Decolorization of Congo red in Microbial Fuel Cell

In this research, three individual conditions (static, shaking and MFC) were tested for Congo Red decolorization. P.aeruginosa MTCC 2582 has showed 96.1% decolourization under MFC condition with 85% COD reduction for the dye (100 µM). Microbial fuel cell of P.aeruginosa can discharge the dual duty of degrading the recalcitrant dye with power generation. To understand the inuence the growth curve, different substrate concentration of glucose (0-20 g/L) were selected to improve the performance of MFC. Results show that a larger open circuit potential of 0.691 V and a maximum power density of 1.9 mW m -2 was possessed for the degradation of 100 µM of dye at 10 g/L of glucose concentration. Further, the selection of optimum concentration of dye (200 µM) increased the open circuit potential to 0.844 V. The degraded metabolites were conrmed using UV-Vis and FTIR analysis. Biolm formation on anode at optimal glucose concentration was studied by using SEM analysis.


Introduction
Textile industry is considered as the one of the major polluting industries by generating huge volume of wastewater with complex toxic compounds (Sen., 2003). Further, it is estimated that 10-15% of the dyes are lost in e uent during the dyeing process in textile industry. Wastewater from textile industry is one of the major sources of aromatic amines getting released into the environment leading to cumulative adverse effects. Another important sector to be considered is energy. Energy plays a critical role in the economic growth, progress and development, as well as poverty eradication and security of any country. In recent years, the industrial development and population growth have led to a surge in the global demand for energy. Present energy resource structure is unsustainable and the pollution control methods are high-energy-consuming and cost intensive.
Thus, it is wise to develop a technology that meets these two severe challenges. Microbial fuel cells (MFCs) are being explored extensively for sustainable energy production, waste disposal and reducing CO 2 emissions [2] . Electricity production from biotechnological application employing the conversion of organic compounds with microbial fuel cells is a concept known for almost 100 years [3] . However, it is only recently that the thoughts of utilizing MFC for practical applications have been entertained with an increase in power output. This was due to the paradigm shift in the exciting discoveries about microbial physiology related to electron transport, and the advancement of fuel cell technologies was established in the past few years [4] .
Microorganisms in MFC can discharge the dual duty of degrading the pollutants and generating power.
Moreover, it yields 50-90% less solids to be [5] . Microbial fuel cell (MFC) is one type of fuel cell, where the conversion of the chemical energy from the fuel to electrical energy is achieved by the catalytic action of microorganisms [6] . It consists of anodic and cathodic compartments separated by a proton exchange membrane (PEM). Bacteria in the anodic chamber oxidize organic matter and transfer electrons to the cathode through an external circuit producing current. For each electron released, an equivalent proton must then diffuse across the proton exchange membrane to the cathode and therefore combine with the electron and oxygen in the cathode to form water [7] . Electric current generation is attributed by separating the microbes from oxygen or any other end terminal acceptor apart from anode [8] for the MFC operation. Some areas where MFCs offer immediate prospect of application are powering gadgets [9] , biosensors [10] and especially in wastewater treatment [11] . It is known that wastewater from textile industry is one of the major sources of aromatic amines in the environment.
Various physical, chemical, biological treatment techniques were employed to remove colour from dye containing wastewaters. The physio-chemical methods which include Fenton's process, oxidation and ion exchange process are not used in developing countries due to their expenses, limited versatility and generation of waste products. Biodegradation has been limited to those compounds that are biodegradable [12] . Not all dye compounds are susceptible to rapid and complete degradation. There are some concerns that the products of biodegradation may be more persistent or toxic than the parent compound. Biodegradation often takes longer time than other treatment methods. Evaluating performance of biodegradation nds it di cult because of no acceptable endpoints for biodegradation treatments.
Due to such drawbacks of the various conventional methods the microbial fuel cells are now used for dye decolourization. Species like Pseudomonas aeruginosa, Pseudomonas uorescens, Proteus hauseri, Geobacter sulfurreducens, Beta Proteobacteria, Trametes versicolor, Shewanella oneidensis and others have be used for dye decolourization and electricity generation in microbial fuel cell ([13]; [14]; [15]; [16]) . For the improved dye degradation e ciency with power production, secondary metabolites of Pseudomonas sp. can work as redox mediator in MFCs [17] . Considering all these points, this study was designed to investigate the e ciency of Pseudomonas aeruginosa for dye decolorization with electricity generation in an MFC system. The objectives of the study were to study the e ciency of decolourization of Congo red dye by Pseudomonas aeruginosa in MFC. MFCs decolourization conditions and electricity production by Pseudomonas aeruginosa were studied in detail. Substrate was regarded as one of the mainly essential factors that affects the power generation in MFCs. MFCs are able to utilize different sources of substrates for production of bioelectricity ([18]; [19]) . The present study also focused on evaluation of MFC performance for different concentrations of substrates at anode chamber.

FEED SOLUTION
In anode compartment, Minimal salt medium (MSM) was used as the feed Solution with the following composition per litre: KH 2 PO 4 -13.60 g; Glucose -10.00 g; Na 2 SO 4 -9.47 g; NaOH -2.33 g; NH 4 Cl - Glucose was added as the energy source to the anode chamber. The salts were of analytical grade and purchased from CDH Chemicals. About 50mM Phosphate buffer (pH 7.0) was used in the cathode compartment as catholyte.

DYE STOCK SOLUTION
The sulfonated azo dye -Congo Red dye (C 32 H 22 N 6 O 6 S 2 Na 2 ) of molecular weight 696.66 of commercial quality from Sisco Research Laboratories Pvt. Ltd., Maharashtra was used and its non-hydrolysed molecular structure is given in Figure.1. 10M dye stock solution was prepared for the reactive dye and was used for further studies.

MICROORGANISM AND ITS CULTURE CONDITION
Pseudomonas aeruginosa MTCC 1274 was obtained from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India and maintained on Nutrient agar medium. The slants of Pseudomonas aeruginosa in Nutrient agar medium was stored at 4 o C in the refrigerator and sub cultured every month. All the culture media, unless otherwise stated, were sterilized at 15 lbs/inch 2 pressure (121ºC) for 15 minutes. Pseudomonas aeruginosa MTCC 1274 was taken from slants and suspended in 0.85% sterile sodium chloride was inoculated in the synthetic feed solution of anode compartment, pH 7.0. Flasks were incubated (32 o C, 150rpm) in a rotary shaker for overnight. Streak plating was done in the minimal salt medium with 1% agar. Samples were withdrawn aseptically at regular time intervals from the freshly inoculated culture and were analysed for cell density at OD 600nm. Growth rate of Pseudomonas aeruginosa on feed solution was prepared as given in 3.2.1. and was investigated. Thus, the maximum decolourization of the sulfonated azo dye mentioned in 3.2.2. during the growth of Pseudomonas aeruginosa in feed solution was studied.

MFC CONSTRUCTION AND ITS OPERATION
The Double Chambered Microbial Fuel cell (DCMFC) consists of two (Anode and Cathode) bottles of 500 mL (18*8 cm) lled with effective volume up to 400mL with graphite electrodes (3.1*1.3 cm). The bottles were joined by a glass bridge containing a proton exchange membrane (Na on TM 117, Dupont Co.) held by a clamp between the attened ends of the two glass tubes (inner diameter 1.8 cm) tted with rubber gaskets. Both Anode and Cathode are made of graphite without any coating. Electrodes was made to be positioned at a distance of 8 cm on either side of PEM (Fig. 2).
The electrodes were made to get attached using copper wire to give external circuit connection. Prior to use electrodes were soaked in 100% ethanol for 1 hour followed by 1M HCL for 1 hour and then in deionized water for a period of 24 h. The anode compartment was lled (400 mL) with MSM (Minimal Salt Medium) containing reactive azo dye as mentioned in 3.2.2. and the cathode compartment was lled with phosphate buffer pH 7.0 as catholyte. The system was operated at room temperature (32 o C). Each chamber was provided with sample port, wire point inputs (top), inlet and outlet ports and anode chamber were sealed to ensure anaerobic microenvironment.
The anode was purged with Nitrogen gas for 20mins at an interval of 24 h to maintain the anaerobic condition in the anode chamber while the cathode was sparged with air (110mL/min) using an aquarium pump. The anodic chamber prior to start-up was inoculated with selectively enriched and adopted Pseudomonas aeruginosa through designed synthetic feed suspension of 1mL (approximately 3X10 6 cells). Constant COD removal and stable voltage generation were considered as indicators for stable operating conditions. The samples were centrifuged at 3500 rpm for 20 min by using a centrifuge (REMI R-8C). This was done for all samples for separation of biomass prior to measurement of absorbance. The chemical oxygen demand (COD) was determined in accordance with the Standard Methods for the Examination of Wastewater treatment. The initial COD of glucose medium was investigated by theoretical chemical oxygen demand on the basis of its stoichiometric reaction with oxygen. The nal COD was measured and COD removal was calculated as; [(COD in -COD out )/ COD in ] ×100%. Where COD in is the in uent COD and COD out is the e uent COD.
FTIR analysis of CR before and after treatment was monitored on a Thermo Scienti c IR 200 FT-IR spectrophotometer. The FTIR spectra were then recorded between 4000 and 500 cm − 1 , at a rate of 16 nm/s.

MICROSCOPIC ANALYSIS
SEM analysis of the bio lm on anode was investigated at high resolution to con rm the bacterial infestation [20] . The anode material was removed at the end of the experiment, was rinsed with the sterile medium (distilled water), and then were immersed in 5% formaldehyde overnight to x the samples. Chips of 1 cm×1 cm were cut for SEM analysis. Before observation the anodic materials were collected and immersed in the 5% formaldehyde overnight for xing samples, followed by washing with detergent. Samples were then dried and prepared by gold sputtering before imaging.

Results And Discussion
3.1 Decolourization performance under Static, Shaking and MFC condition and removal of COD Pseudomonas aeruginosa exhibited higher dye-decolourizing activity when operated under DCMFC condition (80.56%), whereas agitated culture showed less decolourization e ciency (49.25%). At static condition only 16% decolourization of Congo red (CR) was observed after 120 h (Fig. 3). At static condition bacterial growth rate 0.074 h − 1 , this evident that the sulfonated diazo dye Congo red is recalcitrant to bio decolourization under static condition [21] . At shaking condition bacterial growth rate improved to 0.304 h − 1 which resulted in 50% of dye decolourization. The growth rate of bacteria was observed to be 0.688 h − 1 for MFC condition resulted in higher decolourization e ciency of CR dye. The time required for decolourization was decreased with MFC condition. This result proved that the decolourization was not to the adsorption of cells but due to the metabolic activity of bacterial cells [22] .
The average rate of decolourization was increased up to 12 h in MFC condition. On continuous acclimatization in DCMFC 100% decolourization was observed within 48 h. Removal of COD was 85% under MFC condition. Hou et al., 2011 [23] reported 68.9 % removal of COD at MFC condition whereas Isik and Sponza (2003) [24] reported only 57% removal of COD under anaerobic condition while 87% of CR decolourization at aerobic condition.

Effect of glucose concentration on dye decolourization and power density
CR decolourization e ciency and power production by Pseudomonas aeruginosa were investigated under different glucose concentration (0 g/L,5 g/L,10 g/L, 15 g/L and 20 g/L). Initially all MFCs were operated under open circuit condition to evaluate its performance when no load was applied. The OCV was recorded at a time interval of 3 h and the recorded data was averaged for every 24h. Similarly, samples were withdrawn aseptically at regular time intervals from the inoculated MFC and analysed for cell density at OD 600 nm. As shown in Fig. 4, the growth curves of P. aeruginosa cultured under anaerobic condition in MFC displayed a typical three-stage growth cycle: an adaptive phase (0-4h), an exponential growth phase (5-14h), and a stationary phase (after 14h) approximately for all ve sets.
The growth of bacteria was maximum at 10 g/L (0.858 OD at 11th hr). When the glucose concentration was 10g/L the maximum growth rate was expressed. Hence, this concentration was used for further study. Moreover, Fig Concerning the working power densities at 220 ohm external resistance with the duration time of 72 h shows the variation in power densities at different culture time. Therefore, from the growth curve study a culture time of 3 h, corresponding to the middle of log phase would be suggested in this study. From  Fig. 4 shows that the culture time of 4 h, corresponding to the transition of log phase to stationary phase in the growth curve. Therefore, higher the growth in uences the bacteria metabolism and growth in Pseudomonas aeruginosa in MFC [25] . Better power performance was observed in stationary phase of the growth curve and can further be suggested.
Comparing the power performance among different concentration of glucose, 10 g/L of glucose concentration would produce an optimal performance for the studied cases. Further, the MFC operated with 10 g/L of glucose concentration had prolonged stationary phase which improves the electrical activity in the MFC. Previous study by Jafary et al., 2013 [26] investigated that the substrate concentration enhancement resulted in improved MFC performance. But, in this study, with 10 g/L of glucose the DCMFC will produce 1.9 mW m − 2 under the optimal condition of glucose.

Effect of glucose concentration on different dye decolourization
The decolourization activity of Pseudomonas aeruginosa in MFC system was studied using CR at various initial concentrations with different glucose concentrations. DCMFC system inoculated with Pseudomonas aeruginosa can decolourize up to 200 µM of dye concentration, however higher concentration seemed to be toxic for the cell growth in MFC. The dye decolourization was strongly inhibited at 500 µM in the MSM medium. From Table 1, it was evident that a maximum OCV of 0.844 V was observed for the initial concentration 200 µM of dye and 10 g/L of glucose concentration. Further the dye decolourization was also reported to be higher (96.1%).  As for the performance of MFC under 100 µM of dye using 10 g/L of glucose concentration a maximum OCV of 0.691 V and a maximum decolourization of 89.74% was reported. Moreover, for 300 µM of dye concentration, a maximum OCV of 0.392 and a maximum decolorization of 79.94% was observed. These results would indicate that the glucose concentration of 10 g/L and 200 µM seems to be a better choice for better decolourization in MFC. Stable OCV production was achieved during CR decolourization with the presence of 10 g/L of glucose. Further, the dye intermediates of CR might have enhanced the MFC performance after degradation [27] . The test for TSS, TDS and EC were conducted for samples obtained from MFC at optimum condition (200µM dye concentration and 10g/L glucose concentration) and the results are given in table. 2

Degradation analysis
CR colour removal in MFC was con rmed by UV-Visible analysis (200-800 nm) (Fig. 6). CR UV-Visible scanning depicted two peaks. One extended at 340 nm was due to the interaction between the aromatic hydrocarbon /polycyclic aromatic hydrocarbon groups and other chromophore group, and a peak at 495 nm, relative to the azo bond group and a large conjugated system of whole dye molecule. After 72 h treatment, a decrease in the major peak intensity at 495 nm was noted. This indicated that the dye structure especially the azo bond was broken and was degraded during MFC treatment. Surprisingly, the absorbance at 340 nm was also decreased which evident that interaction of the aromatic hydrocarbons and some chromophore group were partially destroyed. These results suggested that MFC treatment is able to degrade the CR dye with a complete breakdown of chromophore group. Unlikely Wang et al, 2017 [28] reported that complete breakdown of aromatic hydrocarbon seemed to be more di cult that the breakdown of azo double bond.
In order to elucidate the nature of the functional groups present in the untreated and treated sample, FTIR analysis was performed. The spectra of decolourized sample reveals shifting of some characteristic bands which depict changes in functional groups after decolourization compared to untreated dye sample (control) (Fig. 7). The FTIR spectrum of untreated sample and treated sample reveals several peaks and stretching as presented in Tables 3 and 4 respectively. FTIR analysis revealed the presence of aromatic compounds in untreated sample and alkenes in treated sample.

Bacterial adhesion on anode
The surface of the anode electrode was visualized by scanning electron microscope SEM (FEI-Quanta FEG 200F) to determine microbial attachment and formation of a bio lm on the anode electrode surface. It was removed after 29 days incubation period. SEM Images of bacterial attachment on Anode from MFC Setup used for decolourization of dye concentration (200µM) along with glucose concentration (10g/L) is represented in Fig. 8. The average size of the microbe was obtained as 1µm.
The formation of a bio lm begun when bacteria sense certain environmental cues and then trigger the transition to life; these environmental signals vary among microbial communities. P. aeruginosa bio lms can be found on any substrate surface suitable for its growth [29] . Some studies [30] have shown that bacteria develop bio lms on surfaces when nutrients are provided at su cient levels in the solution; the cells will then enter back into the solution when the nutrients are scare. This starvation response is a key factor regulating bio lm formation [31] . Changes in environmental factors cause changes in microbial state. Xu found that Desulfovibrio vulgaris adhered to carbon steel surfaces under starvation conditions to acquire extra energy from steel [32] . Additionally, microorganisms are capable of complex behaviours that best bene t their reproduction. Mutual interactions in bio lms result in bio lm formation, which enables sessile organisms to stay on surfaces and exhibit improved growth.
Ilamathi et al., 2019 [17] reported that the presence of P. aeruginosa bio lm on electrode surface strongly improved the redox behaviour when compared to uncolonized electrodes. From the SEM analysis (Fig. 8) and OCV generation (Fig. 5) it is understood that the presence of P. aeruginosa bio lm on electrode surface improved the redox behaviour.

Conclusion
Several concentrations of carbon substrates at the range of 0-20 g/L were studied at different dye concentration at the range of 100-300 µM. The growth curve of bacteria revealed that the growth was maximum at 10 g/L glucose as carbon source. At the optimum growth condition (10 g/L glucose concentration) of P. aeruginosa and 100 µM dye concentration, the decolourization was tested at static, shaking and MFC condition it was found that the maximum decolourization at less time was at MFC condition. The FTIR analysis revealed the presence of aromatic compounds in untreated sample and alkenes in treated sample. From the SEM analysis obtained at optimum condition (10 g/L glucose and 200 µM dye concentration) it was understood that the presence of P. aeruginosa bio lm on electrode surface improved the redox behaviour.

Declarations
The authors declare that there is no con ict of interest.