Waste Activated Sludge Oxidation and Azo Dye Reduction in Microbial Fuel Cell: Optimization of process conditions for high electricity generation and waste treatability

Waste Activated Sludge Oxidation and Azo Dye Reduction in Microbial Fuel Cell: Optimization of process conditions for high electricity generation and waste treatability | 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 Waste Activated Sludge Oxidation and Azo Dye Reduction in Microbial Fuel Cell: Optimization of process conditions for high electricity generation and waste treatability Elif DURNA PİŞKİN, Nevim NevimGENÇ This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2056699/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In microbial fuel cells (MFC), oxidation and reduction processes occur simultaneously. In this study, the operating conditions affecting oxidation-reduction and electricity generation of MFC were optimized using the Taguchi Experimental Design model. Optimization was carried out for maximum power density, coulombic efficiency, azo dye removal and COD removal. With the determined optimum conditions (cathode pH of 3.0, cathode oxygen status of anaerobic, anode substrate of pre-treated, external resistance of 100 Ω, cathode electrode type of plain carbon, cathode electrode surface of 22 cm 2 , cathode conductivity of 20 µs/cm), 177.031 mW/m 2 power density, 7.50% coulombic efficiency, 91.266% azo dye removal efficiency and 21.612% COD removal efficiency were obtained. From the Pareto analysis, it was determined that the power density, coulombic efficiency and COD removal efficiency were most affected by the substrate type at the anode, and the azo dye removal was most affected by the catholyte pH. With the polarization curve, it has been determined that the maximum power density is 145.11 mW/m 2 and the internal resistance of the optimum MFC system is 243.3 Ω. The cyclic voltammogram performed with the optimum experiment was associated with oxidation and reduction reactions. Azo dye electricity production Microbial fuel cell optimization waste activated sludge waste treatment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The microbial fuel cell (MFC) is a bio-catalyzed technology for electricity generation from organic matter. MFC is one of the improved clean technologies with high potential to solve energy needs and waste disposal problems. Real-scale applications of MFCs are limited due to their relatively high investment cost, low power generation, and high internal resistance, and they are complex bioreactors that need to be optimized for higher performance in terms of electricity generation and waste treatment. Therefore, a statistical modelling approach is recommended for improving MFC performance and optimizing waste degradation and power generation simultaneously (Shahi et al. 2021 ). The main constituents of MFCs are electrodes, membranes, and electron donor/acceptor. The most important features of the electrode are good biocompatibility, conduction, chemical stability, and efficient electron transfer between the bacterial and the electrode surface. A Membrane is a physical separator between the anolyte and catholyte, transferring developed protons in the anode to the cathode (Murali et al. 2013 ). Since the power density obtained in MFC is usually very low, because of concentration loss, activation, bacterial metabolism, and high ohmic losses associated with resistance to ion flow through the ion exchange membrane, its commercial applications have also been limited. A high-performance anode material (with excellent electrical conductivity, large surface area, and biocompatibility) is essential to improve the power generations of MFCs (Khalid et al. 2018 ). While external resistance regulates current and voltage, it also indirectly affects the anode potential. The anode potential is an important indicator of energy production in the MFC. It has been stated that the effect of external resistance on substrate removal is more effective than electricity generation (Cai et al. 2020 ). Various waste/wastewater oxidation has been studied in the MFC systems: domestic wastewater (Choi and Ahn 2013 ), dairy wastewater (Cecconet et al. 2018 ), sewage sludge (Karlikanovaite et al. 2020), brewery wastewater (Wen et al. 2010 ), food processing wastewater (Mansoorian et al. 2013 ), swine wastewater (Babanova et al. 2020 ). Waste activated sludge (WAS) is an important source of electrons in MFC systems. Electron acceptors with high redox potential are preferred in MFCs, these offer fast kinetics, be economically valuable, and be readily available. Electron acceptors can be used for bio-energy production in MFCs as well as for bioremediation at the cathode. Considered solely in terms of electricity generation, oxygen is the preferred electron acceptor for MFC scale-up in terms of its sustainability, easy availability, and cost-effectiveness(He et al. 2015 ). Some pollutants cannot be degraded by oxidation anode chamber due to their high redox potential, which can be removed by the reduction in the cathode chamber, thus contributing to the ultimate purification of wastewater and the development of MFC technology. Since many contaminants such as nitro and chlorinated aromatic compounds have positive redox potential, in the cathode chamber they can be good alternatives for electron acceptors (Liu et al. 2009 ). Oxygen is considered the most important electron acceptor. The cathode is suitable for the oxygen reduction reaction as well as for the reduction of biotic or abiotic pollutants (Oon et al. 2017 ).The reduction of oxygen to water in air-cathode MFCs is achieved successfully, besides, the use of strong oxidizers is less sustainable as it will damage the electrode and membrane (Ilamathi and Jayapriya 2018 ).Various electron acceptors are used; ferricyanide (Zain et al. 2015 ), permanganate (Eliato, Pazuki, and Majidian 2016 ), dichromate, persulfate (Pandit et al. 2011 ), nitrogenous compounds(Fang, Min, and Angelidaki 2010 ), metal ions (Wang, Lim, and Choi 2011 ; Zhang et al. 2012 ) and azo dyes (Bakhshian, Kariminia, and Roshandel 2011 ; Mani et al. 2019 ). The chromophoric linkage in the structure of azo dyes at catholyte in MFCs can be reduced by accepting electrons from the anode chamber. Colorless and easily biodegradable aromatic amines are formed as a result of the reduction of azo dye(Ding et al. 2010 ). The wastes from wastewater treatment plants are biologically originated sewage sludge with high organic matter content and can be used as electron donors in MFC systems. Disposal of sewage sludge requires significant operating costs. Sewage sludge contains mixed microbial population and discharged quality treated wastewater. In the stabilization of waste sludge by aerobic/anaerobic digestion processes, microorganisms that cannot adapt to the environment break their cells at the endogenous stage and present their protoplasm as a food source to other microorganisms. In this way, the volume of sludge is reduced and stabilized residue is obtained. There are studies showing that waste activated sludge with high organic content is used as an important electron donor in the MFC process (Ayol et al. 2021 ; Cecconet et al. 2018 ). MFC processes must have high levels of electricity generation as well as good waste remediation. There are limited numbers of studies on the reduction of azo dye under abiotic conditions in cathode chamber. In addition, when the literature is examined, it is seen that the effects of process variables on the performance of oxidation and reduction processes and electricity generation efficiency have been examined individually. In this study, electricity generation and oxidation-reduction potential of waste were investigated by using the activated sludge from the beakers yeast production industry. In a dual-chamber MFC, raw waste activated sludge (WAS) /pre-treated WAS was used as electron donor in the anode chamber and azo dye solution was used as an electron acceptor in the cathode chamber. Optimization of the factors affecting electricity generation, organic matter removal in the anode chamber and decolorization in the cathode chamber were made using the Taguchi Experimental Design approach. The factors affecting the performance of the MFC system were revealed by ANOVA and Pareto analysis. Material And Method WAS and anaerobic sludge were obtained from the anaerobic and aerobic wastewater treatment plant in the factory that produces baker's yeast (PAKMAYA A.Ş Türkiye).WAS taken from the return sludge line of the activated sludge process was used as substrate in the anode chamber and the anaerobic sludge taken from the filled column type anaerobic reactor was used as inoculum. The obtained WAS was used after increasing the solid content by sedimentation. Sunfix Red S3B (SR-S3B-Reactive anionic azo dye) was used as an electron acceptor in the cathode chamber without any pre-treatment. The chemical oxygen demand (COD), mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS), pH values of WAS and anaerobic sludge and the molecular structure of azo dye are given in Table 1 . Table 1 The properties of sludge and azo dye used in the study Experiments were conducted in a dual-chamber, 500 mL volume H-type glass MFC system. The anaerobic sludge and WAS were used both as an inoculum for the enrichment of exoelectrogenic bacteria on surface of the electrode and as an electron donor substrate, respectively. For the formation of an enriched mixture with exoelectrogenic bacteria and biofilm formation on the electrode surface, the anode chamber consists of a 400 ml mixture of 40% (v/v) WAS, 50% (v/v) anaerobic sludge, and 10% (v/v) anaerobic sludge supernatant was fed with sodium acetate (625 mg/L) and nutrient solution every 2 days for 30 days. Solution containing azo dye of 15 mg/L was used as the catholyte medium in all experiments in the experimental design, including the enrichment of exoelectrogens. A certain amount of supernatant from the mixture was removed then nutrients and acetate were added. Biofilm-covered electrodes were used in each experimental run in the experimental design. Fresh WAS as substrate and exoelectrogenic bacteria-rich mixture as inoculum was used at a ratio of 8/1 for each experiment run. During the experiments, WAS was used as raw and pre-treated substrate. The thermochemical pre-treatment process was carried out by adjusting the pH of the WAS to 10 with NaOH and boiling the WAS for 15 minutes. After pre-treatment, the pH of WAS was adjusted to around 7.4. Raw and pre-treated sludge were used with the addition of inoculum and nutrient solution. KCI was used to increase conductivity in experiments with high conductivity compared to the experimental design. The nutrients added for anode chamber at each experiment were 1 mL of NH 4 CI (193.75mg/L), 1 ml of MgCI 2 (62.5 mg/L), 0.5 mL of KCI (81.25 mg/L), 1 mL of NaCI (72.5 mg/L), 5 mL of K 2 HPO 4 (2125 mg/L), 8 ml of KH 2 PO 4 (2600 mg/L). In addition, 0.5 mL of K 2 HPO 4 (212.5 mg/L)and 20 mL of KH 2 PO 4 (6500 mg/L) were added to the cathode chamber as a buffer in each experiment. In all experiments, O 2 has swept away by passing N 2 gas through the anode chamber. According to the experimental design, in certain experiments, the cathode chamber was also swept with N 2 to remove O 2 . When N 2 was passed through the cathode and anode chambers, the chambers were closed throughout the experimental run. In the experiments where the cathode was aerobic, oxygen was supplied to the cathode chamber with the Tetra Aps 400 Aquarium Air pump. When air is pumped into the cathode environment, oxygen and azo dye are together as electron acceptors, and only azo dye becomes an electron acceptor when no air is pumped and an anaerobic environment is formed. Such a factor was included to determine the effect of azo dye and oxygen competing as electron acceptors on the system. The azo dye solution was used as a catholyte in MFC. The anode and cathode chambers are separated by a proton-permeable membrane (DupontTMNafion® 117)with dimension of 9.62 cm 2 treated for 24 hours at 40 0 C with 30% H 2 O 2 . The pre-treated membrane was then preserved in ultrapure water. Plain carbon cloth electrode with the dimension of 22 cm 2 was used in the anode chamber, 0.3 mg/cm² 40% Platinum on Vulcan Carbon Cloth Electrode (Pt CC), and plain carbon cloth electrodes (CC) were used in the cathode chamber according to the experimental design. The circuit between the electrodes is closed using conductive copper wire. The anode electrode was externally connected with the cathode electrode through the resistor. In the MFC system, in which azo dye is used as an electron acceptor, all runs were operated for about 3 days, since the maximum azo dye removal and stabilization of the MFC voltage were observed within the first 3 days. MFC system setup is shown in Fig. 1 . Analysis: Oxidation of the sludge mixture in the anode chamber was monitored by COD removal (%). Volatile solids and COD analysis were performed according to standard methods of 2540 and 5220 respectively (APHA and WEF, 2005). The reduction of azo dye and the generation of aromatic compounds in the cathode chamber were determined by spectrophotometer (Hach Lange DR-6000). The wavelength at which the maximum absorbance is observed, 541 nm, was used for azo dye determination. The calibration curve was created with standard azo dye solutions in the range of 2–30 mg/L, the correlation coefficient (R 2 ) value of the curve was obtained as 0.9998. The removal efficiency of both COD and azo dye was calculated as shown in the Eq. ( 1 ). $$Removal Efficiency \left(\%\right)= \frac{{C}_{0}-{C}_{t}}{{C}_{0}}\times 100$$ 1 where C 0 (mg/L) is the initial COD or azo dye concentration, and C t (mg/L) is the COD or azo dye concentration at reaction time, t (h). Control trials were carried out outside the MFC system at the same catholyte conditions to determine the possible azo dye removal mechanisms (such as adsorption on the carbon electrode surface) other than the electrochemical reduction of azo dye. The output voltage (V) was measured with a digital multimeter (UT181A True RMS Data logging Multimeter) at 10-minute intervals with the software connected to the computer. Voltage is measured over a fixed external resistor (R ext ), while current (I) is calculated from Ohm's law according to Eq. ( 2 ) (Logan et al. 2006 ). $$I={E}_{cell}/{R}_{ext}$$ 2 Where E cell is the voltage measured over an external resistance ( R ext ), I is the electrical current. The power ( P ) generation is calculated according to the Eq. ( 3 ) and was normalized by the surface area of the electrode (I, in square meter). $$P={I*E}_{cell}$$ 3 The Coulombic efficiency (%CE) is expressed as the ratio of the total Coulombs transferred from the substrate to the anode, to the maximum Coulombs that can be transferred. The total Coulombs obtained are determined by integrating the current over time, so that the Coulomb efficiency for a batch mode MFC operation, evaluated over a period t b , is calculated according to the Eq. ( 4 ) (Jia et al. 2008 ; Logan et al. 2006 ). $$CE \left(\%\right)=\frac{8{\int }_{0}^{{t}_{b}}Idt}{F{V}_{an}{\Delta }COD}\times 100$$ 4 where F is the Faraday's constant, Δ COD is the change in COD over time t b and V an is the working volume of the anode chamber. A power curve describing the power density as a function of current density is calculated from the polarization curve. The polarization curve was determined by the voltage drop by different external resistance between 100000 and 10 Ω. Electrochemical characterization of MFC operated at optimum conditions was performed by cyclic voltammetry using a potentiostat (Gamry Reference 3000 Potentiostat). The CV measurement was performed during the period when the highest rates of biodegradation and electricity generation occurred. The reference electrode was placed in the anode chamber and the anode electrode was used as a working electrode against an Ag/AgCl reference electrode while the cathode electrode worked as a counter electrode. CV measurement was performed at 25 mV. Optimization study In this study, the levels of operating conditions affecting oxidation-reduction efficiency and electricity generation potential in the MFC process were optimized using the Taguchi Experimental Design model. Design Expert (version 11) software was used for optimization. Taguchi experimental design methodology is widely used as an advantageous experimental design method, especially due to fewer experiments. The Taguchi design uses the loss function to measure performance characteristics that deviate from the set value. The value of the loss function is converted to the signal-to-noise (S/N) ratio. Optimum operating conditions are calculated from the S/N ratio of the results obtained from the studies designed with the Taguchi (Ross 1996 ). More than one S/N can be defined in the Taguchi Experimental Design Method. In this study, the S/N ratio, which expresses the “higher is better” situation, was used for all responses Eq. ( 5 ) (Aslan and Ünal 2011 ; Ramakrishnan and Karunamoorthy 2006 ). $$\frac{S}{N}=-log10(\frac{1}{n}\sum _{i=1}^{n}\frac{1}{{y}_{i}^{2}})$$ 5 y i is the performance characteristic observed in the i th experiment for a given combination of control factor levels and n is the number of repetitions. The power density, azo dye removal efficiency, coulombic efficiency and COD removal efficiency were determined as response parameters in the optimization. Optimization of the four determined response parameters was performed together as a multi-optimization. The importance weights of all response parameters were accepted as equal. The factors affecting the MFC system and their levels were determined by considering the data in the literature and preliminary trials. The factors and levels used in the experimental design are given in Table 2 . Table 2 Factors and levels determined for Taguchi experimental design Code Parameter Level 1 Level 2 A Cathode pH 3.0 6.05 (pH of raw dye solution) B Cathode Oxygen State Anaerobic Aerobic C Anode Substrate Pre-treated WAS WAS D External Resistance 100 Ω 1000 Ω E Cathode electrode type Pt catalyzed carbon cloth (Pt-CC) Plain carbon cloth (CC) F Cathode Electrode surface area 11 cm 2 22 cm 2 G Cathode conductivity 20 µs/cm (Conductivity of raw dye solution ) 1600µs/cm During the experimental study, the power density, azo dye removal, coulombic efficiency, and COD removal were evaluated according to other factors due to the same anode electrode being used. Results And Discussion Optimization of factors affecting the MFC system In the MFC system, which uses WAS and azo dye solution as the electron donor and electron acceptor, respectively, there are many operating parameters that affect the electricity generation and the treatability of the wastes. In MFC systems, the oxidation of the pollutant in the anode chamber and the reduction of the pollutant in the cathode chamber are equally important as the electricity generation efficiency. In this study, the electricity generation potential was evaluated by calculating the coulombic efficiency and power density, and the treatability of the waste was evaluated by measuring the azo dye and COD removal in catholyte and anolyte, respectively. The Taguchi L8 experiment design, which was created for the optimization of the operating parameters that will provide high waste treatability and high electricity production together, and the response parameters (Power density, coulombic efficiency, azo dye removal and COD removal) obtained from each run are given in Table 3 . Run A B C D E F G Power Density (mW/m 2 ) Coulombic Efficiency (%) Azo Dye Removal (%) COD Removal (%) Table 3 Taguchi experimental design and obtained response parameters 1 3 Aerobic WAS 100 Pt-CC 22 1600 1.556 1.254 69.402 15.064 2 6 Aerobic Pre-treated WAS 1000 Pt-CC 11 1600 100.845 1.792 8.570 23.103 3 6 Anaerobic WAS 100 CC 11 1600 3.584 1.523 65.767 16.095 4 3 Anaerobic Pre-treated WAS 1000 CC 22 1600 92.944 1.773 85.410 22.413 5 6 Aerobic Pre-treated WAS 100 CC 22 20 109.642 8.081 49.956 18.846 6 6 Anaerobic WAS 1000 Pt-CC 22 20 8.797 0.895 13.483 15.865 7 3 Aerobic WAS 1000 CC 11 20 4.995 0.424 81.529 15.00 8 3 Anaerobic Pre-treated WAS 100 Pt-CC 11 20 127.382 6.668 73.182 24.615 As can be seen from Table 3 , depending on the experimental conditions, the maximum values of power density, coulombic efficiency, azo dye removal efficiency, and COD removal efficiency were 109.642 mW/m 2 , 8.081(%), 85.410(%), 24.615(%), respectively. Optimization was made to determine the conditions under which the four response parameters would be highest simultaneously. In order to understand the effect of the variables and to analyze them statistically, ANOVA was performed and coefficients such as F and P were examined (Table 4 ). The F values of the models were determined to be high enough as 267.42, 97.91, 82.83, 34.34 for power density, coulombic efficiency, azo dye removal, and COD removal, respectively. 0.05˂ P-values were determined. P values of the model were determined as 0.0037, 0.0101, 0.0120, and 0.0077 for power density, coulombic efficiency, azo dye removal and COD removal, respectively. P-value ˃ 0.10 means the model and factor terms are not significant, while 0.05˂ P-value shows the significance of the factor and model terms (Singh, Bhunia, and Dash 2019 ). Response parameters are explainable by obtained models if the models are statistically significant. The anode substrate and cathode conductivity for the power density, the anode substrate, cathode conductivity and the external resistance for the coulombic efficiency, the cathode pH, the external resistance and the cathode electrode type for azo dye removal, the anode substrate for COD removal had significant effects. High correlation coefficients were obtained from each model. R 2 and adjusted correlation coefficient (Adj. R 2 ) values for all response parameters were determined to be over 95%. The high R 2 values shows the conformity between the estimated values and experimental data (Rostamiyan et al. 2015 ). The adequate precision is a measure of the range in the predicted response relative to the error of interest and is desired to be greater than 4 (Zinatizadeh et al. 2006 ). Adequate precision values of models for all response parameters were determined to be higher than 4, and indicate an adequate signal. In order for the created statistical model to be compatible, it is desired that the R 2 value should be compatible with the adjusted R 2 and there should not be more than 0.2 difference between the adjusted R 2 and the estimated R 2 (Arslan-Alaton, Tureli, and Olmez-Hanci 2009 ). It can be interpreted that R 2 , predicted R 2 and adjusted R 2 values are compatible. The models are presented in Table 4 . The positive values of the coefficients in equations represent synergistic effects, whereas the negative values represent an antagonistic effect. It may be seen in the model’s equation that all parameters had an antagonistic effect on the power density. According to the model equations, cathode pH and cathode electrode surface area have a synergistic effect on coulombic efficiency. Substrate type and cathode electrode type show a synergistic effect on azo dye removal efficiency. Table 4 ANOVA results obtained for the determined response parameters Power Density Source F-value P-value Significance Fit Statistics Model 267.42 0.0037 Significant R 2 = 0.9985 Adj. R 2 = 0.9948 Predicted R 2 = 0.9761 Adeq Precision = 35.0700 Power density (mW/m 2 ):56.22–51.48×C-4.32×D-3.43×E-2.98×F-6.49×G C 1297.25 0.0008 Significant D 9.15 0.0942 E 5.75 0.1387 F 4.36 0.1722 G 20.59 0.0453 Significant Coulombic Efficiency Model 97.91 0.0101 Significant R 2 = 0.9959 Adj. R 2 = 0.9858 Predicted R 2 = 0.9349 Adeq Precision = 25.6918 Coulombic efficiency (%) :2.80 + 0.2713×A-1.78×C-1.58×D + 0.1996×F-1.22×G A 4.97 0.1555 C 213.37 0.0047 Significant D 168.69 0.0059 Significant F 2.69 0.2426 G 99.84 0.0099 Significant Azo Dye Removal Model 82.83 0.0120 Significant R 2 = 0.9952 Adj. R 2 = 0.9852 Predicted R 2 = 0.9231 Adeq Precision = 23.83 Azo dye removal (%) :55.91–21.47×A-3.55×B + 1.63×C-8.66×D + 14.75×E A 248.25 0.0040 Significant B 6.78 0.1213 C 1.44 0.3536 D 40.44 0.0238 Significant E 117.24 0.0084 Significant COD Removal Model 34.34 0.0077 Significant R 2 = 0.9786 Adj. R 2 = 0.9501 Predicted R 2 = 0.8480 Adeq Precision = 14.5084 COD removal efficiency (%) :18.88-0.8721×B-3.37×C-0.7866×E-0.8282×F B 7.79 0.0684 C 116.22 0.0017 Significant E 6.33 0.0864 F 7.02 0.0770 The values obtained for each response parameter and the predicted values of the model are compatible. Graphs showing the relationships between the actual values and the values predicted by the model are given in the supplementary materials (Supplementary material Fig. S1). Pareto Analysis In MFC system, the operating conditions in the anolyte and catholyte affect the coulombic efficiency, power density, azo dye and COD removal. The effects of the factors on the response parameters are different. The Pareto chart checks for statistical significance by presenting the effect of factors on responses. The Pareto graphs plotted with the t-values of the effects versus the parameters affecting each response are shown in Fig. 2. The t values of the bars in the graphs represent the square root of the F values in the ANOVA. There are two different t-value of effects which are the Bonferroni limit line and the t-value limit line. Factors whose effect is above the Bonferroni line are considered to be extremely significant, while those whose effect is between the Bonferroni line and the t-value limit are considered likely to be significant. Factors whose effect is below the t-value limit are considered statistically insignificant (Asem, Nawawi, and Jimat 2018 ; Boateng, Yang, and Li 2021 ). In Pareto analysis, orange-colored bars indicate positive effects of factors, and blue- colored bars indicate negative effects of factors (Abdulredha, Hussain, and Abdullah 2019). For power density, the t-value of the substrate type factor was above the Bonferroni limit line, which indicates that the substrate type has the highest effect. If the substrate is used without pre-treatment, the power density will decrease. The t value of all other factors is below the t- value limit line, indicating that these factors are statistically insignificant (Fig. 2. (a)). For coulombic efficiency, the t-values of both anolyte substrate and external resistance factors are above the Bonferroni line which indicated that both factors were extremely significant factors. The use of the substrate without pre-treatment and the increase in external resistance negatively affect the coulombic efficiency. The t-value of the catholyte conductivity factor is between Bonferroni and the t-value limit line, indicating that conductivity is an important factor for coulombic efficiency (Fig. 2 (b)). As seen from the graph, pH shows to have the highest effect on the azo dye removal, followed by electrode type and external resistance. The blue color of the pH effect indicates that an increase in pH from 3.0 to 6.05 will decrease the dye removal efficiency. At the same time, it was determined that higher azo dye removal efficiency would be achieved by using the plain carbon electrode (Fig. 2 (c)). It was determined from Fig. 2 (d) that the most effective parameter on COD removal was the substrate type. Pre-treatment of the substrate increased the COD removal efficiency. Evaluation of three-dimensional (3D surface) graphics The effects of operating parameters on response parameters were evaluated with 3D graphics. Thermochemical pre-treatment of WAS in the anode chamber increased the power density from 1.5569 mW/m 2 to 127.382 mW/m 2 when the cathode was anaerobic, the external resistance was 100 ohms, 22 cm 2 PtCC and the cathode conductivity was 20 µs/cm (Fig. 3 (a)). It also increased COD removal from 15–24.6% when the cathode was anaerobic, the external resistance was 100 ohms, 22 cm 2 CC and the cathode conductivity was 20 µs/cm (Fig. 3 (d)). Chae et al.(2009) stated in their study that methanogenesis is compatible with higher resistance conditions, because methanogens are more likely to use the substrate when exoelectrogen activity is reduced. Reducing the external resistance from 600 Ω to 50 Ω increased the coulombic efficiency (CE) from 32–42% (Chae et al. 2009 ). Therefore, lowering the external resistance may be a way to improve MFC performance as it can increase CE by reducing electron losses (Chae et al. 2010 ). In this study, it was observed that the decrease in external resistance increased the coulombic efficiency (Fig. 3. (b)). However, it was observed that the pH change in the cathode chamber did not affect the power density, coulombic efficiency, and COD removal. The conditions with the highest azo dye removal in the cathode chamber were characterized as plain carbon cloth electrode and pH 3. The azo dye removal decreased from 85.41–8.57% when the pH was increased with the cathode being anaerobic, the anode substrate was pre-treated, external resistance was 1000 ohms, 12 cm 2 Pt-CC and the cathode conductivity was 20 µs/cm(Fig. 3(c)). Confirmation experiment and evaluation of catholyte reduction Optimum operating conditions of Taguchi experimental design determined as cathode pH of 3.0, cathode oxygen status of anaerobic, anode substrate of pre-treated, external resistance of 100 Ω, cathode electrode type of plain carbon, cathode electrode surface of 22 cm 2 , cathode conductivity of 20 µs/cm. The presence of O 2 together with the azo dye solution in the cathode chamber, which has a higher electron acceptor property than the azo dye, causes the transfer of electrons to O 2 instead of azo bonds. In the absence of oxygen, the transfer of electrons to the azo bonds in the Cathode chamber is facilitated and higher color removal is achieved. The fact that the cathode oxygen state is anaerobic under optimum conditions also confirms this. A confirmation experiment was performed to verify the optimum MFC conditions. With the confirmation experiment, 177.031 mW/m 2 power density, 7.509% coulombic efficiency, 91.266% azo dye removal efficiency, and 21.612% COD removal efficiency were obtained. It was determined that all response parameters, except power density, were within the 95% confidence interval (Tablo 5). The fact that the power density is above the confidence interval is evaluated positively in terms of MFC performance. The voltage plot measured against 100 Ω resistance at optimum MFC conditions is given in the supplementary material Fig. S2. Table 5 Response parameter values obtained under optimum conditions, and estimated confidence intervals. Response Predicted Mean 95% confidence interval Actual data Power density (mW/m 2 ) 112.101 97.036-127.167 177.031 Coulombic efficiency (%) 7.3033 6.021–8.585 7.509 Azo dye removal efficiency (%) 102.714 88.353-117.075 91.266 COD removal efficiency (%) 21.502 19.278–23.726 21.612 Yusoff et al. obtained a higher COD removal efficiency of up to 85% at the end of 10 days of operation with microwave pre-treated sludge. In the same study, power density and coulombic efficiency were obtained as 42 ± 3 and 6.3%, respectively (Mohd Yusoff et al. 2013 ). Jayashree et al. improved COD removal up to 54% by applying low-temperature thermochemical pre-treatment to dairy WAS. The power density of the MFC had increased from 0.5 W/m 3 (raw sludge) to 0.715 W/m 3 (pre-treated sludge) (Jayashree et al. 2014 ). Ayol et al., on the other hand, obtained a power density of 312.98 mW/m 2 in an enhanced sludge stabilization coupled with amicrobial fuel cell with graphite electrodes (Ayol et al. 2021 ).The reason for the relatively low values obtained in this study can be explained by the application of more moderate operating conditions. In addition, the optimum conditions determined are those that maximize the electricity generation efficiency and the treatability of the waste in the anode/cathode chamber simultaneously. In addition to the anolyte and catholyte conditions, the surface properties of the electrode used to affect the reactions that will take place in the catholyte. It has been extensively accepted that surface-active oxygen functional groups on carbon felt are effective in catalyzing redox reactions of active species and increasing the wettability of carbon felt (Kim et al. 2014 ). The reduction potential of the cathode chamber was evaluated by the removal of azo dye. The catholyte of the MFC system operated under optimum conditions was compared with the control trial. The ultraviolet-visible (UV-VIS) spectrum of the catholyte at the optimum conditions and the control trial show the same trend (Fig. 4 ) Aromatic amines and azo bonds are the two main components in azo dyes. In the spectrophotometric analysis, the absorption peaks in the VIS region correspond to the azo bond, while the absorption peak in the UV region shows the aromatic compounds. Peak variation of aromatic compounds and azo bonds in the UV-VIS spectra indicates the decreasing peak of the azo bond and the increase in aromatic amines. Possible intermediates produced after disruption of the azo bond may be benzene, naphthalene-based sulfonated compounds, and triazine-based compounds (Oon et al. 2017 ). The peak seen at 190 nm in the raw azo dye shifted towards 250–260 nm at the end of 70th hour in the control and MFC process. The peak seen at 290 nm in the raw dye disappeared completely. While the azo dye concentration at the end of the MFC process at 541 nm decreased by 91.26%, a decrease of 49.69% was observed in the control trial. After decolorization, the peak at 247 nm, suggesting that azo dye was reduced to its hydrazine derivative (Ding et al. 2010 ). \(-N=N-\) double bond was reduced to hydrozo or amine Eq. ( 6 )-( 7 )(Liu et al. 2009 ). $$-N=N-+2{e}^{-}+2{H}^{+}\to -NH-NH-$$ 6 $$-N=N-+4{e}^{-}+4{H}^{+}\to -N{H}_{2}+N{H}_{2}-$$ 7 An increase in absorbance density was observed at 265 nm. This indicates that the reduction product (sulfonic acid) associated with the degradation of azo dyes may be present(Liu et al. 2009 ). In the light of this information, it is thought that the change in absorbance of the control experiment may be caused by the reactions of active functional groups on the carbon cloth. In the MFC system, in addition, electrons transmitted from the anode chamber were effective in the azo dye reduction process. Polarization and power curve evaluation The electricity generation potential varies with the characteristics of the electron donor, electron acceptor, electrode material, and catholyte used. The polarization curve was analyzed when stable voltage was obtained in the optimum experiment. The curve is obtained as shown in Fig. 4 with the voltage values obtained across 10 Ω to 100000 Ω external resistance. The maximum power density produced under optimum conditions was obtained as 145.11 mW/m 2 . The internal resistance of the MFC system was obtained as 243.3 Ω. The internal resistance of the MFC system was obtained as 243.3 Ω. Xiao et al. found the internal resistance to be 371 Ω in the two-chamber MFC system using sewage sludge as a substrate (Xiao et al. 2014 ). Maximum power density is not the main goal in MFC systems. In addition, it is aimed to ensure effective waste removal. The high internal resistance value obtained may be due to the fact that WAS was used as a substrate in all experiments instead of an easily degradable substrate, and the higher density of the sludge compared to the MFCs using wastewater increasing electron transport losses. Cyclic voltammetry Cyclic voltammogram is used as an electrochemical technique to describe electron transfer interactions at the electrode-biomass/biofilm-liquor interface in the anode chamber (López Zavala et al. 2019 ). In this study, the CV measurement was carried out under optimum experimental conditions, where the MFC produces a stable voltage. A redox cycle was obtained in the voltammogram shown in Fig. 6 , confirming the important role played by the anaerobic microorganism in transferring electrons to the anode. The current density data were correlated with the oxidation and reduction reactions occurring at the working electrode. The highest anode potential was associated with the largest values of current density corresponding to the oxidation of waste activated sludge. The highest cathode potential corresponded to the lowest negative current density values associated with water reduction and hydrogen gas production. An anodic peak at − 183.2 mV and a cathodic peak at − 181.2 mV were visible in the CV curve. In addition, the small size of the peaks in Fig. 6 may be due to the fact that no redox mediator was used in this study. Mediators have an important effect on the anodic or cathodic current in MFC systems. Certain microorganisms may also support the electron transfer process (Danish Khan et al. 2015 ). Conclusion In this study, besides the electricity generation potential in the MFC system, WAS and azo dye treatment in the anode and cathode chambers, respectively, were investigated. The MFC system was optimized to determine operating conditions that would maximize COD removal in the anode chamber, azo dye removal in the cathode chamber, power density, and coulombic efficiency. Taguchi Experimental Design approach was used in optimization. The results obtained are summarized below; - The relationship between the experimental results and the results predicted by the statistical model was found to be compatible. Optimum MFC operating conditions have been determined as pH of 3.0, cathode oxygen status of anaerobic, anode substrate of pre-treated, external resistance of 100 Ω, cathode electrode type of plain carbon, cathode electrode surface of 22 cm 2 , cathode conductivity of 20 µs/cm. With the confirmation experiment performed under these conditions, 177.031 mW/m 2 power density, 7.50% coulombic efficiency, 91.26% azo dye removal, and 21.61% COD removal were obtained. - It has been observed with the UV-VIS spectrum that the azo bonds of the azo dye that peak in the visible region are broken and the aromatic compounds increase in the UV region under optimum conditions. - In Pareto analysis, it was determined that the anode substrate type was the most effective factor in power density, coulombic efficiency, and COD removal. It has been observed that sludge degradation and electricity generation potential have improved by applying thermochemical pre-treatment to WAS. In the cathode chamber, it was determined that the most effective parameter in the removal of azo dye was the cathode pH, and the highest dye removal could be achieved at pH 3.0. - The polarization curve was performed while the optimum experiment was at a steady state. It has been determined that the maximum power density is 145.11 mW/m 2 and the internal resistance is 243.3 Ω. The cyclic voltammogram performed with the optimum experiment was obtained confirming the oxidation and reduction reactions. Declarations Acknowlegment Authors thank the Kocaeli University Scientific Research Projects coordination, BAP for their financial support to the national project (no: FBA-2021-2672). Confict of interest The authors declare no conflicts of interest. References Abdulredha MM, Hussain SA, Abdullah LC, Separation Emulsion via Non-Ionic Surfactant: An Optimization. Processes 7(6):382. https://doi.org/10.3390/PR7060382. APHA, A. and and WEF (2005) Standard methods for the examination of water and wastewater . 21st ed. Am Public Work Assoc. 21st ed. Washington D.C.: APHA-AWWA-WEF. 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(2013) Bioelectricity generation using two chamber microbial fuel cell treating wastewater from food processing. Enzyme Microb Technol 52(6–7):352–357. https://doi.org/10.1016/j.enzmictec.2013.03.004. Mohd Yusoff MZ et al. (2013) Influence of pretreated activated sludge for electricity generation in microbial fuel cell application. Bioresour Technol 145:90–96. https://doi.org/10.1016/J.BIORTECH.2013.03.003. Murali V et al. (2013) Comprehensive Review and Compilation of Treatment for Azo Dyes Using Microbial Fuel Cells. Water Environ Res 85(3):270–277. https://doi.org/10.2175/106143012X13503213812481. Oon YS et al. (2017) Microbial fuel cell operation using monoazo and diazo dyes as terminal electron acceptor for simultaneous decolourisation and bioelectricity generation. J Hazard Mater 325:170–177. https://doi.org/10.1016/j.jhazmat.2016.11.074. Pandit S et al. (2011) Performance of electron acceptors in catholyte of a two-chambered microbial fuel cell using anion exchange membrane. Bioresour Technol 102(3):2736–2744. https://doi.org/10.1016/j.biortech.2010.11.038. Ramakrishnan R, Karunamoorthy L (2006) Multi response optimization of wire EDM operations using robust design of experiments. Int J Adv Manuf Technol 29(1–2):105–112. https://doi.org/10.1007/s00170-004-2496-6. Ross PJ (1996) Taguchi Techniques for Quality Engineering . Singapore: McGraw Hill Professional. Rostamiyan Y et al. (2015) Using response surface methodology for modeling and optimizing tensile and impact strength properties of fiber orientated quaternary hybrid nano composite. Compos Part B Eng 69:304–316. https://doi.org/10.1016/J.COMPOSITESB.2014.09.031. Shahi A. et al. (2021) A comparative study on the performance of microbial fuel cell for the treatment of reactive orange 16 dye using mixed and pure bacterial species and its optimization using response surface methodology. Sustain Energy Technol Assessments 48:101667. https://doi.org/10.1016/J.SETA.2021.101667. Singh R, Bhunia P. and Dash R.R. (2019) Optimization of organics removal and understanding the impact of HRT on vermifiltration of brewery wastewater. Sci Total Environ 651:1283–1293. https://doi.org/10.1016/j.scitotenv.2018.09.307. Wang Z, Lim B, Choi C (2011) Removal of Hg 2+ as an electron acceptor coupled with power generation using a microbial fuel cell. Bioresour Technol 102(10):6304–6307. https://doi.org/10.1016/J.BIORTECH.2011.02.027. Wen Q et al. (2010) Production of electricity from the treatment of continuous brewery wastewater using a microbial fuel cell. 89(7):1381–1385. https://doi.org/10.1016/J.FUEL.2009.11.004. Xiao B, et al. (2014) Relationship of methane and electricity production in two-chamber microbial fuel cell using sewage sludge as substrate. Int J Hydrogen Energy 39(29):16419–16425. https://doi.org/10.1016/J.IJHYDENE.2014.08.024. Zain SM, et al. (2015) Different Types of Microbial Fuel Cell (MFC) Systems for Simultaneous Electricity Generation and Pollutant Removal. J Teknol 74(3):13–19. https://doi.org/10.11113/JT.V74.4544. Zhang LJ et al. (2012) Bioelectrochemical recovery of ammonia–copper(II) complexes from wastewater using a dual chamber microbial fuel cell. Chemosphere 89(10):1177–1182. https://doi.org/10.1016/j.chemosphere.2012.08.011. Zinatizadeh AAL et al. (2006) Process modeling and analysis of palm oil mill effluent treatment in an up-flow anaerobic sludge fixed film bioreactor using response surface methodology (RSM). Water Res 40(17):3193–3208. https://doi.org/10.1016/J.WATRES.2006.07.005. Additional Declarations No competing interests reported. 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NevimGENÇ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYNCCigQQyXgASDE2EKflDFgLA1QLMxE6GNugWhiI0WLOv/jYg5/z0uT42w8fOPCAwUZ2wwH+Yx/wabGc8SzdsHdbjrHEmbQEoMPSjDccYGaegU+LwY0zZtKM2yoSNzDkGAC1HE4EacHrMIMb579JM84BauF//wGo5T8RWs73sEkzNuQkbpDIAYXYAWJsYTOT7DmWZixx4xnQYQbJxjMPMxsTsOXwM4kfNcly/P3JDx/+qLCT7Tve+BivFgaJBBQTgJhgTPIfIKRiFIyCUTAKRjwAANDMTtctJHdAAAAAAElFTkSuQmCC","orcid":"","institution":"University of Kocaeli","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Nevim","middleName":"","lastName":"NevimGENÇ","suffix":""}],"badges":[],"createdAt":"2022-09-12 12:14:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2056699/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2056699/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":26519194,"identity":"e1116a00-9260-4cd7-9df4-a334e694eff2","added_by":"auto","created_at":"2022-09-15 17:21:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":516090,"visible":true,"origin":"","legend":"\u003cp\u003eDouble chamber MFC system setup\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2056699/v1/b050487b280857d1d9340bac.png"},{"id":26518738,"identity":"17c1ce89-e428-4cc6-a4b0-f572786317f8","added_by":"auto","created_at":"2022-09-15 17:16:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38675,"visible":true,"origin":"","legend":"\u003cp\u003ePareto graphs of response parameters (Power density (a), coulombic efficiency (b), azo dye removal (c), COD \u0026nbsp;removal (d))\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2056699/v1/52f37ec06e049a159e225ff8.png"},{"id":26518130,"identity":"919e3cb1-f3f3-479d-9008-050e1ac770bb","added_by":"auto","created_at":"2022-09-15 17:11:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":357492,"visible":true,"origin":"","legend":"\u003cp\u003e3-D graphics of Power density (a), coulombic efficiency (b), azo dye removal (c), COD removal (d) (for anode substrate -1 refers to pre-treated and 1 refers to raw WAS, for electrode type -1 refers to Pt catalyzed carbon cloth and 1 refers to plain carbon cloth).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2056699/v1/ad6c0aae35f34816d01e2d5c.png"},{"id":26519193,"identity":"2480b4f1-dfa7-4b1f-98d3-7bd52f291338","added_by":"auto","created_at":"2022-09-15 17:21:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34071,"visible":true,"origin":"","legend":"\u003cp\u003eThe wavelength scans of raw azo dye, reduced dye after optimum MFC experiment and the dye after the control trial.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2056699/v1/8c3c8539fcb78f7462d2fa76.png"},{"id":26518134,"identity":"928f82e7-120e-4129-8e3d-1369ea54868e","added_by":"auto","created_at":"2022-09-15 17:11:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43913,"visible":true,"origin":"","legend":"\u003cp\u003ePolarization and power curve of optimum MFC experiment\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2056699/v1/0e0b6519824912de43d3b880.png"},{"id":26518736,"identity":"173101b1-2215-4664-aaeb-1286e0095ea0","added_by":"auto","created_at":"2022-09-15 17:16:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36202,"visible":true,"origin":"","legend":"\u003cp\u003eCyclic voltammogram of the optimum MFC experiment\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2056699/v1/fa5aace0664272167c743014.png"},{"id":27154876,"identity":"554f9ad7-9041-4ac4-b4f3-a8fa0fcb194a","added_by":"auto","created_at":"2022-09-29 21:29:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1465795,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2056699/v1/c6bd6d48-c37f-4c57-ae1c-d39df1a2e7f5.pdf"},{"id":26518735,"identity":"88b3e9f1-2488-477b-8c0d-55541489cd4f","added_by":"auto","created_at":"2022-09-15 17:16:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":138223,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-2056699/v1/39d9cb8daa17a3d36173cea4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Waste Activated Sludge Oxidation and Azo Dye Reduction in Microbial Fuel Cell: Optimization of process conditions for high electricity generation and waste treatability","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe microbial fuel cell (MFC) is a bio-catalyzed technology for electricity generation from organic matter. MFC is one of the improved clean technologies with high potential to solve energy needs and waste disposal problems. Real-scale applications of MFCs are limited due to their relatively high investment cost, low power generation, and high internal resistance, and they are complex bioreactors that need to be optimized for higher performance in terms of electricity generation and waste treatment. Therefore, a statistical modelling approach is recommended for improving MFC performance and optimizing waste degradation and power generation simultaneously (Shahi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main constituents of MFCs are electrodes, membranes, and electron donor/acceptor. The most important features of the electrode are good biocompatibility, conduction, chemical stability, and efficient electron transfer between the bacterial and the electrode surface. A Membrane is a physical separator between the anolyte and catholyte, transferring developed protons in the anode to the cathode (Murali et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince the power density obtained in MFC is usually very low, because of concentration loss, activation, bacterial metabolism, and high ohmic losses associated with resistance to ion flow through the ion exchange membrane, its commercial applications have also been limited. A high-performance anode material (with excellent electrical conductivity, large surface area, and biocompatibility) is essential to improve the power generations of MFCs (Khalid et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile external resistance regulates current and voltage, it also indirectly affects the anode potential. The anode potential is an important indicator of energy production in the MFC. It has been stated that the effect of external resistance on substrate removal is more effective than electricity generation (Cai et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVarious waste/wastewater oxidation has been studied in the MFC systems: domestic wastewater (Choi and Ahn \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), dairy wastewater (Cecconet et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), sewage sludge (Karlikanovaite et al. 2020), brewery wastewater (Wen et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), food processing wastewater (Mansoorian et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), swine wastewater (Babanova et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Waste activated sludge (WAS) is an important source of electrons in MFC systems.\u003c/p\u003e \u003cp\u003eElectron acceptors with high redox potential are preferred in MFCs, these offer fast kinetics, be economically valuable, and be readily available. Electron acceptors can be used for bio-energy production in MFCs as well as for bioremediation at the cathode. Considered solely in terms of electricity generation, oxygen is the preferred electron acceptor for MFC scale-up in terms of its sustainability, easy availability, and cost-effectiveness(He et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome pollutants cannot be degraded by oxidation anode chamber due to their high redox potential, which can be removed by the reduction in the cathode chamber, thus contributing to the ultimate purification of wastewater and the development of MFC technology. Since many contaminants such as nitro and chlorinated aromatic compounds have positive redox potential, in the cathode chamber they can be good alternatives for electron acceptors (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOxygen is considered the most important electron acceptor. The cathode is suitable for the oxygen reduction reaction as well as for the reduction of biotic or abiotic pollutants (Oon et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).The reduction of oxygen to water in air-cathode MFCs is achieved successfully, besides, the use of strong oxidizers is less sustainable as it will damage the electrode and membrane (Ilamathi and Jayapriya \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).Various electron acceptors are used; ferricyanide (Zain et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), permanganate (Eliato, Pazuki, and Majidian \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), dichromate, persulfate (Pandit et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), nitrogenous compounds(Fang, Min, and Angelidaki \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), metal ions (Wang, Lim, and Choi \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and azo dyes (Bakhshian, Kariminia, and Roshandel \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mani et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The chromophoric linkage in the structure of azo dyes at catholyte in MFCs can be reduced by accepting electrons from the anode chamber. Colorless and easily biodegradable aromatic amines are formed as a result of the reduction of azo dye(Ding et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe wastes from wastewater treatment plants are biologically originated sewage sludge with high organic matter content and can be used as electron donors in MFC systems. Disposal of sewage sludge requires significant operating costs. Sewage sludge contains mixed microbial population and discharged quality treated wastewater. In the stabilization of waste sludge by aerobic/anaerobic digestion processes, microorganisms that cannot adapt to the environment break their cells at the endogenous stage and present their protoplasm as a food source to other microorganisms. In this way, the volume of sludge is reduced and stabilized residue is obtained. There are studies showing that waste activated sludge with high organic content is used as an important electron donor in the MFC process (Ayol et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cecconet et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMFC processes must have high levels of electricity generation as well as good waste remediation. There are limited numbers of studies on the reduction of azo dye under abiotic conditions in cathode chamber. In addition, when the literature is examined, it is seen that the effects of process variables on the performance of oxidation and reduction processes and electricity generation efficiency have been examined individually. In this study, electricity generation and oxidation-reduction potential of waste were investigated by using the activated sludge from the beakers yeast production industry. In a dual-chamber MFC, raw waste activated sludge (WAS) /pre-treated WAS was used as electron donor in the anode chamber and azo dye solution was used as an electron acceptor in the cathode chamber. Optimization of the factors affecting electricity generation, organic matter removal in the anode chamber and decolorization in the cathode chamber were made using the Taguchi Experimental Design approach. The factors affecting the performance of the MFC system were revealed by ANOVA and Pareto analysis.\u003c/p\u003e"},{"header":"Material And Method","content":"\u003cp\u003eWAS and anaerobic sludge were obtained from the anaerobic and aerobic wastewater treatment plant in the factory that produces baker\u0026apos;s yeast (PAKMAYA A.Ş T\u0026uuml;rkiye).WAS taken from the return sludge line of the activated sludge process was used as substrate in the anode chamber and the anaerobic sludge taken from the filled column type anaerobic reactor was used as inoculum. The obtained WAS was used after increasing the solid content by sedimentation. Sunfix Red S3B (SR-S3B-Reactive anionic azo dye) was used as an electron acceptor in the cathode chamber without any pre-treatment. The chemical oxygen demand (COD), mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS), pH values of WAS and anaerobic sludge and the molecular structure of azo dye are given in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eThe properties of sludge and azo dye used in the study\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were conducted in a dual-chamber, 500 mL volume H-type glass MFC system. The anaerobic sludge and WAS were used both as an inoculum for the enrichment of exoelectrogenic bacteria on surface of the electrode and as an electron donor substrate, respectively. For the formation of an enriched mixture with exoelectrogenic bacteria and biofilm formation on the electrode surface, the anode chamber consists of a 400 ml mixture of 40% (v/v) WAS, 50% (v/v) anaerobic sludge, and 10% (v/v) anaerobic sludge supernatant was fed with sodium acetate (625 mg/L) and nutrient solution every 2 days for 30 days. Solution containing azo dye of 15 mg/L was used as the catholyte medium in all experiments in the experimental design, including the enrichment of exoelectrogens. A certain amount of supernatant from the mixture was removed then nutrients and acetate were added. Biofilm-covered electrodes were used in each experimental run in the experimental design. Fresh WAS as substrate and exoelectrogenic bacteria-rich mixture as inoculum was used at a ratio of 8/1 for each experiment run. During the experiments, WAS was used as raw and pre-treated substrate. The thermochemical pre-treatment process was carried out by adjusting the pH of the WAS to 10 with NaOH and boiling the WAS for 15 minutes. After pre-treatment, the pH of WAS was adjusted to around 7.4. Raw and pre-treated sludge were used with the addition of inoculum and nutrient solution. KCI was used to increase conductivity in experiments with high conductivity compared to the experimental design. The nutrients added for anode chamber at each experiment were 1 mL of NH\u003csub\u003e4\u003c/sub\u003eCI (193.75mg/L), 1 ml of MgCI\u003csub\u003e2\u003c/sub\u003e (62.5 mg/L), 0.5 mL of KCI (81.25 mg/L), 1 mL of NaCI (72.5 mg/L), 5 mL of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (2125 mg/L), 8 ml of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (2600 mg/L). In addition, 0.5 mL of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (212.5 mg/L)and 20 mL of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (6500 mg/L) were added to the cathode chamber as a buffer in each experiment.\u003c/p\u003e\n\u003cp\u003eIn all experiments, O\u003csub\u003e2\u003c/sub\u003e has swept away by passing N\u003csub\u003e2\u003c/sub\u003e gas through the anode chamber. According to the experimental design, in certain experiments, the cathode chamber was also swept with N\u003csub\u003e2\u003c/sub\u003e to remove O\u003csub\u003e2\u003c/sub\u003e. When N\u003csub\u003e2\u003c/sub\u003e was passed through the cathode and anode chambers, the chambers were closed throughout the experimental run. In the experiments where the cathode was aerobic, oxygen was supplied to the cathode chamber with the Tetra Aps 400 Aquarium Air pump. When air is pumped into the cathode environment, oxygen and azo dye are together as electron acceptors, and only azo dye becomes an electron acceptor when no air is pumped and an anaerobic environment is formed. Such a factor was included to determine the effect of azo dye and oxygen competing as electron acceptors on the system.\u003c/p\u003e\n\u003cp\u003eThe azo dye solution was used as a catholyte in MFC. The anode and cathode chambers are separated by a proton-permeable membrane (DupontTMNafion\u0026reg; 117)with dimension of 9.62 cm\u003csup\u003e2\u003c/sup\u003e treated for 24 hours at 40 \u003csup\u003e0\u003c/sup\u003eC with 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The pre-treated membrane was then preserved in ultrapure water. Plain carbon cloth electrode with the dimension of 22 cm\u003csup\u003e2\u003c/sup\u003e was used in the anode chamber, 0.3 mg/cm\u0026sup2; 40% Platinum on Vulcan Carbon Cloth Electrode (Pt CC), and plain carbon cloth electrodes (CC) were used in the cathode chamber according to the experimental design. The circuit between the electrodes is closed using conductive copper wire. The anode electrode was externally connected with the cathode electrode through the resistor. In the MFC system, in which azo dye is used as an electron acceptor, all runs were operated for about 3 days, since the maximum azo dye removal and stabilization of the MFC voltage were observed within the first 3 days. MFC system setup is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eAnalysis: Oxidation of the sludge mixture in the anode chamber was monitored by COD removal (%). Volatile solids and COD analysis were performed according to standard methods of 2540 and 5220 respectively (APHA and WEF, 2005). The reduction of azo dye and the generation of aromatic compounds in the cathode chamber were determined by spectrophotometer (Hach Lange DR-6000). The wavelength at which the maximum absorbance is observed, 541 nm, was used for azo dye determination. The calibration curve was created with standard azo dye solutions in the range of 2\u0026ndash;30 mg/L, the correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) value of the curve was obtained as 0.9998. The removal efficiency of both COD and azo dye was calculated as shown in the Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equ1\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$Removal Efficiency \\left(\\%\\right)= \\frac{{C}_{0}-{C}_{t}}{{C}_{0}}\\times 100$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003ewhere C\u003csub\u003e0\u003c/sub\u003e(mg/L) is the initial COD or azo dye concentration, and C\u003csub\u003et\u003c/sub\u003e(mg/L) is the COD or azo dye concentration at reaction time, t (h).\u003c/p\u003e\n\u003cp\u003eControl trials were carried out outside the MFC system at the same catholyte conditions to determine the possible azo dye removal mechanisms (such as adsorption on the carbon electrode surface) other than the electrochemical reduction of azo dye.\u003c/p\u003e\n\u003cp\u003eThe output voltage (V) was measured with a digital multimeter (UT181A True RMS Data logging Multimeter) at 10-minute intervals with the software connected to the computer. Voltage is measured over a fixed external resistor \u003cem\u003e(R\u003c/em\u003e\u003csub\u003e\u003cem\u003eext\u003c/em\u003e\u003c/sub\u003e), while current (I) is calculated from Ohm\u0026apos;s law according to Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) (Logan et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equ2\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e$$I={E}_{cell}/{R}_{ext}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eWhere \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003ecell\u003c/em\u003e\u003c/sub\u003e is the voltage measured over an external resistance (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eext\u003c/em\u003e\u003c/sub\u003e), \u003cem\u003eI\u003c/em\u003e is the electrical current.\u003c/p\u003e\n\u003cp\u003eThe power (\u003cem\u003eP\u003c/em\u003e) generation is calculated according to the Eq. (\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) and was normalized by the surface area of the electrode (I, in square meter).\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equ3\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e$$P={I*E}_{cell}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe Coulombic efficiency (%CE) is expressed as the ratio of the total Coulombs transferred from the substrate to the anode, to the maximum Coulombs that can be transferred. The total Coulombs obtained are determined by integrating the current over time, so that the Coulomb efficiency for a batch mode MFC operation, evaluated over a period t\u003csub\u003eb\u003c/sub\u003e, is calculated according to the Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) (Jia et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Logan et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equ4\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e$$CE \\left(\\%\\right)=\\frac{8{\\int }_{0}^{{t}_{b}}Idt}{F{V}_{an}{\\Delta }COD}\\times 100$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003ewhere \u003cem\u003eF\u003c/em\u003e is the Faraday\u0026apos;s constant, \u0026Delta; \u003cem\u003eCOD\u003c/em\u003e is the change in COD over time \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003ean\u003c/em\u003e\u003c/sub\u003e is the working volume of the anode chamber.\u003c/p\u003e\n\u003cp\u003eA power curve describing the power density as a function of current density is calculated from the polarization curve. The polarization curve was determined by the voltage drop by different external resistance between 100000 and 10 Ω.\u003c/p\u003e\n\u003cp\u003eElectrochemical characterization of MFC operated at optimum conditions was performed by cyclic voltammetry using a potentiostat (Gamry Reference 3000 Potentiostat). The CV measurement was performed during the period when the highest rates of biodegradation and electricity generation occurred. The reference electrode was placed in the anode chamber and the anode electrode was used as a working electrode against an Ag/AgCl reference electrode while the cathode electrode worked as a counter electrode. CV measurement was performed at 25 mV.\u003c/p\u003e\n\u003cp\u003eOptimization study\u003c/p\u003e\n\u003cp\u003eIn this study, the levels of operating conditions affecting oxidation-reduction efficiency and electricity generation potential in the MFC process were optimized using the Taguchi Experimental Design model. Design Expert (version 11) software was used for optimization. Taguchi experimental design methodology is widely used as an advantageous experimental design method, especially due to fewer experiments. The Taguchi design uses the loss function to measure performance characteristics that deviate from the set value. The value of the loss function is converted to the signal-to-noise (S/N) ratio. Optimum operating conditions are calculated from the S/N ratio of the results obtained from the studies designed with the Taguchi (Ross \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e). More than one S/N can be defined in the Taguchi Experimental Design Method. In this study, the S/N ratio, which expresses the \u0026ldquo;higher is better\u0026rdquo; situation, was used for all responses Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) (Aslan and \u0026Uuml;nal \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ramakrishnan and Karunamoorthy \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equ5\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ5\" name=\"EquationSource\"\u003e$$\\frac{S}{N}=-log10(\\frac{1}{n}\\sum _{i=1}^{n}\\frac{1}{{y}_{i}^{2}})$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003ey\u003c/em\u003e \u003csub\u003e\u0026nbsp;\u003cem\u003ei\u003c/em\u003e\u0026nbsp;\u003c/sub\u003e is the performance characteristic observed in the i\u003csup\u003eth\u003c/sup\u003e experiment for a given combination of control factor levels and \u003cem\u003en\u003c/em\u003e is the number of repetitions. The power density, azo dye removal efficiency, coulombic efficiency and COD removal efficiency were determined as response parameters in the optimization. Optimization of the four determined response parameters was performed together as a multi-optimization. The importance weights of all response parameters were accepted as equal. The factors affecting the MFC system and their levels were determined by considering the data in the literature and preliminary trials. The factors and levels used in the experimental design are given in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFactors and levels determined for Taguchi experimental design\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCode\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLevel 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLevel 2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCathode pH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.05 (pH of raw dye solution)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCathode Oxygen State\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnaerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnode Substrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePre-treated WAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWAS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExternal Resistance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 Ω\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000 Ω\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCathode electrode type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePt catalyzed carbon cloth (Pt-CC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlain carbon cloth\u003c/p\u003e\n \u003cp\u003e(CC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCathode Electrode surface area\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 cm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22 cm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCathode conductivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 \u0026micro;s/cm (Conductivity of raw dye solution )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1600\u0026micro;s/cm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eDuring the experimental study, the power density, azo dye removal, coulombic efficiency, and COD removal were evaluated according to other factors due to the same anode electrode being used.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003eOptimization of factors affecting the MFC system\u003c/p\u003e\n\u003cp\u003eIn the MFC system, which uses WAS and azo dye solution as the electron donor and electron acceptor, respectively, there are many operating parameters that affect the electricity generation and the treatability of the wastes. In MFC systems, the oxidation of the pollutant in the anode chamber and the reduction of the pollutant in the cathode chamber are equally important as the electricity generation efficiency. In this study, the electricity generation potential was evaluated by calculating the coulombic efficiency and power density, and the treatability of the waste was evaluated by measuring the azo dye and COD removal in catholyte and anolyte, respectively. The Taguchi L8 experiment design, which was created for the optimization of the operating parameters that will provide high waste treatability and high electricity production together, and the response parameters (Power density, coulombic efficiency, azo dye removal and COD removal) obtained from each run are given in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRun\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePower Density\u003c/p\u003e\n \u003cp\u003e(mW/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCoulombic Efficiency (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAzo Dye Removal\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCOD Removal (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTaguchi experimental design and obtained response parameters\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePt-CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.556\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1.254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.402\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.064\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePre-treated WAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePt-CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.845\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1.792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.570\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.103\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnaerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.584\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1.523\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.767\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.095\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnaerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePre-treated WAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1.773\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.413\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePre-treated WAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.846\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnaerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePt-CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.797\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.895\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.865\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.529\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnaerobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePre-treated WAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePt-CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e127.382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.668\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.615\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eAs can be seen from Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, depending on the experimental conditions, the maximum values of power density, coulombic efficiency, azo dye removal efficiency, and COD removal efficiency were 109.642 mW/m\u003csup\u003e2\u003c/sup\u003e, 8.081(%), 85.410(%), 24.615(%), respectively. Optimization was made to determine the conditions under which the four response parameters would be highest simultaneously.\u003c/p\u003e\n\u003cp\u003eIn order to understand the effect of the variables and to analyze them statistically, ANOVA was performed and coefficients such as F and P were examined (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The F values of the models were determined to be high enough as 267.42, 97.91, 82.83, 34.34 for power density, coulombic efficiency, azo dye removal, and COD removal, respectively. 0.05˂ P-values were determined. P values of the model were determined as 0.0037, 0.0101, 0.0120, and 0.0077 for power density, coulombic efficiency, azo dye removal and COD removal, respectively. P-value ˃ 0.10 means the model and factor terms are not significant, while 0.05˂ P-value shows the significance of the factor and model terms (Singh, Bhunia, and Dash \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eResponse parameters are explainable by obtained models if the models are statistically significant. The anode substrate and cathode conductivity for the power density, the anode substrate, cathode conductivity and the external resistance for the coulombic efficiency, the cathode pH, the external resistance and the cathode electrode type for azo dye removal, the anode substrate for COD removal had significant effects. High correlation coefficients were obtained from each model. R\u003csup\u003e2\u003c/sup\u003e and adjusted correlation coefficient (Adj. R\u003csup\u003e2\u003c/sup\u003e) values for all response parameters were determined to be over 95%. The high R\u003csup\u003e2\u003c/sup\u003e values shows the conformity between the estimated values and experimental data (Rostamiyan et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). The adequate precision is a measure of the range in the predicted response relative to the error of interest and is desired to be greater than 4 (Zinatizadeh et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Adequate precision values of models for all response parameters were determined to be higher than 4, and indicate an adequate signal. In order for the created statistical model to be compatible, it is desired that the R\u003csup\u003e2\u003c/sup\u003e value should be compatible with the adjusted R\u003csup\u003e2\u003c/sup\u003e and there should not be more than 0.2 difference between the adjusted R\u003csup\u003e2\u003c/sup\u003e and the estimated R\u003csup\u003e2\u003c/sup\u003e(Arslan-Alaton, Tureli, and Olmez-Hanci \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). It can be interpreted that R\u003csup\u003e2\u003c/sup\u003e, predicted R\u003csup\u003e2\u003c/sup\u003e and adjusted R\u003csup\u003e2\u003c/sup\u003e values are compatible.\u003c/p\u003e\n\u003cp\u003eThe models are presented in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The positive values of the coefficients in equations represent synergistic effects, whereas the negative values represent an antagonistic effect. It may be seen in the model\u0026rsquo;s equation that all parameters had an antagonistic effect on the power density. According to the model equations, cathode pH and cathode electrode surface area have a synergistic effect on coulombic efficiency. Substrate type and cathode electrode type show a synergistic effect on azo dye removal efficiency.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab4\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eANOVA results obtained for the determined response parameters\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"7\"\u003e\n \u003cp\u003ePower Density\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFit Statistics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e267.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9985\u003c/p\u003e\n \u003cp\u003eAdj. R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9948\u003c/p\u003e\n \u003cp\u003ePredicted R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9761\u003c/p\u003e\n \u003cp\u003eAdeq Precision\u0026thinsp;=\u0026thinsp;35.0700\u003c/p\u003e\n \u003cp\u003ePower density (mW/m\u003csup\u003e2\u003c/sup\u003e):56.22\u0026ndash;51.48\u0026times;C-4.32\u0026times;D-3.43\u0026times;E-2.98\u0026times;F-6.49\u0026times;G\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1297.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1722\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eCoulombic Efficiency\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9959\u003c/p\u003e\n \u003cp\u003eAdj. R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9858\u003c/p\u003e\n \u003cp\u003ePredicted R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9349\u003c/p\u003e\n \u003cp\u003eAdeq Precision\u0026thinsp;=\u0026thinsp;25.6918\u003c/p\u003e\n \u003cp\u003eCoulombic efficiency (%) :2.80\u0026thinsp;+\u0026thinsp;0.2713\u0026times;A-1.78\u0026times;C-1.58\u0026times;D\u0026thinsp;+\u0026thinsp;0.1996\u0026times;F-1.22\u0026times;G\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e213.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e168.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0099\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eAzo Dye Removal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9952\u003c/p\u003e\n \u003cp\u003eAdj. R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9852\u003c/p\u003e\n \u003cp\u003ePredicted R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9231\u003c/p\u003e\n \u003cp\u003eAdeq Precision\u0026thinsp;=\u0026thinsp;23.83\u003c/p\u003e\n \u003cp\u003eAzo dye removal (%) :55.91\u0026ndash;21.47\u0026times;A-3.55\u0026times;B\u0026thinsp;+\u0026thinsp;1.63\u0026times;C-8.66\u0026times;D\u0026thinsp;+\u0026thinsp;14.75\u0026times;E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e248.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3536\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e117.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eCOD Removal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9786\u003c/p\u003e\n \u003cp\u003eAdj. R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9501\u003c/p\u003e\n \u003cp\u003ePredicted R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.8480\u003c/p\u003e\n \u003cp\u003eAdeq Precision\u0026thinsp;=\u0026thinsp;14.5084\u003c/p\u003e\n \u003cp\u003eCOD removal efficiency (%) :18.88-0.8721\u0026times;B-3.37\u0026times;C-0.7866\u0026times;E-0.8282\u0026times;F\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0684\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0864\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0770\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThe values obtained for each response parameter and the predicted values of the model are compatible. Graphs showing the relationships between the actual values and the values predicted by the model are given in the supplementary materials (Supplementary material Fig. S1).\u003c/p\u003e\n\u003cp\u003ePareto Analysis\u003c/p\u003e\n\u003cp\u003eIn MFC system, the operating conditions in the anolyte and catholyte affect the coulombic efficiency, power density, azo dye and COD removal. The effects of the factors on the response parameters are different. The Pareto chart checks for statistical significance by presenting the effect of factors on responses. The Pareto graphs plotted with the t-values of the effects versus the parameters affecting each response are shown in Fig. 2. The t values of the bars in the graphs represent the square root of the F values in the ANOVA. There are two different t-value of effects which are the Bonferroni limit line and the t-value limit line. Factors whose effect is above the Bonferroni line are considered to be extremely significant, while those whose effect is between the Bonferroni line and the t-value limit are considered likely to be significant. Factors whose effect is below the t-value limit are considered statistically insignificant (Asem, Nawawi, and Jimat \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Boateng, Yang, and Li \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn Pareto analysis, orange-colored bars indicate positive effects of factors, and blue- colored bars indicate negative effects of factors (Abdulredha, Hussain, and Abdullah 2019). For power density, the t-value of the substrate type factor was above the Bonferroni limit line, which indicates that the substrate type has the highest effect. If the substrate is used without pre-treatment, the power density will decrease. The t value of all other factors is below the t- value limit line, indicating that these factors are statistically insignificant (Fig.\u0026nbsp;2. (a)).\u003c/p\u003e\n\u003cp\u003eFor coulombic efficiency, the t-values of both anolyte substrate and external resistance factors are above the Bonferroni line which indicated that both factors were extremely significant factors. The use of the substrate without pre-treatment and the increase in external resistance negatively affect the coulombic efficiency.\u003c/p\u003e\n\u003cp\u003eThe t-value of the catholyte conductivity factor is between Bonferroni and the t-value limit line, indicating that conductivity is an important factor for coulombic efficiency (Fig.\u0026nbsp;2 (b)).\u003c/p\u003e\n\u003cp\u003eAs seen from the graph, pH shows to have the highest effect on the azo dye removal, followed by electrode type and external resistance. The blue color of the pH effect indicates that an increase in pH from 3.0 to 6.05 will decrease the dye removal efficiency. At the same time, it was determined that higher azo dye removal efficiency would be achieved by using the plain carbon electrode (Fig.\u0026nbsp;2 (c)).\u003c/p\u003e\n\u003cp\u003eIt was determined from Fig. 2 (d) that the most effective parameter on COD removal was the substrate type. Pre-treatment of the substrate increased the COD removal efficiency.\u003c/p\u003e\n\u003cdiv align=\"left\" class=\"colspec\"\u003eEvaluation of three-dimensional (3D surface) graphics\u003c/div\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of operating parameters on response parameters were evaluated with 3D graphics. Thermochemical pre-treatment of WAS in the anode chamber increased the power density from 1.5569 mW/m\u003csup\u003e2\u003c/sup\u003e to 127.382 mW/m\u003csup\u003e2\u003c/sup\u003e when the cathode was anaerobic, the external resistance was 100 ohms, 22 cm\u003csup\u003e2\u003c/sup\u003e PtCC and the cathode conductivity was 20 \u0026micro;s/cm (Fig. 3 (a)). It also increased COD removal from 15\u0026ndash;24.6% when the cathode was anaerobic, the external resistance was 100 ohms, 22 cm\u003csup\u003e2\u003c/sup\u003e CC and the cathode conductivity was 20 \u0026micro;s/cm (Fig. 3 (d)). Chae et al.(2009) stated in their study that methanogenesis is compatible with higher resistance conditions, because methanogens are more likely to use the substrate when exoelectrogen activity is reduced. Reducing the external resistance from 600 Ω to 50 Ω increased the coulombic efficiency (CE) from 32\u0026ndash;42% (Chae et al. \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). Therefore, lowering the external resistance may be a way to improve MFC performance as it can increase CE by reducing electron losses (Chae et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, it was observed that the decrease in external resistance increased the coulombic efficiency (Fig.\u0026nbsp;3. (b)). However, it was observed that the pH change in the cathode chamber did not affect the power density, coulombic efficiency, and COD removal. The conditions with the highest azo dye removal in the cathode chamber were characterized as plain carbon cloth electrode and pH 3. The azo dye removal decreased from 85.41\u0026ndash;8.57% when the pH was increased with the cathode being anaerobic, the anode substrate was pre-treated, external resistance was 1000 ohms, 12 cm\u003csup\u003e2\u003c/sup\u003ePt-CC and the cathode conductivity was 20 \u0026micro;s/cm(Fig.\u0026nbsp;3(c)).\u003c/p\u003e\n\u003cp\u003eConfirmation experiment and evaluation of catholyte reduction\u003c/p\u003e\n\u003cp\u003eOptimum operating conditions of Taguchi experimental design determined as cathode pH of 3.0, cathode oxygen status of anaerobic, anode substrate of pre-treated, external resistance of 100 Ω, cathode electrode type of plain carbon, cathode electrode surface of 22 cm\u003csup\u003e2\u003c/sup\u003e, cathode conductivity of 20 \u0026micro;s/cm. The presence of O\u003csub\u003e2\u003c/sub\u003e together with the azo dye solution in the cathode chamber, which has a higher electron acceptor property than the azo dye, causes the transfer of electrons to O\u003csub\u003e2\u003c/sub\u003e instead of azo bonds. In the absence of oxygen, the transfer of electrons to the azo bonds in the Cathode chamber is facilitated and higher color removal is achieved. The fact that the cathode oxygen state is anaerobic under optimum conditions also confirms this. A confirmation experiment was performed to verify the optimum MFC conditions. With the confirmation experiment, 177.031 mW/m\u003csup\u003e2\u003c/sup\u003e power density, 7.509% coulombic efficiency, 91.266% azo dye removal efficiency, and 21.612% COD removal efficiency were obtained. It was determined that all response parameters, except power density, were within the 95% confidence interval (Tablo 5). The fact that the power density is above the confidence interval is evaluated positively in terms of MFC performance. The voltage plot measured against 100 Ω resistance at optimum MFC conditions is given in the supplementary material Fig. S2.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab5\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResponse parameter values obtained under optimum conditions, and estimated confidence intervals.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResponse\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePredicted Mean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% confidence interval\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActual data\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePower density (mW/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112.101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.036-127.167\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e177.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoulombic efficiency (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.3033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.021\u0026ndash;8.585\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.509\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAzo dye removal efficiency (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.714\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.353-117.075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.266\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCOD removal efficiency (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.278\u0026ndash;23.726\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.612\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eYusoff et al. obtained a higher COD removal efficiency of up to 85% at the end of 10 days of operation with microwave pre-treated sludge. In the same study, power density and coulombic efficiency were obtained as 42\u0026thinsp;\u0026plusmn;\u0026thinsp;3 and 6.3%, respectively (Mohd Yusoff et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Jayashree et al. improved COD removal up to 54% by applying low-temperature thermochemical pre-treatment to dairy WAS. The power density of the MFC had increased from 0.5 W/m\u003csup\u003e3\u003c/sup\u003e (raw sludge) to 0.715 W/m\u003csup\u003e3\u003c/sup\u003e (pre-treated sludge) (Jayashree et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Ayol et al., on the other hand, obtained a power density of 312.98 mW/m\u003csup\u003e2\u003c/sup\u003e in an enhanced sludge stabilization coupled with amicrobial fuel cell with graphite electrodes (Ayol et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).The reason for the relatively low values obtained in this study can be explained by the application of more moderate operating conditions. In addition, the optimum conditions determined are those that maximize the electricity generation efficiency and the treatability of the waste in the anode/cathode chamber simultaneously.\u003c/p\u003e\n\u003cp\u003eIn addition to the anolyte and catholyte conditions, the surface properties of the electrode used to affect the reactions that will take place in the catholyte. It has been extensively accepted that surface-active oxygen functional groups on carbon felt are effective in catalyzing redox reactions of active species and increasing the wettability of carbon felt (Kim et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). The reduction potential of the cathode chamber was evaluated by the removal of azo dye. The catholyte of the MFC system operated under optimum conditions was compared with the control trial. The ultraviolet-visible (UV-VIS) spectrum of the catholyte at the optimum conditions and the control trial show the same trend (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eAromatic amines and azo bonds are the two main components in azo dyes. In the spectrophotometric analysis, the absorption peaks in the VIS region correspond to the azo bond, while the absorption peak in the UV region shows the aromatic compounds. Peak variation of aromatic compounds and azo bonds in the UV-VIS spectra indicates the decreasing peak of the azo bond and the increase in aromatic amines. Possible intermediates produced after disruption of the azo bond may be benzene, naphthalene-based sulfonated compounds, and triazine-based compounds (Oon et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe peak seen at 190 nm in the raw azo dye shifted towards 250\u0026ndash;260 nm at the end of 70th hour in the control and MFC process. The peak seen at 290 nm in the raw dye disappeared completely. While the azo dye concentration at the end of the MFC process at 541 nm decreased by 91.26%, a decrease of 49.69% was observed in the control trial. After decolorization, the peak at 247 nm, suggesting that azo dye was reduced to its hydrazine derivative (Ding et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(-N=N-\\)\u003c/span\u003e\u003c/span\u003edouble bond was reduced to hydrozo or amine Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e)-(\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e)(Liu et al. \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equ6\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ6\" name=\"EquationSource\"\u003e$$-N=N-+2{e}^{-}+2{H}^{+}\\to -NH-NH-$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Equation\" id=\"Equ7\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ7\" name=\"EquationSource\"\u003e$$-N=N-+4{e}^{-}+4{H}^{+}\\to -N{H}_{2}+N{H}_{2}-$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e7\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eAn increase in absorbance density was observed at 265 nm. This indicates that the reduction product (sulfonic acid) associated with the degradation of azo dyes may be present(Liu et al. \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn the light of this information, it is thought that the change in absorbance of the control experiment may be caused by the reactions of active functional groups on the carbon cloth. In the MFC system, in addition, electrons transmitted from the anode chamber were effective in the azo dye reduction process.\u003c/p\u003e\n\u003cp\u003ePolarization and power curve evaluation\u003c/p\u003e\n\u003cp\u003eThe electricity generation potential varies with the characteristics of the electron donor, electron acceptor, electrode material, and catholyte used. The polarization curve was analyzed when stable voltage was obtained in the optimum experiment. The curve is obtained as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e with the voltage values obtained across 10 Ω to 100000 Ω external resistance. The maximum power density produced under optimum conditions was obtained as 145.11 mW/m\u003csup\u003e2\u003c/sup\u003e. The internal resistance of the MFC system was obtained as 243.3 Ω. The internal resistance of the MFC system was obtained as 243.3 Ω. Xiao et al. found the internal resistance to be 371 Ω in the two-chamber MFC system using sewage sludge as a substrate (Xiao et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eMaximum power density is not the main goal in MFC systems. In addition, it is aimed to ensure effective waste removal. The high internal resistance value obtained may be due to the fact that WAS was used as a substrate in all experiments instead of an easily degradable substrate, and the higher density of the sludge compared to the MFCs using wastewater increasing electron transport losses.\u003c/p\u003e\n\u003cp\u003eCyclic voltammetry\u003c/p\u003e\n\u003cp\u003eCyclic voltammogram is used as an electrochemical technique to describe electron transfer interactions at the electrode-biomass/biofilm-liquor interface in the anode chamber (L\u0026oacute;pez Zavala et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, the CV measurement was carried out under optimum experimental conditions, where the MFC produces a stable voltage. A redox cycle was obtained in the voltammogram shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, confirming the important role played by the anaerobic microorganism in transferring electrons to the anode. The current density data were correlated with the oxidation and reduction reactions occurring at the working electrode. The highest anode potential was associated with the largest values of current density corresponding to the oxidation of waste activated sludge. The highest cathode potential corresponded to the lowest negative current density values associated with water reduction and hydrogen gas production. An anodic peak at \u0026minus;\u0026thinsp;183.2 mV and a cathodic peak at \u0026minus;\u0026thinsp;181.2 mV were visible in the CV curve. In addition, the small size of the peaks in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e may be due to the fact that no redox mediator was used in this study. Mediators have an important effect on the anodic or cathodic current in MFC systems. Certain microorganisms may also support the electron transfer process (Danish Khan et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, besides the electricity generation potential in the MFC system, WAS and azo dye treatment in the anode and cathode chambers, respectively, were investigated. The MFC system was optimized to determine operating conditions that would maximize COD removal in the anode chamber, azo dye removal in the cathode chamber, power density, and coulombic efficiency. Taguchi Experimental Design approach was used in optimization. The results obtained are summarized below;\u003c/p\u003e\n\u003cp\u003e- The relationship between the experimental results and the results predicted by the statistical model was found to be compatible. Optimum MFC operating conditions have been determined as pH of 3.0, cathode oxygen status of anaerobic, anode substrate of pre-treated, external resistance of 100 Ω, cathode electrode type of plain carbon, cathode electrode surface of 22 cm\u003csup\u003e2\u003c/sup\u003e, cathode conductivity of 20 \u0026micro;s/cm. With the confirmation experiment performed under these conditions, 177.031 mW/m\u003csup\u003e2\u003c/sup\u003e power density, 7.50% coulombic efficiency, 91.26% azo dye removal, and 21.61% COD removal were obtained.\u003c/p\u003e\n\u003cp\u003e- It has been observed with the UV-VIS spectrum that the azo bonds of the azo dye that peak in the visible region are broken and the aromatic compounds increase in the UV region under optimum conditions.\u003c/p\u003e\n\u003cp\u003e- In Pareto analysis, it was determined that the anode substrate type was the most effective factor in power density, coulombic efficiency, and COD removal. It has been observed that sludge degradation and electricity generation potential have improved by applying thermochemical pre-treatment to WAS. In the cathode chamber, it was determined that the most effective parameter in the removal of azo dye was the cathode pH, and the highest dye removal could be achieved at pH 3.0.\u003c/p\u003e\n\u003cp\u003e- The polarization curve was performed while the optimum experiment was at a steady state. It has been determined that the maximum power density is 145.11 mW/m\u003csup\u003e2\u003c/sup\u003e and the internal resistance is 243.3 Ω. The cyclic voltammogram performed with the optimum experiment was obtained confirming the oxidation and reduction reactions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowlegment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Authors thank the Kocaeli University Scientific Research Projects coordination, BAP for their financial support to the national project (no: FBA-2021-2672).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConfict of interest\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdulredha MM, Hussain SA, Abdullah LC, Separation Emulsion via Non-Ionic Surfactant: An Optimization. Processes 7(6):382. https://doi.org/10.3390/PR7060382.\u003c/li\u003e\n\u003cli\u003eAPHA, A. and and WEF (2005) \u003cem\u003eStandard methods for the examination of water and wastewater\u003c/em\u003e. 21st ed. Am Public Work Assoc. 21st ed. Washington D.C.: APHA-AWWA-WEF.\u003c/li\u003e\n\u003cli\u003eArslan-Alaton I, Tureli G, Olmez-Hanci T (2009) Treatment of azo dye production wastewaters using Photo-Fenton-like advanced oxidation processes: Optimization by response surface methodology. J Photochem Photobiol A Chem 202(2\u0026ndash;3):142\u0026ndash;153. https://doi.org/10.1016/J.JPHOTOCHEM.2008.11.019.\u003c/li\u003e\n\u003cli\u003eAsem M, Nawawi WMFW, Jimat DN (2018) Evaluation of water absorption of polyvinyl alcohol-starch biocomposite reinforced with sugarcane bagasse nanofibre: Optimization using Two-Level Factorial Design. IOP Conf Ser Mater Sci Eng 368(1):012005. https://doi.org/10.1088/1757-899X/368/1/012005.\u003c/li\u003e\n\u003cli\u003eAslan N, \u0026Uuml;nal I (2011) Multi-response optimization of oil agglomeration with multiple performance characteristics. Fuel Process Technol 92(6):1157\u0026ndash;1163. https://doi.org/10.1016/j.fuproc.2010.05.029.\u003c/li\u003e\n\u003cli\u003eAyol A \u003cem\u003eet al.\u003c/em\u003e (2021) Enhanced sludge stabilization coupled with microbial fuel cells (MFCs). Int J Hydrogen Energy 46(57):29529\u0026ndash;29540. https://doi.org/10.1016/J.IJHYDENE.2020.10.143.\u003c/li\u003e\n\u003cli\u003eBabanova S et al. (2020) Continuous flow, large-scale, microbial fuel cell system for the sustained treatment of swine waste. Water Environ Res 92(1):60\u0026ndash;72. https://doi.org/10.1002/wer.1183.\u003c/li\u003e\n\u003cli\u003eBakhshian S, Kariminia HR, Roshandel R (2011) Bioelectricity generation enhancement in a dual chamber microbial fuel cell under cathodic enzyme catalyzed dye decolorization. Bioresour Technol 102(12):6761\u0026ndash;6765. https://doi.org/10.1016/j.biortech.2011.03.060.\u003c/li\u003e\n\u003cli\u003eBoateng ID, Yang XM, Li YY (2021) Optimization of infrared-drying parameters for Ginkgo biloba L. seed and evaluation of product quality and bioactivity. Ind Crops Prod 160:113108. https://doi.org/10.1016/J.INDCROP.2020.113108.\u003c/li\u003e\n\u003cli\u003eCai J, Qaisar M, Sun Y (2020) Effect of external resistance on substrate removal and electricity generation in microbial fuel cell treating sulfide and nitrate simultaneously. Environ Sci Pollut Res 27(1):238\u0026ndash;249. https://doi.org/10.1007/S11356-019-06960-8/FIGURES/7.\u003c/li\u003e\n\u003cli\u003eCecconet D et al\u003cem\u003e.\u003c/em\u003e (2018) Agro-food industry wastewater treatment with microbial fuel cells: Energetic recovery issues. Int J Hydrogen Energy 43(1):500\u0026ndash;511. https://doi.org/10.1016/J.IJHYDENE.2017.07.231.\u003c/li\u003e\n\u003cli\u003eChae KJ et al. (2009) Effect of different substrates on the performance, bacterial diversity, and bacterial viability in microbial fuel cells. Bioresour Technol 100(14):3518\u0026ndash;3525. https://doi.org/10.1016/J.BIORTECH.2009.02.065.\u003c/li\u003e\n\u003cli\u003eChae KJ et al. (2010) Methanogenesis control by employing various environmental stress conditions in two-chambered microbial fuel cells. Bioresour Technol 101(14):5350\u0026ndash;5357. https://doi.org/10.1016/J.BIORTECH.2010.02.035.\u003c/li\u003e\n\u003cli\u003eChoi J, Ahn Y (2013) Continuous electricity generation in stacked air cathode microbial fuel cell treating domestic wastewater. J Environ Manage 130:146\u0026ndash;152. https://doi.org/10.1016/J.JENVMAN.2013.08.065.\u003c/li\u003e\n\u003cli\u003eDanish Khan M, et al. (2015) Bioelectricity Generation and Bioremediation of an Azo-Dye in a Microbial Fuel Cell Coupled Activated Sludge Process. 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Water Res 40(17):3193\u0026ndash;3208. https://doi.org/10.1016/J.WATRES.2006.07.005.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Azo dye, electricity production, Microbial fuel cell, optimization, waste activated sludge, waste treatment","lastPublishedDoi":"10.21203/rs.3.rs-2056699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2056699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn microbial fuel cells (MFC), oxidation and reduction processes occur simultaneously. In this study, the operating conditions affecting oxidation-reduction and electricity generation of MFC were optimized using the Taguchi Experimental Design model. Optimization was carried out for maximum power density, coulombic efficiency, azo dye removal and COD removal. With the determined optimum conditions (cathode pH of 3.0, cathode oxygen status of anaerobic, anode substrate of pre-treated, external resistance of 100 Ω, cathode electrode type of plain carbon, cathode electrode surface of 22 cm\u003csup\u003e2\u003c/sup\u003e, cathode conductivity of 20 \u0026micro;s/cm), 177.031 mW/m\u003csup\u003e2\u003c/sup\u003e power density, 7.50% coulombic efficiency, 91.266% azo dye removal efficiency and 21.612% COD removal efficiency were obtained. From the Pareto analysis, it was determined that the power density, coulombic efficiency and COD removal efficiency were most affected by the substrate type at the anode, and the azo dye removal was most affected by the catholyte pH. With the polarization curve, it has been determined that the maximum power density is 145.11 mW/m\u003csup\u003e2\u003c/sup\u003e and the internal resistance of the optimum MFC system is 243.3 Ω. The cyclic voltammogram performed with the optimum experiment was associated with oxidation and reduction reactions.\u003c/p\u003e","manuscriptTitle":"Waste Activated Sludge Oxidation and Azo Dye Reduction in Microbial Fuel Cell: Optimization of process conditions for high electricity generation and waste treatability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-15 17:11:01","doi":"10.21203/rs.3.rs-2056699/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68c407b4-e2d9-41fd-93d8-998f2d3c05f5","owner":[],"postedDate":"September 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-29T21:29:16+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-15 17:11:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2056699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2056699","identity":"rs-2056699","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}