3.1.1. CE

High CE is desirable in MFCs as it signifies efficient electron transfer from the microbial oxidation of organic substrates to the anode, generating usable electrical current. Factors affecting CE include substrate type, microbial activity, electrode materials, and system design. Several studies showcase how the use of specific materials could affect the CE of MFCs. For example, Chen et al. [57] used sodium citrate as an additive to significantly improve the CE by optimizing the microbial community structure and electron transfer processes. The sodium citrate enhanced not only the electricity generation but also the efficiency of electron recovery in MFCs, which is crucial for improving overall system performance. Yang et al. [58] also explored the simultaneous enhancement of power density and CE in MFCs using a hydrophobic Fe–N4/activated carbon air cathode. Their findings indicated that the hydrophobic cathode significantly improved CE by facilitating better oxygen reduction reactions and minimizing electron losses. Maximizing CE enhances the MFC’s energy conversion efficiency, making it more viable for various applications such as wastewater treatment, energy production from renewable sources, and biosensors. Figure 3 is a graph obtained from [59] depicting the changes in CE and energy efficiency of a MFC over a period of 70 days. From the graph, it can be deduced that the variations in CE suggest that the MFC’s ability to convert substrate into electrical current efficiently changes over time, possibly due to factors like microbial community dynamics, substrate concentration, or operational conditions. The relatively stable but low energy efficiency indicates that while the system consistently produces energy, the proportion of energy captured compared to the theoretical maximum remains low. The sharp decline in CE towards the end of the observation period is due to depletion of nutrients, accumulation of inhibitory byproducts, or changes in the microbial community structure. These deductions confirm that CE translates to MFC performance.

3.1.2. Substrates and Microbes

Different types of microbes and substrates from plant and animal sources have been used in the operation and design of MFCs. As concluded by Gezginci and Uysal [60], Prathiba et al. [61], and Ullah and Zeshan [62], the electrochemical activities of these microbes and substrates translate into the overall performance and power output as they constitute the number of bacteria that can be produced to aid in anaerobic digestion. Confirming this, Sarmin et al. [63] investigated the improvement of power generation in MFCs through effective substrate–inoculum interaction mechanisms. They found that optimizing the interaction between substrates and inoculum can significantly enhance power generation. Similarly, Fadzli et al. [64] also reviewed recent developments in the use of organic substrates and bacterial electrode interactions in MFCs and found that different organic substrates affect bacterial-electrode interactions and energy output overall. The choice of substrate is important as it also directly affects operation parameters such as pH, temperature, power density, and CE [65]. Studies show that catalytic microorganisms such as Escherichia coli, Pseudomonas, Fluoscens, Proteus vulgaris, and Pseudomonas methanica are used primarily in MFCs due to their ability to effectively oxidize organic matter to produce electricity [65]. Another study by Tariq et al. [66] tested the three main types of substrates commonly used in MFCs, namely glucose, acetate, and sucrose, to determine their overall impact on energy generation and CE. Tariq et al. [66] found that maximum power densities (corresponding to maximum voltage measurements) of 31, 53.4, and 52.3 mW/m² were achieved for glucose, acetate, and sucrose, respectively; thus, acetate is more viable for high-performing MFCs. Although this study suggested acetate as the most efficient substrate for MFCs, it is important to recognize that system performance can be affected by many other factors, such as the concentration of substrates, that may change the aforementioned conclusion. For example, David et al. [67] discovered that an MFC system fed a 2,000 mg/L concentration of acetate produces a relatively increased amount of electricity. In a different study by Ullah and Zeshan [62] illustrating the power density (measured in mW/m²) of an MFC over time (hours) using three different substrates: Glucose, Acetate, and Sucrose, as depicted in Figure 4, it was found that all three substrates show a rapid increase in power density within the first 12 hr, indicating that MFCs can quickly metabolize these substrates to generate electricity. Glucose reaches its peak power density later (around 48 hr) and stabilizes at a lower value compared to the other substrates. Acetate peaks early (around 24 hr) and remains fairly stable with slight fluctuations. Sucrose achieves the highest peak power density quickly and remains the most efficient substrate over the observed period. Comparing the efficiency in this study, Sucrose proves to be the most effective substrate for MFC power density, consistently generating the highest values. Therefore, in order to achieve consistent energy production by MFC, one must design an MFC that adjusts to a particular practical substrate concentration stability.

Furthermore, Table 3 presents a comprehensive list of different microbes and substrates commonly used in various MFCs studies, as well as the maximum power or currents recorded in each study.

From Table 3, several observations can be made to understand the functionality and efficiency of various microbes and substrates employed in MFCs. Microbes such as G. sulfurreducens and P. aeruginosa are well-known for their high electrogenic capabilities, producing significant power outputs of 2.5 and 2.2 W/m², respectively. In contrast, microbes like A. succinogenes and P. mirabilis produce lower power outputs of 0.38 mW/m² and 0.5 W/m², respectively. The source of the inoculum also plays a critical role. For example, Geobacter and Shewanella species, often derived from sediment, tend to be more effective in MFCs, suggesting that sediment-derived inocula are rich in electrogenic bacteria. Additionally, microbes like C. beijerinckii and C. butyricum can utilize a broad range of substrates (starch, glucose, lactate, molasses), which might contribute to their versatility in different MFC setups, whereas species like E. coli and G. oxydans are more substrate-specific (glucose), potentially limiting their applicability based on available substrates. The type of substrate affects the efficiency of electricity generation. Glucose is a common substrate used by many microbes, according to Table 3, leading to varying levels of power output. The specific metabolic pathways and efficiencies of these microbes dictate the resultant power or current produced. The inoculum type, ranging from activated sludge and pure cultures to soil and sediment, significantly affects MFC performance. For instance, sludge from wastewater treatment plants (WWTPs) provides a diverse microbial community capable of efficient electricity generation, as seen with E. coli and P. aeruginosa. Mixed cultures derived from environments rich in organic matter (e.g., activated sludge) often outperform pure cultures due to the synergistic interactions within microbial communities, enhancing overall electron transfer processes. It is also evident that single-chamber MFCs, as used for G. sulfurreducens and P. aeruginosa, tend to have higher power outputs compared to dual-chamber systems, which are more common for microbes like C. butyricum and R. ferrireducens. This difference may be attributed to reduced internal resistance and more efficient electron transfer in single-chamber designs. Dual-chamber MFCs are often employed for more controlled environments, possibly leading to more stable but lower power outputs, as they can manage separate anodic and cathodic processes more effectively. There is a significant variability in power outputs across different microbes. High performers like G. sulfurreducens achieve up to 2.5 W/m², whereas others like A. succinogenes produce much lower outputs (0.38 mW/m²). This variability highlights the importance of selecting appropriate microbes based on the desired application and efficiency needs. The performance of each microbe is also dependent on the specific operational conditions, such as temperature, pH, and the presence of electron mediators. Optimizing these conditions can enhance the power output for less efficient microbes. The comparison reveals that while some microbes are inherently more efficient at generating power in MFCs, the choice of substrate, type of inoculum, and MFC design are crucial factors that influence the overall performance. These differences can guide the selection and optimization of MFC systems for various applications, from wastewater treatment to renewable energy production.

3.1.3. Temperature

An increase in temperature results in an increase in the reactions of MFC operation; thus, a higher temperature increases hydrogen-ion diffusions to the cathode, which influences microbial metabolic rates and enzyme activities. As concluded by Ren et al. [86], Gadkari et al. [87], as well as Tremouli et al. [88], higher temperatures generally enhance microbial activity and electron production up to an optimal point, beyond which microbial viability may be compromised. Degefa et al. [89] used a different approach employing multi-parameter optimization for advanced MFC systems to arrive at the same result. In their study, the influence of temperature and low-frequency vibrations was discovered through the laminated structure of an MFC piezoelectric energy harvester. A recent study by Li et al. [90] presented a more specific result. They discovered that, at a 20-degree temperature, the internal resistance of the MFC reached its maximum, while at a 40-degree temperature, the internal resistance decreased to a minimum but restored the increase beyond the 40-degree temperature. This implies that energy generation of MFC increases with increasing temperature as microbial metabolism and membrane permeability improves. Singh and Krishnamurthy [91] also confirmed this by conducting research focused on finding the relationship between cell voltage (V) and current density (A/m²) for a MFC at three different temperatures: 303, 323, and 343 K. As shown in Figure 5, the highest temperature (343 K) shows the highest cell voltages across most current densities, followed by 323 K and then 303 K. At lower current densities (0–2 A/m²), the differences in cell voltage between the three temperatures are more pronounced. As current density increases, the curves converge slightly, but the higher temperature still consistently results in higher voltages. This means increasing temperature generally improves cell voltage at a given current density and is likely due to enhanced microbial activity and reaction kinetics at higher temperatures, which can reduce activation losses and internal resistance. As a result, higher temperatures help maintain better performance at higher current densities, which is crucial for applications requiring sustained high-power output. The study also concludes that operating MFCs at higher temperatures could be beneficial for maximizing voltage and overall energy output. However, practical considerations such as thermal management, stability of the microbial community, and operational costs need to be balanced.

3.1.4. pH

Tang et al. [92] and several other studies have shown that higher voltages are recorded for substrate at neutral pH (pH of 7) than at any other value. A pH of 7 is an indication that the microbes are alive and are actively breaking down the substrate solution under use. Thus, at a neutral pH, higher ATP production is guaranteed and effective electron transfer, translating to higher power output. A study by Li et al. [93] confirmed this by analyzing the effect of pH on bacterial distributions within the cathodic biofilm of MFCs using maltodextrin as the substrate. They found that pH variations significantly influence the composition and activity of the bacterial communities in the cathodic biofilm. Generally, microbes require a pH equal to or close to neutral for optimal growth [88]. This process is known as absorbance. Bagchi and Behera [94] evaluated the effect of anolyte recirculation and anolyte pH on the performance of an MFC employing a ceramic separator. They discovered that maintaining a stable anolyte pH is crucial for sustaining high power output and CE. According to Halim et al. [95], both extremely low and high pH levels can negatively impact power output and microbial viability in MFCs. Acidic conditions disrupt microbial cell membrane integrity, leading to cell lysis and decreased microbial activity, which in turn lowers power output due to the disrupted proton gradient essential for efficient electron transfer. Similarly, alkaline conditions cause denaturation of microbial enzymes and proteins, impairing microbial metabolism and growth and consequently reducing power generation efficiency by affecting the proton gradient and electron transfer processes. As a result, in cases where the pH value does not encourage optimal microbial activity, acid and base dosing or the addition of a chemical buffer could restore the pH to an optimal value. The graph in Figure 6 is an experiment conducted by Puig et al. [96] to assess the impact of pH on MFC performance. Puig et al. [96] studied the result of increasing or decreasing pH and temperature values on power and current densities. They found that a pH 7 at 30°C shows the highest power density among all conditions. At that condition, he records peak values at approximately 1,600 mW/m², around 0.5 mA/cm² current density. The power density sharply increases initially and then decreases after reaching the peak. In contrast, a pH of 7 at 10°C shows a lower power density compared to pH 7 at 30°C and peaks around 200–300 mW/m². The curve is much shorter and lower compared to pH 7 at 30°C, indicating reduced performance at lower temperatures. At a pH of 5 at 10°C, he records the lowest power density, which peaks around 100 mW/m². The curve indicates a very limited power generation capability under these conditions. The graph demonstrates that a neutral pH (pH 7) is more conducive to higher power densities in MFCs compared to a more acidic environment (pH 5). Again, higher temperatures (30°C) result in significantly higher power densities compared to lower temperatures (10°C), indicating that temperature is a crucial factor for microbial activity and MFC efficiency. The study concludes that the best performance of MFC is only observed at pH 7 and 30°C, suggesting that both neutral pH and higher temperatures are optimal for maximizing power density in MFCs.

3.1.5. Organic Loading Rate (OLR)

The OLR defines the capacity of the reactor being used per unit volume or the mass of microorganisms available to decompose substrates. Hence, increasing the OLR results in higher substrate degradation and power generation. Don and Babel [97] studied the effects of organic loading on bioelectricity generation and micro-algal biomass production in MFCs using synthetic wastewater. They found that higher OLRs led to increased bioelectricity production up to an optimal point, beyond which performance declined due to substrate inhibition. Similarly, Gurjar and Behera [98] investigated the bio-electrochemical performance of a ceramic MFC treating kitchen waste leachate, focusing on the effects of OLR and anode electrode surface area. They found that increasing OLR improved power density and CE up to a threshold, after which the performance plateaued or decreased. As seen in Figure 7, Xu et al. [99] demonstrated the relationship between CE and OLR for three different types of MFCs and found that increasing OLR negatively impacts CE across all MFC types, indicating that higher organic loads reduce the efficiency with which the MFCs convert organic matter into electrical energy. Xu et al. [99] recommended that MFC systems need to be designed with an expected OLR for optimization and efficiency purposes. Furthermore, in cases of high OLR, strategies such as pretreatment of the organic matter should be adopted to manage or mitigate its corresponding CE impacts.

3.1.6. Electrode Potential

Electrode potential is crucial for determining the efficiency of electron transfer from the microorganisms to the electrode. Higher electrode potential can enhance the oxidation–reduction reactions, leading to better power generation. Ma et al. [100] show that optimizing the anode potential improves the performance of MFCs by facilitating more efficient electron transfer. Shirpay [101] also investigated the relationship between electrode potential (in mV) and current density (in mA/m²) for MFCs on two different scales, large scale and small scale, and concluded that optimizing electrode potential is crucial for maximizing current density and overall power output. Their study revealed that at an optimal electrode potential, MFCs exhibited higher current densities, indicating improved electron transfer efficiency. This relationship was consistent across both large and small scales, suggesting that precise control of electrode potential can enhance the performance of MFCs irrespective of their size. As seen in Figure 8 by Shirpay [101], both the large-scale and small-scale MFCs show a decrease in potential as the current density increases. This inverse relationship suggests that as the MFC generates more current, the voltage drops. The dotted line with the label “Reducing resistance” suggests an ideal path where reducing internal resistance would maintain higher potentials at given current densities. The actual performance of both the large-scale and small-scale cells deviates from this ideal path, indicating higher internal resistance. The graph highlights the challenges in scaling up MFCs, showing that small-scale cells tend to perform better in terms of maintaining higher potential at increasing current densities. Addressing factors such as internal resistance and optimizing conditions for microbial activity are crucial for improving the performance of large-scale MFCs.

3.1.7. Open Circuit Voltage (OCV)

Higher OCV generally correlates with a higher potential for power generation under load. In the study by Littfinski et al. [102], the impact of OCV on MFC performance was examined using various electrochemical methods. The findings indicated that high OCV values were associated with better electron transfer rates, leading to improved power output. However, prolonged high OCV can stress microbial communities, potentially decreasing cell viability and stability over time. Balancing OCV is essential to optimize performance and maintain microbial health within MFC systems. In some cases, OCV is greatly affected by the type of substrate used. For example, Lawal et al. [103] evaluated the OCVs of MFCs using cow and pig dung as substrates. They found that the OCVs generated from these organic wastes varied significantly, with cow dung generally producing higher OCVs compared to pig dung. The use of pig dung in MFCs is notable due to its unique microbial community composition and nutrient profile, which can influence electron transfer processes. The study concludes that one must select a nutrient-rich substrate selection for a higher MFC performance. The graph in Figure 9 obtained from Choudhury et al. [104] demonstrates the OCV (in Mv) versus time (in hours) for a MFC under different resistances: 5,000, 1,000, and 500 Ω. At 0 to ~36 hr, 5,000 Ω, the voltage increases rapidly, reaching a peak of around 400 Mv. At the peak resistance, the MFC builds up to a high voltage as current flow is limited, resulting in less power loss. Reducing resistance to 1,000 Ω increases current flow, causing an initial voltage spike, but the increased current also leads to higher internal losses, resulting in a lower steady-state voltage compared to the initial phase. Further reducing the resistance to 500 Ω increases current flow even more, but the voltage stabilizes around the same level as with 1,000 Ω, indicating that the cell is operating near its optimal power point. The stable voltage at lower resistance indicates the MFC’s optimal operating range, where it can maintain a consistent performance. This data is crucial for optimizing the design and operational parameters of MFCs for better efficiency and power output.

3.1.8. Treatment Efficiency

Treatment efficiency reflects the MFC’s ability to remove contaminants from the substrate. Higher treatment efficiencies are indicative of better substrate utilization and cleaner effluents. He et al. [105] showed that the efficient substrate treatment enhances the degradation of organic matter, leading to higher electron availability for electricity generation. This results in improved CE, which measures the proportion of electrons captured as electrical current relative to the total electrons released by microbial metabolism. Additionally, optimizing treatment efficiency reduces biofouling and system resistance, thereby maintaining a stable and higher power density. In a different study, Corbella and Puigagut [15] investigated the improvement of treatment efficiency using constructed wetland MFCs (CW-MFCs). They found that the choice of anode material and external resistance significantly influenced the treatment efficiency and power generation of CW-MFCs. According to the discovery of Huang et al. [106], contaminants like phosphorus and Chlorella vulgaris can be removed by frequently adding small amounts of nitrate. They found that this approach significantly boosted phosphorus removal and supported algal growth, enhancing overall treatment efficiency in MFCs. The graph in Figure 10 is an MFC project conducted by Echiegu [107], depicting the relationship between the solid retention time (θₙ) in days and the effluent waste concentration (S) in mg/L, along with the treatment efficiency (eₛ) in percentage, under different conditions of flow and mixing for a microbial treatment system. The graph includes various parameters and two different system dynamics: plug flow and completely mixed systems. For plug flow, treatment efficiency starts high and increases quickly, achieving near-maximum efficiency at shorter solid retention times. For completely mixed systems, treatment efficiency increases steadily with retention time, reaching higher levels at longer retention times. Increased solid retention time generally improves treatment efficiency as microbes have more time to consume the substrate. Also, the plug flow system reaches higher efficiency more quickly compared to the completely mixed system, indicating better performance at shorter retention times. The inset parameters (k, Kₛ, Y, b, S₀, r) indicate the conditions under which the system operates, affecting microbial growth rates, substrate utilization, and overall treatment performance.

3.1.9. Organic Removal Rate (ORR)

3.1.10. HRT

HRT determines the time the substrate stays in the reactor. As established by Xie et al. [113], optimal HRT ensures sufficient time for microbial degradation and electricity generation without overloading the MFC system. This necessitates optimizing HRT for balanced substrate degradation and power output. The impact and application of HRT differ for each MFC type. For continuous flow mode dual-chamber MFC, Ye et al. [114] found that optimizing HRT significantly improved both nutrient recovery rates and electricity generation. Haavisto et al. [115] examined the effect of HRT on continuous electricity production from xylose in an up-flow MFC. They discovered that shorter HRTs enhanced the electricity production rate but reduced the overall xylose removal efficiency. In a different research, Chang et al. [116] explored the effect of HRT on electricity generation using a solid plain-graphite plate MFC in an anoxic/oxic process for treating pharmaceutical sewage. They found that optimal HRTs improved electricity generation while also degrading pharmaceutical compounds. Figure 12 shows the relationship between HRT and Maximum Power Density (in W/m²) for different initial concentrations of acetate used in MFC [117]. It can be observed from the graph that, initially, as HRT increases, the maximum power density also increases for all three initial acetate concentrations. This suggests that at lower HRTs, the contact time between the substrate and the microbial community is insufficient for optimal power generation. Beyond these optimal HRTs, the maximum power density begins to decline. This can be due to several factors, including the depletion of readily available substrates and the accumulation of metabolic byproducts that may inhibit microbial activity. Higher initial concentrations of acetate led to higher maximum power densities. This is because more substrate is available for the microbes to convert into electricity. The optimal HRT increases slightly with higher initial acetate concentrations. This is because the microbes need more time to process the higher substrate levels effectively. Optimal HRTs ensure that microbial interactions with electrodes are maximized (there is a right balance for microbial activity, allowing efficient conversion of organic matter into electricity), improving electron transfer rates and overall power density.

4.1.1. Reactor Configuration and Scaling Up

Recent studies highlight that stack models or modularized systems in MFCs are pivotal for achieving high power density and scalability. For instance, research by Mukherjee et al. [118] and Rabaey and Verstraete [119] demonstrated that stacking multiple MFC units can significantly increase power output per unit area while maintaining operational stability over extended periods. This approach is crucial for applications requiring higher power densities and larger-scale deployment in practical settings. Several recent studies have focused on improving reactor configurations and scaling up MFCs. For example, Tan et al. [120] investigated the integration of a polypropylene biofilm carrier and a fabricated stainless-steel mesh supporting activated carbon in an MFC configuration. They discovered that this innovative design significantly enhanced the MFC’s performance, resulting in higher power density and improved contaminant removal. The enhancements were attributed to the biofilm carrier’s increased surface area, which facilitated greater microbial growth and activity, and the activated carbon’s effective adsorption properties, which improved electron transfer efficiency within the system. In another research, Song et al. [121] focused on recent advances in MFC reactor configurations and coupling technologies for the removal of antibiotic pollutants. They found that innovative reactor designs and hybrid systems significantly improved pollutant degradation and energy recovery. This improvement was achieved through enhanced contact between pollutants and reactive sites within the reactors, increased microbial activity due to optimized environmental conditions, and the integration of coupling technologies that facilitated more efficient electron transfer and energy conversion processes. Also, Rossi and Logan [122] investigated the impact of reactor configuration on the performance of pilot-scale MFCs. They discovered that certain configurations, such as tubular designs and membrane-less systems, offered superior performance in terms of energy recovery and contaminant removal. These studies emphasize the importance of optimizing reactor configurations to enhance efficiency in MFCs and reduce costs.

4.1.2. Membrane Materials: Earthen and Ceramic

Earthen and ceramic membranes are increasingly favored in MFC technologies due to their sustainability and efficiency. Ceramic membranes, in particular, offer superior proton exchange rates and mechanical strength compared to traditional materials like Teflon [123]. Studies have shown that ceramic membranes can operate effectively over long periods with minimal maintenance, making them suitable for commercial MFC applications [123]. Suransh et al. [124] investigated the modification of clayware ceramic membranes to enhance the performance of MFCs. The modification involved incorporating metal oxides into the clayware ceramic membranes. This alteration aimed to improve the membrane’s electrical conductivity, mechanical strength, and overall stability, thereby enhancing the MFCs’ performance and extending their operational lifespan. They identified the primary research gap as the need for cost-effective, durable membrane materials that improve the efficiency and longevity of MFCs. Other studies like Rao et al. [125] and Suransh and Mungray [126] succeeded at solving this cost issue by employing a novel fly ash blended ceramic membrane and reducing the particle size of vermiculite to produce low-cost earthen membranes, respectively. A summary of advanced ceramic-based MFCs has been summarized in the work of Yousefi et al. [127]. Table 4, obtained from their work, illustrates the promising potential of earthen and ceramic materials as separators in MFC technology. These natural materials, such as porcelain, earthen pots, mullite, and red soil clayware, offer a range of benefits for sustainable and cost-effective MFC designs. The varied performance in COD removal, from as low as 41.5% to as high as 96.5%, confirms the importance of factors such as material composition, separator thickness, and porosity. For instance, the dual-chamber MFC with an earthen pot separator shows remarkable efficiency in treating rice mill wastewater, achieving 96.5% COD removal, which highlights its potential for high-performance wastewater treatment applications. In contrast, the single-chamber MFC with a mullite separator demonstrates lower efficiency, suggesting that the specific combination of anode/cathode materials and the nature of the waste stream significantly influence performance.

4.1.3. Wastewater Treatment Advancements

Continuous double-chamber MFC systems have been shown to achieve high removal efficiencies for COD in wastewater treatment [110]. Recent studies have optimized the design of these systems to maximize treatment efficiency while minimizing energy input. For example, researchers have explored novel electrode materials and configurations to enhance pollutant degradation rates and overall system performance [132]. Such advancements are crucial for meeting stringent environmental standards and reducing the environmental footprint of wastewater treatment processes. Additionally, in recent projects, researchers are advancing and optimizing MFC systems by combining wastewater treatments with clean energy production. In the study by Arun et al. [133], they integrate microbial electrolysis cells (MEC) and MFCs for simultaneous wastewater treatment and green fuel (hydrogen) generation. The study demonstrated a novel synergistic system where the byproducts of MECs fuel the MFCs, resulting in higher hydrogen yields and more efficient wastewater treatment. Zahran [134] also investigated iron- and carbon-based nanocomposites as anode modifiers in MFCs for both wastewater treatment and power generation. These nanocomposite anodes significantly increased the power density and treatment efficiency of MFCs by enhancing electron transfer rates and microbial adhesion. Similarly, Chaturvedi and Kundu [135] utilized nanostructured graphene for the same purpose of green energy and wastewater treatment. Their novel use of graphene-based electrodes enhanced conductivity and surface area, leading to improved microbial activity and overall MFC efficiency.

4.1.4. Genetic Engineering for Enhanced Performance

Genetic engineering strategies have revolutionized MFC performance by enabling microbes to produce specific electron-shuttling compounds or enhancing their metabolic pathways for electricity generation [136]. Recent developments include the engineering of microbial consortia to optimize electron transfer efficiency and substrate utilization. For instance, synthetic biology approaches have been employed to design microbial communities with tailored functionalities, such as improved tolerance to environmental stresses and enhanced bioelectricity production [137]. These advancements pave the way for more efficient and sustainable bio-electrochemical systems. Surti et al. [138] reviewed genetic engineering strategies for enhancing bioelectrochemical systems, focusing on improving microbial electron transfer capabilities. Their comprehensive analysis highlighted several key strategies and genetic modifications, including the overexpression of electron transfer proteins such as cytochromes, optimizing metabolic pathways for increased electron flow to the anode, and employing synthetic biology approaches to introduce or enhance electron transfer pathways. Additionally, they discussed the use of mutagenesis and directed evolution to develop microbial strains with superior electron transfer properties, as well as heterologous expression of genes from electroactive microorganisms to impart these capabilities to other microbial hosts. Finally, they emphasized the importance of biofilm engineering to create more stable and conductive biofilms on electrode surfaces. These modifications collectively lead to significant improvements in the performance of bioelectrochemical systems, enhancing power generation and efficiency. Philipp et al. [139] also discussed genetic engineering for enhanced productivity in bioelectrochemical systems, highlighting the critical role of metabolic pathway optimization. They emphasized the importance of fine-tuning metabolic pathways to maximize the efficiency of bioelectrochemical reactions. This involves increasing the availability and flow of electrons to the anode, thereby enhancing the overall energy conversion process. By this method, they addressed the gap of integrating complex genetic circuits into microbial genomes, which are designed to regulate and optimize multiple metabolic processes simultaneously. These complex genetic circuits include synthetic promoters, regulatory elements, and engineered enzymes that coordinate the expression and activity of various metabolic pathways resulting in significant improvement in the productivity and efficiency of bioelectrochemical systems such as MFCs.

4.1.5. Component Modifications for Improved Efficiency

Ongoing research focuses on optimizing MFC components such as anodes and cathodes to improve power output and efficiency. Advanced materials, including carbon-based nanomaterials and metal catalysts, are being investigated for their potential to enhance electrochemical performance and durability under harsh operating conditions [140]. Modifications in the anodic chamber of MFCs are critical for improving bacterial affinity towards the anode and enhancing electrical conductivity. Recent advancements include using polymer or carbon materials as molecular wires to connect biofilms to the anode, resulting in increased power densities from 1,479 to 2,355 mW/m² [141]. Moreover, innovative electrode architectures and fabrication techniques are being explored to achieve higher surface area and better electron transfer kinetics, contributing to overall system efficiency improvements [142]. Hindatu et al. [143], Dessie and Tadesse [144], as well as Savla et al. [145] reviewed various anode modification techniques, such as the use of nanomaterials (nanocomposite-based anode modifiers) and conductive polymers aimed at improving the performance of MFCs. Figure 13 summarizes the results of utilizing nanomaterials for MFC anode modification to aid electron transfer efficiency.

Figure 13(a) illustrates an unmodified anode, where the presence of electroactive bacteria (EAB) is hindered by non-electroactive bacteria (non-EAB) and extracellular polymeric substances, leading to limited electron transfer. In contrast, Figure 13(b) demonstrates a modified anode incorporating nanomaterials, which facilitate the attachment and activity of EAB. These nanomaterials provide a conducive environment for cytochrome c, a key electron transfer protein in EAB, thereby promoting efficient electron transfer. The enhanced electron flow in the modified anode significantly improves the overall performance of the MFC. Though nanomaterials have proven to be better facilitate electron transfer, they are associated with issues related to cost. As a result, some researchers have begun investigating on other materials for MFC component manufacturing. For example, Yaqoob et al. [146] explored alternative materials, such as agricultural waste-derived carbon, which offered a sustainable and economically viable solution for MFC applications while maintaining or enhancing performance. Du et al. [147] also investigated the use of polydopamine as a new modification material to reduce cost and promote anode performance In MFCs. This application of polydopamine coating was found to improve the anode’s hydrophilicity, biocompatibility, and electron transfer properties, leading to faster startup times and enhanced MFC performance while reducing cost. Additionally, techniques such as ammonia gas treatment, heat and acid pretreatments, or anode coating with materials like CNTs or graphene have proven to increase anode surface area and improve performance [148, 149]. As researched by Kadivarian et al. [150] and Zafar et al. [151], cell structure and heat pretreatment of microorganisms impact the performance of MFCs by enhancing electrode conductivity and microbial adhesion, which can boost microbial activity and electron transfer rates. Their study also establishes that the choice and optimization of microorganisms are crucial, as different strains exhibit varying levels of metabolic activity and electron transfer capabilities. Table 5 presents different electrode pretreatment methods currently used in various MFC designs and their respective impacts on power density and CE.

From Table 5, it can be observed that NH₃ gas treatment of carbon cloth generally achieves the highest power densities (up to 1,970 mW/m²) and moderate to high coulombic efficiencies (up to 75%), indicating this method is highly effective for enhancing MFC performance. Comparatively, acetone cleaning and acid treatment provide decent power densities but lower coulombic efficiencies, highlighting a tradeoff between power output and electron transfer efficiency. The different responses to treatment methods across various materials (carbon cloth, carbon mesh, carbon brush) suggest that the choice of electrode material significantly influences the effectiveness of the treatment. NH₃ gas treatment appears more universally effective across different carbon-based materials, while other methods like acetone cleaning and acid treatment show more variability in performance outcomes.

On the other hand, modifications in the cathodic chamber aim to utilize materials with higher redox potentials that can efficiently absorb protons, reducing reduction losses. While platinum (Pt) remains a common cathode material due to its high efficiency, research is increasingly focusing on exploring cost-effective alternatives such as iron-based catalysts, carbon-based materials, and transition metal chalcogenides for their potential to achieve high power outputs at a fraction of the cost of platinum [158]. These alternatives aim to balance performance with affordability, potentially making MFCs more economically viable.

4.1.6. Biosensor Development

MFCs have emerged as promising platforms for biosensor development as a result of their ability to detect various environmental pollutants and biomolecules. Recent advancements include the integration of biorecognition elements with MFC electrodes to create robust and sensitive biosensors for real-time monitoring of water quality parameters, such as BOD and antibiotic residues [21]. These biosensors offer rapid detection capabilities and can be deployed in remote or resource-limited settings, addressing critical challenges in environmental monitoring and public health. Zhou et al. [159] developed an MFC-based biosensor capable of real-time, sensitive toxicity detection by employing advanced anode and cathode materials to enhance electron transfer efficiency and optimizing microbial consortia for increased sensitivity and specificity. They designed the biosensor to monitor changes in output voltage or current correlating with microbial activity inhibition due to toxic substances. Jadhav et al. [160] extended the MFC’s capabilities by integrating sensors for measuring multiple parameters, such as pH and dissolved oxygen, within the MFC setup. This involved modifying electrodes with selective materials, embedding pH and oxygen sensors, and using advanced data acquisition systems to process the MFC’s electrical output along with environmental readings. The integration of MFCs into such biosensors exemplifies significant progress, driven by innovations in bioelectrode design, microbial selection, system miniaturization, and signal processing, resulting in sophisticated, portable biosensors for real-time environmental monitoring. Building on these foundational advancements, further studies have continued to refine and expand the capabilities of MFC biosensors in various environmental applications. For example, Khan et al. [161] addressed the limitations of existing zinc detection methods, which are often complex and costly, by creating a simple, cost-effective, and sensitive MFC-based biosensor for zinc detection. This development showcases the versatility of MFC technology in providing accessible and efficient solutions for heavy metal detection. Similarly, Xiao et al. [162] focused on the hydrodynamic optimization of continuous-flow miniaturized MFC biosensors. They developed an optimized continuous-flow design that improves the efficiency and sensitivity of MFC biosensors, making them more practical for diverse applications. These studies showcase the ongoing advancements in MFC biosensor technology, highlighting innovations in cost reduction, sensitivity enhancement, and practical design improvements.

4.1.7. Innovations beyond Electricity Generation

New MFC designs are driving innovations that enable diverse applications beyond electricity generation, including wastewater treatment, resource recovery, bioremediation, and biohydrogen production. Shabani et al. [25] explored the use of MFC technology for bioremediation. They reviewed various applications of MFCs in treating contaminated environments, explaining their potential for removing pollutants such as heavy metals, hydrocarbons, and dyes while recovering valuable resources like metals and nutrients. The technical innovations included the use of specific electroactive bacterial consortia that can degrade complex pollutants while simultaneously generating electricity. Building on the concept of resource recovery, Ye et al. [163] and Baby and Ahammed [164] conducted feasibility studies on using a double-chamber MFC for nutrient recovery from municipal wastewater. Their systems utilized bioanodes coated with nitrifying bacteria to facilitate the conversion of ammonia to nitrate and biocathodes that supported denitrifying bacteria to convert nitrate to nitrogen gas. The dual functionality of these MFCs was achieved by optimizing the anode and cathode compartments to enhance both electricity generation and nutrient recovery efficiencies, thereby improving the sustainability and economic viability of wastewater management practices. In a different study, Florio et al. [23] investigated biohydrogen production from solid-phase MFC spent substrates. They explored the potential of utilizing byproducts from MFCs, such as organic sludge, for biohydrogen production via dark fermentation and other anaerobic processes. The study involved pretreating the spent substrates to enhance the bioavailability of organic matter, followed by microbial fermentation to produce hydrogen gas. This process demonstrated significant hydrogen yields, presenting a new application for MFC byproducts and contributing to the development of integrated waste-to-energy systems. These advancements are opening up new opportunities for integrating MFCs into biorefinery processes and renewable energy systems, where the efficient conversion of organic substrates into value-added products is paramount. The innovations in microbial selection, electrode materials, system optimization, and multi-functional applications illustrate the significant progress in MFC technology beyond traditional electricity generation.

4.1.8. Mixed Microbial Communities and Biofilm Formation

Research into mixed microbial communities and biofilm formation in MFCs has revealed their crucial role in enhancing electrogenic performance. Biofilms facilitate synergistic interactions between different microbial species, enabling more efficient electron transfer and substrate utilization within MFC electrodes [165]. Recent studies have focused on understanding the dynamics of microbial consortia in biofilms and engineering them for improved stability and performance in diverse environmental conditions. This knowledge is essential for optimizing MFC design and operation to maximize bioelectricity production and wastewater treatment efficiency. Liu et al. [166] investigated how ferric iron affects the microbial community structure of biofilms in MFCs. Their study revealed that ferric iron can significantly influence the composition and function of microbial communities by enhancing the growth of specific electrogenic bacteria, such as Geobacter species, and promoting more robust biofilm formation. Transitioning from the influence of specific elements on biofilm dynamics, Hassan et al. [167] explored the role of microbial consortia in degrading phenol and producing electricity in MFCs. Their research demonstrated that diverse microbial communities, including phenol-degrading bacteria and electrogenic microorganisms, could significantly improve the bioelectrochemical performance of MFCs. With regard to microbial diversity, Cao et al. [168] compared the performance of pure cultures versus mixed microbial communities in the anode of MFCs. Their study showed that mixed communities of electricigens, such as Geobacter and Shewanella species, could enhance MFC performance compared to single-species cultures. This emphasizes the benefits of microbial diversity for effective electricity generation, as different microbes can complement each other’s metabolic capabilities, leading to more efficient substrate utilization and electron transfer. Albarracin-Arias et al. [169] examined the dynamics of microbial communities and their impact on electricity generation in MFCs inoculated with palm oil mill effluent (POME) sludges and pure electrogenic cultures. Their findings highlighted how different microbial inocula affect biofilm formation and electrical output. Specifically, they found that POME sludge inocula promoted the development of diverse and resilient biofilms that enhanced MFC performance. These studies collectively advance the understanding of how mixed microbial communities and biofilm formation contribute to the optimization of MFC technology.

4.1.9. Microbial Catabolic Activities and Biocatalyst Enhancements

Microorganisms like S. oneidensis play crucial roles in enhancing MFC efficiency through their electron transfer capabilities. Recent studies have highlighted that these microorganisms can transport electrons to the electrode surface without the need for mediators, significantly enhancing power output [170]. To further optimize biocatalyst performance, researchers have explored methods such as chemically perforating cell membranes to create pores and channels, which facilitate faster electron transfer and lead to higher power densities in MFCs [147]. Additionally, genetic modifications to increase cell permeability through biosurfactant production have been shown to enhance electron transfer rates by up to 2.5 times, demonstrating significant potential for improving MFC efficiency [171]. Some recent studies on catalyst enhancements include the study by Verma and Mishra [172], who reviewed recent trends in enhancing the performance of yeast as an electrode biocatalyst in MFCs. Their study highlighted various approaches to optimize yeast-based biocatalysts, including genetic and metabolic engineering strategies that improve electron transport chains and cellular respiration rates, thereby boosting MFC efficiency. Also, Chiranjeevi and Patil [136] outlined strategies for enhancing the electroactivity and metabolic functionality of microorganisms in microbial electrochemical technologies. They discussed techniques such as electrode modification with bio-compatible conductive materials and the use of co-cultures to improve the performance of microbial biocatalysts, focusing on both fundamental and applied aspects to enhance MFC efficiency. Similarly, Ummalyma and Bhaskar [173] provided an overview of recent advances in the role of biocatalysts in biofuel cells. Their review covered the latest developments in biocatalyst technology, such as the integration of enzyme cascades and synthetic biology approaches, and emphasized improvements in biocatalyst performance for various biofuel cell technologies. Table 6 summarizes biocatalysts used in MFCs employing novel materials along their respective power densities and substrates.

Table 6 exemplifies significant advancements in MFC technology, particularly highlighting the enhancements in biocatalysts and electrode materials. The use of CNTs combined with various materials like polyaniline, nitrogen-doped CNT, and reduced graphene oxide (rGO) showcases how these modifications lead to substantial increases in power density. For instance, the incorporation of nitrogen-doped CNT/rGO with dual biocatalysts such as E. coli and S. putrefaciens achieves a power density of 1,137 mW/m², emphasizing the potential of mixed microbial cultures to optimize performance. Further, the integration of three-dimensional CNT structures with chitosan microchannels, nano-molybdenum carbide, and graphene oxide-modified carbon paper demonstrates the role of advanced nanomaterials in enhancing microbial adhesion and electron transfer. Notably, the highest power density of 2,668 mW/m² is achieved using graphene-modified stainless-steel mesh with E. coli, highlighting the exceptional conductive properties of graphene and its capacity to form efficient bioelectrochemical interfaces. These innovations indicate a shift towards more efficient, cost-effective, and sustainable MFC systems, driven by the synergistic effects of advanced materials and optimized biocatalyst configurations.

4.1.10. Proton Transfer Membrane (PTM) Innovations

Recent advancements in PTMs for MFCs have focused on enhancing power density and efficiency. Traditional polymeric materials like Nafion, known for their high ionic conductivity and uniform porosity, are costly and have performance limitations [179]. Emerging alternatives include non-Nafion membranes such as polyester cloth and bipolar membranes, which utilize a bilayer cation-exchange layer attached to an anion-exchange layer for improved pH neutrality and performance [180]. Additionally, the development of mediator-less MFCs using metal-reducing bacteria like Shewanella promises cheaper and environmentally friendly energy generation without the need for exogenous electron carriers [181]. Studies by Sirajudeen et al. [182] explored the innovative use of biopolymer composites as proton exchange membranes in MFCs. Their work demonstrated the effectiveness of these biopolymer membranes in generating electricity from real wastewater, showcasing a significant improvement in power density and efficiency compared to traditional materials. Building on this, Neethu et al. [183] developed a novel proton exchange membrane using clay and activated carbon derived from coconut shells. This innovative approach not only enhanced the membrane’s performance but also contributed to sustainability by utilizing low-cost, readily available materials. Their findings showed marked improvements in the overall performance of MFCs, demonstrating the viability of alternative natural materials in MFC applications. Further advancements were made by Saniei et al. [184], who employed a new sulfonated poly (ether ether ketone)-based proton exchange membrane. This membrane was modified with goethite nanoparticles functionalized with tannic acid and sulfanilic acid to enhance performance. The transition from traditional polymeric materials to biopolymers, natural materials, and advanced nanocomposites marks a significant shift towards more efficient, cost-effective, and sustainable PTM technologies for MFCs.

4.2.1. Summary of Challenges in MFC Technology

As earlier discussed, cost remains a major barrier to large-scale deployment of MFC technology. Key cost drivers include expensive materials like platinum for catalysts, ion exchange membranes such as Nafion, and manufacturing processes. Researchers are actively exploring cost-effective alternatives, such as non-precious metal catalysts and polymer-based membranes, to reduce initial investment and operational costs [185]. Scaling up MFCs from laboratory prototypes to practical applications also poses significant challenges. Issues such as maintaining consistent performance across larger systems, optimizing electrode designs for efficient mass transfer, and managing biofilm formation and stability at industrial scales need to be addressed. Advances in reactor design and engineering are critical to achieving scalability without compromising performance [186]. Furthermore, MFC performance can be sensitive to environmental factors such as temperature, pH, and substrate composition. Variations in these parameters can affect microbial activity, electrode performance, and overall energy output. Research focuses on developing robust MFC systems that operate reliably under varying environmental conditions, including extreme temperatures and fluctuating organic loads in wastewater streams [187].

4.2.2. Future Directions in MFC Research

Continued exploration of novel materials, including nanomaterials, conductive polymers, and bio-derived materials, holds promise for improving MFC performance and reducing costs. Advanced manufacturing techniques such as 3D printing and roll-to-roll processing are being investigated to enhance electrode fabrication and assembly processes, enabling scalable production of MFC components [188]. Efforts are underway to integrate MFCs into existing WWTPs and decentralized treatment systems [189]. This integration not only enhances organic pollutant removal efficiency but also generates renewable energy from wastewater streams. Research focuses on optimizing MFC design and operation to maximize energy recovery while meeting effluent quality standards set by regulatory agencies. Beyond electricity generation, MFCs show potential for nutrient recovery and chemical synthesis. Nutrient recovery technologies using MFCs aim to capture and recycle valuable nutrients such as phosphorus and nitrogen from wastewater, contributing to sustainable agricultural practices and reducing nutrient pollution in water bodies. Additionally, MFCs can be tailored for biochemical production through microbial synthesis of value-added chemicals and fuels, expanding their role in biorefinery applications [190].