Removal of Diclofenac Sodium from Wastewater in Microbial Fuel Cell by Anode Modified with MnCo₂O₄

Abstract

Microbial fuel cell (MFC) with a modified anode is one of the new methods to increase MFC efficiency. This study synthesized an anode modified with cobalt manganese oxide (MnCo₂O₄@CF) on carbon felt (CF) by easy hydrothermal method and binder-free. Chemical oxygen demand (COD) was measured with and without diclofenac (DCF). According to SEM results, MnCo₂O₄ was uniformly dispersed on the anode electrode surface. Moreover, the maximum power density in COD (1000 mg/L), 48 h. condition without DCF (726 mA/m²) was 165 ± 0.012 mW/m² and with DCF concentration of 20 mg/L, it was 308 ± 0.013 mW/m² (992 mA/m²). In addition, in the presence of 10 mg/L DCF concentration, the maximum COD removal efficiency was 82% ± 1.93 at 48 h. COD removal efficiency without DCF was 94.67% ± 0.02 at 72 h. After 72 h, the maximum removal efficiency of COD and DCF in the carbon anode was 41% ± 1.15 and 9.5% ± 0.23, respectively. Moreover, the maximum DCF removal efficiency using a MnCo₂O₄ anode was 56% ± 0.55, at 48 h; the initial COD concentration was 500 mg/L, and the DCF concentration was 20 mg/L. This research showed that coating the anode with MnCo₂O₄ could lead to the increased growth of microorganisms on the surface of the anode, decreased load transfer resistance, increased power density, and more removal of COD and DCF. As a result, the performance of fuel cells with modified anode and removal of DCF increased compared to anode with CF-MFC. Thus, the performance of fuel cells with modified anode and removal of DCF increased compared to anode with CF-MFC.

1. Introduction

The average per capita drug consumption worldwide is about 15 gr. This is about 50 to 150 gr per capita in industrialized countries. Medicines are usually used to prevent and treat diseases in humans and animals [1]. After the drug is consumed by humans, most of these compounds are excreted, so the metabolites and unchanged forms of the medicine enter the sewage system. As a result of the incomplete destruction of these compounds in the wastewater treatment plant, they are quickly introduced into the environment [1,2,3]. In the past decades, researchers have identified many drug residues in aquatic environments [4,5,6]. Among pharmaceuticals, diclofenac (DCF) is one of the most widely used painkillers in the world [2,3], which is mostly used in the form of sodium or potassium salt [7]. DCF is one of the non-steroidal anti-inflammatory drugs and has analgesic, antipyretic and anti-inflammatory properties and is used as a pain reliever, anti-arthritis, and anti-rheumatism [2,3,8].

Approximately 940 tons of DCF are consumed worldwide every year [9,10]. DCF has the highest acute toxicity among non-steroidal drugs and is one of the pharmaceuticals which, due to its high consumption and resistance to biodegradation, is mostly detected in water sources; it can be detected in the inlet and outlet of water and wastewater treatment plants, with high concentrations of up to a gram per liter [2,10]. DCF is dangerous for human and animal health. It is considered potentially endocrine-disrupting because it inhibits cholesterol synthesis. In contrast, lethality and teratogenicity were observed in zebrafish embryos exposed to DCF following a 96 h exposure of 480 µg/L. Further, cytological alterations in the liver, kidney, and gills even at 1 g/L in rainbow trout have also been observed [11,12]. Even at the lowest concentration, it has led to cytological changes in the liver and kidneys [9]. In 2019, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) published a notice on the possible health consequences of the DCF presence in surface water and reported the permissible amount of DCF at 0.4 µg/L [3,9,10]. Lethal concentration of DCF is 480 ± 50 µg/L (LC50/96 h) and the effect concentration is 90 ± 20 µg/L (EC50/96 h). In chronic toxicity tests of Daphnia Magna reproduction, the NOEC of DCF was reported to be 1 mg/L, and the LOEC (lowest observed effect concentration) to be 0.2 mg/L [1]. DCF is not completely degraded by conventional water and wastewater treatment methods [13] such as conventional activate-sludge [9,14]. DCF removal efficiency in water and wastewater treatment plants is below 40% [10]. In some texts, the removal efficiency of DCF by wastewater treatment plants has been reported as 21–40% [1,8,15]. Therefore, the concentration of DF in surface water becomes between 1.2 and 4.7 µg/L [8]. As a result, it is necessary to use an effective treatment method to remove DCF from wastewater. To remove DCF from aquatic environments, there are many ways that can be referred to as methods such as advanced oxidation processes (AOPs). This method is a combination of reactive oxidants such as ozone, Fenton, photocatalysis, UV irradiation, and absorbents such as activated carbon [9,10,16]. However, AOPs can decompose resistant molecules into biodegradable intermediates or completely convert them into CO₂, H₂O, and inorganic ions by generating hydroxyl radicals. However, this method has not yet been used on a large scale due to the production of secondary pollution, high cost, creation of toxic intermediates, and high energy consumption [17]. Due to the limitations in using chemical methods, the use of biological methods may be used as an alternative method [18]. Studies show that the microbial fuel cell (MFC) method plays a good role in removing drugs from aquatic environments [19,20]. The MFC is a clean energy process and is a combination of microbial and electrochemical methods [21]. In this method, simultaneously with the decomposition of waste materials, electricity is produced [22,23]. MFC is considered an inventive and environmentally friendly technology to generate electricity along with detoxifying pollutants from wastewater [24]. The removal rate of organic matter in MFC is about 87 to 84% [25]. In addition to the benefits considered for this technology [26,27], it still faces several practical obstacles, such as low output energy and low electron transfer, which limit its applications. In this regard, changing the electrode (anode) is very important because it not only provides active sites for microbial growth but also plays an important role in improving electron transport outside the cell [28,29]. High conductivity, improved electrocatalytic activity, suitable surface, and good biocompatibility are important and necessary features for an anode [30]. These features can be obtained by modifying the electrode, including the use of polymer nanomaterials, metals, and other composite materials [31]. Therefore, due to the exceptional performance of catalysts, cheap price and high abundance, oxides of transition metals such as rubidium [32], iron [33], manganese [34], and cobalt oxides [35] have received wide attention in the field of anode electrode modification [36,37]. The use of other compounds along with these materials improves the electrostatic properties of the electrode. Among these compounds, manganese oxides are one of the most promising electrode materials for electrochemical processes due to their special features such as cheapness, availability, and good environmental nature [38,39]. But due to limitations such as relatively weak electrical conductivity and instability of MnO and Fe₂O₃ and high electrical resistance, they reduce the efficiency of the electron transfer process [34,37]. Therefore, the combination of other metal elements such as RuO₂, NiO, and Co₃O₄ should be used in combination with manganese oxide [40]. Metal oxides prepared by combining manganese and cobalt increase the energy and power density of electricity. Manganese oxidants, including permanganate and manganese dioxide, can act as more effective catalysts in removing organic compounds [37].

Cobalt-based catalysts are one of the most promising basic elements due to the increase in the surface area, reduction in electron transfer resistance, and the ability of high electrochemical and biological activities on the surface of the anode [41]. Therefore, in this study, we tried to use MnCo₂O₄ coated anode because manganese oxide and cobalt are more stable, economical, and environmentally friendly compared to other elements [40,42]. An easy hydrothermal method was used to connect the catalyst on the anode electrode surface in MFC. Since the poor biodegradation of DCF has recently been confirmed by many researchers, the MFC method with an anode modified with MnCo₂O₄ was used in a simple hydrothermal method without the use of a band, and the microbial growth on the anode electrode, voltage and power density, internal resistance, and DCF and COD removal efficiency were investigated, and the results were compared with the control electrode.

2. Experimental Part

2.1. Materials

For the synthesis of Mn-Co oxide catalyst, manganese chloride (MnCl₂), cobalt chloride, and urea (CH₄N₂O) were obtained in analytical grade from Merk, Darmstadt, Germany. Carbon felt was used as cathode and anode. The effective area of each electrode was 2.5 cm × 2.5 cm = 6. 25 cm². DCF (>98% purity) was purchased from Alfa-Aesar.

2.2. Preparation Method of the MnCo₂O₄@CF Anode Electrode

For the synthesis and coating of MnCo₂O₄ on carbon felt, MnCl₂ + 4H₂O (24.89 mg), CoCl₂ + 6H₂O (70.1 mg), and CH₄N₂O (19.1 mg) were dissolved with distilled water and reached a volume of 100 mL. Carbon felt and the prepared solution were poured into the autoclave with Teflon coating and placed in it at 180 degrees for 16 h. Then, the autoclave was brought to room temperature to cool down. The anode coated with MnCo₂O₄@CF was washed with distilled water and placed in a vacuum with a temperature of 60 degrees for 24 h to dry [36]. The number of MnCo₂O₄ deposited on the MnCo₂O₄@CF anode was determined by measuring the difference in weight before and after the coating, and its amount was 0.3 mg. After preparing anode tests (SEM) in different resolutions, (EDS), (EIS), and (CV) were prepared to investigate the characteristics of the MnCo₂O₄@CF anode in terms of the electrode surface.

2.3. MFC System Construction and Operation

In this study, a two-chamber plexiglass reactor with dimensions of 7 cm × 7 cm × 8 cm was used. The volume of each reactor chamber was about 0.4 L. Both chambers were separated by a proton exchange membrane (Nafion^® 117, DuPont Co Wilmington, DE, USA). Two electrodes were connected by a copper wire with 500 Ω external resistance. The system was operated as a batch and with a completely anaerobic anode chamber. At the beginning of the MFC system for bacterial seeding, anaerobic digester sludge from the Shiraz wastewater treatment plant was used. The system was operated as a batch and with a completely anaerobic anode chamber. Prepared sludge with necessary vitamins and minerals, sodium acetate to adjust the COD level (COD 2000 mg/L), and buffer solution was added to the anode chamber of the reactor in suitable proportions. To make an anolyte solution, KCl (0.13 g/L), NH₄Cl (0.31 g/L), Na₂HPO₄ 0.12H₂O (11.3 g/L), NaH₂PO₄·2H₂O (0.54 g/L), and (a small amount of) NaCl were used. Moreover, the amount of voltage produced was measured. After several consecutive cycles and when MFC obtained a stable output voltage and current generated in the reactor, it was confirmed that a biofilm layer was formed on the anode electrode; synthetic wastewater along with DCF sodium (initial COD 500–1000–2000 mg/L) in concentration (10–60 mg/L) was added to the reactor, and the current and voltage production were measured. Output COD (COD tests were performed based on the instructions of the standard method book and using spectrophotometry) and residual DCF sodium were measured using an HPLC device.

Experiments were performed with the CF anode electrode as a control electrode under COD 1000 mg/L, retention time of 48 h, and DCF concentration of 20 mg/L, and the results were compared with the coated electrode. All the tests were repeated three times at a temperature of 25 degrees and a pH of 7.

The collected data were analyzed by Microsoft Excel software (2016). The COD removal efficiency was calculated by Equation (1) COD_i was influent COD, and COD_e was effluent COD.

$$\mathrm{COD}\%=\frac{{\mathrm{COD}}_{\mathrm{i}}-{\mathrm{COD}}_{\mathrm{e}}}{{\mathrm{COD}}_{\mathrm{i}}}\times 100$$

(1)

To make the catholyte solution needed for the cathode compartment, a buffer solution was also used to make the pH of the solution 7. KCl (0.13 g/L), NH₄Cl (0.31 g/L), Na₂HPO₄·12H₂O (11.3 g/L), and NaH₂PO₄·2H₂O (0.54 g/L) were used to make the cathode buffer. The cathode chamber was aerated with an aquarium air pump (ACO-5503, Queensland, Australia). Voltage (V) was measured with an AC, DC auto range selective digital multimeter (VC97, Victor, Iran). Current (I) was calculated using Equation (2)

I = V/R

(2)

• V: Voltage (V)
• R: External resistance in Ω
• I: Current in amps

2.4. Investigation of Cost Estimation of MFC with MnCo₂O₄@CF

In the Iranian market, the cost of buying Plexiglas and tools to make this MFC of dimensions 7 cm × 7 cm × 8 cm was USD 19, and formalin, from the laboratory of the Shiraz Faculty of Health and Medical Sciences, was used to seal the reactor. The cost of preparing the anode and cathode (carbon felt) of dimensions 5 cm × 5 cm was USD 38.46; the cost of buying the Nafion membrane was USD 38.46, and the cost of the connecting wire and chemical materials to make the catalyst for the coating on the anode electrode was USD 19. The total manufacturing cost was almost USD 115.

2.5. Chromatographic Analysis

After filtering the samples using a 0.45 μm filter, they were tested to determine the concentration of DCF by a high-performance liquid chromatography device (HPLC) (AZURA, KNAUER-Germany (Berlin, Germany)) coupled with a UV detector and a C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase for DCF detection was acetonitrile-water (70:30) (v/v) at a flow rate of 1.0 mL/min with a column temperature of 30 °C. DCF detection wavelength and the injection volume were 276 nm and 10 μL, respectively. Therefore, the DCF removal efficiency was formulated as follows (Equation (3)).

η (%) = (1 − C_t/C₀) × 100%

(3)

• C₀ = Initial concentration of DCF.
• C_t = the residual concentration of the DCF after a reaction time.
• η= the DCF removal efficiency%.

With these optimized chromatographic conditions, a typical chromatogram of DCF (Figure 1) was obtained and was found to be a very sharp peak with better resolution within the retention time of 5.95 min.

Calibration Curve Drawing

Different concentrations of DCF were determined by HPLC. The calibration curve was plotted. The calibration curve was found to be linear (Figure 2). The relationship between DCF concentration (C, mg/L) and peak area (y) is shown as y = 17.92x + 11.182, and R² = 0.9995, and x is the DCF concentration, indicating that the peak area has a good linear relationship with DCF concentration.

2.6. Electrochemical Measurements

Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to investigate the electrochemical performance of modified and unmodified electrodes. The analyses were performed using a three-electrode system called µ AUTO LAB III/FRA2 electrochemical analyzer. A MnCo₂O₄@CF anode was used as a working electrode, saturated Ag/AgCl as a reference electrode, and a platinum electrode as an auxiliary electrode.

The power density P (mW/m²) was calculated using the formula P = I × V/A, where I and V are the current and voltage values, respectively, normalized to A, the surface area of the anode electrode (m²). The anode biofilm was analyzed by CV, which provides valuable information about the activity of external electrogenic bacteria and mediators involved in electron transport [36]. CV analysis was performed at different scan rates (10–200 mV/s). EIS testing was performed at a wide frequency range (100 kHz −0.01 Hz), using 50 M phosphate buffer as an electrolyte, and it was carried out under completely anaerobic conditions. CV and EIS results were checked using NOVA and ZView software, respectively. Moreover, the power density of the system will be calculated according to the following formula. To calculate the power density in terms of (mW/m²) and the current density generation, the obtained values were divided by the surface area of the electrode (anode).

3. Results and Discussion

3.1. Characteristics of the Modified Electrode

3.1.1. Electrode Characterization Using SEM and EDS

Electrode surface properties are essential in improving the performance of biochemical systems because they significantly affect microbial growth and adhesion [43]; for this purpose, the surface morphology of the coated and uncoated anodes was analyzed by SEM (Figure 3). Uncoated CF had smooth and clean carbon fibers (Figure 3a), while SEM images confirmed that when the CF was coated with MnCo₂O₄, some crystalline solid particles randomly scattered on the surface of the electrode and had a more irregular and uneven surface (Figure 3b–d), also the increase in the surface roughness of the electrode was significant. which is beneficial for microbial adhesion.

EDS analysis was used to determine the composition of the MnCo₂O₄@CF material, which showed the presence of Mn species (1.36%), Co (2.93%), and O (13.22%) on the surface of the modified electrode (Figure 4). According to previous studies, the synthesis of MnCo₂O₄ on the electrode led to an increase in power density. Therefore, both Mn and Co cations had a positive effect on the catalytic performance [41,44], which can improve the efficiency of MFC.

3.1.2. Cyclic Voltammetry

At the end of the experiment, CV analysis was performed at different scan rates (10 to 200 mV/s) to further evaluate the performance of the anode. CV anode current with MnCo₂O₄ coating improved well in the scan range of +800 to −800 mV. MnCo₂O₄ @ CF (1.125 mA) (Figure 5b) showed better current output compared to (0.4 mA) CF (Figure 5a). As the scan rate increased from 10 to 200 mV/s, the oxidation current increased. As the scan rate increases, the currents also increase, and the potentials of the redox peak change to a more negative or positive position. This phenomenon is mainly because the increase in scan rate also increases the internal diffusion resistance. However, the observation of a good linear relationship between the peak current density and the square root of the scan rate (Figure 5) suggests a reversible and diffusion-controlled electrochemical process on the electrode. Moreover, even at a scan rate above 200 mV/s, the shape of the CV curve of the coated electrode does not deviate significantly, indicating fast electron transfer. MnCo₂O₄ coating leads to a significant increase in anodic current, which can be attributed to electrical conductivity, variable oxidation states, and capacitive behavior of MnCo₂O₄ [38]. The size of the closed area of the CV curve of the electrode is used to determine the capacity of the electrode [36]. In this study, MnCo₂O₄@CF had a larger closed area than the CV of the CF anode, which indicates its capacity characteristics. Pseudo potential behavior, Co/Mn oxides can store electrons, which can lead to improved CV [45]. Higher electrode surface area leads to increased microbial adhesion and substrate flux, which can improve power generation in MFCs. Metal oxides with pseudo-capacitive behavior also can reduce charge transfer resistance. As a result, it improves electron transfer [32,45]. The results of this study are similar to the study by Tahir et al. study (2020) on MnCo₂O₄@CF coated anode electrode [36]; although, in this study, the amount of coated elements on the electrode was much less than in Tahir’s study, showing that small amounts of these elements on the CF electrode can still provide very good results.

3.1.3. Electrochemical Impedance Spectroscopy (EIS)

EIS is used to determine the internal resistance of an electrode. In this study, the EIS test was used to evaluate the electrocatalytic activity of the MnCo₂O₄@CF electrode and check the internal resistance of a three-electrode EIS system. After the EIS test, the results were fitted with ZView software, an equivalent circuit was determined, and the Nyquist curve was presented (Figure 6). The results showed that Rp shows the resistance of the electrode charge transfer (Rp) as well as the charge transfer and the electrode/electrolyte surface phenomenon [46]. In the CF electrode, Rp was 530 Ω, while in MnCo₂O₄@CF, the diameter of the semicircle was 300 Ω and smaller. The smaller diameter of the semicircle means less load transfer resistance. Small charge transfer resistance means a fast electron transfer rate [46]. Moreover, a low Rp value indicates improved electrocatalytic activity [47], which can be attributed to the capacitive property of MnCo₂O₄ particles.

3.2. Effects of the Coated Anode with MnCo₂O₄ @ CF and Uncoated on Voltage Generation

The output voltage of an external resistance of 500 Ω in MFC with modified MnCo₂O₄ @ CF in different CODs (2000, 1000, 500, and 250 mg/L) was investigated for 72 h. As shown in Figure 7, the highest amount of voltage produced in COD 2000 mg/L after 24 h was 261 ± 0.861 mV (218 ± 0.0144 mW/m²). Moreover, with the reduction of COD concentration, the production voltage decreased. At COD concentrations of 250 and 100 mg/L, a rapid voltage drop was observed after 72 h. It may be due to the decrease in the concentration of acetate in the solution, or, since the amount of initial acetate in the solution is low, it may have been quickly consumed by microorganisms in the first 48 h. Thus, after 72 h, there was no food available for microorganisms to produce electricity. According to Figure 7, the COD concentration of the anolyte solution had a direct relationship with the amount of electric current, while it had an inverse relationship with the retention time. As mentioned before, the activity of electrogenic bacteria may decrease due to the consumption of acetate and nutrients by microorganisms because of the amount of electron production and production voltage [48]. This may be due to the reduction of the anolyte level in the anode compartment after 72 h [49].

Anode materials and anode electrode surface composition may affect energy recovery in MFCs by influencing microbial composition, biomass, metabolic behavior, and electron transport properties [32]. Electricity generation in MFC with an anode modified with MnCo₂O₄ catalyst was investigated in the presence of different concentrations (5, 10, 20, 40, and 60 mg/L) of DCF and initial COD of 1000 mg/L (Figure 7).

Moreover, these results were compared with the carbon anode electrode, which was considered the control electrode. As shown in Figure 7, the maximum amounts of voltage produced by the anode modified with MnCo₂O₄ in COD 1000 mg/L and DCF concentration 20 mg/L at times of 48 and 24 h, respectively, were 309 ± 0.35 mV (306 ± 0.02 mW/m², 989 mA/m²) and 310 ± 0.92 mV (992 mA/m², 308 ± 0.013 mW/m²) while in the carbon anode at the concentration of DCF 20 mg/L, COD 1000 mg/L, the maximum amount of voltage produced was 148 ± 0.78 mV (473 mA/m², 70 ± 0.014 mW/m²) 24 h.

After 48 h, the voltage level was 30 mV. The voltage below 50 mV meant the end of the cycle because it indicated the condition that the microorganisms may no longer be active. As a result, zero voltage was considered. Therefore, the amount of voltage generated in the modified anode electrode was much higher than that of the carbon anode under the same conditions.

In the study of the effect of different concentrations of DCF on the output voltage (Figure 7), the results showed that by increasing the concentration of DCF from 5 to 20 mg/L in the anode coated with MnCo₂O₄, the output voltage increased, but with an increase in the concentration of DCF from 40 to 60 mg/L, production voltage decreased. In the retention time of 24 h, the lowest value of the produced voltage was related to the concentration of 60 mg/L DCF (100 ± 0.52 mv), which can be due to the high concentration of DCF in the solution, which has an inhibitory effect and reduces the growth activity of microorganisms. As a result, electron production decreases [50]. Anti-inflammatory drugs such as DCF in 50–100 mg/L have an inhibitory effect on the growth of most Gram-positive and Gram-negative bacteria. The antibacterial action of DCF was due to the inhibition of DNA synthesis [51].

The power density and voltage produced in MFC without DCF (COD 1000 mg/L, 24 h) were 155 ± 0.15 mW/m² and 220 ± 0.83 mV, respectively. Therefore, the generated voltage in the presence of DCF was almost 1.4 times without the presence of DCF, which may be due to the anode coating with MnCo₂O₄, which enriches electrogenetically active bacteria and increases the growth of DCF-decomposing bacteria such as Geobacter, Clostridium, Sedimentobacter, Pseudomonas, and Desulfovibrionas [8]; in addition, it leads to the consumption of DCF as food by microorganisms and electricity generation. In the study by Tahir et al. (2020) 90% of the microorganisms that grew on the anode electrode with MnCo₂O₄ coating included Proteobacteria and Bacteroidetes, which are electrogenic bacteria and produce more electricity [36]. In a study of real wastewater containing DCF with COD 7440 mg/L and Pt-coated Ti Pd/Ir-coated Ti as cathode and stainless steel as an anode in 2019, Amari et al. reported that the production voltage after 4 h in wastewater without DCF was 500 mV and decreased to 400 mV by adding DCF concentration while in the present study, after 4 h with COD less (1000 mg/L), the production voltage was without DCF (200 mV) and with DCF (300 mV) [49].

In the presence of DCF, the production voltage increased, which shows that in this study, the coated anode may cause the growth of species of microorganisms that easily consume DCF and generate electricity. Other studies have also reported increased power generation in MFCs with coated anodes. For example, in the study conducted by Bing Qiu et al. in 2020 [8], the highest power density produced in the Ru/Fe-MFC reactor was 600 mW/m². In Tahir’s study with an anode coated with MnCo₂O₄, the highest power density with sodium acetate consumption was 945 mW/m², and the power generation in the coated anode was three times that of the control anode [36]. The results of these studies are consistent with the present study. Although pt was used in the cathode in Tahir’s study, it was not used in the present study. the amount of voltage produced in the modified anode increased compared to the control anode. In Y C Wu’s study with graphite felt electrode, when acetate was used as a substrate, the production voltage was 570 mV and 300 mV with DCF [52]. Moreover, in the presence of DCF, the output voltage decreased while in the present study, with the addition of DCF up to 20 mg/L, the production voltage increased somewhat and decreased at higher concentrations up to 60 mg/L, which may be due to the type of microorganisms grown on the surface of the anode due to the type of anode coating. In Y C Wu’s study, the production voltage decreased at higher concentrations.

3.3. Efficiency Coated Anode with MnCo₂O₄ @ CF, and Uncoated (CF) in the Removal of COD from Wastewater

COD removal efficiency in different initial COD concentrations (2000, 1000, 500, 250, and 100 mg/L) without the presence of DCF and with the presence of DCF in concentrations (5,10, 20, 40, and 60 mg/L) in COD 1000 mg/L was investigated at different times (24, 48, and 72 h.) with MnCo₂O₄ @ CF MFC (Figure 8). According to

Table 1. COD removal percentage% by MFC white MnCo₂O₄ @ CF electrode.

	COD (mg/L)
Time (h)	2000 (mg/L)	1000 (mg/L)	500 (mg/L)	250 (mg/L)	100 (mg/L)	COD (1000 mg/L) DCF (20 mg/L)
24	53.3 ± 0.19	76.92 ± 1.19	74.4 ± 1.57	58.8 ± 0.35	57.5 ± 1.15	46 ± 1.49
48	85.33 ± 0.23	80.77 ± 0.18	76.75 ± 1.06	70.4 ± 0.27	74.5 ± 1.09	56 ± 1.11
72	94.67 ± 0.02	89.74 ± 0.13	76.75 ± 1.12	80 ± 0.30	75.7 ± 0.55	-

, the removal efficiency of COD without DCF increased by increasing the retention time from 24 to 72 h. The highest COD removal efficiency was 94.67% ± 0.02 with a retention time of 72 h and an initial COD of 2000 mg/L. At an initial COD of 1000 mg/L without DCF, the COD removal efficiency was 80.77% ± 0.018 after 48 h. while by adding DCF, by increasing the DCF concentration from 5 to 10 mg/L, COD removal efficiency was constant at 82% ± 1.93, which was not different from COD removal efficiency without DCF. With the increase of DCF concentration from 10 to 60 mg/L, COD removal efficiency decreased to 25.4% ± 1.16. Zhao et al. (2020) indicated that DCF dosage has a significant effect on COD removal efficiency, so that no effect was observed at concentrations lower than 0.01 mg/L, but the biological removal efficiency was significantly reduced at concentrations higher than 2.0 mg/L DCF [50]. COD (1000 mg/L) removal efficiency in MFC with CF anode was 41% ± 1.15 and was much lower compared to the coated anode. which may be due to the presence of MnCo₂O₄ on the end coat, which caused the growth of microorganisms that increased COD removal efficiency.

In previous studies, the higher removal efficiency of COD has been reported in MFC; for example, Liu et al. (2005) reported removal of more than 99% acetate (initial acetate concentration 800 mg/L) [53] and Zhang et al. (2016) reported graphite fiber brush anode 91%, 1000 Ω and using air cathode MFC [34], while Amair et al. [49] reported 78% COD from synthetic wastewater and 93% from real wastewater using cathode coated with Pt-coated Ti and Pd/Ir-coated Ti and removed steel. Freguia et al. (2007) reported 100% acetate removal using ferricyanide catholyte [54]. In all these studies, electrodes and resistances different from the present study were used. Therefore, different reactor conditions, such as flat and wide-spaced electrodes [53], different amounts of catalyst coating, or the use of platinum compared to the use of CF electrodes alone, may have obtained different results from the present study because all these conditions can affect the development of microbial communities and their response to different situations.

3.4. Efficiency Coated Anode with MnCo₂O₄ @ CF and Uncoated (CF) in the Removal of DCF Sodium from Wastewater

The DCF removal efficiency was investigated at different concentrations (10, 20, 40, and 60 mg/L) with an initial COD of 1000 mg/L at 24 h and 48 h in MnCo₂O₄@CF (Figure 9a). According to Figure 9a, after 48 h, DCF removal efficiency decreased as concentration increased. The highest DCF removal efficiency after 48 h and 10 mg/L DCF concentration was 47.78% ± 0.28, and from 20 to 60 mg/L concentration, DCF removal efficiency decreased with increasing concentration.

High concentrations of DCF may prevent the growth and metabolism of microorganisms in the anode biofilm after 24 h in the environment. As a result, it reduces the removal efficiency. Moreover, Zhao et al., 2020 indicated that there was no effect on biological removal efficiency at low doses with increasing retention time, but in higher doses, the efficiency of biological removal decreases with the increase in retention time [50]. At higher concentrations, DCF may separate during the purification process. It is possible that due to the hydrophobic property of DCF, its biodegradation decreases in higher concentrations, and it is stored in the microbial mass [49,55]. DCF removal efficiency with the modified anode was also compared with the control anode. The results showed that the removal efficiency of DCF at a concentration of 20 mg/L, after 48 h, was 20.07% ± 0.6 while after 72 h, only 9.5% ± 0.23 of DCF was removed by the CF anode (Figure 9b). These results show the effect of the modified anode on the DCF removal efficiency.

To investigate whether the amount of acetate has an effect on the removal rate of DCF or not, the removal rate of DCF was also checked at different concentrations of COD (500, 1000, and 2000 mg/L). To check whether the amount of acetate has an effect on the removal rate of DCF or not, the removal rate of DCF was also investigated at different concentrations of COD (500, 1000, and 2000 mg/L). According to (Figure 9c), the removal efficiency of DCF increased by decreasing the acetate concentration from 2000 to 500 mg/L after 48 h. Moreover, the highest DCF removal efficiency at COD 500 mg/L was 56% ± 0.55 mg/L, which may be forced to use of DCF as a nutrient by decreasing the acetate concentration of the microorganism, and this amount of COD removal is related to DCF. Since DCF is one of the compounds that are difficult to break down by biological processes, many studies have reported the removal of DCF using different types of bioreactors; for example, Y C Wu et al. (2019) reported only 30.73 DCF after two weeks using MFC with graphite felt anode and the cathode coated with platinum [52]. The results of the present study were much better with coated carbon felt and without using platinum in the cathode. Hengduo Xu et al. (2018) have reported the removal of 48.7%–52.6% of DCF using an anode modified with MnO₂, Pd, and Fe₃O₄, and most of this removal was related to Fe₃O₄ metal [56]. The high concentration of DCF with a lower removal rate may be due to the high concentration of DCF, which inhibits the growth and metabolism of microorganisms in the anode biofilm [52]. The aerobic and anaerobic were the reactor. Moreover, the pH of the environment is effective in removing DCF from wastewater [1]. Thus, Zwiener and Frimmel (2003) did not report any decomposition of DCF in a wastewater treatment pilot under aerobic conditions for 55 h and at a concentration of 10 µg/L [57]. Urase and Kikuta (2005) investigated the biodegradation of DCF by activated sludge. They did not observe elimination of DCF in 28 days, neither when DCF was the only carbon source (20 mg/L) nor when milk powder was used [14]. However, in this study, 56% of DCF was decomposed under anaerobic conditions and COD 500 mg/L, which may be due to anaerobic conditions and the existence of microorganisms on the anode covered with MnCo₂O₄ or to some extent the catalyst of MnCo₂O₄.

DCF removal mechanism in MFC with MnCo₂O₄ anode can be through three processes: chemical reduction, microbial reduction, and synergetic reduction of MnCo₂O₄ and microorganisms. Manganese dioxide can act as an effective catalyst in removing organic compounds [37]. As the CV results showed, the electron transfer was higher in the modified anode. This may be another reason for the increased removal of DCFs because the oxidation and reduction current is positively correlated with the rate of electron transfer and chemical reaction on the electrode surface [56,58]; so, it is possible that Manganese cobalt dioxide can act directly in DCF dechlorination. In addition to the above factors, MnO₂ may act on target pollutants through direct catalytic reduction or oxidation. Moreover, according to previous studies, covering the anode with MnO₂ can lead to the creation of a different microbial community compared to the control anode, which may be one of the reasons for removing DCF. In the study by Yuri A. Gorby (2006), MnO₂ anode coating increased the growth of geobacteria on the anode surface, probably because geobacteria can directly use MnO₂ as an electron acceptor [59]. Some geobacteria act as dechlorinating bacteria to remove DCF, and some geobacteria (such as Geobacter metalloreducens and Geobacter grbiciae) can directly oxidize pollutants in the presence of Mn⁺⁴ [36]. In the microbial reduction process, some DCF removals can be assigned to the microbial community on the anode surface, because DCF can be directly reduced by anaerobic microbes. The bacterial community on the modified anode surface plays an important role in the removal of DCF from wastewater.

A variety of dechlorinating bacteria such as Geobacter, Clostridium, Sedimentibacter, Pseudomonas, and Desulfovibrionaceae on the anode surface are responsible for DCF biodegradation. The dechlorination process by bacteria is very slow due to the slow rate of microbial metabolism. Probably, the synergetic effect of MnCo₂O₄ and bacterial activity lead to more removal of DCF.

4. Conclusions

The modified anode with MnCo₂O₄ catalyst on CF was synthesized by a simple hydrothermal method. The amount of voltage produced and the removal of DFC and COD by the modified anode were studied and compared with the control electrode. This anode led to the improvement of MFC performance in charge transfer and production of higher power density and current. This anode was used to remove COD and DCF. At a concentration of 20 mg/L with the modified anode in 48 h and initial COD of 1000 and 500 mg/L, the DCF removal was 20.07% ± 0.6 and 56% ± 0.55, respectively, while in the CF anode, DCF removal efficiency was only 9.5% ± 0.23, at the initial COD of 1000 mg/L in 72 h. The anode coated with MnCo₂O₄ showed better performance in removing DCF than the control anode. The highest COD removal efficiency without the presence of DCF and after 72 h was 94.67% ± 0.02. The power density of the coated anode was 4.39 times that of the carbon anode, increasing current production, increasing anode surface, increasing the growth of microorganisms on the anode surface, and creating a suitable environment for the growth of microorganisms compatible with DCF consumption. The low resistance in charge transfer can be due to the features of anode coated with MnCo₂O₄, which helped to improve MFC performance in power generation and further remove DCF_. These results show the use of MnCo₂O₄ for applications related to DCF removal at high concentrations and better electricity generation.