Microbial Community

Abstract

The microbial community employed as biocatalyst in microbial fuel cells (MFC) play a crucial role in degradation of organic substances and bioelectricity generation. While degradation of organic matters and electrical current generation in MFC technology is predominantly depend on metabolic activities of electroactive bacteria such as Geobacter and Proteobacteria, these bacteria engage in mutual interactions with non-electroactive counterparts within the microbial community. These mutual interactions can modify system performance, which is widely depended on operational conditions, the source of the initial microbial inoculum, substrate diversity and system’s components. Consequently, it is essential to gain a comprehensive understanding of the ecological behavior of microbial communities under diverse conditions to optimize system efficiency. Numerous research studies have delved into the microbial communities under varying circumstances, and the objective of this research is to elucidate the distinctions among microbial communities and investigate the factors that impact their composition.

1. Introduction

The growing needs of our modern world, such as energy, water, and food, have caused significant harm to our environment. As our demand for electricity continues to rise sharply, there’s a pressing need to explore renewable energy sources [1]. One of the emerging technologies garnering substantial interest as a renewable energy source is microbial Fuel Cells (MFC) that use biological catalysts, primarily bacteria, to generate electric energy from organic matter, whether in the environment or waste [2]. MFC work with microorganisms as bio-catalysts that can extract energy from various waste materials and organic compounds [3]. The microorganisms oxidize organic and inorganic substrates at the anode, leading to the generation of electrons. Efficient transfer of these electrons from within the cells to the anode, in anoxic conditions, is vital for electric current production and involves mechanisms such as electron shuttles, mediators, and microbial nanowires [4, 5].

Diverse microorganisms, primarily bacteria from different phylogenetic groups, exhibit the capacity to generate electricity in MFCs without necessitating a mediator. Known as exoelectrogens or electrogenic microorganisms, several bacterial species like Geobacter spp., Shewanella spp., Rhodoferax ferrireducens, among others, have been identified for their electricity-producing abilities within MFCs. Additionally, microalgae, yeast, and fungi have also been reported in these systems [5].

Various factors significantly impact the operational efficiency of MFCs, including reactor configuration, anodic and cathodic materials, membrane type, substrate characteristics, internal resistance, loading rate, pH, buffer solutions, anodophilic populations, temperature, and salinity [5, 6, 7, 8, 9, 10]. Moreover, biological parameters such as inoculation source, pure culture versus consortium, Extracellular Electron Transfer (EET) rate, biofilm growth rate, and composition/activity of exoelectrogen populations play crucial roles since biocatalytic activity drives the MFC process [11, 12, 13, 14, 15, 16, 17].

Mixed microbial communities, particularly in bacterial MFCs, offer several advantages of adaptability and robustness to changing operating conditions [18, 19]. These communities demonstrate resilience against alterations in operating conditions, enabling detailed investigations of individual factors and fostering synergistic interactions among diverse microbes to enhance MFC stability and performance.

The utilization of mixed microbial communities within MFCs presents numerous advantages due to their ability to adapt and endure fluctuations in operating and environmental conditions. Conversely, the initiation phase of MFCs heavily relies on the establishment of biofilms and an enrichment process. The composition and structure of the resultant biofilm and microbial community formation significantly hinge upon various influencing factors. Nevertheless, comprehending the precise impact of these factors on the microbial community’s composition during optimal MFC performance poses a challenging endeavor. This study seeks to investigate and unravel the intricate influence of these factors on the behavior and dynamics of the microbial community.

2. Source of inoculum

Exoelectrogenic communities can be sourced from a variety of origins, including forest soil, anaerobic and aerobic sludge derived from wastewater treatment facilities, sediments, and compost [20, 21, 22, 23, 24]. In addition, isolated strains of model exoelectrogenes like Geobacter spp. and Shewanella spp. can also be utilized [25]. The impact of these diverse bacterial sources on the performance of such systems has been the subject of investigation in numerous studies. The presence of higher concentrations of exoelectrogenes and bacteria that promote biofilm formation can accelerate the development of biofilms on the anode [26]. However, the presence of electron acceptors, such as metal oxides and methanogenic microorganisms within the initial sludge can significantly influence the composition of bacterial communities on the anode and subsequently affect the performance of microbial fuel cells [27, 28]. Therefore, the careful selection of an inoculum source with a high concentration of active exoelectrogenes is of utmost importance.

The diversity of the initial microbial population and the concentrations of exoelectrogenic and non-exoelectrogenic bacteria are significantly influenced by the choice of inoculum source. Nonetheless, it is feasible to improve the inoculum to mitigate potential negative impacts. In a study conducted by Ishii et al., the inoculum was obtained from three distinct sources: (1) Anaerobic sludge from a wastewater treatment plant, (2) Paddy field soil, and (3) Coastal wetland sediments. The bioelectrochemical behavior of these three inocula was examined in a single-chamber fuel cell, and microbial population analysis was carried out using the 16S rRNA method. Paddy field soil displayed the most diverse initial microbial population in comparison to coastal wetland sediments and anaerobic sludge. In this study, an initial external resistance of 510 Ohms was applied, followed by a stepwise reduction to 100 and 25 Ohms to promote the enrichment of exoelectrogenic microorganisms. After 30 days of operation, the highest electric current, measuring 5.4 mA, was produced by the inoculum from coastal lagoon sediments. The analysis of the microbial population revealed that the stepwise enrichment process resulted in a reduction in microbial diversity within these three reactors. As a result, seven dominant bacterial groups emerged, comprising 90% of the microbial population in these cells. Consequently, by enriching the microbial population, it becomes possible to reduce the dependency of microbial fuel cell performance on the specific source of inoculum preparation [29].

While the source of inoculum can initially influence microbial community, over time and with consistent operational conditions, dominant microbial populations tend to converge. In a study conducted by Gao et al., two distinct inocula were examined. One was derived from the aerobic tank of a wastewater treatment plant, while the other originated from the anaerobic treatment reactor handling high sulfur wastewater. Both inocula underwent anaerobic treatment before being inoculated into the system. Bioelectrochemical analyses revealed that the inoculum from aerobic sludge reached maximum voltage more rapidly and exhibited higher voltage and current generation compared to the anaerobic sludge. Examination of the biofilm morphology on the anode biofilm indicated a higher presence of spherical bacteria in the aerobic sludge. Microbial population analysis in the initial stages showed that the aerobic sludge had greater diversity than the anaerobic sludge. However, as time passed and the system operated continuously, the microbial populations in both cells underwent progressive changes, ultimately leading to a convergence in dominant bacterial groups. In both inocula, dominant microbial populations became similar. The microbial population of the aerobic sludge inoculum was characterized by greater complexity and diversity, whereas the microbial population of the anaerobic sludge had a simpler composition. Notably, the anaerobic inoculum contained bromate-reducing bacteria, which are associated with the degradation of complex organic matter and sulfur reduction, reflecting the high sulfur content in the wastewater source. The similarity in dominant microbial populations in both inocula was attributed to their shared substrate. Specifically, Geobacter exoelectrogen species, biofilm-forming Zoogloea, and Acinetobacter species were prominent in both inocula [30].

Nutrient availability in the primary inoculum source significantly influences the diversity and microbial composition of the initial inoculum. In sedimentary Microbial Fuel Cells (SMFC), the sediments serve a dual role as both the substrate and the source of the inoculum, making sediment properties a crucial factor in SMFC performance. In a research study, seven lakes (denoted as S1 to S7) were assessed using SMFC technology. Of these, S1, S2, and S3 were large lakes, while S4 to S7 were smaller local lakes. The research aimed to investigate the geochemical characteristics and the initial microbial communities of each lake and their correlation with the biochemical performance, microbial composition, and microbial population in the SMFC systems. The findings revealed that the highest power output, 25.2 and 23.9 mW/m², was observed in S4 and S5, respectively, and these two systems exhibited the quickest startup times, at 7 and 9 days, respectively. The analysis of sediment characteristics indicated that the high relative yield in S4 and S5 could be attributed to the abundance of carbon and nitrogen sources, signifying higher nutrient levels in these sediments. Furthermore, the removal and degradation of organic matter in S4 and S5 were approximately 1.5 times higher than in the other systems. The initial microbial population in S4 and S5 displayed greater diversity compared to the other systems, and the types of bacteria comprising the populations in S4 and S5 differed from those in the other lakes. All seven SMFC systems exhibited similar voltage levels, ranging from 500 to 600 mV. After 60 days, an examination of the microbial populations on the anode indicated that Proteobacteria were predominant in all samples. However, in S4, only 43% of the population consisted of Proteobacteria, with Pseudomonas bacteria playing a significant role in the microbial population of S4 and S5 [31].

In the selection of the inoculum source, it is essential to consider the specific objectives of employing a fuel cell. For instance, when the aim is the remediation or elimination of a particular pollutant, it becomes possible to isolate microorganisms from the contaminated environment containing the target substance and employ them within a fuel cell system. In a study conducted by Sharma et al., samples were obtained from soil contaminated with hydrocarbons for the purpose of addressing phenanthrene pollution through the use of fuel cells. Following the preparation of the soil sample, it was dissolved in heptamethylnonane, and after an incubation period of 300 days, the mixture underwent filtration and subsequent centrifugation. The centrifuged material was utilized as the primary source of inoculum and introduced into a two-chamber fuel cell. Within this cell, phenanthrene was immobilized on the anode electrode, serving as a substrate for the formation of a biofilm by bacteria capable of decomposing organic matter. The investigation revealed that an increase in the loading concentration of this carbon material on the anode led to a reduction in current production. The maximum power output achieved was 37 mW/m² at a concentration of 2 mg/cm². Upon the analysis of the microbial population, it was observed that electroactive bacteria, notably Pseudomonas, were prevalent within this system, and these bacteria exhibited the capability to degrade phenanthrene, contributing to the pollutant’s removal process [32].

3. Anolyte as substrate

The electrical energy generated by MFC significantly relies on the composition of the organic matter within the anolyte. Microorganisms exhibit the ability to metabolize an array of substrates, spanning from inorganic compounds to intricate organic compounds, utilizing them as sources of energy or carbon. MFC studies have explored various substrates, including simple organic compounds like glucose, ethanol, and acetate, which are easily metabolized, as well as complex organic compounds like starch, chitin, and cellulose. However, while these organic materials are effective, they might not be economically efficient. Alternatively, wastewaters present a viable and cost-effective option. Nevertheless, wastewaters encompass a diverse range of complex organic materials, influencing the microbial population based on the specific organic constituents present within them [33, 34, 35, 36, 37].

In research conducted by sotres et al., a comparison was made between synthetic wastewater and pig slurry as an anolyte. The synthetic wastewater exhibited a power output of 2138 mW/m³, while pig slurry showed 5623 mW/m³. Analysis of microbial diversity revealed that the type of substrate significantly influenced the microbial population. In the synthetic wastewater where acetate was utilized, bacterial groups like Alcaligenaceae, Pseudomonadaceae and Clostridiaceae were among the dominant families. Conversely, in the pig slurry, the Flavobacteriaceae, Chitinophagaceae, Comamonadaceae and Nitrosomonadaceae families stood out as dominant among the microbial groups [38]. Indeed, the composition of the anolyte exerts a significant influence on the microbial community, regardless of the source of inoculum. The specific constituents and composition of the anolyte play a crucial role in shaping and determining the microbial population within the system. Table 1 provides an overview of various anolytes and their influence on the microbial community.

Inoculum	Anolyte	Power output	Microbial community	Ref.
Wastewater sludge	The organic fraction of municipal solid waste (OFMSW)	116 mW/m^2		[16]
moist manure		123 mW/m^2
sediment sample	brewery wastewater (inlet)	8.001 μW/cm^2	Geobacter; Shewanella; Clostridium	[39]
	brewery wastewater (outlet)	1.843 μW/cm^2
sludge	glucose,	0.11 W/m^2	Clostridium; Azospirillum	[40]
	sodium; acetate	0.098 W/m^2	Moheibacter; Azospirillum
	food waste hydrolysate	0.173 W/m^2	Rummeliibacillus; Burkholderia, Enterococcus Clostridium
anaerobic consortia	orange peel waste	358.8 mW/	firmicutes	[41]
sludge	Food waste	422 mW/m^2	Firmicutes; Bacteroidetes; Proteobacteria	[42]
sludge	sea food processing wastewater	05 mW/m^2	Stenotrophomonas	[43]
Swine wastewater	swine; wastewater	1.92 kWh/m^3	Arcobacter; Pseudomonas; Acinetobacter	[44]

Table 1.

Anolytes and their effect on microbial community.

4. Additives

Modifying the anolyte by introducing a range of additives can alter its environmental conditions, consequently enhancing cell performance. This manipulation not only impacts bacterial metabolism but also contributes to improving overall cell efficiency. However, these alterations also exert an influence on the microbial population. Additives such as Cu²⁺, CD²⁺, S²⁻, and surfactants (TWEEN80) have been employed to manipulate the environmental conditions [45, 46, 47].

In a study conducted by Chen et al., they incorporated sodium citrate as an additive, serving dual roles in pH adjustment and electron transfer. This combined addition led to a notable enhancement of 58% compared to the control group, with the output power peaking at a maximum of 785 mW/m². Investigation of the microbial population revealed an increase in microbial diversity following the introduction of citrate, attributed to two primary reasons. Firstly, maintaining a neutral pH balance promotes the growth of a wider array of microorganisms. Secondly, the citrate additive itself serves as a carbon source for microorganisms, stimulating their growth. In the control condition without citrate, Proteobacteria and Bacteroidetes were the dominant species. However, with the inclusion of citrate, the relative abundance of Proteobacteria decreased, while Bacteroidetes and Firmicutes exhibited higher prevalence. Among the Proteobacteria, gammaproteobacteria were predominant in the control condition, yet the addition of citrate diminished their dominance and elevated the relative abundance of alphaproteobacterial [48].

Exoelectrogens transfer the produced electrons to the anode using two methods: direct transfer and transfer facilitated by electron mediators. Additionally, the introduction of artificial electron mediators can enhance electron transfer within exoelectrogenes [49]. These mediators affect the bioelectrochemical properties of MFC and thus affect the microbial population as well. In Lin’s study, they compared two electron mediators, neutral red (NR) and potassium ferricyanide (PFC), in research involving a two-chamber fuel cell fueled by toluene. The introduction of these electron mediators resulted in an increase in voltage, elevating it from 5.53 mV to 109 mV and 2.88 mV for NR and PFC, respectively. Analysis of microbial diversity indicated that these electron mediators play a role in shaping the microbial community’s development. Seven bacterial groups were consistently found in all microbial fuel cells (MFCs). However, the reduced variability in the presence of these bacterial groups in MFCs utilizing NR and PFC respectively could be attributed to potential toxicity linked with these mediators [50].

5. Components

In general, the prevailing belief is that the current generation in an MFC is contingent on both the quantity of biofilm formation on the anode electrode and the level of bioelectrical activity within this biofilm [51]. Furthermore, the anode electrode stands as a pivotal component in determining the overall cost of fuel cell technology, typically constituting about 10% of the total expenses for constructing an MFC [52]. Consequently, the selection of the anode material plays a critical role in influencing both the performance and cost considerations of the battery. Carbon-based materials, such as carbon paper, carbon cloth, graphite, carbon brush, and similar options, are commonly employed in research due to their advantageous electrochemical properties and cost-effectiveness [53]. Variations among these materials pertain to their surface area, porosity, and conductivity, factors that directly impact the absorption rate and growth of electroactive bacteria.

In this investigation, four distinct carbon materials—granular activated carbon (GAC), granular semicoke (GS), carbon felt cube (CFC), and granular graphite (GG)—each possessing respective surface areas of 686 m2/g, 236 m2/g, 0.845 m2/g, and 0.623 m2/g, were assessed within a dual-chambered MFC. The power output was determined as 31.5, 27.9, 26.1, and 23.8 W/m² for GAC, CFC, GG, and GS, respectively. The researchers scrutinized the microbial community present on each electrode using the 16S rRNA method. Notably, GAC demonstrated the highest power output and displayed a dominant presence of 95% Geobacter in its microbial composition. Similarly, the CFC anode exhibited a dominance of Geobacter (43%) and Ruminobacillus (16%). On the GG electrode, the most prevalent phylum was Azospira, constituting 65% of the community. Conversely, the GS anode displayed an abundance of Bacteroidetes (19%), Sinorhizobium (15%), and Synergistes (13%). The outcomes underscored that the greater porosity and improved bioelectrochemical behavior of GAC resulted in a higher power output and an enrichment of electroactive bacteria [54].

The physical and chemical properties of the anode material significantly impact the composition of the electroactive biofilm formed by microorganisms. Altering these properties directly influences the microbial community, its diversity, and enhances the enrichment of microbial populations. Numerous studies have demonstrated that stabilizing metals or metal oxides on carbon materials leads to a notable increase in conductivity, often by two to threefold. This heightened conductivity plays a pivotal role in improving the enrichment of microbial populations [55, 56].

In a study by Xu et al., nanoparticles of MNO², Fe³O⁴, and Pd were loaded on carbon cloth treated with 5% Nafion. These modified carbon cloth materials served as anodes in a two-chamber cell. They were then compared against a control anode consisting of bare carbon cloth. The initial inoculum for the MFC was obtained from activated sludge sourced from a wastewater treatment plant. Synthetic wastewater was used as the substrate for the MFC. Electrochemical indicated that Pd, MNO2, and Fe3O4 exhibited power output of 824, 782, and 728 mW/m², respectively. These values surpassed the power output of the control, which utilized bare carbon cloth with a power output of 680 mW/m². Anodes treated with MNO2 and Fe3O4 particularly facilitated the enrichment of Geobacter, constituting for 73% of the microbial coverage. On the Pd-treated anode, Geobacter and sphaerochaeta bacteria were the predominant species, comprising 38% and 16% of the microbial composition, respectively [57].

In a different study, bio-synthesized gold nanoparticles (bio-Au) were utilized independently as well as in combination with multi-walled carbon nanotubes (MWCNTs) to modify carbon cloth electrodes. Overall, the incorporation of gold nanoparticles significantly enhanced the bioelectric behavior of the MFC. Specifically, BioAu/MWCNT showed the best performance compared to unmodified carbon cloth. The BioAu/MWCNT modification resulted in a 140% improvement in system startup time and a 56% increase in system output power, with the maximum power output reaching 178 mw. In this anode modified with gold nanoparticles and MWCNT, the bacterial groups Gammaproteobacteria and Negativicutes were among the dominant species [58].

In conclusion, the modification of the anode improves its physiochemical characteristics, creating a significant preference for EAB over fermentative bacteria. Despite not notably impacting bacterial diversity, the increased presence of electroactive bacteria on the modified electrodes resulted in better MFC system performance. Table 2 provides a summary of the modifications made to the anodes.

Anode modification	Inoculum	Power output	Microbial community	Ref.
Graphene oxide (GO)	active sludge	124.58 mW/m^2	increase in the relative ratio of; Proteobacteria, but a decrease of Firmicutes	[59]
tungsten carbide	anaerobic sludge	3.26 W/m^2	Geobacter; Geothrix; Pseudomonas	[60]
CF/N-CNT/PANI		3.8 W/m ^3	Desulfuromonas; Geobacter	[61]
poly(3,4-ethylenedioxythiophene)	sludge		Geobacter	[62]
Graphene oxide (GO) and carbon nanotubes (CNTs)	—	—	Pseudomonas Thauera; Diaphorobacter; Tumebacillus Lysobacter	[63]

Table 2.

Anode modifications and dominant microorganism.

Certain environmental conditions, such as elevated COD levels, high salinity, and the toxicity of wastewater, can hinder the performance of microbial biofilms. To enhance performance under such challenging circumstances, bacteria can be securely immobilized on the anode surface to mitigate the adverse effects of these harsh conditions and prevent a decline in MFC performance. Various methods for immobilizing bacteria on the anode have been reported, including adsorption, covalent binding, and entrapping. Through these methods, a substantial concentration of microorganisms can be firmly anchored to the anode’s surface. Among these different stabilization techniques, the entrapping method stands out due to its stability and enhanced strength [64].

In a study conducted by Leo et al., they employed the entrapping method to immobilize inoculum obtained from a municipal wastewater treatment plant onto carbon cloth using agarose gel. They examined the impact of varying concentrations of sodium acetate (ranging from 1 to 20 g/l) and compared it to non-immobilized carbon cloth as control. The stabilization of bacteria led to a 60% and 70% increase in output power for concentrations of 5 and 10 g/liter, respectively. Through an analysis of the microbial community, they demonstrated that stabilizing the inoculum on carbon cloth resulted in a 60% prevalence of Geobacteria, whereas the control anode exhibited a Geobacteria prevalence of 40% [65].

The membrane constitutes a pivotal factor that influences both the bioelectrochemical behavior and the microbial community within the developed biofilm on the anode electrode of a MFC [66, 67]. Serving as a barrier between the electrodes and compartments of the MFC, it plays a crucial role in upholding the anaerobic condition on the anode and preventing the undesired leakage of organic substances from the anolyte to the catholyte [68, 69]. Any leakage of anolyte substrate to the catholyte adversely impacts the kinetics of oxygen reduction. Furthermore, oxygen leakage into the anolyte promotes the proliferation of aerobic or facultative anaerobic bacteria, leading to the wastage of substrate and influencing the kinetics of electron production [70]. Additionally, the adhesion of microorganisms to the membrane’s surface results in biofouling affecting overall performance of the system [71]. Consequently, the type of membrane emerges as a key determinant affecting the overall performance of the system, consequently influencing the microbial population dynamics.

They modified a polymeric membrane (SPEEK) with nano silver, ranging from weight percentages of 2.5 to 10 mg/cm-2, and assessed its performance in a tubular Microbial Fuel Cell (MFC). The treatment with nano silver showed no substantial difference in water absorption levels, yet it notably enhanced proton ion transfer rates and reduced internal resistance and oxygen transfer rates. Examining various weight percentages for the treated membranes, it was found that the membrane treated with 7.5% by weight of silver exhibited the highest output power compared to other treatments. Subsequently, a comparison was made between the output power and microbial population of SPEEK7.5 with Nafion 117 and the non-treated SPEEK membrane, where the maximum output power recorded was 156 mw/m2. The presence of silver nanoparticles exhibited an antibacterial property, resulting in reduced biofouling on the membrane, with only silver-resistant bacteria thriving in the environment. The analysis identified 33 bacterial groups, with over 6000 bacterial species falling under Proteobacteria and Firmicutes [72].

6. Operational conditions

The oxygen present in the anolyte can significantly impact the functioning of MFC. As the performance of MFCs relies on exoelectrogenic bacteria, the presence of oxygen and aeration can alter their performance by influencing these microorganisms. Exoelectrogens respond differently to the presence of oxygen. For instance, Geobacter, being an obligate anaerobe, is highly susceptible to oxygen exposure. Even minimal contact with oxygen can inhibit its respiration and decrease power generation [73]. Conversely, facultative anaerobes like Shewanella can generate electricity under both aerobic and anaerobic conditions, although the quantity produced varies [74]. While several studies have demonstrated that even minimal oxygen ingress to the anode can diminish MFC performance, higher concentrations of oxygen can actually enhance performance [75].

In an investigation by Quan et al., a MFC was employed, and a combination of aerobic and anaerobic sludge served as the inoculum. The researchers noted that the output voltage was greater under anaerobic conditions compared to aerobic conditions. Additionally, they observed prolonged cycles in the anaerobic setup. These findings suggest that the presence of aerobic conditions encouraged the proliferation of bacteria detrimental to the system’s performance. Analysis of the microbial population revealed that aeration led to a decline in diversity, with species such as Bacteroidetes and others becoming dominant [76].

External resistance is one of the factors influencing the performance of MFC. In some studies, it has been shown that the use of external resistance has increased the removal of COD [77]. Given that the anode functions as the final electron acceptor, adjusting the external resistance can impact system performance by directing electron flow towards the cathode and facilitating the regeneration of the electron acceptor. This electron flow alteration can influence the diversity and composition of the microbial community [78]. Understanding the effect of external resistance on the microbial community provides a holistic understanding of exoelectrogen performance. External resistance prompts bacterial growth, with higher resistance leading to increased biomass. However, as resistance intensifies, there’s a reduction in current generation [79]. Consequently, it’s crucial to explore the appropriate amount of external resistance to optimize system performance.

Katuri et al., conducted a study exploring the influence of external resistance on the composition of the anode biofilm and microbial population. They employed a two-chamber MFC, utilizing glucose as a substrate, and varied the resistances from 100 to 50 kΩ. Their observations revealed that as resistance increased, the current production in the system decreased, and the system reached peak performance at a slower rate. Interestingly, they found that the highest production of biomass was achieved with 25 and 50 kΩ resistors, surpassing even the conditions of open circuit voltage. The investigation of microbial diversity showed that with the increase in resistance from 100 ohms to 50 kΩ, bacterial groups increased from 15 to 27. 100 Ohm resistance showed the simplest microbial diversity [80].

Continuous operation stands as a vital requirement for effective and economical wastewater treatment processes. In this context, the Organic Loading Rate (OLR) holds importance, and its control is influenced by the Hydraulic Retention Time (HRT). HRT, a critical operational parameter, directly affects OLR, subsequently impacting the production of electricity in MFCs [81, 82].

The study conducted by Haavisto et al. utilized a continuous two-chamber system supplied with xylose. Their observations revealed a significant impact of HRT variations on electricity generation. Decreasing the HRT from 3.5 days to 1 day resulted in an increase in system voltage from 344 mV to 480 mV. However, further reduction in HRT to 0.75 days and 0.17 days led to decreased voltages of 218 mV and 156 mV, respectively. Moreover, when adjusting the external resistance, the highest current production of 2460 mA/m² was achieved with an HRT of 1 day. Additionally, HRTs exceeding 1 day displayed Chemical Oxygen Demand (COD) levels below 0.4 g. L⁻¹/day, indicating insufficient substrate availability for the metabolism of microorganisms in the system. The decrease in Hydraulic Retention Time (HRT) led to the elimination of bacteria that were not attached to the biofilm. Consequently, there were notable shifts in microbial population diversity throughout the experiment. When the HRT was reduced to 0.5 days, there was an increase in the xylose content within the anolyte. This increase correlated with a significant rise in Christensenella, a type of bacteria adept at fermenting xylose. During the fermentation process of xylose, acetate, propionate, and butyrate are generated as byproducts, serving as carbon sources for exoelectrogenic bacteria. However, the heightened concentration of the substrate led to excessive proliferation of xylose-fermenting bacteria. This overgrowth, in turn, resulted in decreased current production within the system [83].

Studies have demonstrated that fluctuations in temperature significantly affect the initiation and continuous operation of MFCs. For instance, MFCs operating below 15°C experienced prolonged startup times and limited electricity production within a month of operation. However, recent research has highlighted successful startup and sustained functioning of both MFCs and Microbial Electrolysis Cells (MECs) even at extremely low temperatures, such as 4°C. These studies uncovered the existence of distinct psychrophilic populations within the anode biofilms, supporting extracellular electron transfer in MECs at lower temperatures [84, 85].

In the study conducted by Mei et al., MFC reactors were initially operated at 25°C to establish a biofilm on the anode surface. Following biofilm formation, the performance of the system was evaluated across temperatures of 10, 20, and 30°C over six cycles. Their findings demonstrated a correlation between decreasing temperature and increased cycle duration, as well as a decrease in power output. The highest output power, reaching 894 mW/m², was achieved at 30°C. Moreover, the maximum COD removal of 93% was observed at 30°C, with reduced COD removal at lower temperatures.

Throughout all tested temperatures, Proteobacteria, Bacteroidetes, and Firmicutes were the dominant bacterial groups. Specifically, Proteobacteria accounted for 61%, 65%, and 45% at temperatures of 10, 20, and 30°C, respectively. Bacteroidetes prevalence was 17%, 20%, and 25.5% at 10, 20, and 30°C, while Firmicutes constituted 6% at 10 and 20°C, rising to 11% at 30°C [86]. The study indicated that biofilm formation, metabolic activity, and extracellular electron transfer in MFCs are influenced by temperature.

7. Biodegradation

The potential of MFC technology extends beyond bioenergy production, delving into bioremediation, marking a promising avenue for efficient and sustainable environmental cleanup. Biodegradation serves as a sustainable resolution to combat persistent pollutant contamination. While physical-chemical methods exhibit rapid treatment efficacy, they often entail high costs and the potential generation of harmful by-products, diverging from the principles of Green Remediation. Bioremediation, on the other hand, harnesses the inherent capability of natural microbial communities to break down contaminants. This approach aligns with environmental sustainability, offering economic and social advantages while restoring ecosystems [86].

The composition of the microbial community is impacted by the substances that need to be biologically degraded. The recognition of resistance genes and clarification of degradation mechanisms, such as efflux pumps and modifying factors, emphasize the importance of biodegradation within MFCs. It underscores the complex interplay between the primary microorganisms present on the anode and the particular substrates utilized in MFCs. Grasping these associations is crucial for enhancing the efficiency of MFCs in breaking down pollutants, highlighting the essential role of biodegradation in the sustainable remediation of polluted water [87, 88, 89]. Table 3 summarizes various research studies conducted in the field of biodegradation, focusing on the dominant microbial populations responsible for reducing pollution related to specific pollutants.

Pollutant	Efficiency (%)	Power Output	Dominant Bacteria	Ref.
Food wastes	95.9	56 mW/m2	Geobacter; Bacteroides	[90]
vanadium	76.8	19 ± 11 mW/m	Deltaproteobacteria; Bacteroidetes Spirochaete	[91]
Copper	98.3%	10.2 W m-3	Proteobacteria; Bacteroidetes; Actinobacteria; Acidobacteria	[92]
sulfamethoxazole	87.52	1186.2 mW/m2	Proteobacteria; Bacteroidetes	[93]
nitrogen	100	6.3 W/m3	Nitrosomonas; Nitratireductor; Acidovorax	[94]
p-nitrophenol	81		Corynebacterium; Comamonas; Chryseobacterium; Rhodococcus	[95]
bisphenol and ibuprofen	78.7–63.2, respectively	28.63 mW m2	Chlorobi; Proteobacteria; Firmicutes; Bacteroidetes	[96]
Congo red decolorization	97%	41.1 mW/m2	Proteobacteria; Bacteroidetes; Actinomycetes and Firmicutes	[97]
polychlorinated biphenyls	(37.5	108.89 mW/m2	Geobacter and Pseudomonas Longilinea Desulfofustis)	[98]

Table 3.

Pollutant removal and dominant bacterial community.

8. Conclusion

Microbial fuel cells have a dual function of electrical power generation and treating a variety of wastewater that contain both organic and inorganic pollutants. As a form of biological technology, these cells utilize microbial catalysts to drive bioelectrochemical reactions. Mixed microbial populations are predominantly employed due to their exceptional adaptability to diverse environmental conditions. While certain bacterial groups may enriched and dominated based on system function and the type of wastewater utilized, this is contingent upon various factors. The primary source of the initial microbial community significantly influences microbial community development and can be selected based on the intended application of the MFCs. Additionally, organic and inorganic substances within the anolyte create conditions favoring specific species that metabolize these substances, influencing bacterial metabolism and their dominance. Alterations in MFC components, such as electrodes and membrane modifications can influence electrochemical properties, which impact biological properties and the enrichment of the microbial community. Environmental parameters like temperature, light, aeration and external resistance also exert considerable influence over the microbial community. Thus, it is essential to perceive and explore MFCs as a form of biological technology in order to enhance their performance and target specific applications.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.