Fe₃O₄ nanoparticle synthesis was carried out by adopting a solvothermal method, as previously reported along with some moderations in the procedure (Zhang et al., 2020b). 1 g of FeCl₃.6H₂O was added to 20 ml of ethylene glycol, and a clear solution was obtained upon stirring. After that in the acquired solution, 10 ml of ethylene diamine and 3 g of sodium acetate were added and stirred at ambient temperature for 30 min. The mixture was then poured into a Teflon-lined autoclave having 50 ml capacity and was heated in an oven at 180°C for 8 h. The resultant black-colored product was washed repeatedly with distilled water/ethanol, followed by desiccation to gain a dry product. This drying led to the formation of a magnetic iron powder.

The formation of Fe₃O₄ nanoparticles, their purity, size, and morphology were affirmed by the help of various analytical techniques. Dimensions, crystal phase, and size of the nanoparticles were evaluated by X-ray diffraction, using an X’pertpro (PANalyatical) XRD model. FT-IR spectroscopy of the nanoparticles in the scanning range 4,000–600 cm⁻¹, using the Cary 630 Agilent FT-IR spectrometer, was performed to check the purity of the nanoparticles. Morphological characterization was carried out by transmission electron microscopy (TEM) (Tecnai G² F20 U-TWIN), with an accelerating voltage of 200 kV. The size and morphology of the nanoparticles were also assessed by SEM microscopy (Model JSM5910, JEOL, Japan), with 30kV energy, a maximum magnification of 300,000x, and 2.3 nm resolving power, while the purity and elemental ratio of iron and oxygen in nanoparticles were estimated by using EDX (JSM5910) (INCA200/Oxford instruments, United Kingdom).

The coating of nanoparticles on the surface of electrodes was effectively achieved by dipping CF anodes in a Teflon-based autoclave in the solution of Fe₃O₄ sustained at 180°C for 20 h. The autoclave was cooled down to room temperature (25°C), and deionized water was used to wash the Fe₃O₄-coated anodes. The surface-modified electrodes were then placed in the vacuum oven at 60°C for 24 h for the purpose of drying. The amount of Fe₃O₄ deposited on the surface of Fe₃O₄@CF was measured as a count of difference in the weight of electrodes before and after the process of coating that was estimated to be 8.3 mg, suggesting that CF exhibited high potential for Fe₃O₄ coating by this methodology.

Tannery wastewater was obtained from local tanneries in the tannery zone, Sialkot, Pakistan, in 1-L glass bottles, which was mixed in a large 5-L beaker to get the representative sample. The samples were stored at 4 C before the physicochemical characterization and used in the MFC as the anolyte. Physicochemical compositions of the tannery wastewater are provided in Table 1. The initial microbial inoculum was also collected from a tannery wastewater treatment plant in the tannery zone, Sialkot, Pakistan. The collected sludge was pretreated by removing the foreign particles (metals) by sieving, and the final sludge obtained was stirred at 150 rpm for 1 h to obtain uniform sludge in the liquid phase. Afterward,the processed sludge was introduced into two 1-L Erlenmeyer flasks sealed with stoppers.

Selective enrichment of the active SRB cultures was attained by Postgate B medium (Postgate, 1984) that used lactate as the electron donor and carbon source. The main components of the medium were given as follows (in g/L): sodium lactate (3.5); K₂HPO₄ (0.5), NH₄Cl (1.0), FeSO₄.7H₂O (0.5), MgSO₄.7H₂O (2.0), CaSO₄ (1.0), yeast extract (1.0), ascorbic acid (0.1), and thioglycolic acid (0.1). The culture was also supplemented with bromoethanesulfonic acid (BESA) (3.2 g/L) during the enrichment phase for inhibiting the methanogens that can consume the lactate medium. The pH of the growth medium at the start was kept at 7.5 ± 0.1 using 1 N NaOH or HCl solution. Furthermore, the sterilization of the medium was carried out for 20 min at 15 psi and 120°C. Anaerobic conditions were maintained by purging N₂ gas in the medium before inoculation. 1-L bottles were used for enrichment which were kept inside the incubator and were shaken using a rotary shaker at 120 rpm and 30°C. The enrichment was confirmed by the reduction of sulfate to sulfide, signified by change in the media color to black. The enrichment of SRB culture was further enhanced by subculturing every week using a fresh medium after decanting 70% of the supernatant from the culture bottle. After a period of 4 weeks, a culture potentially having a dense population of mixed SRBs was achieved. This enriched SRB culture was then used in MFCs. A non-enriched culture was used as the control.

Two-chamber MFC systems were employed in this research. Each anodic and cathodic chamber had a working volumetric capacity of 200 mL. The anode used in the experimental work was 5-cm × 5-cm carbon felt (CF) (Alfa Aesar, United States) with 3.18 mm thickness or CF coated with Fe₃O₄, while platinum-fabricated carbon cloth was utilized as the cathode (1.00 mg cm⁻² 20 wt% Pt (5 × 5 cm); Fuel Cell Earth, Wakefield, United States). A minimum separation was ensured between the electrodes for reducing the internal resistance (R_int) using a proton (H⁺) exchange membrane (Nafion^® 117, Dupont Co., United States). The proton exchange membrane was treated with H₂O₂, sulfuric acid, and deionized water to improve MFC performance. The external resistor with an external resistance (R_ext) of 500 Ω was used to complete the circuit by linking the electrodes. The temperature of the MFC system was sustained at 30°C using a water bath. The anolyte medium of tannery wastewater was diluted to the COD levels of 500 mg/L and COD/SO₄ ²⁻ of 2.0 by using nutrients with compositions as follows: NaHCO₃, 420 mg L⁻¹; CaCl₂, 15 mg L⁻¹; and 1 ml of trace mineral solution. (NH₄)₂SO₄, MgSO₄.7H₂O, and MnSO₄·H₂O were also added as nutrients to maintain consistent initial sulfate concentration. The MFC medium was sparged with nitrogen. A magnetic stirrer was used in the anodic chamber for stirring, and anaerobic conditions of the anodic chamber were maintained by purging N₂ gas. In the cathodic chamber, 0.1 M phosphate buffer with pH 7.0 was added as the catholyte, and O₂ (from air) was delivered via an air pump equipped with a regulator. Ports were provided to each chamber for feeding, withdrawing samples, and withdrawing the ultimate treated medium. For the MFC setup, 20% sludge and 80% anolyte mixture were used to sustain the growth of the biofilm on the anodic electrode; 50% medium was decanted for the first three runs and superseded with sludge and fresh medium with the same ratio (1:4). After the third cycle, only the anolyte was added for subsequent cycles.

The voltage was measured using a multimeter that was attached across the anodic and cathodic electrodes, and voltage data were monitored at regular intervals. The logged voltage (V) was utilized to calculate the current (I) using Ohm’s law (I = V/R). The power density (P), standardized to the anodic surface area facing the anodic chamber, was measured using the equation P = V²/(R_ext.A), where A (m²) = anodic surface area, R_ext (Ω) = the external circuit resistance, and V [Volts] = MFC potential. By changing the R_ext step by step from 10 KΩ to 10 Ω, the polarization curve was acquired. Every resistor was retained for 30 min, and the voltage was stable.

COD (chemical oxygen demand) was calculated by the standard method reported previously (Greenberg et al., 1992). Percentage removal of COD was acquired by the following relation: C O D   r e m o v a l   %   =     ( C O D 0 − C O D f ) C O D f , where COD ₀ and COD _f represent the COD measured at time t = 0 and final time t = f, respectively. Sulfates were measured using the standard APHA method.

The Coulombic efficiency (CE) of the MFC was estimated by the relation given as follows: C E % = 8 × I × Δ t Δ C O D × V an × 96,485 ×   100.

Here, I (Amp) is the average current generated, ∆t (sec) is the time period for which the experiment was run, while the ∆COD is the COD change from the initial value to the final value, V_an (L) is the wastewater volume which has been treated in the anodic chamber, number 8 in the equation is the constant against M.W. of the oxygen molecule (32 g/mol), and four electrons swapped per reduced oxygen molecule. Electrochemical experiments were repeated atleast twice (n = 2).

First, the characterization of synthesized Fe NPs was performed. The FT-IR spectrum of synthesized nanoparticles was obtained in the range of 4,000–600 cm⁻¹ to elucidate the formation of the nanomaterial and its purity. Figure 1A shows the FT-IR spectrum of the subjected nanoparticles. Two main peaks at 3,266 cm⁻¹ and at 650 cm⁻¹ are obtained which corresponds to the -OH bond stretching vibration and Fe–O bond vibrations, respectively. The FT-IR spectrum of the synthesized nanoparticles was in alignment with previously reported studies (Mishra et al., 2014; Touqeer et al., 2020). The morphology and surface of the synthesized nanoparticles was elucidated by SEM, as shown in Figure 1B. SEM images at different magnification levels exhibit that nanoparticles of very small size and uniform spherical morphology have been produced through a hydrothermal process. Few clumps/aggregations were also observed in the SEM images which might be due the magnetic character of Fe₃O₄ nanoparticles. Since the purity and morphology of the nanomaterial are crucial factors which could affect the cell performance, the elemental analysis of synthesized nanoparticles was done through the EDX. The purity and percentage composition of elements were obtained by the EDX plot, which is presented in Figure 1C. The atomic percentage of nanoparticles obtained by EDX is given in the table of Figure 1C. The ratio between atomic percentages of iron and O₂ is similar to the atomic ratio of iron and O₂ in Fe₃O₄, which affirms the formation of Fe₃O₄ nanoparticles. The morphology of nanoparticles was also determined by TEM using an accelerating voltage of 200 kV. From Figure 1D at different magnifications, one can clearly see a homogeneous morphology of Fe₃O₄ nanoparticles. The particles were somehow agglomerated due to the magnetic behavior of the nanoparticles. Furthermore, nanoparticles were spherical in nature with the appearance of mesopores in them which can be seen from the TEM images. The crystallinity and phase angle of the nanoparticles were estimated by XRD. The XRD plot of the synthesized nanoparticles is in good agreement with magnetite JPCDS Card No 019–0629. The obtained two theta values attribute to the hkl values of (220), (311), (400), (422), 511), and (440), respectively (Figure 1E). The sharp peaks and absence of any extraneous peaks affirms the purity and high crystallinity of the subjected nanoparticles, respectively. The crystal shape corresponding to the identified peaks (JPCDS Card No 019–0629) is a cubic spinel shape. The results obtained by XRD analysis match with that of previously reported results by other researchers (Xing et al., 2021). Once the nanoparticle synthesis was confirmed, it was coated on CF using the dip-and-dry method. Since surface properties of electrodes can substantially influence the operation of MFCs, especially bacterial growth and its adhesion to the electrodes (Yu et al., 2017), SEM analysis was conducted to confirm the morphology of the modified anode surface (Figure 1F). The images produced by SEM clearly revealed very smooth and clean fibers in the unmodified CF electrode. Conversely, a much irregular and rougher fiber surface in the modified electrode was observed, indicating that the presence of iron particles on the CF structures (Fe₃O₄@CF anode) promotes roughness on the CF electrodes. It is well-known that a rough surface assists additional microbial adhesion/colonization and ultimately enhanced electron transfer (Quan et al., 2015).

Voltage production by MFCs with the non–SRB-enriched CF electrode, SRB-enriched CF electrode, and SRB-enriched Fe NP–modified CF electrode (Fe₃O₄@CF electrode) was compared. Non-enriched and SRB-enriched bacteria were added to the respective MFCs. The microbial attachment and development of the biofilm on the electrode surface of the anodic chamber took place, and relatively stable voltage was generated (at 500 Ω) in the fourth week of MFC operation with tannery wastewater as the electron donor (at COD/SO₄ ²⁻ of 2.0) in all cases (Figure 2A). In the fed batches, once the electron donors had been substantially consumed, the voltage started to decline and then attained the stable state again after the addition of fresh anolyte containing tannery wastewater, nutrients, and sulfate. In comparison to SRB non-enriched culture CF (0.39 V), SRB-enriched CF produced almost 15% higher voltage (0.45 V) (Figure 2A), suggesting the presence of more electroactive bacteria. A further substantial increase in voltage production was observed in the case of the SRB-enriched Fe₃O₄@CF electrode that was 1.53 times greater than the control without SRB-enriched culture and CF only (Figure 1A). This strongly suggests that the Fe₃O₄@CF electrode promotes electron transfer to the anode likely due to higher conductivity of Fe₃O₄ which can be used as conduit by SRB-like biogenic iron compounds (Murugan et al., 2018). At a tannery wastewater strength of 500 mg/L (COD) and COD/SO₄ ²⁻ ratio of 2.0, a maximum power density and corresponding current density achieved were 124 ± 8 mW/m² and 388 ± 24 mA/m², respectively, obtained from power and polarization curves (Figure 2B). The achieved CE percentage was 24.6%, and the R_int calculated on the basis of the slope curve technique was 330 Ω. In comparison, while a maximum power density and corresponding current density achieved with SRB-enriched CF were 166 ± 11 mW/m² and 449 ± 24 mA/m², respectively, they were 309 ± 13 mW/m² and 612 ± 25 mA/m², respectively, for the SRB-enriched Fe₃O₄@CF electrode, clearly indicating the significance of electrode modification for enhancing maximum power density. Moreover, the enhanced power output suggested better bacterial growth and attachment on the Fe₃O₄@CF anode. Such facts have been reported in previous studies, where current generation enhancement was correlated with the bacterial biofilm growth (Reguera et al., 2006). Since in the mixed microbial community, many different microbes can play different roles including organic degradation and direct or mediated transfer of electrons from microbes to the electrode surface, the advantage associated with the presence of SRB is to outcompete methanogens that result in the suppression of CH₄ formation in SRB-enriched cultures (Dar et al., 2008), and hence, improved MFC performance can be achieved as more electrons are available for transfer to electrodes. While electrons are transferred to the anode either directly via electrogens by c-type cytochrome or electrically conductive pili (nanowires), or indirectly via redox mediators (Lovley and Nevin, 2011), the SRB group also comprised species having c-type cytochromes that aid in DET to the anode, excluding the requirements of an exterior mediator (Kang et al., 2014). Apparently, there is a disadvantage linked with SRBs as part of the electron produced in the MFC was consumed for sulfate reduction, but the presence of continuous electron donors in real wastewater systems can help surmount this drawback to a considerable extent. Given that sulfates are reduced to sulfides by SRBs in the MFC, sulfides can be further oxidized to elemental sulfur and eventually assist in electricity production. Sulfide oxidation for current generation has already been well-investigated while treating sulfate-/sulfide-rich wastewater using SRBs (Lee et al., 2014). The soluble mediator-like sulfides allow all cells to contribute to current generation whether they are located in the biofilm or in the planktonic phase, and hence, planktonic cells may also play an important role in overall current generation. Based on the results, main possible steps encompassed in electricity generation, tannery wastewater biodegradation, and sulfate removal in this wotk are shown in Figure 2C. A comparison of MFC performance for different anode modified electrodes is also provided in Table 2.

MFC performance in relation to COD removal and sulfate reduction was evaluated in all three MFC systems. A consistent initial COD and sulfate concentration were maintained at 500 mg/L and 250 mg/L, respectively, in all these MFCs. The removal percentage was observed for 72 h by taking samples after intervals of 12 h. A 53 ± 3.1% COD removal was achieved in the current study with non–SRB-enriched CF (Figure 3A). In comparison, SRB-enriched CF showed 10% higher COD removal (63 ± 3.6%), indicating the importance of SRB in degrading complex compounds in tannery wastewater. Nevertheless, COD removal was further enhanced with the SRB-enriched Fe₃O₄@CF electrode where 75 ± 2.1% COD removal was accomplished (Figure 3A). The accelerated biodegradation of tannery wastewater in the MFCs with SRB enriched-Fe₃O₄@CF can be attributed to the anode electrode which provides a pathway for electron transfer to the cathode via an external circuit that can eventually boost the metabolic rate of microbes in the presence of these non-exhaustible electron acceptors (Mohanakrishna et al., 2020; Hu et al., 2018). Furthermore, the improvement in tannery wastewater biodegradation rates attributed to SRB cultures suggests the enrichment of microbes which were highly specific to the biodegradation of recalcitrant pollutants in tannery wastewater. SRB cultures performing more specific and efficient functions in anaerobic systems have already been shown in previous studies. In terms of sulfate reduction, a similar trend was observed as with COD removal. Highest sulfate removal was demonstrated by the MFC with SRB-enriched Fe₃O₄@CF (88 ± 4.1%), followed by SRB-enriched CF (78 ± 2.6%) and non–SRB-enriched CF (51 ± 3.7%) (Figure 3B). While theoretical calculations showed that the COD/SO₄ ^2- ratio of 0.67 is sufficient for completely removing the organics (as COD) by SRBs during sulfate reduction (Rasool et al., 2015), it was reported earlier that in real systems, a COD/SO₄ ^2- ratio of 1.7–2.5 is optimum for microbial growth while reducing sulfates in fed batches in the presence of SRBs (Miran et al., 2018). Therefore, in this study, an optimal concentration of COD/SO₄ ^2- ratio of 2.0 was selected to avoid any limitations as a low COD/SO₄ ^2- ratio can result in inferior MFC performance, as reported by Ghangrekar et al. (Ghangrekar et al., 2010). Furthermore, good repeatability of MFC cycles in this work indicates the robustness of electroactive SRB for COD and sulfate removal.