Anode surface modification with reduced graphene oxide (rGO) and molybdenum (Mo) enhances microbial diversity and chemical oxygen demand (COD) removal in microbial fuel cells

Anode surface modification with reduced graphene oxide (rGO) and molybdenum (Mo) enhances microbial diversity and chemical oxygen demand (COD) removal in microbial fuel cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anode surface modification with reduced graphene oxide (rGO) and molybdenum (Mo) enhances microbial diversity and chemical oxygen demand (COD) removal in microbial fuel cells Habib Akyazı, Fatma Çiğdem Güldür, Ebru Beyzi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7472748/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2025 Read the published version in Environmental Science and Pollution Research → Version 1 posted 6 You are reading this latest preprint version Abstract This study aimed to investigate the effects of anode surface modifications on microbial community composition and chemical oxygen demand (COD) removal efficiency in microbial fuel cells (MFCs). Four different anode electrodes were fabricated: bare nickel foam (NF), reduced graphene oxide-coated nickel foam (NF/rGO), and NF/rGO modified with 30 wt% and 50 wt% molybdenum (Mo). These electrodes were tested in a single-chamber, membraneless, air-cathode MFC. Surface morphology was characterized using scanning electron microscopy (SEM), and microbial diversity was assessed through 16S rRNA metagenomic sequencing. Distinct microbial profiles were observed across the electrode types. The NF anode supported high abundances of Mesoterricola sediminis (22.2%), Klebsiella pneumoniae (10.1%), and other facultative species. The NF/rGO electrode promoted colonization by Cutibacterium acnes (8.1%) and Paracidovorax avenae (5.4%). On the NF/rGO/30%Mo electrode, notable species included Escherichia coli (8.4%) and Salmonella enterica (6.0%). The NF/rGO/50%Mo anode exhibited the highest microbial diversity, with species such as Streptomyces sp. RerS4 (6.9%) and Micromonospora endophytica (6.5%) being predominant. The highest COD removal efficiency (88.58%) was achieved using the NF/rGO/50%Mo anode. These findings demonstrate that molybdenum-modified rGO coatings enhance both microbial colonization and electrochemical performance, offering a promising strategy for improving MFC efficiency in wastewater treatment applications. Microbial fuel cell Anode modification Molybdenum loading Reduced graphene oxide coating COD removal Microbial community Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction In recent years, bioelectrochemical systems (BES) have emerged as innovative technologies that utilize the electrochemical activities of microorganisms to generate energy and remove pollutants. These systems contribute not only to the reduction of environmental pollution loads but also to simultaneous electricity production. BES can be used in a variety of applications such as fuel cells (for electricity generation), electrolyzers, desalination, and chemical synthesis (Jayabalan et al. 2019). In these systems, microorganisms grow under anaerobic conditions and degrade organic compounds—including various pollutants—producing and releasing electrons during metabolic processes. These electrons are transferred to terminal electron acceptors via different mechanisms, resulting in electricity generation (Borello et al. 2021). Therefore, microorganisms play a critical dual role in BES: organic matter conversion and electricity generation through extracellular electron transfer (Khoirunnisa et al. 2021). Microbial fuel cells (MFCs) are one of the most widely studied BES types, in which electricity is produced through the oxidation of organic matter by microorganisms (Dege and Danış 2020). A key factor in MFC development is the presence of microbial species capable of generating electrons and transferring them beyond the cell surface (Gorby et al. 2006; Indriyani et al. 2024). The operation of MFCs relies on electroactive microorganisms, known as exoelectrogens, which form biofilms on the anode electrode and have the ability to transfer electrons extracellularly, playing a crucial role in electricity generation (Logan and Regan 2006). Both pure and mixed microbial cultures have been used in MFC studies (Erensoy and Çek 2020). Compared to pure cultures, mixed cultures offer several advantages, including higher resistance to environmental disturbances, broader substrate utilization, greater substrate consumption, and higher power density (Nevin et al. 2008; Vamshi Krishna and Venkata Mohan 2016). However, the development of non-electrogenic bacteria such as fermenters and methanogens within the biofilm may reduce the system’s efficiency (Kim et al. 2005; Michie et al. 2011; Velasquez-Orta et al. 2011; Vamshi Krishna and Venkata Mohan 2016) Therefore, the type of microorganisms used as biocatalysts is critically important for the efficient performance of MFCs. To date, hundreds of electroactive strains have been isolated and used in MFCs. According to the NCBI Taxonomy database, numerous strains isolated from MFCs are cataloged to better understand their diversity and similarities (Erensoy and Çek 2020). Recent studies have focused on enhancing MFC efficiency by targeting gene expression related to cytochrome and pili production, which influence biofilm morphology and electron transfer capacity (Perchikov et al. 2024). In MFCs, the anode chamber is where microbial proliferation occurs. Here, organic matter is degraded by anaerobic bacteria to produce electrons and protons required for metabolic reactions (Mohan et al. 2008). The material and structural characteristics of the anode electrode significantly influence microbial growth, adhesion, substrate degradation, and electron transfer efficiency. As a result, various materials—such as metals, carbon-based substances, and hybrid composites—have been investigated for use as anode electrodes. However, current research indicates that existing anode designs have yet to achieve optimal performance for large-scale applications (Logan et al. 2006; Wang et al. 2009; Dege and Danış 2020). The surface properties of the anode directly affect microbial adhesion, biofilm formation, and extracellular electron transfer, thereby determining overall system efficiency. Flat electrode surfaces with limited surface area and inadequate porosity hinder microbial attachment and electron transport (Zou et al. 2016). In contrast, porous and three-dimensional electrode architectures provide microenvironments that promote microbial colonization and enhance electron transfer efficiency (Zou et al. 2016; Chong et al. 2019). The surface structure and morphology, electrical conductivity, mechanical strength, and corrosion resistance of electrodes used in microbial fuel cells (MFCs) are critical factors influencing anode performance. An ideal anode material should possess high surface area (Oh and Logan 2006), excellent electrical conductivity (Malvankar et al. 2012; Zhang et al. 2020), low internal resistance (Zhao et al. 2006), strong corrosion resistance, appropriate mechanical durability, and suitable porosity. Moreover, biocompatibility is essential to ensure that microorganisms can effectively adhere to the anode surface and establish robust electrical connections (Perchikov et al. 2024). In the literature, numerous composite anode materials have been developed using carbon-based, metal-based, or hybrid structures (Akyazı et al. 2025). Among these, carbon materials are the most commonly used due to their high surface area, chemical stability, corrosion resistance, and biocompatibility. Typical carbon-based anode materials include carbon cloth, carbon paper, carbon felt, carbon fiber, carbon brush, carbon rods, carbon mesh, carbon grids, and graphite (Liu et al. 2004; Oh et al. 2004; Cheng and Logan 2007; Logan et al. 2007; Wang et al. 2009; Thung et al. 2016; Bian et al. 2018; Akyazı et al. 2025). For example, carbon cloth provides excellent porosity and mechanical strength but is relatively expensive (Santoro et al. 2011; Guerrini et al. 2014; Dumitru and Scott 2016). Carbon mesh are inexpensive and commercially available, yet they exhibit low conductivity and mechanical stability (Santoro et al. 2014; Dumitru and Scott 2016; Wu et al. 2017). Carbon paper is rigid, brittle, and flat, which may limit its application (Guo et al. 2012; Santoro et al. 2014; Dumitru and Scott 2016). Graphite plates offer high conductivity and high cost but have limited surface area compared to porous materials (Dumitru and Scott 2016). Carbon fiber outperforms many others due to its three-dimensional architecture and large surface area (Chen et al. 2011). Additionally, nanostructured carbon materials such as carbon nanotubes, graphene, and graphene oxide are considered highly promising due to their exceptional conductivity and mechanical strength (Yazdi et al. 2016). In particular, graphene and its derivatives are frequently employed in MFCs owing to their high surface area (~ 2630 m²/g) and strong biocompatibility (Yuan and He 2015; Zhuang et al. 2015; Akyazı et al. 2025). A study evaluating 14 different metals (Al, Fe, Cu, Ti, Ni, Zn, Zr, Mo, Nb, Ag, In, Sn, Ta, and W) as anode materials reported that Mo, W, Fe, and Sn were the most effective in supporting biofilm formation. Among these, molybdenum (Mo) demonstrated outstanding performance due to its structural simplicity and long-term stability in microbial fuel cell systems. Furthermore, both Mo and W were found to be compatible with Geobacter species, leading to superior biofilm development and the highest current densities recorded among all tested materials (Yamashita and Yokoyama 2018). The performance of MFCs depends on both the microbial activity in the anode chamber (Calli et al. 2006; Oh and Logan 2006) and the material composition of system components (Ozcan 2013). Microbial activity directly influences electricity generation and the removal of chemical oxygen demand (COD), which is further affected by factors such as bacterial species and activity levels, temperature, pH, oxygen availability, nutrient concentrations, and reaction time (Aghababaie et al. 2015; Nosek and Cydzik-Kwiatkowska 2020; Borello et al. 2021; Malekmohammadi and Mirbagheri 2021). In this study, different anode electrode materials were developed and the microbial community and electrochemical performance of these anodes were investigated. To achieve this, reduced graphene oxide (rGO) was synthesized and coated onto three-dimensional nickel foam (NF) structures. Subsequently, the surface was further modified with molybdenum (Mo) to promote the attachment of electroactive bacterial species. Four types of anode electrodes were fabricated: NF, NF/rGO, NF/rGO/30%Mo, and NF/rGO/50%Mo. These electrodes were tested in a single-chamber, membraneless, air-cathode MFC system to evaluate their efficiency in synthetic wastewater treatment and to characterize the microbial communities that developed specifically on the anode surfaces. 2. Materials and Methods To improve the anode performance of microbial fuel cells (MFCs), reduced graphene oxide (rGO) was chemically synthesized and coated onto a three-dimensional porous nickel foam (NF) structure. To further enhance the surface adhesion of electroactive microbial species, molybdenum (Mo) was loaded onto the anode surface. The fabricated electrodes were then tested in a single-chamber, membraneless, air-cathode MFC system along with biofilm enrichment procedures. 2.1. Preparation and Characterization of Anode Electrodes rGO-coated nickel foam (NF/rGO) electrodes were synthesized in order to achieve high electrical conductivity and a large surface area. As detailed in a previous study (Akyazı et al. 2025), graphene oxide (GO) was first obtained by oxidizing graphite via the Hummers method, and was then chemically reduced to obtain rGO. The nickel foam (NF) was cut into the required dimensions, treated with acid solution to remove surface oxides, and subsequently coated with rGO using hydrothermal synthesis. To further enhance electrochemical performance and microbial adhesion, Mo was loaded onto the surface of rGO-coated electrodes at two different concentrations (30 wt% and 50 wt%), yielding hybrid anodes: NF/rGO/30%Mo and NF/rGO/50%Mo. Post-experimental SEM imaging revealed the presence of various bacterial colonies on the electrode surfaces. Characterization results confirmed that the synthesized anodes exhibited uniform coating and suitable porosity for microbial colonization (Akyazı et al. 2025). 2.2. Reactor Design One of the most critical parameters affecting MFC performance is the reactor configuration. In this study, a single-chamber, membraneless, air-cathode microbial fuel cell design was adopted due to its high efficiency, eco-friendly properties, broad applicability, low cost, and ease of assembly. A cubic reactor (4 cm × 4 cm × 6.25 cm) with a total working volume of 100 mL was fabricated from transparent plexiglass due to its affordability, chemical inertness, ease of fabrication, and resistance to corrosion. Inlet and outlet channels (1 cm in diameter) were incorporated into the reactor for sampling and fluid transfer. The anode electrodes used in the study were: (1) nickel foam (NF), (2) NF/rGO, (3) NF/rGO/30%Mo, and (4) NF/rGO/50%Mo. A carbon cloth cathode loaded with 20 wt% platinum was used as the cathode, obtained from Protek Group. All experiments were conducted using an external resistance of 100 Ω. The anode electrodes were circular (3.8 cm diameter, 1.6 mm thickness), and the distance between the anode and cathode was fixed at 4 cm. 2.3. Operation of the MFC Reactors In this study, the treatment efficiency of the microbial fuel cell (MFC) system equipped with fabricated hybrid anode electrodes was evaluated under laboratory conditions using synthetic wastewater. For this purpose, the components of the synthetic wastewater used as the substrate were first prepared according to the composition presented in Table 1. The trace element solution added to the wastewater was formulated as described in Table 2. Performance assessments were based on chemical oxygen demand (COD) removal. A 10% (v/v) inoculum was added to the synthetic wastewater described in Table 1. The inoculum consisted of a mixed microbial culture obtained from the anaerobic sludge digester tank of the Tatlar Wastewater Treatment Plant, located in Ankara, Türkiye. The reactors were operated simultaneously under identical conditions, and their performance was evaluated by determining the COD removal achieved by the biomass in each cell. COD measurements were performed before reactor start-up and at the end of the 7th day of operation. The measurements were conducted using a Hach DR1900 Portable Spectrophotometer. Test kits (Hach LCK 514) capable of measuring COD concentrations in the range of 100–2000 mg/L O₂ were used in the analysis. A 2 mL sample collected from each reactor was added to the pre-prepared test kits. The test tubes were then incubated in a thermal reactor at 148 °C for 2 hours. After cooling to room temperature, the tubes were inserted into the spectrophotometer for measurement. All MFC reactors were operated in batch mode at room temperature (22 ± 2 °C), and the pH was maintained between 6.3 and 7.2 throughout the experimental period. Table 1 Composition of Synthetic Wastewater Component Amount Unit Glucose 1.87 g/L NH₄Cl 1.5 g/L KCl 0.6 g/L MgCl₂ 0.2 g/L CaCl₂ 0.1 g/L KH₂PO₄·H₂O 0.3 g/L K₂HPO₄ 0.3 g/L EDTA 0.001 g/L Citric acid 0.003 g/L Trace element solution 10 mL/L Table 2 Composition of the Trace Element Solution Component Amount Unit Na₂EDTA·2H₂O 0.750 g/L FeCl₃·6H₂O 0.097 g/L MnCl₂·4H₂O 0.041 g/L ZnCl₂ 0.005 g/L CoCl₂·6H₂O 0.002 g/L Na₂MoO₄·2H₂O 0.004 g/L 2.4. Determination of Microbial Diversity Microbial species that developed on the anode electrodes used in the system were analyzed. The 16S rRNA-based next-generation sequencing (NGS) metagenomic analysis was performed by SuGenomik Biotechnology Co., Ltd. The sequential steps carried out during the analysis are outlined below. Genomic DNA was extracted using the SuSpin Bacterial Fecal/Soil DNA Isolation Kit (Cat. No.: NA01B100) provided by SuGenomik Biotechnology. The quantity of the isolated DNA was determined fluorometrically using a Qubit 3.0 Fluorometer. The V5–V7 regions of the 16S rRNA gene were amplified using 799F and 1191R primer pairs on a SimpliAmp Thermal Cycler. The primer sequences used in the study were as follows: 799F: 5'-AACMGGATTAGATACCCKG-3' and 1191R 5'-ACGTCATCCCCACCTTCC-3' The PCR conditions were: 95 °C for 5 minutes (initial denaturation), followed by 35 cycles of: 95 °C for 30 seconds (denaturation) 53 °C for 30 seconds (annealing) 72 °C for 30 seconds (extension) A final extension was conducted at 72 °C for 2 minutes, and the PCR was completed by lowering the temperature to 4 °C. Before sequencing, the V5–V7 amplicon products were purified using the Qiagen “Qiaseq Beads Clean-Up Kit” (Cat. No.: 180795). Library preparation was conducted using the “Qiaseq FX DNA Library Prep Kit” (Cat. No.: 1120146), and indexing was performed using the “Qiaseq UDI Y-Adapter Kit A (96)” (Cat. No.: 180312). Library concentrations were measured using the Qubit™ dsDNA HS Assay Kit (ThermoFisher Scientific, USA). Sequencing was performed on the Illumina iSeq100 platform in paired-end mode (2×150 bp). The raw reads obtained in FASTQ format were taxonomically classified into OTU categories using the Kraken metagenomic system. 3. Results and Discussion In this study, a single-chamber, air-cathode, membraneless MFC was employed, in which the anode electrode was modified using four different configurations: Nickel Foam (NF), NF/rGO, NF/rGO/30% Mo, and NF/rGO/50% Mo. The microbial communities that developed on these anode surfaces were characterized, and their impact on COD removal efficiency was investigated. 3.1. SEM Images of the Anode Electrodes The SEM images of the microbial communities that developed on the synthesized anode electrodes—1-NF, 2-NF/rGO, 3-NF/rGO/30% Mo, and 4-NF/rGO/50% Mo—whose performances were tested in a single-chamber, membraneless, air-cathode microbial fuel cell, are presented in Fig. 1. Upon examination of Fig. 1, it is evident that various types of bacterial communities developed on the anode electrodes after the operation. These bacterial communities and their diversity were analyzed in detail in the following section on biological analyses and results. 3.2. Biological Analyses and Results According to the 16S NGS metagenomic analysis results, the classes with the highest read counts obtained from the samples taken from the anode electrode surfaces using 799F–1191R primers targeting the V5–V7 regions of the 16S rRNA gene are presented in Table 3. Table 3 Bacterial classes with the highest sequence reads obtained from the anode electrode samples Class NF (%) NF/rGO (%) NF/rGO/ %30 Mo NF/rGO/ %50 Mo Gammaproteobacteria 27.26 7,72 48,44 16,87 Alphaproteobacteria 26.89 58,57 17,42 13,63 Bacilli 16.74 9,47 12,44 22,19 Holophagae 12.86 - - - Actinomycetes 6.95 8,5 7,06 22,95 Betaproteobacteria 4.04 5,52 4,86 15,04 Flavobacteriia 0.69 0,78 - 0,76 Clostridia 0.63 2,21 2,09 3,01 Bacteroidia - 2,53 2,75 1,93 Spirochaetia - - 0,78 0,76 Upon examining the results, the major bacterial classes found in all biofilm samples were Gammaproteobacteria, Alphaproteobacteria, Bacilli, Actinomycetes, Betaproteobacteria, and Clostridia. In the NF and NF/rGO/30% Mo anodes, Gammaproteobacteria were found in high abundance (27.26% and 48.44%, respectively), while Alphaproteobacteria dominated the NF/rGO anode (58.57%), and Betaproteobacteria were prominent on the NF/rGO/50% Mo anode (22.95%). In contrast, Flavobacteriia and Spirochaetia were detected in low abundance across all anodes (Table 3). The 25 families with the highest read counts obtained from the anode electrode samples are presented in Fig. 2. The relative abundance data of the top 25 bacterial species with the highest read counts obtained from samples collected from the anode electrode surfaces are presented in Fig. 3. As a result of the biological analyses, a high microbial diversity was observed on the NF electrode surface, with dominant families including Sphingomonadaceae (22.0%), Holophagaceae (14.6%), and Enterobacteriaceae (9.3%). In particular, Holophagaceae was distinctly detected on this electrode surface but was not identified on the other electrode types. This family is represented by the genus Geothrix , and certain strains within this genus are known to produce electricity in pure culture microbial fuel cells (MFCs) (Miyahara et al., 2013). Indeed, Bond and Lovley (2005) reported that Geothrix fermentans can completely oxidize organic compounds and reduce electrodes, thereby generating electricity. Their study demonstrated that microorganisms can employ different mechanisms for electron transfer to electrodes, either through direct cell–electrode contact or via soluble electron shuttles (Bond and Lovley, 2005). When the findings obtained at the family level were analyzed at the species level, relatively high abundances of Mesoterricola sediminis (22.2%), Klebsiella pneumoniae (10.1%), Bacillus amyloliquefaciens (5.6%), Lactobacillus jensenii (5.1%), Bacillus paralicheniformis (4.7%), and Cutibacterium acnes (4.3%) were observed on the NF electrode surface. Notably, M. sediminis was detected in high abundance exclusively on the NF electrode, suggesting that this species either could not grow or was suppressed on rGO- or Mo-modified surfaces. This bacterium was isolated by Itoh et al. (2023) from river sediments in Okinawa, Japan. The genus Mesoterricola consists of Gram-negative and aerobic bacteria, commonly found in natural environments such as soil and sediments, and is typically adapted to mesophilic temperature conditions (Itoh et al. 2023). To the best of our knowledge, the current study is the first to report the presence or use of M. sediminis in microbial fuel cell (MFC) studies. The NF/rGO electrode was notably dominated by the Sphingomonadaceae family, which accounted for 50.7% of the microbial community. This indicates a low microbial diversity at the family level, with a single group prevailing on the electrode surface. The Sphingomonadaceae family is particularly known for its biofilm-forming capacity, a trait that contributes to efficient electron transfer through stable biofilm formation in microbial fuel cells (MFCs) (Bhadra et al. 2024). It is suggested that the rGO coating may have facilitated the adhesion of this family to the electrode surface, thereby promoting its dominance. Other microbial families detected on the electrode surface included Comamonadaceae (4.2%), Propionibacteriaceae (4.3%), and members grouped as "Others" (12.4%). In contrast, the Holophagaceae family, which was found in high abundance on the NF electrode, was not detected on this surface, suggesting that the rGO modification may have inhibited the growth of certain microbial families. Despite the limited diversity at the family level on the NF/rGO electrode, a more microdistributed structure was observed at the species level. The total relative abundance of numerous low-abundance species classified under the "Others" category was 49.3%, indicating the presence of a diverse range of non-dominant species coexisting on the electrode surface. In other words, while family-level diversity was low, species other than those from Sphingomonadaceae belonged to many different families in small proportions. Notable species on this surface included C. acnes (8.1%), K. pneumoniae (5.7%), L. jensenii (5.3%), and Paracidovorax avenae (5.4%). This suggests that the NF/rGO electrode supported a broader and more homogeneous colonization pattern. Among these, C. acnes is a Gram-positive, anaerobic bacterium that has not been widely utilized in microbial fuel cell (MFC) systems. On the other hand, K. pneumoniae is a Gram-negative, facultatively anaerobic bacterium that is frequently studied for various biotechnological applications such as bioremediation and biofuel production. Studies have shown that K. pneumoniae can secrete electrochemically active compounds capable of transferring electrons to anode electrodes (Deng et al. 2010). Furthermore, Thulasinathan et al. (2020) reported that co-cultures of Serratia marcescens and K. pneumoniae enhanced power generation in MFC systems (Thulasinathan et al. 2020). The NF/rGO/30% Mo electrode was characterized by a microbial community structure predominantly composed of the Enterobacteriaceae family (33.5%). This family includes microorganisms with high energy production potential in microbial fuel cells (MFCs), such as K. pneumoniae , Citrobacter freundii , Kluyvera spp., S. marcescens , and Enteric Gp68 (Leung 2020). Additionally, families such as Sphingomonadaceae (14.8%), Moraxellaceae (6.6%), Lactobacillaceae (3.8%), and Comamonadaceae (3.3%) were also detected on this electrode surface and appeared to contribute to a supportive microbial structure in conjunction with Enterobacteriaceae . In contrast, the Holophagaceae family, which was dominant on the NF electrode, was not detected on this surface. This suggests that both the rGO coating and molybdenum supplementation exert selective and inhibitory effects on certain microbial groups. Specifically, the Mo addition appears to create an electrochemical microenvironment that favors the colonization of microorganisms with high biotechnological potential, such as those from the Enterobacteriaceae family. This structure at the family level was also consistent with the species-level analysis. On the NF/rGO/30% Mo electrode, species belonging to or associated with the Enterobacteriaceae family— Escherichia coli (8.4%), K. pneumoniae (7.4%), L. jensenii (6.0%), and Salmonella enterica (6.0%)—were detected at relatively high abundances. This suggests that Gram-negative enteric bacteria are particularly well adapted to colonize this surface. Additionally, the presence of C. acnes (5.6%) and Staphylococcus aureus (4.1%), which originate from human sources and represent different phylogenetic lineages, indicates that this electrode surface supports a moderate level of microbial diversity. These findings suggest that the addition of molybdenum not only promotes specific bacterial families but may also influence the microsurface in a way that accommodates microorganisms from diverse origins. Among these, E. coli is a Gram-negative, facultative anaerobe commonly found in the gastrointestinal tract of humans and animals. In its native form, E. coli does not effectively transfer electrons directly to electrodes in MFC systems. However, several studies have demonstrated that E. coli can be genetically modified or enhanced via cytochrome c expression to facilitate interactions with electrodes. Such modifications allow E. coli to more efficiently transfer electrons, thereby improving MFC performance (Davis and Higson 2007; Du et al. 2007; Aghababaie et al. 2015). These results underscore the importance of evaluating not only the presence but also the functional potential of microbial species detected on molybdenum-modified electrodes. The NF/rGO/50% Mo electrode emerged as the anode surface with the highest microbial diversity. At the family level, the “Others” category accounted for a substantial proportion (15.2%), indicating the presence of numerous microbial families with low abundance. No single microbial family was found to dominate this electrode surface; instead, families from different phylogenetic groups—such as Comamonadaceae (10.9%), Streptomycetaceae (9.5%), Sphingomonadaceae , and Staphylococcaceae (both 8.7%)—were detected at comparable levels. This suggests that the surface provided an open, balanced, and multifunctional environment for microbial colonization. Among these, the Comamonadaceae family is known for its facultative anaerobic nature and its ability to degrade both organic and inorganic substrates, contributing to chemical oxygen demand (COD) removal (Quan et al. 2012). The absence of a single dominant microbial group on the electrode surface suggests that the 50% molybdenum incorporation may have altered the microsurface properties in a way that promotes a microecological environment open to high microbial diversity. A similar pattern was observed at the species level for the NF/rGO/50% Mo electrode. The "Others" category accounted for 35.3%, indicating the co-existence of numerous low-abundance species. Noteworthy species detected on this electrode surface included Streptomyces sp. RerS4 (6.9%), M. endophytica (6.5%), C. acnes (6.7%), and L. jensenii (6.2%), each exhibiting moderate relative abundance. This balanced distribution of species, without a single dominant microorganism, reflects a colonization pattern that supports microbial diversity. The coexistence of species from different phylogenetic origins suggests that the electrode surface provides a favorable environment for diverse microbial communities. One of the species present on this surface, S. aureus , is a Gram-positive and facultative anaerobic bacterium (Keyes 2014). Although this species is not commonly used in microbial fuel cell (MFC) applications, Flimban et al. (2019) reported Staphylococcus as the most dominant bacterial genus in an MFC reactor designed for cellulose degradation (Flimban et al. 2019). When evaluated at the family level, the NF/rGO/50% Mo electrode displayed a distinctly different microbial composition compared to the other electrodes. For instance, while the NF electrode was dominated by families such as Sphingomonadaceae (22.0%), Holophagaceae (14.6%), and Enterobacteriaceae (9.3%), the NF/rGO electrode exhibited a low-diversity structure dominated primarily by Sphingomonadaceae (50.7%). The NF/rGO/30% Mo electrode, on the other hand, presented a moderate level of diversity with a strong presence of Enterobacteriaceae (33.5%). In contrast, the 50% Mo-modified electrode did not show dominance by any single microbial family, instead revealing a richer and more balanced community structure. This suggests that the surface of this electrode supports the development of a more stable microbial ecosystem. Similarly, the NF/rGO/50% Mo electrode also exhibited distinct differences at the species level compared to the other electrodes. For instance, while certain species such as M. sediminis were predominantly detected only on the NF electrode surface, specific species like K. pneumoniae and E. coli were dominant on the NF/rGO and 30% Mo-modified anodes. In contrast, the absence of any distinctly dominant species on the 50% Mo-modified electrode suggests a more balanced microbial distribution and a stable diversity. Consequently, increasing the molybdenum content to 50% appears to limit the dominance of specific species, thereby promoting the development of a more homogeneous, resilient, and potentially functionally diverse microbial community. This condition may offer significant advantages in terms of long-term stability and functional diversity in MFC systems. Overall, the species-level findings clearly demonstrate that chemical modifications applied to the anode electrodes exert significant influence on microbial diversity, species selectivity, and dominance patterns. The unmodified NF electrode provided a more natural and diverse microbial environment, whereas the incorporation of rGO and especially molybdenum altered the physicochemical properties of the surface, thereby either promoting or suppressing the development of specific microbial species. These changes directly impacted the structural composition of the biofilm formed on the electrode surface and should be carefully considered as potential engineering strategies to enhance the performance of microbial fuel cells. According to Fig. 3, certain bacterial species were commonly detected on all electrode surfaces. These include L. jensenii , C. acnes , K. pneumoniae , Staphylococcus haemolyticus , S. aureus , and P. avenae . On the other hand, some species were found exclusively on specific electrode types. For example, M. sediminis was detected only on the NF electrode surface, while S. sp. RerS4 and M. endophytica were observed solely on the NF/rGO/50% Mo electrode. These electrode-specific distributions of bacterial species clearly highlight the influence of surface chemistry on microbial selectivity. The chemical modification of electrode surfaces has emerged as a critical factor influencing the adaptability of microbial species and their energy generation potential (Borole et al. 2011; Thapa et al. 2024). Notably, species such as E. coli (Zou et al. 2008; Thapa et al. 2024) , K. pneumoniae (Zhang et al. 2009; Deng et al. 2010; Thulasinathan et al. 2020) , S. aureus (Bhuvaneswari et al. 2013; Tahernia et al. 2020) and S. haemolyticus (Flimban et al. 2019) have been utilized for energy production in microbial fuel cells (MFCs) with various electrode modifications (Fan et al. 2021; Cheng et al. 2023). In our study, C. acnes and K. pneumoniae were specifically detected on the NF/rGO electrode modified with rGO. On the Mo-enriched NF/rGO/30% Mo electrode, E. coli , S. aureus , and C. acnes were observed. The presence of these species has also been supported by previous studies, which reported their effectiveness in energy production and their ability to promote microbial growth (Zou et al. 2008; Thapa et al. 2024). On the NF/rGO/50% Mo electrode, K. pneumoniae and S. aureus were detected. K. pneumoniae is a significant species capable of metabolizing carbon sources to generate electrons, and it can benefit from electron transfer properties (Zhang et al. 2009; Deng et al. 2010; Thulasinathan et al. 2020). E. coli , on the other hand, is widely used in MFC systems due to its amenability to genetic manipulation and biofilm-forming ability (Zou et al. 2008; Thapa et al. 2024) . The high abundance of E. coli observed on the NF/rGO/30% Mo electrode surface suggests that this surface provides a favorable microenvironment for its growth. S. aureus is well known for its ability to form biofilms, a trait that makes it a potentially electroactive species in MFC systems (Akiyama et al. 1998; Smith et al. 2008; Keyes 2014). Tahernia et al. (2020) reported that this species can exhibit electroactive properties (Tahernia et al. 2020). Similarly, Bhuvaneswari et al. (2013) demonstrated that S. aureus is capable of direct electron transfer on carbon felt anodes and can generate bioelectricity from cellulose (Bhuvaneswari et al. 2013). Other studies conducted on MFC systems have demonstrated that a wide range of microorganisms can colonize anode surfaces. For instance, in a study using carbon cloth-based anodes, Almatouq et al. (2020) reported that the dominant microbial phyla were Bacteroidetes , Proteobacteria , and Firmicutes (Almatouq et al. 2020). Similarly, Hemdan et al. (2023) examined biofilms formed on polyaniline-coated anodes and found that electroactive phyla, particularly Proteobacteria , Firmicutes , and Bacteroidetes , were most abundant. At the class level, Gammaproteobacteria , Clostridia , and Bacilli were dominant, while at the genus level, Pseudomonas , Clostridium , Enterococcus , and Bifidobacterium were reported in high abundance on polyaniline anodes (Hemdan et al. 2023). In addition, a study conducted by Nguyen et al. (2021) compared anodic microbial communities in carbon-coated MFC systems, both membrane and membraneless, operated under open-air conditions to evaluate the effect of sunlight. In both systems, the dominant microbial phyla were Proteobacteria , Firmicutes , and Synergistetes , with the genus Rhodopseudomonas being the most abundant. Sunlight was found to have a positive effect on the growth of this genus. Moreover, the membraneless MFCs exhibited a richer microbial diversity compared to membrane-based systems, which was attributed to differences in reactor design and the extent of sunlight exposure (Nguyen et al. 2021). In a dual-chamber microbial fuel cell (MFC) using wetland sediment in the anode compartment, 16S metagenomic sequencing was used to investigate microbial diversity in anodic biofilms. The dominant taxa in the anodic biofilm were primarily Clostridiales and Burkholderiales (class β-Proteobacteria), with the fermentative genus Achromobacter also found in high abundance (Singh and Kaushik 2021). The structure of the microbial community in the anode compartment of MFC systems is influenced by several factors, including the anode material and architecture, type of substrate, pH level, ambient temperature, and the presence or absence of a membrane in the reactor (Aghababaie et al. 2015). The anode material is a critical factor that affects biofilm formation, substrate oxidation, and electron transfer, thereby directly impacting MFC performance. Carbon-based materials (e.g., carbon cloth, carbon paper, carbon fabric), which offer high conductivity, large surface area, and biocompatibility, are frequently preferred as anode materials (Dege and Danış 2020). The type and concentration of the substrate directly influence the structure and activity of the microbial community. In particular, organic acids can alter system pH, thereby affecting microbial composition and performance (Piskin and Genc 2023). Temperature is another crucial factor that affects microbial activity and biofilm development, with optimal temperature ranges required for efficient microbial function (Min et al. 2008; Michie et al. 2011; Tee et al. 2017). Proton exchange membranes are commonly used to separate the anode and cathode compartments in MFCs. Membrane use prevents oxygen diffusion from the cathode to the anode chamber, maintaining anaerobic conditions and thus influencing microbial community structure (Sevda et al. 2023). Each of these factors significantly affects the microbial community structure in the anode compartment and, consequently, the overall performance of MFC systems. Therefore, the findings of this study reveal both overlapping and divergent aspects when compared with similar research in the literature. Comparisons regarding electrode types, surface modifications, and experimental conditions highlight their respective impacts on microbial diversity, offering valuable insights for system design and optimization strategies. 3.3. Performance Measurements in the Reactors Performance evaluations were conducted based on chemical oxygen demand (COD) removal efficiencies. The COD removal performances obtained from the reactors are presented graphically in Fig. 4. When the microbial communities in these reactors are evaluated in terms of COD removal, it is observed that the reactor using the NF/rGO/50% Mo anode electrode achieved the highest COD removal efficiency. In this reactor, species such as L. jensenii , C. acnes , P. avenae , S. sp. RerS4 , and M. endophytica were found to be dominant at relative abundances between 6–7%, and these species are thought to have positively contributed to COD removal. In contrast, in the reactor with the lowest COD removal efficiency—using the NF/rGO anode electrode— C. acnes was the most abundant species at 8.08%, while Bacillus wiedmannii and Cystobacter fuscus were exclusively found in the NF/rGO electrode. Additionally, B. paralicheniformis was not observed to develop on electrodes containing molybdenum. 4. Conclusion In this study, nickel foam (NF), reduced graphene oxide (rGO)-coated NF, and molybdenum (Mo)-enriched NF/rGO anode electrodes were used in a microbial fuel cell (MFC), and the microbial community and COD removal efficiencies of these electrodes were investigated. The results revealed that the NF/rGO/50% Mo anode electrode exhibited the highest COD removal efficiency (88.58%). Dominant bacterial species identified on this anode included L. jensenii , C. acnes , P. avenae , S. sp. RerS4 , and M. endophytica . The microbial community analysis revealed that the composition of bacteria developing on the anode electrode surface is directly influenced by the electrode material. While the Gammaproteobacteria class was found in the highest abundance on the NF and NF/rGO/30% Mo anodes, Betaproteobacteria was more dominant on the NF/rGO/50% Mo anode. In addition, distinct microbial species were identified across different electrodes. Notably, M. sediminis was detected exclusively on the NF anode. Since this species has not been previously reported in MFC systems, its identification represents one of the novel contributions of this study. The biological analysis results indicate that the microbial community structures of the NF and NF/rGO anodes are similar to each other, whereas the NF/rGO/30% Mo and NF/rGO/50% Mo anodes host more distinct and diverse microbial communities. In particular, the NF/rGO/50% Mo electrode stands out as the most effective anode material in terms of both energy generation and wastewater treatment due to its unique bacterial diversity. In conclusion, Mo-enriched anode electrodes have been found to offer advantages in terms of organic matter conversion and electricity generation in microbial fuel cells (MFCs). The NF/rGO/50% Mo electrode was observed to enhance both electron transfer capacity and COD removal efficiency by promoting biofilm formation. The findings highlight that surface modification of anode electrodes is a critical factor for improving MFC efficiency, and that Mo-doped rGO coatings can provide significant enhancements in this regard. Accordingly, future studies should focus on optimizing Mo doping levels, investigating interactions with different microbial communities in detail, and evaluating their long-term effects on MFC performance. Declarations Acknowledgements This study was carried out with the support of the Gazi University Scientific Research Projects Coordination Unit (Project No. FCD-2023-8721). The authors would like to thank for the financial support provided. Funding This work was supported by the Gazi University Scientific Research Projects Coordination Unit (Project No. FCD-2023-8721). Authors’ Contributions All authors contributed to the study’s conception and design. The experiments were conducted by Habib Akyazı, who also prepared the first draft of the manuscript. The biological studies were carried out and the biological results interpreted by Ebru Beyzi; overall supervision was provided by Çiğdem Güldür. Material preparation and data collection, as well as data analysis, evaluation, and interpretation, were performed jointly by all authors; the manuscript was critically reviewed by all authors, who approved the final version. Ethical Approval This is not applicable. Consent to Participate This is not applicable. Consent to Publish This is not applicable. Competing Interests The authors declare no competing interests. Data availability All data supporting the findings of this study are available within the paper. References Aghababaie M, Farhadian M, Jeihanipour A, Biria D (2015) Effective factors on the performance of microbial fuel cells in wastewater treatment–a review. 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Int J Hydrogen Energy 33:4856–4862. https://doi.org/10.1016/j.ijhydene.2008.06.061 Cite Share Download PDF Status: Published Journal Publication published 21 Nov, 2025 Read the published version in Environmental Science and Pollution Research → Version 1 posted Editorial decision: Major Revision 07 Oct, 2025 Reviewers agreed at journal 19 Sep, 2025 Reviewers invited by journal 18 Sep, 2025 Editor invited by journal 17 Sep, 2025 Editor assigned by journal 04 Sep, 2025 First submitted to journal 02 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7472748","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516853971,"identity":"57eab6ce-d7d2-4985-a4d4-31c1d51fa005","order_by":0,"name":"Habib Akyazı","email":"","orcid":"","institution":"Ankara University Beypazarı Vocational School","correspondingAuthor":false,"prefix":"","firstName":"Habib","middleName":"","lastName":"Akyazı","suffix":""},{"id":516853972,"identity":"d0463c15-ac97-4dd2-b5ad-6f0cc71b14c8","order_by":1,"name":"Fatma Çiğdem Güldür","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYHACxgM8DAxyINYBBgZm4vSAtBgjtLARqSWxAcImQgt//xqDA2/b7qXPdz/+8ABDhXVig3zvA7xaJG68MTg4t604d+OZhIQDDGfSExvY2A3wajGQOGNwmLctIXdjQ8KBA4xth4FaCLgMpiXdsP9hwwHGf8Ro4e8Ba0mQl0hmOMDYQIQWiRtsBQfnnEsw3CDxjOFAwrF04za2NPxa+PsPb3zwpixBXr4//fGHDzXWsv3Mx/BrYZBIgLrwAJAAsQnHJP8BCC3fQFDpKBgFo2AUjFQAAJfFSjyH3tixAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4404-6882","institution":"Gazi University Faculty of Engineering: Gazi Universitesi Muhendislik Fakultesi","correspondingAuthor":true,"prefix":"","firstName":"Fatma","middleName":"Çiğdem","lastName":"Güldür","suffix":""},{"id":516853973,"identity":"6bd6e1b2-6089-4fae-94bc-8df8affe8a16","order_by":2,"name":"Ebru Beyzi","email":"","orcid":"","institution":"Gazi University Vocational School of Health 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1","display":"","copyAsset":false,"role":"figure","size":777706,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of the microbial communities observed on the anode electrodes after operation: a) NF, b) NF/rGO, c) NF/rGO/30% Mo, d) NF/rGO/50% Mo\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7472748/v1/f56f66ddb0078c9201f0418d.png"},{"id":92444851,"identity":"f8c2098a-b089-4eea-91aa-49cf6b75878f","added_by":"auto","created_at":"2025-09-29 19:56:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108496,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance percentages of the 25 most prevalent bacterial families and the “Others” category identified on the anode electrode surfaces\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7472748/v1/ff2a48cef5a076f3cc815a1a.png"},{"id":92445580,"identity":"7abc7212-baad-4a0d-9099-419a0e45be3b","added_by":"auto","created_at":"2025-09-29 20:04:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121384,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance percentages of the 25 most prevalent bacterial Species and the “Others” category identified on the anode electrode surfaces\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7472748/v1/f32b829b854f403ee7b03c5e.png"},{"id":92444854,"identity":"8779573e-5578-4dd2-a291-ec4edccb0972","added_by":"auto","created_at":"2025-09-29 19:56:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44710,"visible":true,"origin":"","legend":"\u003cp\u003eCOD removal efficiency in the reactors\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7472748/v1/4ff6adc04d1a7fa2283a4a36.png"},{"id":96651321,"identity":"c5619991-7b05-4cc9-8ce5-4114873aa155","added_by":"auto","created_at":"2025-11-24 16:14:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1716901,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7472748/v1/cb6b707b-675d-4e92-97a9-116a13cc831e.pdf"}],"financialInterests":"","formattedTitle":"Anode surface modification with reduced graphene oxide (rGO) and molybdenum (Mo) enhances microbial diversity and chemical oxygen demand (COD) removal in microbial fuel cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, bioelectrochemical systems (BES) have emerged as innovative technologies that utilize the electrochemical activities of microorganisms to generate energy and remove pollutants. These systems contribute not only to the reduction of environmental pollution loads but also to simultaneous electricity production. BES can be used in a variety of applications such as fuel cells (for electricity generation), electrolyzers, desalination, and chemical synthesis (Jayabalan et al. 2019). In these systems, microorganisms grow under anaerobic conditions and degrade organic compounds\u0026mdash;including various pollutants\u0026mdash;producing and releasing electrons during metabolic processes. These electrons are transferred to terminal electron acceptors via different mechanisms, resulting in electricity generation (Borello et al. 2021). Therefore, microorganisms play a critical dual role in BES: organic matter conversion and electricity generation through extracellular electron transfer (Khoirunnisa et al. 2021).\u003c/p\u003e\u003cp\u003eMicrobial fuel cells (MFCs) are one of the most widely studied BES types, in which electricity is produced through the oxidation of organic matter by microorganisms (Dege and Danış 2020). A key factor in MFC development is the presence of microbial species capable of generating electrons and transferring them beyond the cell surface (Gorby et al. 2006; Indriyani et al. 2024). The operation of MFCs relies on electroactive microorganisms, known as exoelectrogens, which form biofilms on the anode electrode and have the ability to transfer electrons extracellularly, playing a crucial role in electricity generation (Logan and Regan 2006).\u003c/p\u003e\u003cp\u003eBoth pure and mixed microbial cultures have been used in MFC studies (Erensoy and \u0026Ccedil;ek 2020). Compared to pure cultures, mixed cultures offer several advantages, including higher resistance to environmental disturbances, broader substrate utilization, greater substrate consumption, and higher power density (Nevin et al. 2008; Vamshi Krishna and Venkata Mohan 2016). However, the development of non-electrogenic bacteria such as fermenters and methanogens within the biofilm may reduce the system\u0026rsquo;s efficiency (Kim et al. 2005; Michie et al. 2011; Velasquez-Orta et al. 2011; Vamshi Krishna and Venkata Mohan 2016) Therefore, the type of microorganisms used as biocatalysts is critically important for the efficient performance of MFCs. To date, hundreds of electroactive strains have been isolated and used in MFCs. According to the NCBI Taxonomy database, numerous strains isolated from MFCs are cataloged to better understand their diversity and similarities (Erensoy and \u0026Ccedil;ek 2020). Recent studies have focused on enhancing MFC efficiency by targeting gene expression related to cytochrome and pili production, which influence biofilm morphology and electron transfer capacity (Perchikov et al. 2024).\u003c/p\u003e\u003cp\u003eIn MFCs, the anode chamber is where microbial proliferation occurs. Here, organic matter is degraded by anaerobic bacteria to produce electrons and protons required for metabolic reactions (Mohan et al. 2008). The material and structural characteristics of the anode electrode significantly influence microbial growth, adhesion, substrate degradation, and electron transfer efficiency. As a result, various materials\u0026mdash;such as metals, carbon-based substances, and hybrid composites\u0026mdash;have been investigated for use as anode electrodes. However, current research indicates that existing anode designs have yet to achieve optimal performance for large-scale applications (Logan et al. 2006; Wang et al. 2009; Dege and Danış 2020). The surface properties of the anode directly affect microbial adhesion, biofilm formation, and extracellular electron transfer, thereby determining overall system efficiency. Flat electrode surfaces with limited surface area and inadequate porosity hinder microbial attachment and electron transport (Zou et al. 2016). In contrast, porous and three-dimensional electrode architectures provide microenvironments that promote microbial colonization and enhance electron transfer efficiency (Zou et al. 2016; Chong et al. 2019).\u003c/p\u003e\u003cp\u003eThe surface structure and morphology, electrical conductivity, mechanical strength, and corrosion resistance of electrodes used in microbial fuel cells (MFCs) are critical factors influencing anode performance. An ideal anode material should possess high surface area (Oh and Logan 2006), excellent electrical conductivity (Malvankar et al. 2012; Zhang et al. 2020), low internal resistance (Zhao et al. 2006), strong corrosion resistance, appropriate mechanical durability, and suitable porosity. Moreover, biocompatibility is essential to ensure that microorganisms can effectively adhere to the anode surface and establish robust electrical connections (Perchikov et al. 2024).\u003c/p\u003e\u003cp\u003eIn the literature, numerous composite anode materials have been developed using carbon-based, metal-based, or hybrid structures (Akyazı et al. 2025). Among these, carbon materials are the most commonly used due to their high surface area, chemical stability, corrosion resistance, and biocompatibility. Typical carbon-based anode materials include carbon cloth, carbon paper, carbon felt, carbon fiber, carbon brush, carbon rods, carbon mesh, carbon grids, and graphite (Liu et al. 2004; Oh et al. 2004; Cheng and Logan 2007; Logan et al. 2007; Wang et al. 2009; Thung et al. 2016; Bian et al. 2018; Akyazı et al. 2025). For example, carbon cloth provides excellent porosity and mechanical strength but is relatively expensive (Santoro et al. 2011; Guerrini et al. 2014; Dumitru and Scott 2016). Carbon mesh are inexpensive and commercially available, yet they exhibit low conductivity and mechanical stability (Santoro et al. 2014; Dumitru and Scott 2016; Wu et al. 2017). Carbon paper is rigid, brittle, and flat, which may limit its application (Guo et al. 2012; Santoro et al. 2014; Dumitru and Scott 2016). Graphite plates offer high conductivity and high cost but have limited surface area compared to porous materials (Dumitru and Scott 2016). Carbon fiber outperforms many others due to its three-dimensional architecture and large surface area (Chen et al. 2011). Additionally, nanostructured carbon materials such as carbon nanotubes, graphene, and graphene oxide are considered highly promising due to their exceptional conductivity and mechanical strength (Yazdi et al. 2016). In particular, graphene and its derivatives are frequently employed in MFCs owing to their high surface area (~\u0026thinsp;2630 m\u0026sup2;/g) and strong biocompatibility (Yuan and He 2015; Zhuang et al. 2015; Akyazı et al. 2025).\u003c/p\u003e\u003cp\u003eA study evaluating 14 different metals (Al, Fe, Cu, Ti, Ni, Zn, Zr, Mo, Nb, Ag, In, Sn, Ta, and W) as anode materials reported that Mo, W, Fe, and Sn were the most effective in supporting biofilm formation. Among these, molybdenum (Mo) demonstrated outstanding performance due to its structural simplicity and long-term stability in microbial fuel cell systems. Furthermore, both Mo and W were found to be compatible with \u003cem\u003eGeobacter\u003c/em\u003e species, leading to superior biofilm development and the highest current densities recorded among all tested materials (Yamashita and Yokoyama 2018).\u003c/p\u003e\u003cp\u003eThe performance of MFCs depends on both the microbial activity in the anode chamber (Calli et al. 2006; Oh and Logan 2006) and the material composition of system components (Ozcan 2013). Microbial activity directly influences electricity generation and the removal of chemical oxygen demand (COD), which is further affected by factors such as bacterial species and activity levels, temperature, pH, oxygen availability, nutrient concentrations, and reaction time (Aghababaie et al. 2015; Nosek and Cydzik-Kwiatkowska 2020; Borello et al. 2021; Malekmohammadi and Mirbagheri 2021).\u003c/p\u003e\u003cp\u003eIn this study, different anode electrode materials were developed and the microbial community and electrochemical performance of these anodes were investigated. To achieve this, reduced graphene oxide (rGO) was synthesized and coated onto three-dimensional nickel foam (NF) structures. Subsequently, the surface was further modified with molybdenum (Mo) to promote the attachment of electroactive bacterial species. Four types of anode electrodes were fabricated: NF, NF/rGO, NF/rGO/30%Mo, and NF/rGO/50%Mo. These electrodes were tested in a single-chamber, membraneless, air-cathode MFC system to evaluate their efficiency in synthetic wastewater treatment and to characterize the microbial communities that developed specifically on the anode surfaces.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eTo improve the anode performance of microbial fuel cells (MFCs), reduced graphene oxide (rGO) was chemically synthesized and coated onto a three-dimensional porous nickel foam (NF) structure. To further enhance the surface adhesion of electroactive microbial species, molybdenum (Mo) was loaded onto the anode surface. The fabricated electrodes were then tested in a single-chamber, membraneless, air-cathode MFC system along with biofilm enrichment procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1. Preparation and Characterization of Anode Electrodes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003erGO-coated nickel foam (NF/rGO) electrodes were synthesized in order to achieve high electrical conductivity and a large surface area. As detailed in a previous study (Akyazı et al. 2025), graphene oxide (GO) was first obtained by oxidizing graphite via the Hummers method, and was then chemically reduced to obtain rGO. The nickel foam (NF) was cut into the required dimensions, treated with acid solution to remove surface oxides, and subsequently coated with rGO using hydrothermal synthesis.\u003c/p\u003e\n\u003cp\u003eTo further enhance electrochemical performance and microbial adhesion, Mo was loaded onto the surface of rGO-coated electrodes at two different concentrations (30 wt% and 50 wt%), yielding hybrid anodes: NF/rGO/30%Mo and NF/rGO/50%Mo.\u003c/p\u003e\n\u003cp\u003ePost-experimental SEM imaging revealed the presence of various bacterial colonies on the electrode surfaces. Characterization results confirmed that the synthesized anodes exhibited uniform coating and suitable porosity for microbial colonization (Akyazı et al. 2025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Reactor Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne of the most critical parameters affecting MFC performance is the reactor configuration. In this study, a single-chamber, membraneless, air-cathode microbial fuel cell design was adopted due to its high efficiency, eco-friendly properties, broad applicability, low cost, and ease of assembly.\u003c/p\u003e\n\u003cp\u003eA cubic reactor (4 cm \u0026times; 4 cm \u0026times; 6.25 cm) with a total working volume of 100 mL was fabricated from transparent plexiglass due to its affordability, chemical inertness, ease of fabrication, and resistance to corrosion. Inlet and outlet channels (1 cm in diameter) were incorporated into the reactor for sampling and fluid transfer. The anode electrodes used in the study were: (1) nickel foam (NF), (2) NF/rGO, (3) NF/rGO/30%Mo, and (4) NF/rGO/50%Mo. A carbon cloth cathode loaded with 20 wt% platinum was used as the cathode, obtained from Protek Group. All experiments were conducted using an external resistance of 100 \u0026Omega;. The anode electrodes were circular (3.8 cm diameter, 1.6 mm thickness), and the distance between the anode and cathode was fixed at 4 cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Operation of the MFC Reactors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the treatment efficiency of the microbial fuel cell (MFC) system equipped with fabricated hybrid anode electrodes was evaluated under laboratory conditions using synthetic wastewater. For this purpose, the components of the synthetic wastewater used as the substrate were first prepared according to the composition presented in Table 1. The trace element solution added to the wastewater was formulated as described in Table 2.\u003c/p\u003e\n\u003cp\u003ePerformance assessments were based on chemical oxygen demand (COD) removal. A 10% (v/v) inoculum was added to the synthetic wastewater described in Table 1. The inoculum consisted of a mixed microbial culture obtained from the anaerobic sludge digester tank of the Tatlar Wastewater Treatment Plant, located in Ankara, T\u0026uuml;rkiye.\u003c/p\u003e\n\u003cp\u003eThe reactors were operated simultaneously under identical conditions, and their performance was evaluated by determining the COD removal achieved by the biomass in each cell. COD measurements were performed before reactor start-up and at the end of the 7th day of operation.\u003c/p\u003e\n\u003cp\u003eThe measurements were conducted using a Hach DR1900 Portable Spectrophotometer. Test kits (Hach LCK 514) capable of measuring COD concentrations in the range of 100\u0026ndash;2000 mg/L O₂ were used in the analysis. A 2 mL sample collected from each reactor was added to the pre-prepared test kits. The test tubes were then incubated in a thermal reactor at 148 \u0026deg;C for 2 hours. After cooling to room temperature, the tubes were inserted into the spectrophotometer for measurement.\u003c/p\u003e\n\u003cp\u003eAll MFC reactors were operated in batch mode at room temperature (22 \u0026plusmn; 2 \u0026deg;C), and the pH was maintained between 6.3 and 7.2 throughout the experimental period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Composition of Synthetic Wastewater\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eComponent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmount\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eUnit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGlucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNH₄Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eKCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMgCl₂\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCaCl₂\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eKH₂PO₄\u0026middot;H₂O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eK₂HPO₄\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEDTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCitric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTrace element solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003emL/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eComposition of the Trace Element Solution\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eComponent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmount\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eUnit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNa₂EDTA\u0026middot;2H₂O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFeCl₃\u0026middot;6H₂O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMnCl₂\u0026middot;4H₂O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eZnCl₂\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCoCl₂\u0026middot;6H₂O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNa₂MoO₄\u0026middot;2H₂O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Determination of Microbial Diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrobial species that developed on the anode electrodes used in the system were analyzed. The 16S rRNA-based next-generation sequencing (NGS) metagenomic analysis was performed by SuGenomik Biotechnology Co., Ltd. The sequential steps carried out during the analysis are outlined below.\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted using the SuSpin Bacterial Fecal/Soil DNA Isolation Kit (Cat. No.: NA01B100) provided by SuGenomik Biotechnology. The quantity of the isolated DNA was determined fluorometrically using a Qubit 3.0 Fluorometer.\u003c/p\u003e\n\u003cp\u003eThe V5\u0026ndash;V7 regions of the 16S rRNA gene were amplified using 799F and 1191R primer pairs on a SimpliAmp Thermal Cycler. The primer sequences used in the study were as follows:\u003c/p\u003e\n\u003cp\u003e799F: 5\u0026apos;-AACMGGATTAGATACCCKG-3\u0026apos; and 1191R 5\u0026apos;-ACGTCATCCCCACCTTCC-3\u0026apos;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe PCR conditions were:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e95 \u0026deg;C for 5 minutes (initial denaturation),\u0026nbsp;\u003c/li\u003e\n \u003cli\u003efollowed by 35 cycles of:\u003cul style=\"list-style-type: circle;\"\u003e\n \u003cli\u003e95 \u0026deg;C for 30 seconds (denaturation)\u003c/li\u003e\n \u003cli\u003e53 \u0026deg;C for 30 seconds (annealing)\u003c/li\u003e\n \u003cli\u003e72 \u0026deg;C for 30 seconds (extension)\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/li\u003e\n \u003cli\u003eA final extension was conducted at 72 \u0026deg;C for 2 minutes,\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eand the PCR was completed by lowering the temperature to 4 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eBefore sequencing, the V5\u0026ndash;V7 amplicon products were purified using the Qiagen \u0026ldquo;Qiaseq Beads Clean-Up Kit\u0026rdquo; (Cat. No.: 180795). Library preparation was conducted using the \u0026ldquo;Qiaseq FX DNA Library Prep Kit\u0026rdquo; (Cat. No.: 1120146), and indexing was performed using the \u0026ldquo;Qiaseq UDI Y-Adapter Kit A (96)\u0026rdquo; (Cat. No.: 180312). Library concentrations were measured using the Qubit\u0026trade; dsDNA HS Assay Kit (ThermoFisher Scientific, USA). Sequencing was performed on the Illumina iSeq100 platform in paired-end mode (2\u0026times;150 bp).\u003c/p\u003e\n\u003cp\u003eThe raw reads obtained in FASTQ format were taxonomically classified into OTU categories using the Kraken metagenomic system.\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eIn this study, a single-chamber, air-cathode, membraneless MFC was employed, in which the anode electrode was modified using four different configurations: Nickel Foam (NF), NF/rGO, NF/rGO/30% Mo, and NF/rGO/50% Mo. The microbial communities that developed on these anode surfaces were characterized, and their impact on COD removal efficiency was investigated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1. SEM Images of the Anode Electrodes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SEM images of the microbial communities that developed on the synthesized anode electrodes\u0026mdash;1-NF, 2-NF/rGO, 3-NF/rGO/30% Mo, and 4-NF/rGO/50% Mo\u0026mdash;whose performances were tested in a single-chamber, membraneless, air-cathode microbial fuel cell, are presented in Fig. 1.\u003c/p\u003e\n\u003cp\u003eUpon examination of Fig. 1, it is evident that various types of bacterial communities developed on the anode electrodes after the operation. These bacterial communities and their diversity were analyzed in detail in the following section on biological analyses and results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Biological Analyses and Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the 16S NGS metagenomic analysis results, the classes with the highest read counts obtained from the samples taken from the anode electrode surfaces using 799F\u0026ndash;1191R primers targeting the V5\u0026ndash;V7 regions of the 16S rRNA gene are presented in Table 3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Bacterial classes with the highest sequence reads obtained from the anode electrode samples\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"502\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClass\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003eNF (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003eNF/rGO (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eNF/rGO/ %30 Mo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eNF/rGO/\u003c/p\u003e\n \u003cp\u003e%50 Mo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eGammaproteobacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e27.26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;7,72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003e48,44\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e16,87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eAlphaproteobacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e26.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e58,57\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e17,42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e13,63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eBacilli\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e16.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e9,47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e12,44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e22,19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eHolophagae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e12.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eActinomycetes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e6.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e8,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e7,06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e22,95\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eBetaproteobacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e5,52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e4,86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e15,04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eFlavobacteriia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e0,78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0,76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eClostridia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e2,21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2,09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3,01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eBacteroidia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e2,53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2,75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1,93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eSpirochaetia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 93px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0,78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0,76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eUpon examining the results, the major bacterial classes found in all biofilm samples were Gammaproteobacteria, Alphaproteobacteria, Bacilli, Actinomycetes, Betaproteobacteria, and Clostridia. In the NF and NF/rGO/30% Mo anodes, Gammaproteobacteria were found in high abundance (27.26% and 48.44%, respectively), while Alphaproteobacteria dominated the NF/rGO anode (58.57%), and Betaproteobacteria were prominent on the NF/rGO/50% Mo anode (22.95%). In contrast, Flavobacteriia and Spirochaetia were detected in low abundance across all anodes (Table 3).\u003c/p\u003e\n\u003cp\u003eThe 25 families with the highest read counts obtained from the anode electrode samples are presented in Fig. 2.\u003c/p\u003e\n\u003cp\u003eThe relative abundance data of the top 25 bacterial species with the highest read counts obtained from samples collected from the anode electrode surfaces are presented in Fig. 3.\u003c/p\u003e\n\u003cp\u003eAs a result of the biological analyses, a high microbial diversity was observed on the NF electrode surface, with dominant families including \u003cem\u003eSphingomonadaceae\u003c/em\u003e (22.0%), \u003cem\u003eHolophagaceae\u003c/em\u003e (14.6%), and \u003cem\u003eEnterobacteriaceae\u003c/em\u003e (9.3%). In particular, \u003cem\u003eHolophagaceae\u003c/em\u003e was distinctly detected on this electrode surface but was not identified on the other electrode types. This family is represented by the genus \u003cem\u003eGeothrix\u003c/em\u003e, and certain strains within this genus are known to produce electricity in pure culture microbial fuel cells (MFCs) (Miyahara et al., 2013). Indeed, Bond and Lovley (2005) reported that \u003cem\u003eGeothrix\u003c/em\u003e fermentans can completely oxidize organic compounds and reduce electrodes, thereby generating electricity. Their study demonstrated that microorganisms can employ different mechanisms for electron transfer to electrodes, either through direct cell\u0026ndash;electrode contact or via soluble electron shuttles (Bond and Lovley, 2005).\u003c/p\u003e\n\u003cp\u003eWhen the findings obtained at the family level were analyzed at the species level, relatively high abundances of \u003cem\u003eMesoterricola sediminis\u003c/em\u003e (22.2%), \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (10.1%), \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e (5.6%), \u003cem\u003eLactobacillus jensenii\u0026nbsp;\u003c/em\u003e(5.1%), \u003cem\u003eBacillus paralicheniformis\u0026nbsp;\u003c/em\u003e(4.7%), and \u003cem\u003eCutibacterium acnes\u003c/em\u003e (4.3%) were observed on the NF electrode surface. Notably, M. sediminis was detected in high abundance exclusively on the NF electrode, suggesting that this species either could not grow or was suppressed on rGO- or Mo-modified surfaces. This bacterium was isolated by Itoh et al. (2023) from river sediments in Okinawa, Japan. The genus \u003cem\u003eMesoterricola\u003c/em\u003e consists of Gram-negative and aerobic bacteria, commonly found in natural environments such as soil and sediments, and is typically adapted to mesophilic temperature conditions (Itoh et al. 2023). To the best of our knowledge, the current study is the first to report the presence or use of \u003cem\u003eM. sediminis\u003c/em\u003e in microbial fuel cell (MFC) studies.\u003c/p\u003e\n\u003cp\u003eThe NF/rGO electrode was notably dominated by the \u003cem\u003eSphingomonadaceae\u003c/em\u003e family, which accounted for 50.7% of the microbial community. This indicates a low microbial diversity at the family level, with a single group prevailing on the electrode surface. The \u003cem\u003eSphingomonadaceae\u003c/em\u003e family is particularly known for its biofilm-forming capacity, a trait that contributes to efficient electron transfer through stable biofilm formation in microbial fuel cells (MFCs) (Bhadra et al. 2024). It is suggested that the rGO coating may have facilitated the adhesion of this family to the electrode surface, thereby promoting its dominance. Other microbial families detected on the electrode surface included \u003cem\u003eComamonadaceae\u003c/em\u003e (4.2%), \u003cem\u003ePropionibacteriaceae\u003c/em\u003e (4.3%), and members grouped as \u0026quot;Others\u0026quot; (12.4%). In contrast, the \u003cem\u003eHolophagaceae\u003c/em\u003e family, which was found in high abundance on the NF electrode, was not detected on this surface, suggesting that the rGO modification may have inhibited the growth of certain microbial families.\u003c/p\u003e\n\u003cp\u003eDespite the limited diversity at the family level on the NF/rGO electrode, a more microdistributed structure was observed at the species level. The total relative abundance of numerous low-abundance species classified under the \u0026quot;Others\u0026quot; category was 49.3%, indicating the presence of a diverse range of non-dominant species coexisting on the electrode surface. In other words, while family-level diversity was low, species other than those from \u003cem\u003eSphingomonadaceae\u003c/em\u003e belonged to many different families in small proportions. Notable species on this surface included \u003cem\u003eC. acnes\u003c/em\u003e (8.1%), \u003cem\u003eK. pneumoniae\u003c/em\u003e (5.7%), \u003cem\u003eL. jensenii\u003c/em\u003e (5.3%), and \u003cem\u003eParacidovorax avenae\u003c/em\u003e (5.4%). This suggests that the NF/rGO electrode supported a broader and more homogeneous colonization pattern. Among these, \u003cem\u003eC. acnes\u003c/em\u003e is a Gram-positive, anaerobic bacterium that has not been widely utilized in microbial fuel cell (MFC) systems. On the other hand, \u003cem\u003eK. pneumoniae\u003c/em\u003e is a Gram-negative, facultatively anaerobic bacterium that is frequently studied for various biotechnological applications such as bioremediation and biofuel production. Studies have shown that \u003cem\u003eK. pneumoniae\u003c/em\u003e can secrete electrochemically active compounds capable of transferring electrons to anode electrodes (Deng et al. 2010). Furthermore, Thulasinathan et al. (2020) reported that co-cultures of \u003cem\u003eSerratia marcescens\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e enhanced power generation in MFC systems (Thulasinathan et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe NF/rGO/30% Mo electrode was characterized by a microbial community structure predominantly composed of the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family (33.5%). This family includes microorganisms with high energy production potential in microbial fuel cells (MFCs), such as \u003cem\u003eK. pneumoniae\u003c/em\u003e, \u003cem\u003eCitrobacter freundii\u003c/em\u003e, \u003cem\u003eKluyvera\u003c/em\u003e spp., \u003cem\u003eS. marcescens\u003c/em\u003e, and \u003cem\u003eEnteric Gp68\u003c/em\u003e (Leung 2020). Additionally, families such as \u003cem\u003eSphingomonadaceae\u003c/em\u003e (14.8%), \u003cem\u003eMoraxellaceae\u003c/em\u003e (6.6%), \u003cem\u003eLactobacillaceae\u003c/em\u003e (3.8%), and \u003cem\u003eComamonadaceae\u003c/em\u003e (3.3%) were also detected on this electrode surface and appeared to contribute to a supportive microbial structure in conjunction with \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. In contrast, the \u003cem\u003eHolophagaceae\u003c/em\u003e family, which was dominant on the NF electrode, was not detected on this surface. This suggests that both the rGO coating and molybdenum supplementation exert selective and inhibitory effects on certain microbial groups. Specifically, the Mo addition appears to create an electrochemical microenvironment that favors the colonization of microorganisms with high biotechnological potential, such as those from the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family.\u003c/p\u003e\n\u003cp\u003eThis structure at the family level was also consistent with the species-level analysis. On the NF/rGO/30% Mo electrode, species belonging to or associated with the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family\u0026mdash;\u003cem\u003e\u0026nbsp;Escherichia coli\u003c/em\u003e (8.4%), \u003cem\u003eK. pneumoniae\u003c/em\u003e (7.4%), \u003cem\u003eL. jensenii\u003c/em\u003e (6.0%), and \u003cem\u003eSalmonella enterica\u003c/em\u003e (6.0%)\u0026mdash;were detected at relatively high abundances. This suggests that Gram-negative enteric bacteria are particularly well adapted to colonize this surface. Additionally, the presence of \u003cem\u003eC. acnes\u003c/em\u003e (5.6%) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (4.1%), which originate from human sources and represent different phylogenetic lineages, indicates that this electrode surface supports a moderate level of microbial diversity. These findings suggest that the addition of molybdenum not only promotes specific bacterial families but may also influence the microsurface in a way that accommodates microorganisms from diverse origins.\u003c/p\u003e\n\u003cp\u003eAmong these, \u003cem\u003eE. coli\u003c/em\u003e is a Gram-negative, facultative anaerobe commonly found in the gastrointestinal tract of humans and animals. In its native form, \u003cem\u003eE. coli\u003c/em\u003e does not effectively transfer electrons directly to electrodes in MFC systems. However, several studies have demonstrated that \u003cem\u003eE. coli\u003c/em\u003e can be genetically modified or enhanced via cytochrome c expression to facilitate interactions with electrodes. Such modifications allow \u003cem\u003eE. coli\u003c/em\u003e to more efficiently transfer electrons, thereby improving MFC performance (Davis and Higson 2007; Du et al. 2007; Aghababaie et al. 2015). These results underscore the importance of evaluating not only the presence but also the functional potential of microbial species detected on molybdenum-modified electrodes.\u003c/p\u003e\n\u003cp\u003eThe NF/rGO/50% Mo electrode emerged as the anode surface with the highest microbial diversity. At the family level, the \u0026ldquo;Others\u0026rdquo; category accounted for a substantial proportion (15.2%), indicating the presence of numerous microbial families with low abundance. No single microbial family was found to dominate this electrode surface; instead, families from different phylogenetic groups\u0026mdash;such as \u003cem\u003eComamonadaceae\u003c/em\u003e (10.9%), \u003cem\u003eStreptomycetaceae\u003c/em\u003e (9.5%), \u003cem\u003eSphingomonadaceae\u003c/em\u003e, and \u003cem\u003eStaphylococcaceae\u003c/em\u003e (both 8.7%)\u0026mdash;were detected at comparable levels. This suggests that the surface provided an open, balanced, and multifunctional environment for microbial colonization.\u003c/p\u003e\n\u003cp\u003eAmong these, the \u003cem\u003eComamonadaceae\u003c/em\u003e family is known for its facultative anaerobic nature and its ability to degrade both organic and inorganic substrates, contributing to chemical oxygen demand (COD) removal (Quan et al. 2012). The absence of a single dominant microbial group on the electrode surface suggests that the 50% molybdenum incorporation may have altered the microsurface properties in a way that promotes a microecological environment open to high microbial diversity.\u003c/p\u003e\n\u003cp\u003eA similar pattern was observed at the species level for the NF/rGO/50% Mo electrode. The \u0026quot;Others\u0026quot; category accounted for 35.3%, indicating the co-existence of numerous low-abundance species. Noteworthy species detected on this electrode surface included \u003cem\u003eStreptomyces\u003c/em\u003e sp. RerS4 (6.9%), \u003cem\u003eM. endophytica\u003c/em\u003e (6.5%), \u003cem\u003eC. acnes\u003c/em\u003e (6.7%), and \u003cem\u003eL. jensenii\u003c/em\u003e (6.2%), each exhibiting moderate relative abundance. This balanced distribution of species, without a single dominant microorganism, reflects a colonization pattern that supports microbial diversity. The coexistence of species from different phylogenetic origins suggests that the electrode surface provides a favorable environment for diverse microbial communities.\u003c/p\u003e\n\u003cp\u003eOne of the species present on this surface, \u003cem\u003eS. aureus\u003c/em\u003e, is a Gram-positive and facultative anaerobic bacterium (Keyes 2014). Although this species is not commonly used in microbial fuel cell (MFC) applications, Flimban et al. (2019) reported \u003cem\u003eStaphylococcus\u003c/em\u003e as the most dominant bacterial genus in an MFC reactor designed for cellulose degradation (Flimban et al. 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen evaluated at the family level, the NF/rGO/50% Mo electrode displayed a distinctly different microbial composition compared to the other electrodes. For instance, while the NF electrode was dominated by families such as \u003cem\u003eSphingomonadaceae\u003c/em\u003e (22.0%), \u003cem\u003eHolophagaceae\u003c/em\u003e (14.6%), and \u003cem\u003eEnterobacteriaceae\u003c/em\u003e (9.3%), the NF/rGO electrode exhibited a low-diversity structure dominated primarily by \u003cem\u003eSphingomonadaceae\u003c/em\u003e (50.7%). The NF/rGO/30% Mo electrode, on the other hand, presented a moderate level of diversity with a strong presence of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e (33.5%). In contrast, the 50% Mo-modified electrode did not show dominance by any single microbial family, instead revealing a richer and more balanced community structure. This suggests that the surface of this electrode supports the development of a more stable microbial ecosystem.\u003c/p\u003e\n\u003cp\u003eSimilarly, the NF/rGO/50% Mo electrode also exhibited distinct differences at the species level compared to the other electrodes. For instance, while certain species such as \u003cem\u003eM. sediminis\u003c/em\u003e were predominantly detected only on the NF electrode surface, specific species like \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e were dominant on the NF/rGO and 30% Mo-modified anodes. In contrast, the absence of any distinctly dominant species on the 50% Mo-modified electrode suggests a more balanced microbial distribution and a stable diversity. Consequently, increasing the molybdenum content to 50% appears to limit the dominance of specific species, thereby promoting the development of a more homogeneous, resilient, and potentially functionally diverse microbial community. This condition may offer significant advantages in terms of long-term stability and functional diversity in MFC systems.\u003c/p\u003e\n\u003cp\u003eOverall, the species-level findings clearly demonstrate that chemical modifications applied to the anode electrodes exert significant influence on microbial diversity, species selectivity, and dominance patterns. The unmodified NF electrode provided a more natural and diverse microbial environment, whereas the incorporation of rGO and especially molybdenum altered the physicochemical properties of the surface, thereby either promoting or suppressing the development of specific microbial species. These changes directly impacted the structural composition of the biofilm formed on the electrode surface and should be carefully considered as potential engineering strategies to enhance the performance of microbial fuel cells.\u003c/p\u003e\n\u003cp\u003eAccording to Fig. 3, certain bacterial species were commonly detected on all electrode surfaces. These include \u003cem\u003eL. jensenii\u003c/em\u003e, \u003cem\u003eC. acnes\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e, \u003cem\u003eStaphylococcus haemolyticus\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eP. avenae\u003c/em\u003e. On the other hand, some species were found exclusively on specific electrode types. For example, \u003cem\u003eM. sediminis\u003c/em\u003e was detected only on the NF electrode surface, while \u003cem\u003eS. sp. RerS4\u003c/em\u003e and \u003cem\u003eM. endophytica\u003c/em\u003e were observed solely on the NF/rGO/50% Mo electrode. These electrode-specific distributions of bacterial species clearly highlight the influence of surface chemistry on microbial selectivity.\u003c/p\u003e\n\u003cp\u003eThe chemical modification of electrode surfaces has emerged as a critical factor influencing the adaptability of microbial species and their energy generation potential (Borole et al. 2011; Thapa et al. 2024). Notably, species such as \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003e(Zou et al. 2008; Thapa et al. 2024)\u003cem\u003e, K. pneumoniae\u0026nbsp;\u003c/em\u003e(Zhang et al. 2009; Deng et al. 2010; Thulasinathan et al. 2020)\u003cem\u003e, S. aureus\u0026nbsp;\u003c/em\u003e(Bhuvaneswari et al. 2013; Tahernia et al. 2020) and \u003cem\u003eS. haemolyticus\u0026nbsp;\u003c/em\u003e(Flimban et al. 2019) have been utilized for energy production in microbial fuel cells (MFCs) with various electrode modifications (Fan et al. 2021; Cheng et al. 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our study, \u003cem\u003eC. acnes\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e were specifically detected on the NF/rGO electrode modified with rGO. On the Mo-enriched NF/rGO/30% Mo electrode, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eC. acnes\u003c/em\u003e were observed. The presence of these species has also been supported by previous studies, which reported their effectiveness in energy production and their ability to promote microbial growth (Zou et al. 2008; Thapa et al. 2024).\u003c/p\u003e\n\u003cp\u003eOn the NF/rGO/50% Mo electrode, \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e were detected. \u003cem\u003eK. pneumoniae\u003c/em\u003e is a significant species capable of metabolizing carbon sources to generate electrons, and it can benefit from electron transfer properties (Zhang et al. 2009; Deng et al. 2010; Thulasinathan et al. 2020). \u003cem\u003eE. coli\u003c/em\u003e\u003cem\u003e, on the other hand, is widely used in MFC systems due to its amenability to genetic manipulation and biofilm-forming ability\u0026nbsp;\u003c/em\u003e(Zou et al. 2008; Thapa et al. 2024)\u003cem\u003e.\u003c/em\u003e The high abundance of \u003cem\u003eE. coli\u003c/em\u003e observed on the NF/rGO/30% Mo electrode surface suggests that this surface provides a favorable microenvironment for its growth.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e is well known for its ability to form biofilms, a trait that makes it a potentially electroactive species in MFC systems (Akiyama et al. 1998; Smith et al. 2008; Keyes 2014). Tahernia et al. (2020) reported that this species can exhibit electroactive properties (Tahernia et al. 2020). Similarly, Bhuvaneswari et al. (2013) demonstrated that \u003cem\u003eS. aureus\u003c/em\u003e is capable of direct electron transfer on carbon felt anodes and can generate bioelectricity from cellulose (Bhuvaneswari et al. 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOther studies conducted on MFC systems have demonstrated that a wide range of microorganisms can colonize anode surfaces. For instance, in a study using carbon cloth-based anodes, Almatouq et al. (2020) reported that the dominant microbial phyla were \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, and \u003cem\u003eFirmicutes\u0026nbsp;\u003c/em\u003e(Almatouq et al. 2020). Similarly, Hemdan et al. (2023) examined biofilms formed on polyaniline-coated anodes and found that electroactive phyla, particularly \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eBacteroidetes\u003c/em\u003e, were most abundant. At the class level, \u003cem\u003eGammaproteobacteria\u003c/em\u003e, \u003cem\u003eClostridia\u003c/em\u003e, and \u003cem\u003eBacilli\u003c/em\u003e were dominant, while at the genus level, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, and \u003cem\u003eBifidobacterium\u003c/em\u003e were reported in high abundance on polyaniline anodes (Hemdan et al. 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, a study conducted by Nguyen et al. (2021) compared anodic microbial communities in carbon-coated MFC systems, both membrane and membraneless, operated under open-air conditions to evaluate the effect of sunlight. In both systems, the dominant microbial phyla were \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eSynergistetes\u003c/em\u003e, with the genus \u003cem\u003eRhodopseudomonas\u003c/em\u003e being the most abundant. Sunlight was found to have a positive effect on the growth of this genus. Moreover, the membraneless MFCs exhibited a richer microbial diversity compared to membrane-based systems, which was attributed to differences in reactor design and the extent of sunlight exposure (Nguyen et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn a dual-chamber microbial fuel cell (MFC) using wetland sediment in the anode compartment, 16S metagenomic sequencing was used to investigate microbial diversity in anodic biofilms. The dominant taxa in the anodic biofilm were primarily \u003cem\u003eClostridiales\u003c/em\u003e and \u003cem\u003eBurkholderiales\u003c/em\u003e (class \u0026beta;-Proteobacteria), with the fermentative genus \u003cem\u003eAchromobacter\u003c/em\u003e also found in high abundance (Singh and Kaushik 2021).\u003c/p\u003e\n\u003cp\u003eThe structure of the microbial community in the anode compartment of MFC systems is influenced by several factors, including the anode material and architecture, type of substrate, pH level, ambient temperature, and the presence or absence of a membrane in the reactor (Aghababaie et al. 2015). The anode material is a critical factor that affects biofilm formation, substrate oxidation, and electron transfer, thereby directly impacting MFC performance. Carbon-based materials (e.g., carbon cloth, carbon paper, carbon fabric), which offer high conductivity, large surface area, and biocompatibility, are frequently preferred as anode materials (Dege and Danış 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe type and concentration of the substrate directly influence the structure and activity of the microbial community. In particular, organic acids can alter system pH, thereby affecting microbial composition and performance (Piskin and Genc 2023). Temperature is another crucial factor that affects microbial activity and biofilm development, with optimal temperature ranges required for efficient microbial function (Min et al. 2008; Michie et al. 2011; Tee et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProton exchange membranes are commonly used to separate the anode and cathode compartments in MFCs. Membrane use prevents oxygen diffusion from the cathode to the anode chamber, maintaining anaerobic conditions and thus influencing microbial community structure (Sevda et al. 2023). \u0026nbsp;Each of these factors significantly affects the microbial community structure in the anode compartment and, consequently, the overall performance of MFC systems.\u003c/p\u003e\n\u003cp\u003eTherefore, the findings of this study reveal both overlapping and divergent aspects when compared with similar research in the literature. Comparisons regarding electrode types, surface modifications, and experimental conditions highlight their respective impacts on microbial diversity, offering valuable insights for system design and optimization strategies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Performance Measurements in the Reactors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePerformance evaluations were conducted based on chemical oxygen demand (COD) removal efficiencies. The COD removal performances obtained from the reactors are presented graphically in Fig. 4.\u003c/p\u003e\n\u003cp\u003eWhen the microbial communities in these reactors are evaluated in terms of COD removal, it is observed that the reactor using the NF/rGO/50% Mo anode electrode achieved the highest COD removal efficiency. In this reactor, species such as \u003cem\u003eL. jensenii\u003c/em\u003e, \u003cem\u003eC. acnes\u003c/em\u003e, \u003cem\u003eP. avenae\u003c/em\u003e, \u003cem\u003eS. sp. RerS4\u003c/em\u003e, and \u003cem\u003eM. endophytica\u003c/em\u003e were found to be dominant at relative abundances between 6\u0026ndash;7%, and these species are thought to have positively contributed to COD removal. In contrast, in the reactor with the lowest COD removal efficiency\u0026mdash;using the NF/rGO anode electrode\u0026mdash;\u003cem\u003eC. acnes\u003c/em\u003e was the most abundant species at 8.08%, while \u003cem\u003eBacillus wiedmannii\u003c/em\u003e and \u003cem\u003eCystobacter fuscus\u003c/em\u003e were exclusively found in the NF/rGO electrode. Additionally, \u003cem\u003eB. paralicheniformis\u003c/em\u003e was not observed to develop on electrodes containing molybdenum.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, nickel foam (NF), reduced graphene oxide (rGO)-coated NF, and molybdenum (Mo)-enriched NF/rGO anode electrodes were used in a microbial fuel cell (MFC), and the microbial community and COD removal efficiencies of these electrodes were investigated.\u003c/p\u003e\n\u003cp\u003eThe results revealed that the NF/rGO/50% Mo anode electrode exhibited the highest COD removal efficiency (88.58%). Dominant bacterial species identified on this anode included \u003cem\u003eL. jensenii\u003c/em\u003e, \u003cem\u003eC. acnes\u003c/em\u003e, \u003cem\u003eP. avenae\u003c/em\u003e, \u003cem\u003eS. sp. RerS4\u003c/em\u003e, and \u003cem\u003eM. endophytica\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe microbial community analysis revealed that the composition of bacteria developing on the anode electrode surface is directly influenced by the electrode material. While the \u003cem\u003eGammaproteobacteria\u003c/em\u003e class was found in the highest abundance on the NF and NF/rGO/30% Mo anodes, \u003cem\u003eBetaproteobacteria\u003c/em\u003e was more dominant on the NF/rGO/50% Mo anode. In addition, distinct microbial species were identified across different electrodes. Notably, \u003cem\u003eM. sediminis\u003c/em\u003e was detected exclusively on the NF anode. Since this species has not been previously reported in MFC systems, its identification represents one of the novel contributions of this study.\u003c/p\u003e\n\u003cp\u003eThe biological analysis results indicate that the microbial community structures of the NF and NF/rGO anodes are similar to each other, whereas the NF/rGO/30% Mo and NF/rGO/50% Mo anodes host more distinct and diverse microbial communities. In particular, the NF/rGO/50% Mo electrode stands out as the most effective anode material in terms of both energy generation and wastewater treatment due to its unique bacterial diversity.\u003c/p\u003e\n\u003cp\u003eIn conclusion, Mo-enriched anode electrodes have been found to offer advantages in terms of organic matter conversion and electricity generation in microbial fuel cells (MFCs). The NF/rGO/50% Mo electrode was observed to enhance both electron transfer capacity and COD removal efficiency by promoting biofilm formation. The findings highlight that surface modification of anode electrodes is a critical factor for improving MFC efficiency, and that Mo-doped rGO coatings can provide significant enhancements in this regard. Accordingly, future studies should focus on optimizing Mo doping levels, investigating interactions with different microbial communities in detail, and evaluating their long-term effects on MFC performance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was carried out with the support of the Gazi University Scientific Research Projects Coordination Unit (Project No. FCD-2023-8721). The authors would like to thank for the financial support provided.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Gazi University Scientific Research Projects Coordination Unit (Project No. FCD-2023-8721).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study\u0026rsquo;s conception and design. The experiments were conducted by Habib Akyazı, who also prepared the first draft of the manuscript. The biological studies were carried out and the biological results interpreted by Ebru Beyzi; overall supervision was provided by \u0026Ccedil;iğdem G\u0026uuml;ld\u0026uuml;r. Material preparation and data collection, as well as data analysis, evaluation, and interpretation, were performed jointly by all authors; the manuscript was critically reviewed by all authors, who approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003eThis is not applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u0026nbsp;\u003c/strong\u003eThis is not applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAghababaie M, Farhadian M, Jeihanipour A, Biria D (2015) Effective factors on the performance of microbial fuel cells in wastewater treatment\u0026ndash;a review. 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Int J Hydrogen Energy 33:4856\u0026ndash;4862. https://doi.org/10.1016/j.ijhydene.2008.06.061\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microbial fuel cell, Anode modification, Molybdenum loading, Reduced graphene oxide coating, COD removal, Microbial community","lastPublishedDoi":"10.21203/rs.3.rs-7472748/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7472748/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to investigate the effects of anode surface modifications on microbial community composition and chemical oxygen demand (COD) removal efficiency in microbial fuel cells (MFCs). Four different anode electrodes were fabricated: bare nickel foam (NF), reduced graphene oxide-coated nickel foam (NF/rGO), and NF/rGO modified with 30 wt% and 50 wt% molybdenum (Mo). These electrodes were tested in a single-chamber, membraneless, air-cathode MFC. Surface morphology was characterized using scanning electron microscopy (SEM), and microbial diversity was assessed through 16S rRNA metagenomic sequencing. Distinct microbial profiles were observed across the electrode types. The NF anode supported high abundances of \u003cem\u003eMesoterricola sediminis\u003c/em\u003e (22.2%), \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (10.1%), and other facultative species. The NF/rGO electrode promoted colonization by \u003cem\u003eCutibacterium acnes\u003c/em\u003e (8.1%) and \u003cem\u003eParacidovorax avenae\u003c/em\u003e (5.4%). On the NF/rGO/30%Mo electrode, notable species included \u003cem\u003eEscherichia coli\u003c/em\u003e (8.4%) and \u003cem\u003eSalmonella enterica\u003c/em\u003e (6.0%). The NF/rGO/50%Mo anode exhibited the highest microbial diversity, with species such as \u003cem\u003eStreptomyces sp.\u003c/em\u003e RerS4 (6.9%) and \u003cem\u003eMicromonospora endophytica\u003c/em\u003e (6.5%) being predominant. The highest COD removal efficiency (88.58%) was achieved using the NF/rGO/50%Mo anode. These findings demonstrate that molybdenum-modified rGO coatings enhance both microbial colonization and electrochemical performance, offering a promising strategy for improving MFC efficiency in wastewater treatment applications.\u003c/p\u003e","manuscriptTitle":"Anode surface modification with reduced graphene oxide (rGO) and molybdenum (Mo) enhances microbial diversity and chemical oxygen demand (COD) removal in microbial fuel cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 19:56:13","doi":"10.21203/rs.3.rs-7472748/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-10-07T11:35:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-20T01:51:07+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-18T05:33:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2025-09-17T15:43:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T05:45:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2025-09-02T06:21:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"521da40f-c413-4904-aa89-b4afe84ce3e3","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:13:07+00:00","versionOfRecord":{"articleIdentity":"rs-7472748","link":"https://doi.org/10.1007/s11356-025-37243-0","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2025-11-21 15:58:39","publishedOnDateReadable":"November 21st, 2025"},"versionCreatedAt":"2025-09-29 19:56:13","video":"","vorDoi":"10.1007/s11356-025-37243-0","vorDoiUrl":"https://doi.org/10.1007/s11356-025-37243-0","workflowStages":[]},"version":"v1","identity":"rs-7472748","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7472748","identity":"rs-7472748","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}