Methylene blue (MB) is a textile dye that can be fatal to aquatic life, plants, and human health when discharged into the environment without treatment. A cheese whey-microbial fuel cell (CW-MFC) is a device that generates electricity from the degradation of cheese whey by microbial activity. The microbial activity of the CW-MFC during electricity production was able to decolorize MB. In this study, 50 ppm of MB was used to evaluate the decolorization capability of bacteria of the CW-MFC. A bacterial consortium present in the bioanode of the CW-MFC showed good MB decolorization in both the ex situ and in situ operations. Ex situ operation performed outside the CW-MFC reactor showed 92.2% MB decolorization within 18 h, while the in situ operation conducted inside the CW-MFC reactor showed 97.1% MB decolorization within the same timeframe. The maximum decolorization performance was achieved at pH 4 and 37 °C. The treated MB exhibited very little or no toxicity in the germination, rooting, and shooting of Oryza sativa compared to the untreated MB. Thus, the CW-MFC can be used as a promising technique to decolorize and remove the toxic effects of MB-contaminated wastewater, and the treated wastewater can be applicable for irrigation purposes.

  • Bacterial consortium on the bioanode of the cheese whey-microbial fuel cell (CW-MFC) was developed for methylene blue (MB) decolorization with simultaneous electricity generation.

  • CW-MFC was capable of decolorizing MB in both ex situ and in situ operations.

  • More than 90% decolorization was achieved in continuous mode at a flow rate of 0.025 ml/min of MB (50 ppm).

  • The treated MB showed no phytotoxicity against Oryza sativa.

Various synthetic dyes are being used in different industries, and their applications are nowadays increasing in multiple areas, such as the textile, leather, paper, and plastic industries (Al-Tohamy et al. 2022). Synthetic dyes are considered a major source of water pollution and pose harmful effects on the environment and human health (Fobiri 2022). It is reported that most of the unused dyes are disposed of because of inefficient dyeing processes in a nearby aquatic ecosystem or landfill without proper treatment by the dyeing industries (Kishor et al. 2021). In addition to high chemical oxygen demand and deep color, dyeing wastewaters are highly toxic and hardly show biodegradability (Natarajan et al. 2011). The wastewaters containing synthetic dyes have become a major concern in terms of environmental pollution and water toxicity, and thus, they must be treated effectively before being discharged into the nearby rivers or environment (Wang et al. 2022). MB, a heterocyclic aromatic synthetic dye, is widely used in dyeing cotton, nylon, leather, and paper, as well as in coloring plastics and oils (Rafatullah et al. 2010; Oladoye et al. 2022). Discharging textile wastewater containing 10–50 ppm MB into the environment can have devastating environmental effects, including disturbance to aquatic life, reducing soil fertility and crop productivity, and harming human health (Chung 2016; Berradi et al. 2019). So, it is vital to treat MB-containing wastewater effectively before discharging it into the environment. Physical, chemical, and biological methods are mainly employed to eliminate textile dyes in wastewater. Various physicochemical methods have been used for the decolorization or removal of textile dyes from wastewater. These include adsorption, flotation, coagulation, filtration, photocatalysis, electrolysis, plasma-coupled treatment, and Fenton process (Melgoza et al. 2009; Li et al. 2019; Wu et al. 2019; Zhou et al. 2019; Dihom et al. 2024; Singh & Yadav 2024). Among these methods, mainly photocatalysis, adsorption, and plasma treatment have become popular in dye decolorization. However, the applications of these methods on large-scale dye degradation are limited owing to their operational complexity, low economic feasibility, and disposal problems, especially in developing countries. However, biological methods have many advantages in decolorizing textile wastewater owing to their cost-effective operation, environmental friendliness, high degradation efficiency, and lower energy requirements (Sharma et al. 2016; Eslami et al. 2017; Bharti et al. 2019). Despite the disadvantages of sensitivity to environmental conditions and limitations in treating certain contaminants, biological methods can be combined with different physicochemical methods for the efficient decolorization of textile dyes. Many studies used microorganisms, especially bacteria, for the degradation of MB. Some researchers have looked at how pure forms of bacteria like Bacillus subtilis MTCC441, Rhodococcus strain UCC 0003, Staphylococcus aureus, and Acinetobacter pittii can break down MB (Upendar et al. 2017; Maniyam et al. 2018; Ogunlaja et al. 2020; Ayed et al. 2022). In contrast, other studies have employed bacterial co-culture and communities of anaerobic sludge for dye decolorization (Lade et al. 2015; Das & Mishra 2017; Zhu et al. 2018; Haque et al. 2021). However, conventional biological methods require special preparation for microbial growth, such as growth media, which is expensive, and precautions for maintaining a pure bacterial culture that limits the use of them for decolorization. So, there is a need to design the biological treatment processes in such a way that they can use wastewaters as substrates for microbial activity in dye decolorization plants without maintaining pure culture procedures. Microbial fuel cell (MFC) is a technique that can produce electricity using microbes from organic substrates (Rabaey & Verstraete 2005). One study reported that an MFC with Geo sulfurreducens as a bioanode was capable of decolorizing Congo Red (CR) and generating electricity using sodium acetate as a growth medium (Ma et al. 2023). Some research groups employed MFCs (in the form of a single-chamber MFC, double-chamber MFC, and constructed wetland-MFC) to decolorize textile dyes such as New Coccine, X-3B, Reactive black 5, and Remazol Yellow with a natural microbial consortium (Oon et al. 2018; Joksimovic et al. 2022; Teoh et al. 2023; Xie et al. 2024). To our knowledge, there is no study on the decolorization of MB by MFCs with simultaneous electricity generation. A CW-MFC can be used as a promising, feasible technique for decolorizing MB. The CW-MFC is a device that can use cheese whey wastewater (CWW) as a substrate for microbial activity in the anode chamber for MB decolorization and electricity generation. In this study, we have developed a natural microbial consortium on the bioanode of the CW-MFC for the decolorization of MB. The microbial community, especially the bacterial consortium of the bioanode of the CW-MFC, has shown good decolorizing activity for MB in both the ex situ and in situ experiments.

Construction and operation of CW-MFC

A CW-MFC was constructed using polycarbonate blocks with specified holes and rubber gaskets, as in our previous study (Mahato et al. 2021). The anode and cathode chambers were separated by a proton exchange membrane (PEM, Nafion-117). Graphite felts were used as both anode and cathode electrodes connected by a Ti wire with an external resistor (Figure 1). Injection needles were hooked up tightly across the rubber gaskets for inserting and withdrawing the solution from the chambers. The anode chamber of the CW-MFC was filled with anolyte (only cheese whey) and then inoculated with mixed bacterial suspension. However, the cathode chamber of the CW-MFC was filled with KMnO4 as a catholyte. The volume of the anolyte and catholyte was 40 ml for each. The CW-MFC was operated under open and closed circuits with an external resistor of 1,000 Ω at 37 °C. The anode and cathode chambers were operated under anaerobic and aerobic conditions, respectively. The anodic and cathodic solutions were replaced when the voltage output dropped to a lower level for each batch, and this replacement cycle was continued as needed. The voltage generated by the CW-MFC was recorded by a digital multimeter.
Figure 1

Typical cheese whey-microbial fuel cell (CW-MFC).

Figure 1

Typical cheese whey-microbial fuel cell (CW-MFC).

Close modal

Decolorization of MB dye

The decolorization of MB dye by the CW-MFC was conducted as follows:
  • (i) First, a bacterial consortium in the bioanode of the CW-MFC was developed for steady-state voltage production. To achieve this state, the CW-MFC was operated successively in batch modes with the replacement of the anolyte and catholyte as needed. When the maturation of a bioanode and steady-state voltage were achieved, the CW-MFC was operated for the decolorization of MB. The concentration of MB used in the decolorization experiment was 50 ppm unless otherwise stated. The voltage produced by the CW-MFC during MB decolorization was continuously recorded until more than 90% MB decolorization was achieved. During this time, the catholyte was replaced several times to keep the voltage generation almost at a steady state. The decolorization of MB was evaluated by both ex situ and in situ experiments in batch and continuous modes and was measured by a UV–visible spectrophotometer. The percentage of MB decolorization was calculated by the following equation:
    where A′ is the absorbance of the dye before treatment and A is the absorbance of the dye after treatment.
  • (ii) The ex situ batch mode decolorization was conducted by adding the dye to the effluent of the CW-MFC outside the anode chamber (Figure 2(a)). The treated whey drawn out of the anode chamber of the CW-MFC was mixed up with MB at a final concentration of 50 ppm in a glass beaker and then taken into the screw-cap test tubes. The sealed test tubes were subsequently placed in an incubator at 37 °C. Afterward, the test tubes were successively taken out of the incubator one by one at different time intervals (in a range of 1–18 h), and the decolorization was measured by a UV–visible spectrophotometer. The effects of physicochemical parameters such as temperature, pH, and various concentrations on the decolorization of MB were also evaluated to obtain optimum decolorization performance. To evaluate the effect of pH on decolorization, the experiments were conducted under acidic (pH 4), neutral (pH 7), and basic (pH 10) conditions. To monitor pH changes during the operation, a small amount of the sample from the reactor was frequently taken out with the increment of time and measured by a pH meter. Then, the expected pH value of the reactor solution was adjusted by adding HCl or NaOH with proper mixing without disturbing other parameters. For evaluating the effect of temperature on decolorization, a controllable heating system was used to make the surrounding environment at a desired temperature of about 25 , 37 , and 40 °C monitored by a thermometer. To determine the duration of time required for the decolorization of MB at various concentrations, the decolorization experiments were also conducted at 10, 20, and 50 ppm of MB.

  • (iii) To conduct ex situ continuous mode decolorization, two plastic containers, one filled with sterile whey connected to the anode chamber by an infusion set pipe and another one filled with MB solution connected to the decolorization glass reactor by another infusion set pipe, were used for continuous flow of whey and MB, respectively, during the operation period. The opening of the whey-containing container was connected to the anode chamber through an infusion set pipe. Another pipe was used to connect the outflow of the treated whey from the anode chamber to the decolorization glass reactor (Figure 2(b)). The MB-containing container was directly connected to the decolorization glass reactor by an infusion set pipe. The treated whey and MB at a final concentration of 50 ppm from the reservoirs were added dropwise to the decolorization glass reactor. The decolorization reaction was conducted under almost anaerobic conditions at 37 °C and pH 4 with continuous stirring. The decolorized effluent from the reactor was collected through an outlet pipe at various time intervals for UV–visible spectrophotometric readings.

  • (iv) In the case of in situ batch mode decolorization, the sterile CWW was mixed with MB at a final concentration of 50 ppm and then inserted into the anode chamber of the CW-MFC (Figure 2(c)). The operating pH and temperature of the CW-MFC were maintained at pH 4.0 and 37 °C during decolorization, respectively. The samples from the anode chamber were collected at various periods (1–18 h) for UV–visible spectrophotometric readings during batch mode operation.

  • (v) For in situ continuous mode decolorization, MB mixed with sterile whey was continuously inserted into the anode chamber at different hydraulic retention times. The whey–dye mixed solution was first taken into a plastic container and then inserted into the anode chamber of the CW-MFC through an infusion set pipe from that reservoir in a dropwise manner. The operating parameters such as pH, temperature, and concentration were similar to the above in situ batch mode decolorization. At different time intervals, the treated effluent from the anode chamber was collected through an outlet pipe for UV–visible spectrophotometric analysis to evaluate the decolorization of MB obtained by the in situ continuous mode operation (Figure 2(d)).

Figure 2

Experimental setup for the decolorization of MB. Ex situ batch (a) and continuous (b) mode operations; in situ batch (c) and continuous (d) mode operations.

Figure 2

Experimental setup for the decolorization of MB. Ex situ batch (a) and continuous (b) mode operations; in situ batch (c) and continuous (d) mode operations.

Close modal

Phytotoxicity assay

To evaluate the toxic effects of MB and the treated effluent on the germination, rooting, and shooting of the rice variety Oryza sativa (BRRI dhan 98), a phytotoxicity assay was conducted against O. sativa seeds. Six Petri plates were used as containers to germinate the O. sativa seeds. In each Petri plate, 20 healthy seeds were placed on the bed of Whatman filter paper and were incubated at room temperature for a period. All Petri plates were supplemented with water, untreated MB, treated MB, untreated whey, treated whey, and untreated whey with MB as media for germinating seeds. Initially, 5 ml of each of these solutions was poured into the Petri plates, and then healthy seeds of O. sativa were placed on them. Petri plates were covered by transparent wrapping paper with an air-passing system, and 1 ml of each media solution was added to the respective Petri plates daily. After 6 days, the percentage of germination (%), shoot length, and root length were recorded to evaluate the effects of treated and untreated MB and whey on O. sativa compared to water.

Generation of voltage by the CW-MFC

The CW-MFC was inoculated with a sample of bacteria collected from a long-run MFC. It was then run in open circuit mode for 1 h and subsequently in a closed-circuit mode with a 1-kΩ resistor for at least 6 h for each batch. Such an operation enhanced the attachment of electrogenic bacteria to the anode and their growth and multiplication. To make a mature biofilm on the anode of the CW-MFC, seven successive batches were carried out in the above mode. The short-time operation in a closed-circuit mode with available substrate and catholyte reduced the time needed for bioanode maturation compared to the exhausted condition. As batch numbers increased, the open circuit potential (OCP) generated by the CW-MFC gradually increased. The OCPs of the additional batches after batch number seven were almost the same (about 680 mV). After achieving stable OCP, the CW-MFC was operated in a closed-circuit mode with a 1-kΩ resistor for at least 18 h. The closed-circuit potential (CCP) or voltage produced by the CW-MFC sharply dropped at the point of transition from an open circuit to a closed circuit. Afterward, the voltage dropout rate gradually slowed down and eventually attained a steady state (Mahato et al. 2021). To maintain the stable voltage, the cathode chamber was frequently replaced with KMnO4. The steady-state voltage generated by the CW-MFC in this operation was stable within a range of 310–255 mV with a slight decrease in potential after 16 h (Figure 3(a)). When the voltage dropped below 230 mV, the anolyte was replaced for a new batch cycle of decolorization. After completing the ex situ experiment, the CW-MFC was operated for in situ decolorization of MB. In the case of in situ batch mode decolorization, MB mixed with whey was used as the anolyte. During in situ MB decolorization, the stable voltage generated by the CW-MFC was in the range of 300–240 mV, which was comparatively slightly lower than that of the CW-MFC without MB (Figure 3(b)). This lower voltage production by the CW-MFC could be due to the extraction of some electrons by MB molecules from the electrogenic bacteria of the bioanode instead of the direct transfer of all electrons to the anode material. This result was consistent with a previous study that explained that G. sulfurreducens was capable of decolorizing CR in the MFC and the voltage generated by the MFC was lower than that of the MFC without CR (Ma et al. 2023). One previous study used MB as a mediator in the MFC to increase electricity production by Escherichia coli due to the limitation of direct electron transfer of this strain, where MB accepted electrons from E. coli and then transferred them to the anode electrode (Montoya-Vallejo et al. 2023). Another study found that MB facilitated the electron transport to produce electricity by MFC inoculated with Saccharomyces cerevisiae (Rahimnejad et al. 2011). Although it was evident that MB could enhance MFC performance only in pure or co-culture MFC, there was no report for a mixed culture or natural microbial consortium MFC where MB acted as a mediator. This research showed that a natural bacterial consortium of the bioanode of the CW-MFC decolorized MB while producing electricity at the same time. In addition, the voltage production by the CW-MFC during continuous mode operation for both ex situ and in situ decolorization of MB was almost like the respective batch mode operation. From the above discussion, it is apparent that MB can be used as a mediator only for a pure culture MFC but not for a natural bacterial consortium MFC. The CW-MFC can be a promising approach for decolorizing MB-containing textile wastewater with simultaneous energy production.
Figure 3

Generation of voltage by CW-MFC at 1 kΩ during ex situ (a) and in situ (b) batch mode operations. The arrows indicate the replacement of catholyte.

Figure 3

Generation of voltage by CW-MFC at 1 kΩ during ex situ (a) and in situ (b) batch mode operations. The arrows indicate the replacement of catholyte.

Close modal

Ex situ decolorization of MB

The natural microbial population, especially the bacterial consortium present in the anode chamber of the CW-MFC, exhibited good MB decolorization activity under anaerobic conditions. Ex situ batch mode decolorization of MB, occurring outside the CW-MFC with bacterial species, showed a gradual increment in decolorization rate with increasing incubation periods (Figure 4). The percentage of decolorization achieved in ex situ mode after 18 h was 92.2%. Initially, the decolorization rate within 1 h was 20%, which was very low compared to that after 18 h. This was due to the adaptation of bacteria to the environment created after the addition of MB. During this time, bacteria were prepared for the expression of enzymes and other metabolites involved in the decolorization process. This pattern of decolorization was consistent with a previous report, which found that the expression of azoR1 gene was very high after the exposure of Pseudomonas species to azo dyes, resulting in profound azoreductase activity for azo dye decolorization (Elfarash et al. 2017). Perhaps the adsorption and absorption of dye by bacterial cells and low catalytic activity were attributed to a slow decolorization rate within 1 h. However, after 2 h of incubation, the decolorization rate greatly increased up to 63.2%. The sharp increase in MB decolorization was obtained due to the increase in bacterial cell mass production and the synthesis of responsible enzymes and reducing metabolites. The decolorization of MB with incubation times following 8 h exhibited a slow mood of increment compared to the previous incubation periods. This happened due to the gradual decline in substrate concentration and the number of bacterial cells. Another probable reason was a diminished chance of contact between MB molecules and bacterial cells at the end of the operation when the dye concentration was too low to be decolorized.
Figure 4

Time-course decolorization of MB at 50 ppm by ex situ batch mode operation. UV–visible spectrum (a) and percentage of decolorization (b) of MB.

Figure 4

Time-course decolorization of MB at 50 ppm by ex situ batch mode operation. UV–visible spectrum (a) and percentage of decolorization (b) of MB.

Close modal
Physiochemical factors such as pH, temperature, and concentration play vital roles in the biodecolorization of MB. These factors affect the growth and efficacy of bacteria involved in decolorization. The effects of these parameters on the ex situ batch mode decolorization were evaluated, and the results were subsequently implicated for the other operations. To evaluate the effect of pH on decolorization, the decolorization reactions were operated under acidic (pH 4), neutral (pH 7), and basic (pH 10) conditions. It was shown that the acidic condition exhibited the highest MB decolorization, followed by neutral and basic conditions. The decolorization of MB achieved under acidic, neutral, and basic pH conditions after 18 h of incubation was 95, 83, and 71%, respectively (Figure 5(a)). This result indicated that the decolorization of MB by the CW-MFC effluent was more effective in an acidic environment than in a neutral or basic one. The acidic condition provided available protons that facilitated the reduction of MB by the electron-donating exoelectrogenic bacteria in the anodic effluent, resulting in higher decolorization. This result was consistent with a previous study that reported that increased decolorization of methyl red by suspended and co-immobilized bacteria in acidic pH than in an alkaline one (Sharma et al. 2016).
Figure 5

Effects of pH at 4.0, 7.0, and 10.0 (a) and temperature at 25, 37, and 40 °C (b) on ex situ batch mode decolorization of MB.

Figure 5

Effects of pH at 4.0, 7.0, and 10.0 (a) and temperature at 25, 37, and 40 °C (b) on ex situ batch mode decolorization of MB.

Close modal

To optimize the operation temperature, various temperatures (25 , 37 , and 40 °C) were applied during incubation periods. Variation in incubation temperatures caused variation in decolorization rate. It was observed that the decolorization of MB increased with increasing temperature from 25 to 37 °C (Figure 5(b)). However, at 40 °C, the decolorization was less than at 37 °C. The percentage of the decolorization of MB was 68, 95, and 82% at the incubation temperatures of 25 , 37 , and 40 °C, respectively. Maximum decolorization achieved at 37 °C was due to the suitable temperature for the growth and enzymatic activity of bacteria (Sharma et al. 2016). The reduction in the decolorization efficiency at 40 °C might be the partial inhibition of enzymatic activity involved in decolorization and/or degradation reactions (Elfarash et al. 2017). From the above discussion, it is apparent that the decolorization of MB takes place not only by electron transfer from electrogenic bacteria to MB but also by other enzymes and reducing agents produced by bacteria because a little fluctuation of pH or temperature greatly influences the rate of decolorization. Thus, the decolorization of MB by the CW-MFC occurs by the complex reactions. The plausible mechanism of MB decolorization by the CW-MFC is the complex reactions where the reduction of MB takes place by accepting electrons from electrogenic bacteria and other reducing agents, followed by the degradation with the help of enzymatic catalysis of other bacteria (Sharma et al. 2016; Pham et al. 2022; Xie et al. 2024).

Usually, MB present in different wastewaters varies in concentration due to the variation of its dilution in water. The incubation period needed for decolorization depended on the concentration of MB. The time required for more than 90% decolorization was 4, 8, and 18 h at concentrations of 10 ppm, 20 ppm, and 50 ppm, respectively (Figure 6). This result implies that the wastewater containing a lower concentration of MB needs less time for decolorization than higher concentrations; additionally, for the same incubation period, the percentage of decolorization is higher for lower concentrations of MB than for higher concentrations (Sharma et al. 2016; Bharti et al. 2019). This may happen because the same number of bacteria supply a larger amount of electrons and other reducing agents to the lower concentration of MB compared to the higher concentration of MB.
Figure 6

Time requirement for the decolorization of MB at a concentration of 10 ppm, 20 ppm, and 50 ppm.

Figure 6

Time requirement for the decolorization of MB at a concentration of 10 ppm, 20 ppm, and 50 ppm.

Close modal
This study also focused on the ex situ continuous mode decolorization. In continuous mode operation, MB was continuously supplied to the reaction chamber at flow rates of 0.05 ml/min and 0.025 ml/min. The percentage of MB decolorization was determined using a UV–visible spectrophotometer after an operation period of 18 h. At a flow rate of 0.05 ml/min, 67.3% decolorization was obtained. However, when the flow rate was reduced from 0.05 ml/min to 0.025 ml/min, the decolorization of MB achieved by the ex situ continuous mode was 91.8% (Figure 7). This result suggests that the decolorization rate increased with an increase in hydraulic retention time of MB molecules in the reaction chamber (Manu & Chaudhari 2003). From the above results, it was evident that the effluent of the CW-MFC could effectively decolorize MB in both ex situ batch and continuous mode operations without further requiring a new medium.
Figure 7

Decolorization of MB by ex situ continuous mode operation at flow rates of 0.050 ml/min and 0.025 ml/min. UV–visible spectrum (a) and percentage of decolorization (b) of MB.

Figure 7

Decolorization of MB by ex situ continuous mode operation at flow rates of 0.050 ml/min and 0.025 ml/min. UV–visible spectrum (a) and percentage of decolorization (b) of MB.

Close modal

In situ decolorization of MB

In the case of the in situ batch mode operation, the decolorization of MB almost exhibited a similar trend to the Ex situ batch mode, but the percentage of decolorization was remarkably higher than that of the ex situ one. This result was attributed to the presence of a larger number of bacteria in the case of in situ operation than that of the ex situ one because the decolorization reaction was accomplished by both the suspended and biofilm cells (Sharma et al. 2016). The percentage of MB decolorization by the in situ batch mode operation gradually increased with the extension of the operation period from 1 to 18 h (Figure 8). The in situ batch mode operation showed 97.1% decolorization of MB in 18 h.
Figure 8

Time-course decolorization of MB at 50 ppm by in situ batch mode operation. UV–visible spectrum (a) and percentage of decolorization (b) of MB.

Figure 8

Time-course decolorization of MB at 50 ppm by in situ batch mode operation. UV–visible spectrum (a) and percentage of decolorization (b) of MB.

Close modal
After completing the in situ batch mode operation, the experiment was designed for the in situ continuous mode operation. To evaluate MB decolorization by the in situ continuous mode operation, the MB mixed with whey was continuously provided into the anode chamber at flow rates of 0.05 ml/min and 0.025 ml/min. After 18 h of operation at a flow rate of 0.05 ml/min, the MB decolorization obtained was 74.1%, while the decolorization at a flow rate of 0.025 ml/min was 97.0% (Figure 9). This result implies that the decolorization of MB by the Inin situ continuous mode dependend on the hydraulic retention time of MB molecules in the anode chamber (Manu & Chaudhari 2003). The in situ continuous mode exhibited better performance in decolorization than the ex situ mode at the same flow rate. The results of this study indicate that the CW-MFC can be used to decolorize MB in both ex situ and in situ modes. However, the in situ mode for decolorization is more cost-effective than the ex situ mode because it requires no additional setup. Thus, the CW-MFC technique can be used as a prestep process for treating MB-containing textile wastewater at the point of source where it is disposed of in combination with other treatment methods. The information of this study will also help to decentralize the conventional wastewater treatment technology.
Figure 9

Decolorization of MB by in situ continuous mode operation at flow rates of 0.050 ml/min and 0.025 ml/min. UV–visible spectrum (a) and percentage of decolorization (b) of MB.

Figure 9

Decolorization of MB by in situ continuous mode operation at flow rates of 0.050 ml/min and 0.025 ml/min. UV–visible spectrum (a) and percentage of decolorization (b) of MB.

Close modal

Phytotoxicity assay

A phytotoxicity test was done to observe the facts of whether the treated wastewater was safe for irrigation of rice paddy. The phytotoxic effect of untreated and treated MB on O. sativa (BRRI dhan 98) was evaluated in this study. The assay was conducted twice to ensure accuracy, and the average values of two individual results were recorded. The untreated MB and the untreated whey remarkably affected the germination (%), root length, and shoot length of O. sativa (Figure 10). However, the treated ones showed very little or no toxicity to the seeds of O. sativa. The relative sensitivity of O. sativa to the treated and untreated MB (50 ppm) and the treated and untreated whey is presented in Table 1. In the phytotoxicity assay, the untreated MB at 50 ppm exhibited 57% germination of O. sativa, while the treated MB showed 100% germination similar to water (Figure 10). The average lengths of the roots of O. sativa after a period of incubation with the untreated and treated MB were 5 and 75 mm, respectively. Interestingly, the average length of root obtained for the treated MB was even bigger (75 mm) than that (70 mm) for water. Thus, the treated MB effluent can have a growth-promoting effect on the root length of O. sativa with no germination inhibition. This might be because of the good effects of metabolic products when whey is used as a growth medium and MB is broken down by a group of bacteria (Elfarash et al. 2017). In the case of shoot length, the untreated MB also showed little inhibitory effects on O. sativa. The intensity of the inhibitory effect on shoot length was eliminated when MB was treated with the bacterial consortium. Thus, it is apparent that the untreated MB at 50 ppm badly affects the germination, root length, and shoot length of the rice variety O. sativa ‘BRRI dhan 98’ (Yu et al. 2015). However, the proper treatment of MB with CW-MFC could eliminate the inhibitory effects of MB on O. sativa. A previous study found that MB was harmful to Tunisian Saragolla plants, but the harmful effects were lessened when MB was treated with S. aureus (Ayed et al. 2022). In addition, the effects of the untreated whey, the untreated whey with untreated MB, and the treated whey without MB on the germination, rooting, and shooting of O. sativa were also tested. The results indicated that the untreated whey (CWW) had high toxicity to the germination, rooting, and shooting of O. sativa. So, whey must be treated before being disposed of into the water bodies. The inhibition of the germination, rooting, and shooting of O. sativa was more vigorous when the untreated whey and MB were present together than alone only. Interestingly, the toxicity of both MB and whey to O. sativa was eliminated when MB and whey were treated by the ex situ and in situ operations of CW-MFC in this study. Thus, the industrial effluents containing MB can be detoxified by CW-MFC, and the treated wastewater can be used in irrigation applications.
Table 1

Phytotoxic effects of untreated and treated MB and CWW on germination, rooting, and shooting of Oryza sativa (BRRI dhan 98)

ParametersUntreated
Treated
WaterMBCWWMB + CWWCWWMB
Germination (%) 100 57 15 10 100 100 
Root length (mm) 70 75 75 
Shoot length (mm) 75 55 25 23 67 65 
ParametersUntreated
Treated
WaterMBCWWMB + CWWCWWMB
Germination (%) 100 57 15 10 100 100 
Root length (mm) 70 75 75 
Shoot length (mm) 75 55 25 23 67 65 
Figure 10

Phytotoxicity assay of MB against Oryza sativa (BRRI dhan 98).

Figure 10

Phytotoxicity assay of MB against Oryza sativa (BRRI dhan 98).

Close modal

This study was conducted to develop a natural bacterial consortium in the bioanode of CW-MFC as a promising technique for decolorizing MB with simultaneous electricity generation. Both the ex situ and in situ operations of CW-MFC showed good capability to decolorize MB by more than 90% within 18 h of incubation, while in situ treatment exhibited better decolorization performance than ex situ one. If MB is treated with a group of bacteria from the bioanode of the CW-MFC, it will no longer be harmful to O. sativa plants. So, the CW-MFC technique can be used as a viable option for the decrease of toxicity of MB-containing wastewater, and the treated effluent can directly be applied for the irrigation of paddy.

This research work was funded by the Rajshahi University Research Project (Grant number A-1253/5/52/R.U./Science-27/2020-2021) and the Faculty of Science at the University of Rajshahi, Bangladesh.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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