Biodegradation of Congo Red Using Co-Culture Anode Inoculum in a Microbial Fuel Cell

Abstract

Congo red is an azo dye widely used as a colouring agent in textile industries. It is a serious threat due to its carcinogenic effects. Its degradation has been challenging due to its complex yet stable structure. The present study was aimed to investigate the effective degradation of Congo red by bioremediating bacteria isolated from different environments. To investigate predominant microorganisms that degrade Congo red and its functions in microbial fuel cells (MFCs), strains isolated from cow dung (Enterococcus faecalis SUCR1) and soil (Pseudomonas aeruginosa PA1_NCHU) were used as a co-culture inocula. The remarkable results establish that E. faecalis as an excellent microbial source for the biological degradation of dye-contaminated wastewater treatment alongside bioactive treating wastewater with varied concentrations of congo red dye. The highest efficiency percentage of dye degradation was 98% after 3 days of incubation at pH 7 and 37 °C, whereas findings have shown that the decolorization at pH 5 and 6 was lower at 66% and 83.3%, respectively, under the same incubation conditions. Furthermore, the co-culture of E. faecalis SUCR1 and P. aeruginosa at a 1:1 ratio demonstrated improved power generation in MFCs. The maximum power density of 7.4 W/m³ was recorded at a 150 mg L⁻¹ concentration of Congo red, indicating that the symbiotic relation between these bacterium resulted in improved MFCs performance simultaneous to dye degradation.

1. Introduction

Approximately 10–15% of dyes from various textile industries are released directly into water bodies [1]. India is the world’s second-largest producer of paint and artificial pigments. Thus, the total amount of dye discharged, considering the various dyes, and artificial coloring agents cross the benchmark of over 80,000 tons of annual production. Because of their low cost and simple methods, azo dyes are widely utilized in the textile and paper industries to make the market more lucrative and thus gain a more significant economic output [2]. The large volume of waste in these industries severely threatens the environment and ecosystem. Many of these dyes, such as Congo red, are made with a benzidine ring, which has been popularly regarded as a carcinogenic compound [3]. Industries’ demand for dye colorants is constantly increasing [4]. Textile dyes significantly contribute to environmental contamination, aesthetic degradation, eutrophication, and alarming concerns regarding the aquatic ecosystem. Even low concentrations of dye pigments, such as 10–50 mg L⁻¹, can contribute to aesthetic pollution [5].

To tackle the degradation of residual azo dyes in wastewater, three widely employed methods are: physical, chemical, and biological. Adsorption, flocculation, electrocoagulation, precipitation, ozonation, and irradiation are examples of physical and chemical treatments [6]. Physical methods, such as adsorption, ion exchange, and membrane filtering, have been used to remove the dyes. Such physical methods have two significant drawbacks: they are most effective when the amount of wastewater is minimal and can be further broken down into a few smaller components difficult to digest. A solute (adsorbate) is bound to a solid surface by forces of attraction during the molecular process of adsorption (adsorbent) [7]. Ion exchange may be used to remove anions and cations from dyeing process discharge by running sewage over beds of ion exchange resins, in which certain undesired cations or anions in the polluted water are swapped for sodium or hydrogen ions inside the resin. Advanced oxidation is a synthetic decolorization procedure that incorporates the combined effect of many chemical oxidations. It is an excellent decolorization procedure because it can eliminate toxic compounds and dyes in unusual settings. Unfortunately, this is an expensive operation that results in undesirable metabolic effects. It is also pH-dependent, which implies that it requires specific pH values and therefore only works in specific settings [8]. However, this technique failed to degrade the dyes, and ultimately there is always a chance of the dyes leaching. In addition, the major drawback is that they also produce the humongous quantities of sludge, leading to a secondary contamination. Apart from these technologies being highly complex, they are extravagantly costly while also resulting in the discharge of waste, which becomes the primordial cause of secondary contamination [9].

The most widely used method for dye degradation is chemical, starting with advanced oxidation techniques that have the disadvantage of causing precipitated sludge but can oxidize a wide range of chemicals, including inorganic compounds found in industrial water. Additionally, there are incredibly powerful chemical oxidation processes that can even use ozone molecules to disassemble dye molecules with double bonds and intricate aromatic rings [10]. However, utilizing these substances results in negative side effects, and are high expensive. Finally, there are numerous associative improved oxidation techniques. It can also be divided into a few smaller pieces. A synthetic decolorization process called advanced oxidation integrates the cumulative impact of numerous chemical oxidations [11] It is pH-dependent, which suggests that it needs particular pH ranges and can only operate in certain environments. Due to the electrochemical reduction method’s low yield in contaminant breakdown when compared to direct and indirect electro-oxidation techniques, it has only been explored in a few studies [12]. This method is particularly well adapted for the cleanup of highly colored wastewaters, such as residual pad-batch color baths that include reactive dyes. The reduction of the dye results in the production of hydrazine and amino compounds overall. It is a practical method for removing both soluble and insoluble dyes, but because it produces hazardous chemicals and needs energy, the procedure is more expensive and less efficient than other methods due to higher flow rates. In order to remove colors from waste water, the Fenton method requires the use of Fenton’s reagent, which is a catalyst and H₂O₂ mixture [13]. Ozonation is a very effective way to remove color. This technique employs ozone, a gaseous product of oxygen that is utilized to remove dye particles. This system’s main benefit is that it does not produce any silt [10]. However, ozone is only employed in a few specific situations because of its short lifespan (about 20 min), the production of hazardous byproducts, instability, and expensive expense. Biological approaches are the newest entry in the field of dye degradation. A sustainable method to remove dye from textile waste with the lowest possible cost and shortest possible working time is using biological approaches for the degradation of dye through biological phenomena, such as bioremediation. The use of basic biological technology to break down wastewater from the textile industry has been successful. Biological degradation is more economical, environmentally secure, and results in less sludge than other procedures. Due to the connection, namely the chromophoric group, being broken, it enables dyestuffs to break down into less hazardous inorganic compounds and aids in decolorization. It is subdivided into smaller pieces. Adsorption is a cheap and effective way to remove dye from water. Activated carbon is the primary component utilized in the adsorption process. However, its extensive cost and renewal concerns preclude its widespread use. On the other hand, a different technique for bio-adsorptive color removal is adsorption by biomaterial.

Conventional methods use either microbes or immobilized enzymes as catalysts for the degradation of dyes. The performance of the enzymes, such as azo reductase, tyrosinase, lignin peroxidase, and laccase, has been investigated. Azo reductases help in catalyzing the reductive cleavage (that is, the triple nitrogen bonds of azo dyes) to produce the colorless aromatic amine products [14]. Lignin peroxide plays an important role in catalyzing the reduction of dyes, such as Procion Brilliant Blue HGR, Acid Red 119, Ranocoid Fast Blue, and Navidol Fast Black [15]. Laccase helps in decolorizing the azo dyes without involving the direct cleavage of the azo bonds. This helps in avoiding the development of toxic aromatic amines. Tyrosinase enzymes help in catalyzing the oxidation of phenols [16]. Manganese peroxides also help in decolorizing azo dyes and phthalocyanine complexes in Mn²⁺ unconventional manner. This treatment in the presence of enzymes helps in the catastrophic destruction of the chromophoric groups and in the chemical nature and structure of the dye [17].

A diverse range of microbes, such as bacteria, fungus, actinomycetes, algae, and others, can decolorize the azo dyes. When certain microorganisms are present, the decolorization efficacy of dyes is also dependent on the surrounding conditions and metabolic processes. As a result, it is critical to comprehend the mechanisms involved in the decolorization methods and to research the destruction by various microorganisms in order to provide a proper environment for microorganisms that contribute towards the decolorization of these dyes to live [18]. The anaerobic dye destruction of effluent is a potential approach, since it is an unspecific azo-dye degradation process that is quite straightforward. During direct enzymatic reactions, unspecific azo-reductase bacteria catalyze the azo dye reduction. The majority of the bacteria that breakdown dyes in aerobic settings cannot use the dye as a carbon source and instead need another carbon source. Only a few bacteria can use azo compounds as their only carbon source and thrive. Some of these bacteria, such as Pigmentiphaga kullae K24 and Xenophilus azovorans KF 46, can break –N=N– bonds and use amines to grow. Aerobic bacteria have oxidoreductive enzymes and can symmetrically or asymmetrically cleave the dye molecules. In addition, they have the potential to initiate reactions such as deamination, desulfonation, hydroxylation, etc.; thus, anaerobic bacteria can degrade a variety of dye structural forms. Bioaccumulation is the process of toxicant uptake by living cells, whereas biosorption is the passive reception of toxins by dead or inactive biological materials. A notable advantage of biosorption over bioaccumulation is that live organisms are not necessary for the continuous treatment of particularly dangerous effluents.

Microbial fuel cells (MFCs) are bioelectrochemical systems that use microorganisms to transform chemical energy into electricity [19] using bacteria as catalysts. Electrons are produced by microbial oxidation of the organic substrate. These free electrons are then passed through external load before reducing into cathode. The fundamental mechanism of producing electricity depends on electrochemically active bacteria in MFCs [20]. These inocula include electroactive microorganisms that can be seen evolving during MFC operation (

Table 1. List of microorganisms efficiently able to degrade Congo red dye.

Microorganism	References
Rhodopseudomonas palustris	[23]
Oudemansiella canarii	[24]
Phanerochaete chrysosporium	[25]
Pichia sp.	[26]
Shewanella xiamenensis BC01	[27]
Dietzia sp. DTS26	[28]
Aspergillus niger	[29]
Acinetobacter baumannii YNWH 226	[30]
Rhodococcus strain UCC 0010	[31]
Chryseobacterium geocarposphaerae DD3	[32]

). The anode has been treated with electroactive bacteria that can harvest electrons. Inside the anolyte, they break down organic molecules, producing protons and CO₂ as byproducts [21]. Oxygen reduced into water molecules in the cathode compartment, wherein electrons are produced in the anode through an electrical circuit [22].

Co-cultures favor the symbiotic relationships between microbial species that drive MFC functioning while providing opportunities to improve technical applications. Co-culturing has shown an excellent outcome in drug delivery and waste degradation [33]. Overall, this allows a higher level of throughput testing and provides a more extraordinary view for monitoring the actions and effects of cell–cell and cell–substrate interactions [34]. A study reported that certain cultures thrive well and deliver greater levels of practical metabolic activities as per desires only when co-cultured with another species and not in a monoculture population; thus, this can be utilized as a critical aspect to increase the overall efficiency of the process [35]. The major factors affecting MFC performances include dye concentration, pH, anolyte ionic concentration, etc.

In the present study, the co-culture method was employed to degrade Congo red in the anode chamber of the MFC. Bacteria were isolated, identified, and used as biocatalysts at the anode chamber. Furthermore, the specified co-cultures of the isolated Enterococcus faecalis SUCR1 and Pseudomonas aeruginosa PA1_NCHU were explored to assess their performance in MFCs and wastewater treatment containing different concentrations of Congo red (10, 50, 75, 100, 150 mg/L). The effect of parameters such as dye concentration and anolyte pH were evaluated on dye removal percentage and power output.

2. Results

2.1. Isolation and Screening of Pseudomonas Aeruginosa PA1_NCHU and Enterococcus faecalis SUCR1

The enrichment and isolation of cultures were carried out by streaking on minimal salt agar plates for five to ten generations. The 16S rRNA sequence showed them to be 99.9% closely identical to Enterococcus faecalis 27688 and Pseudomonas aeruginosa PA1_NCHU PA1_NCHU from cow dung and soil samples, respectively. The BLAST program was used to compare bacterial DNA sequences in order to determine which species in the database were the most closely related. The query sequence and the species with accession number LR962620.1 had the best match for Enterococcus faecalis 27688 and species with accession number CP093395.1 had the best match for Pseudomonas aeruginosa PA1_NCHU (Figure 1). Additionally, matches were found for several additional species with the accession numbers CP045045.1, CP040898.1, LR962820.1, LR962316.1, MG763919.1 KX373594.1, KX373591.1, KX373589.1, and KX373586.1 for Enterococcus faecalis. Similarly, matches were found for several additional species with the accession numbers CP093358.1, CP093356.1, CP093357.1, CP093355.1, CP093354.1, CP093028.1, CP093030.1, CP093032.1, and CP093031.1 for Pseudomonas aeruginosa PA1_NCHU. In order to determine the species with the greatest phylogenetic link to the query sequence, phylogenetic analysis was carried out (Figure 1).

2.2. Effect of pH

The influence of pH on Congo red decolorization through the desired bacteria was observed in the pH ranging of 5.0 and 8.0 (Figure 2).

The bacterium showed over 98% decolorization with an optimum dye concentration of 100 mg/L at pH 7. At pH 7, it showed a maximum decolorization efficiency of 98% whereas at pH 8 it was around 83.5% in a similar condition after three days of incubation at 37 °C. While the decolorization at pH 5 and 6 were comparatively lower with 66% and 83.3%, respectively. These results are in agreement with Pearce et al., Chan and Kuo (2000), and Shah (2014), who reported that neutral pH are more favorable for azo dye decolorization [15,16]. The incubation of the isolates in the presence of Congo red at pH 5, 6, 7, and 8 showed that the pH of the growth media influences their development. Interestingly, growth was more significant at pH 8 and 7 than at pH 5 and 6.

2.3. Effect of Dye Concentration

The concentration of the dye is an essential aspect of bacterial textile dye degradation. The degradation performance of Congo red by E. faecalis SUCR1 was investigated by culturing the bacteria in the medium with different concentrations of Congo red (50, 75, and 100 mg L⁻¹). The highest growth was seen in mixtures with 100 mg L⁻¹ dye, which was observed to be highly related to its degradation (>90% at 100 mg L⁻¹). The dye removal efficiency for Congo red was 78.9, 82.1, 95.7, and 84.0 % for 50, 75, 100, and 150 mg L⁻¹, respectively (Figure 3). The caulomic efficiency (CE) was found to be higher in MFCs with 100 mg/L dye concentration; on the other hand, both coulombic efficiency and dye removal percentage was reduced at a concentration above 100 mg/L, indicating substrate inhibition. Low substrate concentration reduced power output as well as CE.

At a 150 mg L⁻¹ Congo red concentration, the bacterial growth was observed to be affected, as evidenced by reduced efficiency with dye degradation. It was observed that increasing the concentration of dye inhibited microbial activity, thereby resulting in a slow rate of dye degradation even after prolonged incubation. This adverse effect could be due to dye toxicity to the cells by blocking the enzyme activities. Previous studies have also shown an adverse connection between dye content and the degree of removal efficiency in synthetic minimal media and textile wastewater, which was comparable to our results. The highest dye removal efficiency was observed at 100 mg L⁻¹ substrate concentration, with different concentrations of dye chosen for further research for power generation in MFCs.

2.4. Congo Red Degradation by E. faecalis SUCR1

The dye decolonization could be due to adsorption or degradation in the system. The dyes are exclusively adsorbed on the surface of bacterial cells, which are then predicted to be degraded by bacterial enzymes. Due to the low dye content in the medium and the inefficiency of E. faecalis to degrade dyes in traces, dye decolorization was at its lowest at 10 mg/L. Dye was used as carbon source for microbial metabolism, at low concentration of dye, power generation was found low. Therefore, when aiming to optimize dye concentration, 100 mg/L was found to be optimum. To test the decolorization effectiveness of E. faecalis SUCR1, different concentrations of Congo red (50 mg/L, 75 mg/L, 100 mg/L, and 150 mg/L) were used. As shown in Figure 4, the decolorization of Congo red began within 24 h of inoculation and the rate of decolorization accelerated with an increase in incubation time. This observation suggested that E. faecalis SUCR1 is effective at decolorizing and degrading synthetic azo dyes with a concentration of up to 100 mg L⁻¹. The maximum degradation of 95% was observed within two days of incubation.

2.5. Power Generation in MFC

For polarization studies, data were taken after five days of MFC operation as power density curves. The performance of the half-cell electrodes in terms of current densities is shown in Figure 5. The anodic half-cell potential increased due to the oxidative degradation catalysed by bacteria, which can be seen to be increasing over time. MFCs with 50 and 75 mg/L dye concentration showed an analogous trend with similar outputs. Similarly, MFCs with 100 and 150 mg/L showed alike values, whereas the half-cell cathode potential was the same in all cells. This is obvious due to the common reactions with same conditions taking place in cathode chamber. The maximum power density was observed at 150 mg/L MFC, which was 7.3 mW/m³, slightly higher than 100 mg/L MFC (7.2 mW/m³). Dye concentration enhanced the amount of power generated in Figure 4. The lowest power density of 3.8 mW/m³ was observed with a 10 mg/L dye concentration (

Table 2. Some reference comparisons regarding the performance of MFC.

Dye	Bacteria	Decolorization Efficiency	Output Produced	External Resistor	References
Reactive Blue	Pseudomonas aeruginosa	90%	2004 µW/m2	220 Ω	[37]
Methyle Orange	Pseudomonas aeruginosa	94%	4100 µW/m2	220 Ω	[37]
Congo red	Pseudomonas aeruginosa	80%	586 µW/m2	220 Ω	[37]
Acid orange	Shewanella oeidensis	93%	-	NA	[38]
Acid black 172	Aspergillus	88%	133 mA/m2	NA	[39]
Acid black 172	Trichoderma	85%	129 mA/m2	NA	[39]
Remazol brilliant blue R	Pleurotus ostreatus	80–90%	180.5 mW/m2	NA	[40]
Congo red	Enterococcus fecalis + Pseudomonas aeruginosa	98%	7.3 W/m3 (223.21 mW/m2)	50 Ω	This study

). Dutta et al. also showed significant work in the degradation of methylene blue [36]. The order of power production was found to be increasing with the increase in the concentration of dye in the medium. The major reason for this could be the metabolites released during the Congo red degradation, which acted as electron mediators in shuttling electrons between the bacterium and the electrode. This assumption is supported by the anode potential, which fluxed with dye concentration in MFCs.

2.6. Electrochemical Impedance Spectroscopy Studies (EIS)

EIS is mainly used to assess the quality of electrode materials, biofilm development, and the kinetics of chemical processes. Electrochemical characteristics at the electrode interfacial layer are studied using Nyquist plots (imaginary impedance vs. actual impedance). EIS is applied to understand internal losses, such as ohmic resistance, charge transfer resistance, and diffusion transfer resistance [41]. A potentiostat was used in this approach to measure a wide frequency range (100 kHz to 1 MHz). The results of this approach are displayed on Nyquist or Bode graphs. The Nyquist plot compares imaginary impedance to original impedance. The graph depicts impedance at different frequencies. They do not reveal the frequency utilized to record a particular instant, which is a significant issue. The X-axis represents impedance vs. frequency, while the Y-axis represents the phase angle and impedance absolute values. A well-defined semicircle in the high-frequency band followed by a straight line in the lower frequency range in MFCs speaks about the internal resistance of the system. At the highest frequency point, the resultant resistance is most significant. The diameter of the semicircles can be used to determine each electrode’s charge transfer resistance (R_ct). Interfacial interactions between the catalyst and reactant or electrolyte are directly connected to the Rct value.

The internal resistance of MFC containing 150 mg/L Congo red dye concentration (37.5 Ω) was lower than the MFC with a 10 mg/L dye concentration (223.611 Ω). Similarly, the MFCs with a dye concentration of 50 mg/L, 75 mg/L, and 100 mg/L were lower (125 Ω, 102.78 Ω, and 45.78 Ω, respectively) than the MFCs containing a 10 mg/L Congo red dye concentration, as shown in Figure 6.

3. Discussion

The electrogenic bacteria, E. faecalis, can degrade Congo red simultaneously to power production in MFC. The UV-Vis spectroscopy analysis of the degradation product indicates that E. faecalis SUCR1 has the highest Congo red dye degradation efficiency at pH 7 and a concentration of 100 mg L⁻¹. A study reported that E. faecalis SUCR1 may thrive in extreme conditions. It was shown that Enterococcus sp. was remarkably adaptable metabolically, able to use a wide variety of non-typical substrates, such as chlorpyrifos, pentaerythritol tetranitrate, 2,4,6-trinitrotoluene, and phosphonate. Recently, ref. [42] reported the importance of Enterococcus sp. in dairy industries increasing the biotechnological value of this strain. Previous studies have indicated that E. faecalis 27688_1#112 is an effective bacterium for the breakdown of Congo red and many other azo dyes, and researchers have also shown that this bacterium may have significant applications in the bioremediation of textile effluent. The co-culture of Pseudomonas aeruginosa PA1_NCHU and Enterococcus faecalis SUCR1 resulted in symbiotic adaptation and metabolic activities.

4. Materials and Methods

4.1. Sample Collection

Fresh cow dung was procured from a local cow shelter for the purpose of isolation of the bacteria, which would serve the purpose of the degradation of Congo red dye using its own metabolic pathways. The soil sample was collected from the depths of the ground, ensuring the presence of a substantial amount of moisture so that microorganisms are able to be present and survive.

4.2. Isolation and Screening of Microorganisms

Cow dung was collected in a sterile container using an autoclaved spatula and other materials and were transported to the laboratory by maintaining the temperature of the container at 4 °C. A sample comprising 1 g of cow dung and soil was suspended in 10 mL sterile sodium chloride solution (0.85% w/v) in separate test tubes and made into a homogenous solution. Of thus prepared suspension, 1 mL was taken and added to another test tube along with 9 mL of sterile sodium chloride solution (0.85% w/v). This process was sequentially repeated twice separately for both soil and cow dung, giving a 10⁻² serial dilution. The prepared serial dilution of the soil sample was spread on commercially available selective media plates to grow Pseudomonas species. Pseudomonas isolation agar base (SRL) has been used to isolate Pseudomonas. However, the serial dilution of the cow dung sample was spread onto Nutrient agar plates prepared in nutrient agar media. The final pH of both growth media was adjusted to 7.0. Media was then supplemented with 100 mg L⁻¹ of dye tested. All plates were incubated at 37 °C for 2–3 days. A bacteria colony surrounded by decolorized zones was selected and streaked on nutrient media plates containing 100 mg L⁻¹ of Congo red [43]. The plates were incubated under the same conditions to demonstrate their ability to decolorize Congo red. On nutrient agar, the pure culture was stored and kept at 4 °C. The biochemical and molecular characterization of the strains thus isolated was performed (by 16S rRNA sequencing by Sandors Lifesciences Pvt, Hyderabad, India. ltd. Hyderabad, AP, India) for both bacterial colonies. 16S rDNA sequences of the bacterial isolates were submitted to GenBank, and the results thus obtained were identified as Pseudomonas aeruginosa PA1_NCHU and Enterococcus faecalis SUCR1, respectively. Additionally, Gram staining was performed to study bacterial morphology [44].

4.3. Decolorization Protocol

A bacterial isolate pre-culture was created by cultivating a single colony in 10 mL of broth containing 1% sodium chloride and yeast extract at 37 °C for 24 h in a shaker incubator. The culture was incubated overnight at 37 °C under the same conditions. Of pre-culture, 5 mL was inoculated into 200 mL of minimal media (MM) containing 100 mg L⁻¹ of Congo red dye in the flask. To quantify bacterial growth, 10 mL culture were taken at different time intervals, and their cell densities were quantified at 600 nm in an UV/Visible spectrophotometer. Bacterial cells were then separated by centrifugation at 15,000 rpm for 20 min, and the supernatant was separated to determine the concentration of a respective dye through a spectrophotometer. The absorption of Congo red dye was determined at 497 nm. The medium without dye and inoculum represented a blank, and the medium with dye but no inoculum acted as a control. The following formula was used to calculate dye decolorization efficiency (%):

$$Decolorization\left(\%\right)=\frac{A0-A}{A0}\times 100$$

where A0 is the initial absorbance of media before inoculation, and A is the absorbance of decolorized media.

4.4. Growth Media and Conditions

An inoculated culture of a strain with the activity of interest to us was subsequently cultured under various experimental settings to examine the impact of factors pH and concentration on dye decolorization and the growth of bacteria. The experiment was run at pH 5, 6, 7, and 8, along with substrate concentrations of 10, 50, 75, 100, and 150 mg/L, respectively. The minimal medium used to study the effect of pH containing 100 mg L⁻¹ of dye, 1% yeast extract, and 1% sodium chloride in 4 different flasks. Furthermore, the pH of the growth media was adjusted to 6, 7, and 8 using 1 M HCl or NaOH before the inoculation cultures were incubated at 37 °C under shaking conditions at 150 rpm so that optimization could be performed. Upon optimization from pH range 5 to 8, observations were made and discussed in the results. After the optimization of the pH was complete, the growth medium used to examine the effect of substrate concentration was 1% yeast extract and 1% sodium chloride, and the substrate concentration was varied between 50 mg L⁻¹, 100 mg L⁻¹, 150 mg L⁻¹, and 200 mg L⁻¹. Thus, the inoculated cultures were maintained at the optimized pH of 7 and incubated in the shaking incubator at 37 °C and 150 rpm, respectively, for 24 h to obtain appropriate results. The MFCs were operated at about 37 °C under batch mode with repetitive cycle (each cycle = 36 h). In order to perform anolyte pH optimization before adding inoculum, the initial anolyte pH was adjusted by weak acid or base solution to the desired values. The optical density of 0.8 was used for each MFC experiments using the isolated microbes (Pseudomonas aeruginosa PA1_NCHU and Enterococcus faecalis SUCR1) with 1:1 volume. Of inoculum, 20 mL was used per MFC. Different concentrations of Congo red dye-blended MSM (minimal salt media) was used as an anolyte [45]. The composition of MSM broth was (g/L): dye; peptone 5; Na₂HPO₄ 2.4; K₂HPO₄ 2.0; NH₄NO₃ 0.1; MgSO₄ 0.01; and CaCl₂ 0.01 with pH 7.0. During anolyte pH optimization, pH was changed using mild HCl and NaOH.

4.5. Microbial Fuel Cell (MFC) Design and Inoculation

Five air-cathode single chamber MFCs were prepared using plexiglass with a total volume of 250 mL, while the anode was made of 6 × 6 cm carbon felt, the cathode was exposed to open air, and the entire circuit was wired using stainless steel [46]. The anode chamber had two ports at the top, one for electrode terminal and the other for reference electrode (Ag/AgCl, saturated KCl; +197 mV, Equiptronics, India) and sampling. The following event was to check for dye degradation and simultaneous current production by these microbes, using an MFC with a 1:1 mixture of the respective isolated bacterial culture (Pseudomonas aeruginosa PA1_NCHU and Enterococcus faecalis SUCR1). The electrochemical analysis of MFC was performed through polarization and power density curves by using the variable resistor method (10–10 k Ω) after the OCV was steady. The SS mesh used in the present study was of the SS-304 type with 50×50 number of openings per square inch [47]. The wire used in the SS mesh had diameter of 0.17 mm. It was connected with a concealed copper wire as the cathode terminal. Thus, the concealed copper wires were used to connect the external resistance to close the circuit [48]. The inter-electrode distance was kept constant at about 2.5 cm in all the experiments [49]. The anodes were placed equidistantly from the MCA. The additional ports were sealed with clamped tubes to ensure anaerobic environment.

The current output was recorded using a digital multi-meter (HTC 830 L). A data recorder and a variable external resistance stage were used in the polarization experiment [50]. The associated power was measured for at least 30 min with an impedance range of 20,000 to 5 (Agilent 34970A, Selangor Malaysia). Anode normalized power density and volumetric power output P_d (W/m³ and mW/m²) = EI/A, where E and I are voltage and current, respectively, coupled to precisely imposed loads, and A is the anode surface area, was used to determine the power density in relation to the anticipated anode surface area. The slope of the linear portion of the voltage vs. the current graph was used to compute the inherent resistance of the MFC. Coulombic efficiency was calculated elsewhere [51]. The anodic dye degradation in various anolytes were studied using electrochemical techniques including electrochemical impedance spectroscopy (EIS). In the EC lab electrochemical interrogations, each MFC’s platinum rod and anode served as the counter and operational electrodes for the 3-electrode setup utilized to examine MFCs. Measurements were made of the voltage and current associated with the Ag/AgCl (+197 mV vs. SHE) counter electrode. Alternating current (AC) at a frequency of 100 kHz to 100 MHz and a voltage intensity of 5 mV was used to accomplish the EIS. For the purpose of calculating the charge transfer resistance (Rct) and solution resistance, the EIS spectra were modelled in an equivalent network (Rs). The COD values of the anolyte were measured using a COD measurement instrument set (TOPLAB INDIA PVT LTD, Mumbai, India) [50].