Modiﬁcation of Graphite Sheet Anode with Iron (II, III) Oxide-Carbon Dots for Enhancing the Performance of Microbial Fuel Cell

: The present study explores the use of carbon dots coated with Iron (II, III) oxide (Fe 3 O 4 ) for its application as an anode in microbial fuel cells (MFC). Fe 3 O 4 @PSA-C was synthesized using a hydrothermal-assisted probe sonication method. Nanoparticles were characterized with XRD, SEM, FTIR, and RAMAN Spectroscopy. Different concentrations of Fe 3 O 4 - carbon dots (0.25, 0.5, 0.75, and 1 mg/cm 2 ) were coated onto the graphite sheets (Fe 3 O 4 @PSA-C), and their performance in MFC was evaluated. Cyclic voltammetry (CV) of Fe 3 O 4 @PSA-C (1 mg/cm 2 ) modiﬁed anode indicated oxidation peaks at − 0.26 mV and +0.16 mV, respectively, with peak currents of 7.7 mA and 8.1 mA. The ﬂuxes of these anodes were much higher than those of other low-concentration Fe 3 O 4 @PSA-C modiﬁed anodes and the bare graphite sheet anode. The maximum power density (Pmax) was observed in MFC with a 1 mg/cm 2 concentration of Fe 3 O 4 @PSA-C was 440.01 mW/m 2 , 1.54 times higher than MFCs using bare graphite sheet anode (285.01 mW/m 2 ). The elevated interaction area of carbon dots permits pervasive Fe 3 O 4 crystallization providing enhanced cell attachment capability of the anode, boosting the biocompatibility of Fe 3 O 4 @PSA-C. This signiﬁcantly improved the performance of the MFC, making Fe 3 O 4 @PSA-C modiﬁed graphite sheets a good choice as an anode for its application in MFC.


Introduction
Bioelectricity generated by electrochemically active bacteria (EAB) has gained much attention, with promising significance for renewable energy generation, wastewater remediation, and metallic nanoparticle fabrication [1,2]. In such fuel cells, waste disposal can be converted to electricity with the aid of EAB that colonizes on the anode surface. On the surface of the anode, biodegradable waste is oxidized and generates electrons, protons, and other by-products, protons pass through the cation exchange membrane from anode to cathode, and electrons via an outer circuit, electrons, and protons combined with oxygen at the cathode to complete the reaction [3]. Anode plays a major role in determining the power output of the MFC [4]. However, sluggish EAB biofilm growth impedes the development of the anode half-cell potential and causes voltage loss; consequently, the MFC productivity has not achieved its full potential, resulting in decreased power output [5]. MFC power interruptions occur due to undesirable physicochemical conditions for bacterial consortiums and system architecture [6,7]. The anode composition has the largest impact on energy output in MFCs because it combines microbiology and electrochemistry. The anode's conductance and biocatalytic efficiency must be improved to reduce potential losses. In MFCs, graphite disc anodes are treated with Au and Pd nanoparticles, for example, outperformed untreated anodes in biocompatibility and power generation [8]. As a result, changing the anode makeup might be a good technique for dealing with overpotential problems [9]. Anode remodeling alters the physical and chemical characteristics of the anode, allowing for improved microbial adhesion by increasing the electrochemically specific surface area and enhancing electron transport due to the increased permeability of the anode [10].
Interesting and informative research has been conducted in order to bring about positive changes in MFC anodes [11]. For example, a carbon cloth anode treated with formic acid boosted the ideal power density of a single-chambered MFC by 38.1 percent (from 611.56 mW/m 2 to 877.935 mW/m 2 ) [12]. Likewise, electrolysis of the carbon fiber anode in nitric acid followed by rinsing in aqueous ammonia gave a noteworthy power density of 3.20 ± 0.05 W/m 2 for two-chambered MFCs [13]. Chaetoceros phytoplankton and lauric acid were used as methanotrophic inhibitors in MFCs implanted with mixed fermented sludge in the anodic compartment [14]. Rajesh et al. found 45.18% and 11.6% optimum coulombic efficiency, respectively [15]. Spontaneous elimination of AQS diazonium cations generated in situ immobilized an electron transfer accelerator, anthraquinone-2sulfonic acid (AQS), onto the surface of a graphite sheet anode [16]. They used AQS in MFC, which showed a maximum power density increasing from 967 ± 33 mW/m 2 to 1872 ± 42 mW/m 2 .
Entities including neutral red [17] and lignin [18] have also been used to improve the bio-catalytic effectiveness of the anode in MFCs. Anode characteristics like transmittance and the percentage surface area available for bacterial attachment are crucial, and both can be improved using graphene to improve electronic conductivity. A family of adaptable nanomaterials called carbon quantum dots (C-dots) has not yet been described in MFCs. Prior attempts at creating C-dots from coconut shell were said to include surface functional groups for hydroxyl and carboxyl. The C-dots' capacity to absorb and transport electrons was made possible by the existence of these ligands on a composite. C-dots were added as a suspension to the anode compartment of an MFC, which increased maximum power density by 22.5 percent. Compared to methylene blue, a common electron shuttle used in MFCs, C-dots demonstrated superior efficiency as electron shuttles [19]. Most of the EABs are metal reducing in natural condition. One of the primary limiting phases of MFC performance that requires attention is the sluggish exocellular electron transfer (EET) process in the anode. Most of the time, EET is improved by employing a modified anode. Anodes in MFCs are often made of biocompatible conductive carbon paper/cloth, graphite, and stainless steel. These materials, however, lack electrochemical activity for anode microbial processes. As a result, it is critical to create appropriate anode materials for efficient bacterial adhesion and electron collecting capabilities for better EET. The electron transfer efficiency of the outermost cytochromes (Omcs) [Omc C and Omc A] may be increased in the presence of redox-active biocompatible metal oxides and/or hydroxides. However, there have been relatively few reports on the C-dots metal oxides modified anode in MFC to date. As a result, the incorporation of C-dots and Iron oxide might address the shortcomings of the electrode materials, i.e., non-capacitive anode.
In the present research work, Fe 3 O 4 @PSA-C (0.25 mg/cm 2 , 0.5 mg/cm 2 , 0.75 mg/cm 2 , and 1 mg/cm 2 ) were used to change graphite sheet anodes. Fe 3 O 4 @PSA-C assigned for Iron oxide @ sodium poly-acrylate-Carbon dot where Iron oxide (Fe 3 O 4 ) is synthesized with sono-chemical method in presence of sodium poly-acrylate and after chemical sonication process with C-dots, finally we got Fe 3 O 4 @PSA-C-dots nanocomposite. The performance of MFCs using these anodes was compared to that of MFCs using a raw graphite sheet as an anode. Higher amount of Fe 3 O 4 @PSA-C like 1.25 mg/cm 2 was showing minute increase/ no increase in power density as compared to 1 mg/cm 2 which can be neglected hence the amount of Fe 3 O 4 @PSA-C was taken till the highest power density observed. This study aimed to examine if changing the anode to Fe 3 O 4 @PSA-C may enhance MFC power density (PD) at a fair cost to create a replacement anode composition that could improve MFC performance. The phase composition and crystallite size were determined using an X-ray Diffraction (XRD) technique to find the structure of nanoparticles. Figure 1 shows the XRD pattern of the as-synthesized nanoparticles, which shows the typical diffraction peaks for the synthesized nanocomposites as a pure phase with no secondary phases. Figure 1a depicts the XRD diffraction peak for C-dots at 2θ value 18 • consist of (002) plane. Figure   The assigned XRD peaks for Fe 3 O 4 @PSA-C functionalized hetero-structure nanoparticles were confirmed to have the presence of all validated positions of Fe 3 O 4 @PSA and C-dots as given in Figure 2b [20,21]. The calculated dimension of the magnetic nanoparticle crystallites at room temperature using the Debye Scherrer equation, based on the broadening of the most intense peak (311-35.58), was recorded in the XRD graph ( Figure 1). The average crystallite size of the produced nanoparticles is 10 nm computed using the Debye-Scherrer equation [22,23]. show that synthesized samples are spherical agglomerated structures due to the composition of numerous small nanoparticles and their particle size in the range of 10 nm to 30 nm. The particle size decreased due to sodium polyacrylate (C 3 H 3 NaO 2 ) n as CA, which chelates the Fe ions during the sonochemical process. The compositions of Fe 3 O 4 @PSA and Fe 3 O 4 @PSA-C nanocomposite was determined by X-ray energy-dispersive spectroscopy (EDS), as shown in Figure 2c [24]. Cow milk-based carbon dots contain various chemical compositions with polarities [25]. The band between 3100-3600 cm −1 shows the presence of O-H and N-H bonds, whereas the bands around 2800 and 2900 cm −1 are attributed to the C-H bond. The peaks at 1400 cm −1 ,1550 cm −1 , and 1630 cm −1 were assigned to C=C, N-H, and C=O starch bonds with π-π* and n-π* transitions. The FTIR spectra peaks of Fe 3 O 4 @PSA-C nanocomposite represent the slightly shifted band at 1400 cm −1 ,1550 cm −1, and 1630 cm −1 ascribed to C=C, N-H, and C=O bonds due to the surface interaction between the functional moieties of nanocomposites. The peaks at 1100 cm −1 and 1300 cm −1 show the interaction between Fe-O bonds in Fe 3 O 4 @PSA and the carbonyl group present in C-dots. All such results confirm the hetero-structure nanocomposite of Fe 3 O 4 @PSA-C [26].

RAMAN Spectroscopy
In Figure 4, the Raman spectroscopy technique was used to identify the modes of C-dots, Fe 3 O 4 @PSA-C and Fe 3 O 4 @PSA nanoparticles. The cation distribution, nonstoichiometry, and defect conditions influence the results of Raman modes. The estimated values of polarized intensities also help to identify the Fe 3 O 4 @PSA Raman modes. The weak anti-stokes spectrum is present in the Fe 3 O 4 @PSA Raman band around 195 cm −1 . The ascribed vibrational spectrum of the observed modes was based on polarization induced due to the orientation of Fe 3 O 4 nanoparticles like T2g at 193 cm −1 ; T1u at 201 cm −1 ; 390 cm −1 and 450 cm −1 ; Eg at 296 cm −1 , T2g at 500 cm −1 and 590 cm −1 whereas, A1g at 660 cm −1 as shown in Figure 5. The bands around 1340 cm −1 and 1600 cm −1 in Raman spectra were assigned to D-band and G-bands for C-dots. The presence of sp3 and sp2 hybridized carbon atoms shows C-dots nanoparticles' graphitic nature. In Fe 3 O 4 @PSA-C spectra, the presence of T2g, T1u, Eg, T2g, A1g, D, and G vibrational modes with a modest frequency shift, shows that the Fe 3 O 4 and C-dots were well coupled in the hetero-structure nanomaterial [27].

Optical Properties
The optical properties of functionalized nanoparticles depend on their electronic characteristics and energy bandgaps (Eg). These optical properties of pristine samples were analyzed by the UV-Visible absorption spectra shown in Figure 6. The energy bandgap (Eg) was calculated using 'Tauc's relation.' where A refers to constant, hν for photon energy, Eg for band gap, and α for absorption coefficient [22]. The bandgap was determined from the (αhν) 2 vs. hν curve by drawing an extrapolation of the data point to the photon energy axis where (αhν) 2 = 0, giving the optical band gap Eg. Figure 3 shows the energy bandgap (Eg) values for Fe 3 O 4 @PSA and Fe 3 O 4 @PSA-C synthesized materials. The Eg values for Fe 3 O 4 @PSA were estimated to be around 2.4 eV and 2.25 eV for Fe 3 O 4 @PSA-C nanocomposite [24]. The bandgap was reduced after the composition of C-dots due to the agglomerated particle size and functionalization of the surface of the composite nanomaterial. The composition of C-dots results in a charge imbalance, which triggers the presence of oxygen vacancies in prepared samples. The variation in the angle of Fe-O-Fe towards 180 • also affects their electronic structure and creates an impurity band. This impurity band enhances charge transfer between the carriers by alteration of the energy bandgap. The substitution of C-dots in pristine Fe 3 O 4 @PSA shows lower energy band gap values, which resulted in improved optical properties of the Fe 3 O 4 @PSA-C nanocomposite.

Microbial Electrochemical Study
MFCs having anode with different concentrations of Fe 3 O 4 @PSA were operated in a fed-batch mode. Following the biofilm development throughout the ninth batch rotation, the anode's electrochemical kinetics were studied in operational MFCs. The CV approach has been widely utilized to evaluate the biocompatibility of anode components for electro-catalyzing substrates using microbes as biocatalysts [28]. In this study, the functionalization of anodes enhanced with various components like raw graphite sheet and different amounts of Fe 3 O 4 @PSA-C mixture was assessed only based on the MFCs' electrochemical properties. Five MFC system were built and named; for instance, MFC having raw graphite as anode was named as MFC-1, followed by MFC with increasing amount of Fe 3 O 4 @PSA-C 0.25 mg/cm 2 , 0.50 mg/cm 2 , 0.75 mg/cm 2 and 1 mg/cm 2 as MFC-2, MFC-3, MFC-4 and MFC-5, respectively (Table 1). Figure 6a summarizes the output of the cyclic voltammetry (CV) evaluation of the various anode components employed in these tests. By using a linearly cycled potential sweep among two or more preset values, cyclic voltammetry is an electrochemical method for determining how much current flows through a redox-active solution. The rates of electronic-transfer events, the energy levels of the analyte, and the thermodynamics of redox reactions can all be easily ascertained using this method. The Fe 3 O 4 @PSA-C (1 mg/cm 2 ) composite exhibited separate redox maxima, which were determined to be greater than the Fe 3 O 4 @PSA-C (0.25 mg/cm 2 , 0.5 mg/cm 2 , and 0.75 mg/cm 2 ) and reference anode, as per CV. During the oxidative examination of CV, two oxidation maxima at −0.26 mV and +0.16 mV at current values of 7.7 mA and 8.1 mA were recorded in MFC utilizing Fe 3 O 4 @PSA-C (1 mg/mL). Similarly, MFC having Fe 3 O 4 @PSA-C (0.75 mg/cm 2 ) also reported two oxidation peaks at −0.40 mV and +0.02 mV at current values of 5.7 mA and 6.8 mA. A single oxidation spike was observed at −0.12 mV with a current of 3.7 mA in the case of MFC having Fe 3 O 4 @PSA-C 0.5 mg/cm 2 and −0.2 mV with a current of 4.8 mA in the case of MFC having Fe-0.25 mg/cm 2 , respectively. Still, the reference anode showed no enhanced oxidation spike, perhaps because of poor anode kinematics.
The oxidation of essential metabolites may have caused the initial oxidation spike at −0.26 mV. Furthermore, electroactive enhancers such as Fe 3 O 4 @PSA-C-treated electrodes have been discovered to boost the electronic conductivity of the electroactive species, resulting in greater signal responsiveness [29]. FCDMA, for instance, had several oxidation spikes that were not hazy, while other anodes had crushed maxima. Moreover, the oxidation potential in the Fe 3 O 4 @PSA-C-modified anode was substantially greater than in the control anode, which might be attributable to decreased oxidation overpotential failure due to the FCDMA anode's super-hydrophilicity. In contrast, as opposed to the control, the Fe 3 O 4 @PSA-C (0.25 mg/cm 2 and 0.5 mg/cm 2 ) had greater efficacy and generated more oxidative potential.
Solution resistance (R s ), charge transfer resistance of the electrode (R ct ), and diffusion resistance (R d ) have all been demonstrated to be effective in assessing anodic impedance in MFCs using EIS [30]. The dielectric characteristics of materials, and the complex resistance are determined through electrochemical impedance spectroscopy (EIS). This is determined by the interaction of the external field with the specific sample's dipole moment, which is often expressed by the permittivity. Additionally, it is thought of as an experimental method for describing electrochemical systems. With this technique, system impedance is measured across a range of frequencies. As a result, a frequency response involving energy storage and dissipation characteristics is provided. The majority of the time, Nyquist plots are used to visually represent the data obtained by electrochemical impedance spectroscopy. The EC-Lab V11.36 tool was used to create the EIS evaluation findings. A calculated Nyquist plot of hypothetical impedance vs. real impedance is shown in Figure 6b. The width of the semi-largest circle may be used to compute R ct readings. Any electrode's R ct score is a statistic that represents the electrode's oxidation or reduction activation energy. A higher R ct value suggests a larger potential barrier for redox processes and vice versa. As a result, a change in R ct 's value might impact anode dynamics. Improved electrochemical rates resulted in a lower R ct of 52.96 and 33.45 for electron transport in MFCs using Fe 3 O 4 @PSA-C (0.75 mg/cm 2 and 1 mg/cm 2 ), approximately 6.9-fold lower than the control anode (230 Ω). The R ct of the Fe 3 O 4 @PSA-C (0.25 mg/cm 2 and 0.5 mg/cm 2 ) was 164.46 and 103.14, respectively, 2.22 times lower than the control anode.
In the investigation, Fe 3 O 4 @PSA-C (0.75mg/cm 2 and 1 mg/cm 2 ) has been transformed into highly conductive oxide in the anode during MFC operation, enhancing extracellular electron transport [31]. Furthermore, the magnitudes of the Fe 3 O 4 @PSA-C-0.25 mg/cm 2 , 0.5 mg/cm 2 , 0.75 mg/cm 2 , and 1 mg/mL R s were found to be almost equivalent (~18 ohm), showing that the mixture should be conceptually similar.

Power Generation
The power output reached a stable state after two fed-batch periods. At infinite impedance, the maximum OCV improved from 701 ± 23 mV in MFC-1 to 726 ± 20 mV in MFC-2, 746 ± 22 mV in MFC-3, 765 ± 25 mV in MFC-4, and 771 ± 28 mV in MFC-5 (reported as open-circuit voltage, OCV). Stronger electrogenic biofilm formation on the anode in MFCs using Fe 3 O 4 @PSA-C and other assessed MFCs resulted in enhanced OV and OCV, leading to enhanced electrogenic yield.
When the entire anodic chamber volume and actual anode surface area were normalized, polarisation experiments were performed to establish the appropriate power density. Figure 7a shows that MFC-1 with an unmodified anode had a lower power density of 285.01 ± 10 mW/m 2 , compared to MFC-2 (0.25 mg/ cm 2 Fe 3 O 4 @PSA-C)which had a power density of 334.41 ± 6 mW/m 2 (4.86 ± 0.8 W/m 3  These results were superior to those previously reported for single-chambered MFCs with various anode promoters. Despite this, the MFC-2 and MFC-3 with lower Fe 3 O 4 @PSA-C altered anode had a lower inbuilt impedance than the MFC-1, which might be due to the reduced carbon dots' lower charge transmission obstacle. Because Fe 3 O 4 @PSA-C on the anode side worked as a final electron acceptor for extracellular electron transport, reduced the internal impedance, and decreased the anode's conductance (Table 1).   Figure 7b. Using carbon dots in conjunction with Fe 3+ can increase the anode's surface area on a micro/nanoscale, allowing additional cell adhesion and catalytic regions. The Fe 3 O 4 -carbon dot modified anode-based MFC's increased cell adhesion and catalytic regions were combined with increased energy output. Moreover, because most electricigens employ hydrophilic redox messengers, the interface between the anode contacts region and the biofilm impacts the MFC's energy output [32,33]. Increased microbe adhesion to the anode elevated anode voltage, resulting in improved electrogenic productivity in MFCs using Fe 3 O 4 @PSA-C modified anode (FCDMA).

Synthesis of Fe-Carbon Dots
Materials Required: Iron Chloride (FeCl 3 ·6H 2 O); Iron Sulphate (FeSO 4 ·7H 2 O); Ammonium Hydroxide (NH 4 OH); Tri-Sodium Citrate (Na 3 C 6 H 5 O 7 ) as capping agent (CA) and Cow milk. All chemicals were purchased from Sigma Aldrich. The Fe 3 O 4 @PSA-C hetero-structure was synthesized by the hydrothermal-assisted probe sonication method. The C-dots synthesis process was performed using the hydrothermal method, whereas further Fe 3 O 4 @PSA-C nanoparticle synthesis was done using the sonochemical method (Probe Sonicator), as shown in Figure 8.
Synthesis of C-dots: For the synthesis of C-dots, a fixed amount of cow milk was taken for the hydrothermal treatment at 120 • C for 4 h in a 100 mL stainless steel autoclave [25]. After cooling at room temperature, the sample was filtered and washed continuously using double distilled water until it reached pH-7. The C-dots filtered particles were dried at 60 • C, and the prepared C-dots nanoparticles became ready for characterization and participated in the Fe-C-dot synthesis process. For the synthesis of C-dots, we have used the cow milk. The Amul cow milk was purchased from the local market. The specifications and content of the Amul cow milk are given below (Table 2).  The C-dots were synthesized using the Amul cow milk many times in our lab; further the XRD, FTIR, and Raman measurements were performed. The results are reproducible.
Synthesis of Fe 3 O 4 @PSA-C: The whole sonication synthesis process was divided into three steps, and the prepared solutions in different steps were marked as solution A, Solution B, and F-Cdots. A certain amount of FeCl 3 ·6H 2 O and FeSO 4 ·7H 2 O (1:1 ratio) were dissolved in 15 mL of DD water to prepare solution A. The prepared solution was kept in the probe sonicator system (Labman Pro650, Labman Scientific Instruments Pvt. Ltd., Chennai, India) with frequency 20-25 kHz and 6 mm probe. The settings for the probe sonicator are pulse rate (3 sec on/off), power 585 watt and the cut off temperature 65 • C. Subsequently, a 7 mL NH 4 OH solution was added to the prepared solution and sonicated for the next 5 min under the same conditions. The prepared solution was marked as Solution A. Parallelly; solution B was prepared in 10 mL of DD water using a certain amount of sodium polyacrylate (C 3 H 3 NaO 2 ) n as CA. Finally, solution B and 0.2 gm C-dots (pre-synthesized using cow milk) were mixed with solution A under vigorous sonication for 10-15 min. The resultant solution was filtered and continuously washed with DD water and acetone till pH-7 was achieved. The prepared sample was dried in an oven at 70 • C for 12 h to obtain Fe 3 O 4 @PSA-C hetero-structure nanoparticles in pure blackish powder form [34].

Characterization of Fe-Carbon Dots
The prepared samples were structurally characterized by an X-Ray diffractometer (XRD, MiniFlex 600, Rigaku, Japan). The measurements were carried out at room temperature using a CuK α radiation source (λ = 1.5406 Å, operated at 40 KV and 40 mA). The data were collected in the range 2θ = 7 • to 90 • with a step size of 0.02 • and 1.2 s count time at each step. Scanning electron microscopic images were recorded by using a Zeiss Ultra scanning electron microscope (SEM, JSMIT200, JEOL, New Delhi, India) operated at 10-15 kV. Raman spectroscopy was carried out in the backscattering configuration (LabRAM HR, HORIBA India Private Limited, Kolkata, India) with a charge-coupled device detector and 532 nm Laser excitation sources. The Laser power was kept below 10 mW in order to avoid any sample heating. The vibrational properties were characterized by Fourier Transformed Infrared (FTIR) spectroscopy performed on a spectrum BX-II spectrophotometer (PerkinElmer, New Delhi, India) with a spectral resolution of 1 cm −1 . The FTIR measurements of all the samples were done in the reflectance method in the wavenumber range of 400-4000 cm −1 at room temperature. The optical properties of the synthesized samples were analyzed by using a LABMAN-900S UV-visible spectrometer (Labman Scientific Instruments Pvt. Ltd., Chennai, India).

Construction of Electrodes
A titanium horn edge sonicator was used to disperse the Fe 3 O 4 -carbon dots in 50 mL of deionized water (Piezo-U-Sonic, Kolkata, India). After 3 h of sonication, the composite mixture was transferred to 0.5 mL of 5% polyvinyl alcohol (PVA) and sonicated for another 30 min. The Fe 3 O 4 @PSA-C concentrations in the composite mixture were kept at 0.25 mg/cm 2 , 0.5 mg/cm 2 , 0.75 mg/cm 2 and 1 mg/cm 2 , respectively. Clumps of graphite sheet with an approximate contact area of 9 cm 2 (3.0 cm × 3.0 cm) were washed in 1 M HCl. These pieces were sonicated for 30 min with distilled water to remove adhering particles, then rinsed multiple times using distilled water and 35% ethanol, followed by thermal treatment in a muffle furnace for over 30 min at 400 • C. A fabricated graphite sheet was submerged in the combination above of Fe-Carbon dots in a new Petri plate overnight at 60 • C to generate Fe 3 O 4 @PSA-C-modified anodes (FCDMA). A raw graphite sheet anode was made using the same procedure but without including Fe 3 O 4 @PSA-C in the solution. The redesigned anodes were heated in a hot air oven at 60 • C for 24 h. Measurement of dried anodes provided the correct surface mass deposition of Fe 3 O 4 @PSA-C mixture of raw graphite sheet.
The MFCs' air cathodes were constructed using stainless steel (SS) wire mesh potential collectors (6 cm × 6 cm). Pt-C catalysts (0.5 mg/cm 2 , Sigma-Aldrich, St. Louis, MO, USA) and 33.33 L/cm 2 PDMS (Polydimethylsiloxane) adhesive were combined in acetone and coated on SS wire mesh. Fabricated air cathodes were heated for 6 h at 80 • C in a muffle furnace before being stored in a desiccator for MFCs [35].

MFC Fabrication and Operation
Acrylic sheets were used to make five comparable single-chambered MFCs with a 30 mL anodic chamber size. In such MFCs, four FCDMA (with different amounts of Fe 3 O 4 @PSA-C) and a raw graphite sheet anode (reference) were utilized, with the rest of the constituents remaining the same. MFC-1, MFC-2, MFC-3, MFC-4, and MFC-5 were assigned to MFCs that used raw graphite sheet, Fe 3 O 4 @PSA-C (with 0.25 mg/cm 2 , 0.5 mg/cm 2 , 0.75 mg/cm 2 , and 1 mg/cm 2 ) covered anodes, respectively. The anodic chamber and cathode of MFCs were separated by a 16 cm 2 cation exchange membrane (Ultrex, Ventura, CA, USA), which allowed H + ions to move from the anodic chamber to the air-cathode interaction junction. High-quality concealed copper wires were employed to connect the anode and cathode over a 100 Ω applied load. Shewanella putrefaciens MTCC 8104, an EAB, was used as an inoculant in the anodic chamber [36]. Twenty milliliters of inoculum was used as an inoculant in the anodic chamber of each MFC [37].Synthetic effluent containing 3 g/L sodium acetate and an adequate feeding media were supplied to the MFCs [36,38]. At the top of the anode chamber, there were two ports: one for the electrode terminal and the other for the reference electrode (Ag/AgCl, saturated KCl; +197 mV, Equiptronics, Mumbai, India) for sampling [39]. In the testing, five single-chambered MFCs with varied customized anodes were employed. However, the performance results may be trusted because the tests were done in triplicate for many feed gaps. These MFCs were kept in a batch mode with a three-day fresh eating interval at ambient temperatures varying from 33 to 37 • C.

Computation and Analysis
A digital multi-meter was utilized to record current output daily (HTC 830L). The polarisation experiment was conducted with the help of a data logger and a variable external resistance stage. The impedance ranged from 20,000 Ω to 5 Ω, and the related power was measured for at least 30 min (Agilent 34970A, Selangor Darul Ehsan, Malaysia). The power density in proportion to the expected surface area of the anode was calculated using Pd (mW/m 2 ) = EI/A, in which E and I are voltage and current, respectively, connected to specifically applied loads, and A is the anode surface area. The inherent resistance of the MFC was calculated using the current interruption method [39].

Analyses of Improved Anodes
The physical characterization of several anodes after remodeling and during biofilm growth on the relevant anodes while performing was carried out using a scanning electron microscope (SEM, ZEISS, Oberkochen, Germany).

Electrochemical Analysis
Electrochemical methods like electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were used to investigate the anodic degradation kinetics of the different anodes used. Each MFC's platinum rod and anode were used as counter and operating electrodes in EClab electrochemical interrogations to analyze MFCs with a 3-electrode setup. The voltage and current related to the Ag/AgCl (+197 mV vs. SHE) counter electrode w measured. The CV of the anodes was achieved by scanning the administered voltage from −1 V to +1 V at a frequency of 10 mV/s. The EIS was performed using alternating current (AC) at a rate of 100 kHz to 100 MHz and a voltage intensity of 5 mV. The EIS spectra were modeled in an analogous network to determine charge transfer resistance (R ct ) and solution resistance (R s ).

Conclusions
The present study used carbon dots coated Iron (II, III) oxide (Fe 3 O 4 @PSA-C) as a biocompatible anode to enhance power generation in a microbial fuel cell (MFC).
The Fe 3 O 4 @PSA-C hetero-structure was synthesized by the hydrothermal-assisted probe sonication method. The C-dots synthesis process was performed using the hydrothermal method, whereas further Fe 3 O 4 @PSA-C nanoparticle synthesis was completed using the sonochemical method. Nanoparticles were characterized with XRD, SEM, FTIR, and RAMAN Spectroscopy. The structural characterizations confirm the desired nanoparticles' pure phase formation with crystallite sizes ranging between 10-30 nm. Different concentrations of Fe 3 O 4 @PSA-C were coated onto graphite sheets and used as anodes in a single-chambered air cathode MFC. A graphite sheet anode enhanced with a Fe 3 O 4 @PSA-C combination was used to examine the building of a more bio-compatible anode for boosting MFC efficiency. Power density in MFCs using Fe 3 O 4 @PSA-C was 1.54 times greater than in MFCs using raw graphite sheet anodes in a Shewanella -grown MFC.
The physical characterization of Fe 3 O 4 @PSA-C showed that the high surface area and several active sites were responsible for increased adhesion and low activation energy for the electroactive bacteria, thereby increasing the extracellular electron transfer. The coupling of Fe 3 O 4 with carbon dots enhanced hydrophilicity with only a few sheets of flat Fe 3 O 4 @PSA-C, generating stronger stickiness and a broad contact surface for microorganisms to cling on the anode, resulting in better anode dynamics and efficiency in MFC. As a result, an anode made of carbon-based materials coated with Fe 3 O 4 @PSA-C may be employed in MFCs to improve coulombic efficiency and collect more energy while eliminating sewage in the anode compartment. The Fe 3 O 4 @PSA-C-modified bio-anodes were shown to boost the redox peak current during CV (Figure 6a). The im-proved redox profile with modified bioanodes is thought to be owing to EAB's intrin-sic capacity to transport electrons from its OmcC to insoluble Fe(III) metal centers of Fe 3 O 4 @PSA-C. A capacitive anode modifier may provide a capacitive bridge between the EAB and the anode, allowing the regulated passage of electrons from the EAB to the anode.
As a consequence, the larger the capacitance of the anode modifier, the better the efficiency. The various valence metal centers of Fe 3 O 4 @PSA-C may act as a redox cou-ple to link the EAB and anode interfaces. Furthermore, it is hypothesized that the con-stant interconversion of Fe(II) to Fe(III) contributes to transient electron storage through microbial or electrochemical reduction/oxidation, resulting in an increase in transient charge storage on Fe 3 O 4 @PSA-C loaded anodes. EIS analysis was used at OCP to investigate the interfacial charge transfer activity of several bioanodes [40]. Nyquist charts depict the impedance characteristics of all electrodes ( Figure 5). CPE was uti-lized to analyze the varied resistive and capacitive properties of bioanodes using an equivalent circuit consisting of R s , R ct , and Z w . The presence of Fe +3 and C-dot in re-dox active Fe 3 O 4 @PSA-C might be responsible for improved charge transfer and consequently decreased R ct (Figure 6b). The half-cell potential measurements show that at the same current, all MFCs with bare graphite sheet anodes have equivalent half-cell potentials (Figure 7b, curve with solid symbol). Thus, the discrepancies in power generation between these five MFC reactors were caused by variances in cathode potentials (Figure 6b, curve with open symbol).