ABSTRACT
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.
HIGHLIGHTS
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.
INTRODUCTION
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.
MATERIALS AND METHODS
Construction and operation of CW-MFC
Decolorization of MB dye
- (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)).
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.
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.
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.
RESULTS AND DISCUSSION
Generation of voltage by the CW-MFC
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.
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.
Ex situ decolorization of MB
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.
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.
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.
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.
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).
Time requirement for the decolorization of MB at a concentration of 10 ppm, 20 ppm, and 50 ppm.
Time requirement for the decolorization of MB at a concentration of 10 ppm, 20 ppm, and 50 ppm.
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.
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.
In situ decolorization of MB
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.
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.
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.
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.
Phytotoxicity assay
Phytotoxic effects of untreated and treated MB and CWW on germination, rooting, and shooting of Oryza sativa (BRRI dhan 98)
Parameters . | Untreated . | Treated . | ||||
---|---|---|---|---|---|---|
Water . | MB . | CWW . | MB + CWW . | CWW . | MB . | |
Germination (%) | 100 | 57 | 15 | 10 | 100 | 100 |
Root length (mm) | 70 | 5 | 2 | 2 | 75 | 75 |
Shoot length (mm) | 75 | 55 | 25 | 23 | 67 | 65 |
Parameters . | Untreated . | Treated . | ||||
---|---|---|---|---|---|---|
Water . | MB . | CWW . | MB + CWW . | CWW . | MB . | |
Germination (%) | 100 | 57 | 15 | 10 | 100 | 100 |
Root length (mm) | 70 | 5 | 2 | 2 | 75 | 75 |
Shoot length (mm) | 75 | 55 | 25 | 23 | 67 | 65 |
CONCLUSION
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.
ACKNOWLEDGEMENTS
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.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
CONFLICT OF INTEREST
The authors declare there is no conflict.