18]. This process not only produces hydrogen but also enables the simultaneous treatment of organic waste, offering the double benefit of energy production and waste management. Additionally, as in other innovative biotechnologies like the production of polyhydroxyalkanoates (PHAs) or even in the use of membrane bioreactors (MBRs) [19,20], ammonia reduction can occur at the cathode, allowing for nitrogen removal from wastewater, further enhancing resource recovery [21]. Consequently, MECs are positioned as a sustainable and integrative solution in the energy transition landscape, where waste valorization and renewable energy production can synergistically reduce environmental burdens. Their integration into the hydrogen economy could fundamentally alter the market dynamics by providing a scalable and environmentally friendly alternative to fossil fuel-based hydrogen production. As governments and industries increasingly prioritize sustainability, technologies like MECs are likely to gain prominence. Their potential to reduce dependency on non-renewable resources while mitigating the environmental impact of energy production is a critical factor in the ongoing discourse surrounding climate change and energy security.

In summary, by embracing advanced technologies such as MECs, the EU could aim to catalyze a paradigm shift in energy production, one that prioritizes environmental integrity, resource efficiency, and long-term sustainability, thus offering a promising pathway to a cleaner energy future, that not only embodies the spirit of innovation but also represents a tangible step toward a sustainable, low-carbon economy.

In this view, the objective of the present work is to assess the efficiency of the MEC system by continuously supplying a synthetic substrate to the anodic chamber. Specifically, following an initial acclimation period, the system was optimized to achieve consistent hydrogen and current production at a minimal energy cost while keeping a stable anodic overpotential and minimizing cathodic overvoltage over the long term.

Furthermore, in contrast to the conventional potential used in these systems equal to +0.2 V vs. SHE selected as the break-even point based on previous studies [22,23], a potential of +0.3 V vs. SHE was imposed, to assess the possibility of enhancing electric current production while balancing the energy consumption, thus preventing higher energy consumption. This study sets the stage for a deeper exploration of MEC technology, its operational principles, and its potential role in revolutionizing green hydrogen production in an era defined by environmental urgency and technological progress.

2. Materials and Methods

2.1. Microbial Electrolysis Cell Set Up

The laboratory-scale MEC, illustrated in Figure 1, comprised two identical Plexiglas chambers (each with a volume of 0.86 L and internal dimensions of 17 cm × 17 cm × 3 cm). A CMI International cation exchange membrane (Membrane International, Ringwood, NJ, USA) was installed between the two reactor chambers, sealed with butyl rubber gaskets, thus permitting the migration of protons. The cathode chamber consisted of two sheets of 316 stainless steel (RS components), each with a surface area of 176.46 cm², while granular graphite with a diameter < 4 mm (Faima srl, Milan, Italy) was used as filler for the bioanode.

Notably, the graphite granules functioned both as the electrode material and as a biocompatible substrate for microbial biofilm development. The anodic chamber was operated under continuous flow conditions at 2.9 L/d, corresponding to an organic loading rate (OLR) of 4 g COD/L⋅d and a hydraulic retention time (HRT) of 0.3 days. The feeding solution was stored in a tedlar bag and delivered to the reactor via tygon tubing using a peristaltic pump. The composition of the feeding solution was as follows: 1.250 g COD/L (comprised of Glucose 0.680 g/L, Sodium Acetate 0.211 g/L, Peptone 0.276 g/L, and Yeast Extract 0.150 g/L); and a mineral medium with a composition reported elsewhere [24,25]. A second tedlar bag was employed to collect the anodic effluent. After a period without pH control, from the 17th day of operation to the end of the experiment, the pH of the feeding solution, was maintained at around pH 7.5 through a buffer solution of sodium bicarbonate.

Regarding the cathodic chamber, it was filled with a 0.5 M NaCl solution and operated in batch mode, with daily removal of the catholyte to offset the water electro-osmotic diffusion through the cation exchange membrane (CEM). Additionally, a 0.30-L sampling glass chamber—equipped with sampling ports sealed by butyl rubber stoppers and aluminum crimps—was positioned above each chamber to facilitate the collection of both liquid and gaseous phases. Throughout the entire experimental period, the MEC temperature was maintained at 25 ± 1 °C.

To monitor and control the potential of individual electrodes, an Ag/AgCl reference electrode (immersed in a saturated KCl solution with E°’ = 199 mV vs. the standard hydrogen electrode, SHE) was placed in each MEC chamber. To integrate the graphite granules into the electrical circuit, two graphite rods (serving as a collectors) were immersed into the chambers and connected via titanium wires to a potentiostat (IVIUM Technologies, Eindhoven, The Netherlands). A potential of +0.3 V vs. SHE was applied to the anode, designating it as the working electrode. For consistency, all voltage measurements reported in this manuscript are referenced against the SHE.

2.2. Start-Up of the Reactor

The bacterial consortium used as inoculum for the bioanode consisted of an activated sludge composed of mixed microbial cultures (MMCs) from a full-scale wastewater treatment plant. Before inoculation, this sludge was washed down with the same mineral medium that made up the reactor feed and aerated, thus removing any residual sCOD. The sludge was composed of 4.42 gVSS/L of solids. A 300 mL volume (35% v/v) was introduced into the anodic chamber, operated in batch configuration for 8 days by using a concentrated spike of the same synthetic feeding solution and by using a +0.3 V vs. SHE as a working potential. Following this stage, the acclimatization of the inoculum occurred, and the establishment of the biofilm on the anode began. Subsequently, the reactor was placed in continuous flow mode through the transition of the anode electrolyte, using the synthetic C-solution outlined in Section 2.1.

2.3. Analytical Techniques

The outlet gaseous flow rate was obtained by using a Ritter^® milligas counter, while gas composition and concentrations (O₂, H₂, CO₂, and CH₄) were determined by drawing a 50 μL gaseous sample with an airtight syringe and subsequently injecting it into a Dani Master GC (Milan, Italy) gas chromatograph equipped with a thermal conductivity detector (TCD). The total and soluble chemical oxygen demand (totCOD and solCOD) were quantified by digesting the unfiltered and filtered (0.2 µm) samples, respectively, at 160 °C using commercial Spectroquant kit tests (Macherey-Nagel, Düren, Germany) in conjunction with a UV–visible spectrophotometer (Nanocolor Vario4, Macherey-Nagel, Germany) operated at a wavelength of 605 nm. The electric current intensity and charge were recorded automatically via the Ivium n-Stat potentiostat software 4.1194 (Ivium Technologies B.V., Eindhoven, Netherlands), while electrode potentials were determined by using an Amprobe AM-520-EUR multimeter (Everett, WA, USA). Thus, throughout the operation period of the MEC, it was possible to monitor the potential difference (ΔV) established between the two compartments as a function of the different generated current values, as it was possible to determine the polarization curve of the system.

Suspended solids (total TSS and volatile VSS) concentrations were determined in accordance with Standard Methods and subsequently converted to COD units by using a conversion factor of 1.42 g COD/g obtained from the complete oxidation of heterotrophic biomass, using the C₅H₇O₂N formula [26]. The conductivity and the pH values were measured through an SI Analytics HandyLab680 (Fisher Scientific, Wien, Austria).

2.4. Experimental Parameters and Calculation Methods

In order to calculate the removed COD (mg/Ld), the following equation was employed where COD_in and COD_out (mg/L) represent the influent and effluent COD concentrations, and Fin and Fout (L/d) denote the influent and the effluent flow rates, respectively. The result was then normalized by the anodic chamber volume (0.86 L).

$${COD}_{removed}\left({\displaystyle \frac{mg}{L d}}\right)={\displaystyle \frac{Fin \ast CODin-Fout\ast CODout}{V}}$$

(1)

The coulombic efficiency was determined according to the following equation:

$$CE\left(\%\right)=100 \ast {\displaystyle \frac{meqi}{meqCOD }}$$

(2)

in which meqi represents the cumulative charge that has passed in the circuit and meqCOD represents the theoretical cumulative charge which could have been generated by the oxidation of the removed COD.

The hydrogen production rate (rH₂) was determined using the following relationship:

$${rH}_2 \left({\displaystyle \frac{eq}{d}}\right)={\displaystyle \frac{d \left(meq{H}_2\right) }{d \left(d\right)}}$$

(3)

where meqH₂ represents cumulative equivalents of hydrogen produced, mmolH₂ represents cumulative moles of hydrogen produced, meqH₂ = 2 mmolH₂, and d corresponds to the duration of the experimental period (in days).

The cathodic coulombic efficiency (CCE) was determined as the ratio among the cumulative H₂ produced expressed in meq and the cumulative H₂ that might be produced with the electric current flowing in the circuit:

$$CCE\left(\%\right)=100 \ast {\displaystyle \frac{meq{H}_2}{meqi}}$$

(4)

The overall energy efficiency was evaluated using the following equation:

$$\eta E={\displaystyle \frac{\left|{\mathsf{\Delta }G}^0{H}_2\right| CCE}{2\left|\Delta V\right|F}}$$

(5)

in this expression, ΔG⁰_H2 is the standard Gibbs free energy for the combustion of hydrogen (kJ/mol), CCE represents the cathodic coulombic (capture) efficiency, 2 are the mole of electrons required to obtain a mole of hydrogen (eq/molH₂), ΔV (V = J/C) is the potential difference established between the two chambers, and F is Faraday’s constant (96,485 C/eq).

The energy consumptions were calculated according to the following equation:

$$EC{H}_2 \left({\displaystyle \frac{Wh}{Nd{m}^3{H}_2}} \right)={\displaystyle \frac{24\left|\Delta V\right|i}{r{H}_2\ast 24.4 }}$$

(6)

where 24 are the number of hours in a day (h/d), ΔV (V), and i (A) denote the average potential variance and the average electric current measured through the working period, respectively. rH₂ corresponds to the hydrogen production rate defined in mol/d, and 24.4 is the volume (L/mol) occupied by a mole of gas at a 25 °C temperature.

3. Results and Discussion

The reactor was conducted for 26 days without any interruption. After a batch-acclimatization period, the anodic parameters were stated as stable as possible to assess the system performance during a steady-state period. The interest of the study was to follow the stabilization of the reactor, thus evaluating its behavior and its running over the long term. During the steady-state period, the results obtained remained stable, with a current produced by the electro biofilm characterized by few fluctuations, which was expected in a long-term investigation. However, it was observed that the pH conditions established in the anodic chamber play a key role in enhancing system performance.

3.1. Start Up of the Bioanode

In Figure 2, the current generated by the electroactive biofilm in the MEC system is shown over the course of 26 days. During the initial eight days, the system was operated in batch mode to allow the biomass in the anode chamber to acclimate. Biological activity was assessed through the addition of concentrated spikes of synthetic substrate by measuring the COD consumed and obtaining an average current of 15.45 ± 0.03 mA.

Following this acclimation period, the anode chamber was switched to continuous operation at unchanged working conditions for 16 days (i.e., OLR of 2.0 gCOD/Ld, anodic potential controlled at +0.3 V vs. SHE), resulting in a marked increase in current production, with peaks reaching approximately 90 mA and an average value of 78.3 ± 0.2 mA from day 9 to day 14. However, between days 14 and 17, a substantial decline in the current was observed, reaching 35 ± 0.2 mA, due to a drop in the anode chamber pH to around 4.5. To address this issue, the pH of the synthetic feed was adjusted to approximately 7.5 using a sodium bicarbonate buffering solution. Hence, stable system performance was restored, enabling a nine-day steady-state operation phase characterized by an average current of 99 ± 2 mA. These findings highlight the critical role of pH control in maintaining optimal performance of electroactive biofilms in MEC applications [27].

3.2. Anodic and Cathodic Chambers Performance During Steady-State Operation

During the steady-state operation, all the characteristic parameters remained roughly constant. Figure 3a shows the results obtained for the anodic compartment. In these working days, this latter was continuously fed with a substrate solution containing around 584 ± 5 mgCOD/L (≈1.97 gCOD/Ld), the effluent had an average value of 342 ± 4 mgCOD/L (≈1.15 gCOD/Ld), resulting in a COD abatement standing at around 40–45% and a CE going from 64% to 67%, with a maximum of 69%.

Throughout the experimental period, the anodic performance remained essentially unchanged, demonstrating the effective achievement of a plateau condition for the system. Furthermore, this result also demonstrates the resilience of the bioelectric film, which was able to overcome the inconvenience due to the pH decrease in the anode chamber, which is fundamental behavior for processes that require sustained operation over extended periods, as in wastewater treatment cases. Regarding the cathodic chamber, the pH moved to stable values of 9.10 ± 0.5, leading to a constant cathodic potential and, consequently, a constant energetic consumption. It is significant to mention that with this value of pH > 9, most hydrogenophilic microbial metabolisms are usually inhibited, avoiding acetate production and preventing the occurrence of methanogenesis phenomena in favor of greater hydrogen production [28,29].

The obtained pH value agrees with the catholyte used, as the NaCl solution was chosen not only for its cost-effectiveness but also for its lack of buffering power, allowing for alkalinization of the cathode chamber. Indeed, by considering the mineral medium used in the feeding solution of the anodic chamber, a high concentration of K₂HPO₄ (4 g/L) was present to maintain pH stability around physiological values. Given the use of a weak buffer ([HPO₄²⁻] = 23 mM), alkalinization of the cathodic compartment was inevitable due to proton consumption for hydrogen production. However, despite the relatively high concentration of cations present in the mineral medium (calcium, magnesium, potassium, and sodium), their migration through the CEM from the anodic chamber—necessary to maintain electroneutrality—did not effectively counterbalance the generated alkalinity. This was due to the production of very weak conjugated acids, which had a negligible impact on the pH in the cathodic chamber. Hereafter, the main results characterizing this chamber in terms of current and hydrogen produced, cathodic capture, and energy efficiencies are shown in Figure 3b. As with the results obtained for the bioanode, there were no noticeable fluctuations in cathode performance over the operation days. The oxidation of the substrate solution at the anode generated an electric current ranging from 91 mA to 108 mA, which was directly correlated with the steady production of hydrogen in the cathodic compartment, with the averaged value of 44.0 ± 1.0 mmol/d during the steady-state period. In these days, the conversion yield of the current in hydrogen (CCE) increases from 70% to a maximum of 77% (on day 21st), and with an average value of potential equal to 2.64 ± 0.03 V vs. SHE, a value of ηE between 81% and 85% were attained.

Figure 4 shows the average values in terms of COD removed anodic coulombic efficiency, current produced, cathodic capture, and energy efficiency during the 9-day steady state. Regarding the bioanode chamber, the removal of 0.82 ± 0.01 gCOD/Ld was achieved, with an average COD abatement equal to 41.5 ± 0.7% and an average production of current equal to 99 ± 2 mA, with a cathodic current density of 0.55 ± 0.01 mA/cm² and a CE of 66.7 ± 0.7%. Less than the amount was used for the maintenance of its essential functions; this result means that bioanode COD consumption was not entirely devolved into current production, probably due to the use of a mixed microbial consortium that can limit process performance with lower efficiency when compared to the pure cultures use. Indeed, as already found in the literature, when pure culture was used, higher efficiency was achieved. As an example, in Kadier et al. [30], where Geobacter sulfurreducens as inoculum was used, a genus renowned for its electroactive capacities, capable of producing elevated power densities at mild temperatures [31], a COD removal equal to 94 ± 2% with a CE of 75 ± 4% was obtained.

Meanwhile, regarding the cathodic chamber, an average CCE of 73.9 ± 0.7 and an ηE of 82.9 ± 0.5 were obtained. Although the cathodic capture efficiency did not reach 100%, this was likely due to an underestimation of hydrogen production. This discrepancy may be attributed to the potential loss of the dissolved hydrogen at the system outlet and leakage through the tubing line, which is not entirely gas-tight, especially under alkaline conditions. Furthermore, as reported in the literature [18,32], CEM is not entirely gas-impermeable, potentially allowing hydrogen to back-diffuse into the anodic compartment where its consumption by hydrogenophilic bacteria would take place.

In Figure 5, the polarization curve of the MEC reactor, obtained during the whole working period, is shown. In detail, through this curve, it is possible to illustrate the kinetic behavior of the electrochemical system, describing the relationship between the potential difference established between the two chambers and the current generated at a set operating potential (i.e., +0.3 V vs. SHE). The observed linear trend indicates that as the current increases, the potential difference also rises.

From an electrochemical standpoint, this behavior can be attributed to internal resistance within the biofilm. According to Ohm’s law (V = IR), a higher current flow through a given resistance produces a larger drop in voltage. Consequently, when more current is generated, the system requires a higher potential difference to overcome the increased resistive losses in the biofilm. This is reflected in the slope of the linear regression, which represents the overall resistance. The high coefficient of determination (R² ≈ 0.98) confirms the strong correlation between these variables, emphasizing the role of biofilm resistance in influencing the electrochemical performance. Within this study, the ΔV varied due to the different currents produced during the several periods characterizing the 26 days of reactor operation, with minimum values characterizing the acclimatization and pH drop period, and maximum values reached during the steady-state period.

In addition, the polarization curve falls within the potentiostatic techniques, along with anodic kinetics (not reported here), by which it is possible to identify the optimal potential range for the working electrode in the potentiostatic mode. This approach helps pinpoint the most energetically favorable and least energy-intensive conditions for conducting the process.

3.3. Comparison of the Research Performance with Literature Research

The main results obtained during the steady-state period are shown in

Table 1. Comparison of MEC results with the literature in the frame of H₂ production.

Ref. Study	System Configuration	Inoculum	Current Density (A/m3)	CODremoval Efficiency (%)	CE (%)	CCE (%)	ηE (%)	H2 Production Rate (m3/m3d)
This study 1	Abiotic cathode	MMCs	99 ± 2	42 ± 1	67 ± 1	74 ± 1	83 ± 1	1.28
[23] 2	Abiotic cathode	MMCs	59 ± 5	61 ± 13	51 ± 3	86 ± 3	68 ± 5	0.62
[30]	Biotic cathode	Pure culture	312 ± 9	94 ± 2	75 ± 4	89 ± 4	94 ± 3	4.18
[33]	Membrane-free	MMCs	56	- 3	73	87	60	0.69

¹ The results obtained in this study are reported to be abbreviated to the nearest whole number. ² Case study “B”—cathodic chamber with phosphate buffer in batch configuration and initial pH 5.2. ³ Assuming all substrate (composed by only acetate) was consumed.

, which compares them with other literature studies.

In the study conducted by Cristiani [23], the same MEC configuration was used, inoculated with MMCs, and continuously fed with the same synthetic substrate composition. However, a key difference lies in the catholyte composition, composed of phosphate buffer. Despite the higher COD consumption reported in Cristiani’s study, the current output was greater in the present work, probably due to the higher applied working potential of 0.3 V vs. SHE compared to the 0.2 V vs. SHE used by Cristiani. This is consistent with the obtained CE values, while the lower CCE of 74% observed in this study, as discussed in Section 3.2, might be attributed to an underestimation of the actual hydrogen production. Although energy efficiency (83%) and hydrogen production rates (1.28 m³/m³d) were higher in the present study, the overall energy cost must also be considered. In this regard, energy consumption was found to be 6.32 KWh/Nm³H₂, compared to the lower value of 5.22 KWh/Nm³H₂ reported by Cristiani, in line with the lower ΔV of 2.0 V observed in this latter respect to 2.64 V found here.

In contrast, Hu [33] employed a single-chamber, membrane-free configuration to reduce potential losses linked with the membrane and enhanced energy recovery, but still using MMCs. Interestingly, the results obtained in this setup were very similar to those reported by Cristiani, despite the different system configurations. A possible explanation could be the similar pH conditions imposed, with a pH of 5.8 in Hu’s study and 5.2 in Cristiani’s cathode chamber, both achieved using a concentrated phosphate buffer solution. On the contrary, in the present study, an alkaline cathodic pH of 9 was maintained. This further confirms that electrolyte conditions, particularly pH and conductivity, cover a pivotal role in defining the activation of specific metabolic pathways in electroactive bacteria, ultimately influencing the total efficiency of the MEC system. The electrolyte pH can directly influence the electroactive microorganism’s activity, which is why it is often adjusted to control the potentials of oxidation and reduction at the electrodes [21].

The results obtained in Kadier’s study can be explained by pure bacterial strain use, which were capable of achieving high energy density values, along with catalytic materials (Nichel mesh cathode) designed to enhance catalytic activity and increase the hydrogen production rate (4.18 m³/m³d) [30]. This suggests that the combination of pure cultures and optimized electrode materials can significantly improve MEC performance. However, such approaches may come with higher operational costs and, potentially, several operational limitations, such as the use of specific substrates or sterile conditions.

Overall, this comparative analysis highlights how variations in operating conditions, such as working potential, pH, and cell configuration, strongly influence both hydrogen production performance and overall energy balance. While a higher working potential can enhance the current output, it also increases energy consumption, requiring careful optimization to balance efficiency and cost. Additionally, the electrolyte environment appears to be a key factor in driving bacterial metabolic activity, further emphasizing the importance of pH control in MEC systems.

4. Conclusions

The present work confirmed the potential of microbial electrolysis cells (MECs) as a viable and sustainable technology for hydrogen production, while emphasizing the importance of working parameters such as pH control, applied potential, and reactor configuration. Under the steady-state phase, the system demonstrated robust and efficient performance, achieving an averaged current of 99 ± 2 mA, a hydrogen production rate of approximately 44.0 ± 1.0 mmol/d, and a cathodic capture efficiency of 73.9 ± 0.7%. A key finding was the establishment of an alkaline pH (>9) in the cathodic chamber, which effectively suppressed methanogenic activity, thereby favoring hydrogen production.

The system also proved resilient, with the electroactive biofilm recovering functionality after a temporary acidification event in the anodic chamber, demonstrating its robustness for long-term operation. Additionally, the polarization curve revealed that biofilm-associated internal resistance significantly contributes to increased voltage requirements at higher current levels, emphasizing the importance of optimizing working potential to balance hydrogen yield and energy input.

Despite minor hydrogen losses attributed to retro diffusion through the cation exchange membrane and system leakage, the reactor achieved a maximum energy efficiency (ηE) of 85%, further supporting its promise as a sustainable and cost-effective biohydrogen production technology.

These findings underscore the high potential of MECs, not only in controlled experimental settings but also in future scalable applications. Further research should focus on enhancing microbial community compositions, explore advanced membrane materials, refine reactor geometry, and implement integrated energy management strategies. These developments could significantly improve overall performance, reduce operating costs, and accelerate the transition of MEC technology from laboratory-scale systems to real-world, industrial biohydrogen production platforms.