Hydrogen production in single-chamber microbial electrolysis cells using Ponceau S dye

Abstract

In this study, Ponceau S dye, which is one of the hazardous dyes found in industrial wastewater, was examined for hydrogen production in single chamber-free membrane-free microbial electrolysis cells at different concentrations (10–40 mg L⁻¹). A gas content analysis (hydrogen, carbon dioxide, and methane) was measured daily using gas chromatography to determine the effects of the Ponceau S on hydrogen production levels. Hydrogen was successfully produced in the presence of Ponceau S dye, but the gas production levels were affected by the concentrations of Ponceau S. The maximum hydrogen production was measured as 18 mL at a concentration level of 20 mg L⁻¹. Decolorization ratios of Ponceau S were in the range of 85–100%. Hydrogen production rates increased in the presence of Ponceau S (20 mg L⁻¹); however, yield (%) of the production decreased when compared to the control group. The percentage of COD removal was 94.78% in the presence of 40 mg L⁻¹ of Ponceau S. In conclusion, hydrogen can be generated using wastewaters contaminated with azo dyes such as Ponceau S, and decolorization of the dye can be achieved, simultaneously.

Introduction

Microbial electrolysis cells (MEC) are novel biotechnological tools for hydrogen production from renewable materials. Several organic compounds are utilized as carbon sources by exoelectrogenic microorganisms for hydrogen production in MECs. Recently, many studies have focused on the use of wastewaters and organic wastes for hydrogen production in MECs because of the fact that these organic compounds are renewable, cheap, and available in large amounts. Approximately, 700,000 tons of dyes are used worldwide every year, mainly in the textile and cosmetic industry (Zollinger 1987; Kumru et al. 2012). Azo dyes are dyes that contain one and/or azo bonds (N=N) in their structure. They make up about 80% of the dye industry (Wang et al. 2013). Around 15% of the dyes used are released to the environment, especially to water resources, during various processes. It has been determined that there is dye waste in concentrations of 10–200 mg L⁻¹ in the wastewaters coming from various industries such as textile industry (Bisschops and Spanjers 2008). The textile industry emits high volumes of wastewater without applying any treatment or any treatment to the water source around it, and affects the entire ecosystem. Azo dyes in wastewaters are potentially toxic and can remain without deterioration in nature for a long time if no treatment is applied. These wastes can increase the pH, biological oxygen demand (BOI), and chemical oxygen demand (COD) ratios in the water sources (Singh et al. 2015; Catal et al. 2018).

Approximately 86% of energy needs worldwide has been provided by fossil fuels such as oil, coal, and natural gas. However, these fossil fuels have deleterious effects on the environment and living organisms (Lu 2016; Logan and Rabaey 2012). In this respect, researchers have been trying to find alternative renewable energy resources. In the search for sustainable and renewable energy sources, wastewater containing different carbon sources draw attention (Moiseyev et al. 2014; Catal et al. 2019). The use of wastewater for hydrogen production has become a popular subject in science (Katuri 2019). The clean treatment of wastewaters is important to find a possible approach for providing energy needs from renewable and cheap resources. The amount of electrical energy used for the treatment of wastewater accounts for three percent of total electrical energy use. However, the amount of energy obtained by wastewater treatment can be approximately three times higher than the amount of electricity spent for treatment (Lu and Ren 2016). So, MECs may help decreasing the wastewater treatment plants’ operational costs with additional value of producing hydrogen.

Hydrogen is an alternative renewable energy source which can be used as a fuel, and it is environmentally friendly. The energy content of hydrogen per kg is at a 120 MJ/kg ratio which is higher when compared to other gaseous fuels such as methane (CH₄) and ethanol. In addition, hydrogen can be obtained from domestic waste and biomass substrates, which leads to the development of new technologies for hydrogen production (Catal 2015; Catal 2016). Any organic substance containing carbohydrate, protein, or oil can be used as a substrate for biohydrogen production through microorganisms (Leicester 2020; Catal et al. 2018; Hu et al. 2018). Microbial electrochemical cells are systems that can produce energy from resources such as wastewater or lignocellulosic biomass (Abourached 2014). Microbial electrolysis cell (MEC) technology, a type of microbial electrochemical cells, can generate hydrogen from organic compounds, and can be used simultaneously in wastewater treatment (Wilson and Kim 2016). Microbial electrolysis cells consist of microorganisms and electrodes. Microorganisms form an electrochemically active biofilm structure on the anode surface utilizing organic substrates (Catal et al. 2017; Kadier 2016). During the production of hydrogen using MECs, methane and carbon dioxide can be produced through microbial activities. The electrons coming from the degradation of substrates are transferred to the anode, and the protons are released into the solution; then the electrons cross the cathode and merge with the protons present in the solution. These processes end with hydrogen production at the cathode. Microbial electrolysis processes cannot happen spontaneously, because the reactions are endothermic. Therefore, a potential should be applied to overcome the thermodynamic barrier (Catal 2016; Kadier et al. 2016). Various environmental contaminants have been evaluated for the production of hydrogen in MECs (Leicester 2020).

Ponceau S dye is commonly found in industrial wastewaters and is hazardous to the ecosystem. Various strategies including photocatalytic degradation, nanosized ZnO, smart water-based ferrofluid, and photo-fenton processes and several nanomaterials were reported to degrade and decolorize the contamination caused by Ponceau S (Patil and Shrivastava 2011; Marathe and Shrivastava 2015; Laftani et al. 2019; Ruan et al. 2019; Chen et al. 2020). However, microbial electrolysis cell technology has not been examined, and the information about the effects of azo dyes in wastewaters on hydrogen generation in MECs is limited based on our literature research.

In this study, effects of Ponceau S on hydrogen production in MECs were examined for the first time as a model study. Gas productions including hydrogen, methane, and carbon dioxide were measured using gas chromatography (GC). Decolorization of Ponceau S in MECs and COD removals were investigated. Hydrogen production rates, actual ratio, and yield (%) levels were also calculated. The cytotoxicity of Ponceau S was assessed using cell viability assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT assay).

Materials and methods

Chemicals

Ponceau S dye [3-hydroxy-4-[[2-sulfo-4- [(4-sulfophenyl) diazenyl] phenyl] diazenyl] naphthalene-2,7-disulfonic acid] was obtained from VWR (CAS No: 6226-79-5, Solon, USA). Sodium acetate trihydrate, 99% [NaOOCCH₃-3H₂O], was obtained from VWR (Alfa Aesar, CAS 6131-90-4, Karlsruhe, Germany). D ( +)-Glucose [C₆H₁₂ O₆] was obtained from VWR (Alfa Aesar, Karlsruhe, Germany). Sodium acetate trihydrate was used as a carbon source in MEC. Ponceau S dye was dissolved in sodium acetate trihydrate buffer and prepared in three different concentrations as 10 mg L⁻¹, 20 mg L⁻¹ and 40 mg L⁻¹.

Construction and inoculation of microbial electrolysis cells (MECs)

Single-chamber microbial electrolysis cells (MECs) were prepared according to previous report (Catal et al. 2015). Four MECs were used in the study. Glass serum bottles (15 mL) were used in the construction of MECs to investigate hydrogen production. Anode (Lot: 14032102, FuelCells, Texas, USA, 4 cm²) and cathode (CTO32414, FuelCells, Texas, USA, 4 cm²) were placed inside the MECs. MECs were inoculated with a mixed microbial culture enriched from a local wastewater treatment plant (Pasakoy Advanced Biological Wastewater Treatment Plant, Istanbul, Turkey) using sodium acetate as the sole carbon source. A sodium phosphate buffer (pH 7.0) was prepared as stock in 2 L according to the previous description (Lovley and Phillips 1988). 8.19 g L⁻¹ sodium phosphate dibasic (Na₂HPO₄, Calbiochem, Cat: 567550, Darmstadt, Germany) and 6.601 g L⁻¹ sodium dihydrogen phosphate (NaH₂PO₄.2H₂O, Alfa Aesar, Cat: A11316, Karlsruhe, Germany) were prepared. For the 50 mM inoculum solution, 0.675 g sodium acetate trihydrate was dissolved in 80 mL, and 100 mM sodium phosphate buffer (NaPO₄, pH 7). They were then passed through gas mixture containing nitrogen and carbon dioxide (80:20) for 10 minutes to remove air from inside. The MECs were applied 0.7 V of applied voltage and 2.2 Ω external resistance and were operated at 30 ± 2 °C (n = 2). No potential was applied to the control groups (n = 2). One milliliter of the gas sample was taken from the MECs with a Hamilton needle (Hamilton Company, HAM-81317, Switzerland).

GC analyses

The gases produced by MECs was measured using the GC instrument (Agilent, 7820A; J&W Scientific, USA), which is a thermal conductivity detector (AGT-64332A) and a column with argon as the carrier gas (113–3133 GS-CARBONPLOT, 30 m, 0.32 mm, 3 mm, J&W Scientific, USA). Conditions during the gas analysis were as follows: column flow 2.8 mL min⁻¹, 185 ℃ heater (Catal et al. 2015).

Decolorization analysis

The following Ponceau S concentrations were examined in the study; 0 mg L⁻¹, 10 mg L⁻¹, 20 mg L⁻¹, 40 mg L⁻¹. Sodium acetate trihydrate (50 mM) was used as the sole carbon source. The effluents from the MECs were used to measure the decolorization of Ponceau S using a UV–VIS Spectrophotometer (Shimadzu Europe UV-2600, A116654, Japan). The decolorization was calculated as follows:

$$\text{The percentage of color removal}=\left(\left(\text{A1}-\text{A2}\right)/\text{A1}\ast 100\right)$$ 1

where A1 is the absorbent of the solutions entering the reactor, and A2 is the absorbent of the solutions coming out of the MECs.

Cytotoxicity analysis

Ponceau S dye and ECV 304 human endothelial cell line were used in cytotoxicity assay using MTT method. For MTT, the test was performed to measure the cytotoxic activity of the dyes on the cells. Eight mg of Ponceau S dye was weighed and dissolved in 100 mL NaPO₄ buffer, and a stock solution was prepared. Thus, the concentration of stock solutions was adjusted to 80 mg L⁻¹. Then, stock solutions were prepared as 40 mg L⁻¹, 20 mg L⁻¹, 10 mg L⁻¹, and 5 mg L⁻¹ and applied to the cell lines. 90 µL of cell-medium mixture was placed in each plate well to make 104 cells. The cells were kept in a 37 °C 5% CO₂ incubator for 24 h, and afterwards 10 µL of the dye solutions prepared in appropriate doses were added to each well. Cells were kept in 37 °C 5% CO₂ incubator for 24 h to be treated with dye solution. After the period was filled, 30 µL of MTT (Invitrogen, Cat No: 1899778, USA) prepared in sterile PBS was added to each well and incubated for four h in a 37 °C 5% CO₂ incubator for 4 h. After waiting, all liquids were drawn from the wells without touching the cells and added from 90 µL of a 50% DMSO-50% isopropanol mixture (BDH, Cat No: BDH1115-4LP). Spectrophotometer (Thermo MultiScan Go, USA) absorbance measurements were taken at 570 nm after waiting for 45 min at room temperature with aluminium (Enisoglu-Atalay et al. 2018; Cebecioglu et al. 2019).

Chemical oxygen demand (COD) analysis

COD analysis was performed on samples collected from MECs before and after the operation (American Public Health Association 1992). Solutions to be used in the analysis were prepared. 0.5 mL of each sample, 0.3 mL of the lysing solution, and finally 0.7 mL of sulfuric acid reagent were added to the glass tubes. The mouth of the glass tubes was covered with parafilm and aluminium foil and vortexed for 30 s. Then, it was boiled with a magnetic heater stirrer (Stuart, SD162) at 150 ℃ for 2 h. Color variation was observed in the samples. After the glass tubes were cooled at room temperature, the UV–VIS Spectrophotometer (Shimadzu, UV-2600) absorbance at 600 nm was measured. Standard graphics were prepared, and the percentage COD was calculated according to the standard method.

Results and discussion

Hydrogen production in MECs

Figure 1 shows the effects of three different concentrations of Ponceau S on the gas production in MECs (Fig. 1). MECs were fed with sodium acetate trihydrate (50 mM) for 35 days without inoculation of the dye solution in the first batch. After producing hydrogen gas (H₂), the total gas production was measured, daily. Hydrogen gas production was observed in all the experimental groups, where an external voltage (0.7 V) was applied, while there was no hydrogen production in the control groups. The total amount of accumulated hydrogen gas was 25 mL in 35 days with sodium acetate trihydrate (50 mM). Along with hydrogen gas, MECs produced also 25 mL of methane (CH₄) gas and 10 mL of carbon dioxide (CO₂) gas (Fig. 1a). Only nine mL of CO₂ gas was accumulated in the control MECs. In the next batch of MEC operation, Ponceau S dye was introduced into MECs at the concentration of 40 mg L⁻¹ for 13 days. The total amount of hydrogen gas produced at the end of the 13th day after dye addition was low (around one mL). Along with hydrogen gas, methane (CH₄) gas production was not observed in MECs, and methane was suppressed. The amount of carbon dioxide (CO₂) gas produced was 4.5 mL. In the controls without voltage, hydrogen gas and methane gas production were not observed, and only 4 mL of CO₂ gas accumulated. Then, the concentration of Ponceau S decreased to 10 mg L⁻¹ in the next batch, and MECs were operated for 15 days. The total amount of hydrogen gas produced at the end of the 15th day after dye inoculation was 6 mL. The amount of carbon dioxide (CO₂) gas produced was 12 mL. There were no hydrogen or methane production, and only 14 mL of carbon dioxide was produced. In the last batch of operation, the concentration of Ponceau S was increased to 20 mg L⁻¹, and MECs were operated for 22 days. The total amount of hydrogen gas produced at the end of the 15th day after dye inoculation was 18 mL. MECs also produced one mL of methane gas and four mL of carbon dioxide gas. As shown in Fig. 1b, hydrogen or methane gas productions were not observed in the controls, and only 16 mL of accumulated carbon dioxide gas was produced (Fig. 1b).

Fig. 1.

Total gas accumulation (pH 7.0) by mixed culture in membrane-free MEC using 10 mM sodium acetate (external resistance of 2.2 Ω, 0–40 mg L ⁻¹) (a). Total gas accumulation (pH 7.0) by mixed culture in membrane-free MEC using 10 mM sodium acetate of control groups (b)

Hydrogen production rates increased in the presence of Ponceau S (20 mg L⁻¹); however, yield (%) of the production decreased when compared to the control group (Table 1). These results indicate that hydrogen gas can be produced in the presence of Ponceau S which is found in various industrial wastewaters.

Table 1.

Decolorization ratios and performance results of MECs in the presence of Ponceau S (0–40 mg L⁻¹)

Concentration (mg L−1)	Decolorization (%)	pH	Hydrogen production (m3/day/m3)	Actual ratio (mol H2/mol acetate)	Yield (% max)
0	–	7.5	0.038	8.18	205
40	85	7.5	0.005	0.38	10.0
20	94	7.5	0.053	6.37	159
10	100	7.5	0.037	2.31	58.0

Hou et al. (2017) reported both decolorization of methyl orange, an azo dye, and simultaneous hydrogen production in single chamber MECs. They reported that hydrogen production decreased from 98 to 76% in 12 h by increasing from 50 mg L⁻¹ to 300 mg L⁻¹. While it was 113 mL at 50 mg L⁻¹ for hydrogen production, it was reported as 68 mL at 300 mg L⁻¹. Li et al. (2016) reported a system integrated with MFC-MEC to increase performance in addition to microbial fuel cells (MFC) for which decolorization performances have been previously reported and evaluated the effect of various key factors on the reactor performance. It was reported that the decolorization rate of the combined MFC-MEC systems using acetate as the carbon source showed an improvement of 37–75% compared to the single MFC. Huang et al. (2018) investigated the decolorization of the azo dye type Congo red using membrane-free microbial electrolysis cells (MFMEC). The decolorization time was reported as 42 h, and reactors were applied 0.3 V, 0.6 V, and 0.9 V, respectively. In MFMEC, all color removal efficiency reached 90%, and the decolorization rates in 24 h were 88%, 85%, and 87%, respectively. When the pH analysis of the samples taken from MEHs before and after the operation was performed, it was seen that the pH value increased from 7 to 7.5 depending on the fermentation processes. It is thought that another reason for the suppression of methane in experiments may be the increasing pH value. According to the researches, it has been reported that the ideal conditions for the production of methanogen bacteria are neutral pH value (Chen et al. 2002; Van Hanndel and Lettinga 1994). pH analysis was performed by taking 1 mL of samples from MEHs at different concentrations before and after the operation. According to the results, it was seen that the pH value increased from 7 to 7.5 depending on the fermentation processes. Especially in the operation performed with a dye solution at a concentration of 20 mg L⁻¹, it was suggested that the pH value increases as the reason for the suppression of methane generation. According to the study, it has been reported that the ideal conditions for the production of methanogen bacteria are neutral pH value (Chen et al. 2002; Van Hanndel and Lettinga 1994).

Azo dyes can show different levels of strength to decolorization depending on their azo bond, chromophore, and auxochrome structures. Therefore, the structure of the dye is an important parameter in the selection of the decolorization method (Gürses et al. 2016). Absorbance measurements of the samples from microbial electrolysis cells were measured, and decolorization rates were calculated in accordance with the previously mentioned formula. As shown in Table 1, the maximum decolorization from the dye solutions infused into the MEC reactors was observed at a concentration of 100% and 10 mg L⁻¹. At the 40 mg L⁻¹ concentration, the decolorization rate was calculated as 85% for the experimental groups and 97% for the control group. At 20 mg L⁻¹ concentration, it was calculated as 94% in the experimental groups and 100% in the control groups. According to these results, decolorization decreased as the concentration of the dyes in different concentrations inoculated with MEC increased. Likewise, in experimental groups with 0.7 V electricity, a lower rate of decolorization was observed compared to control groups with 0 V electricity. According to the results of the study (Fig. 2), it was determined that Ponceau S, which is a diazo dye in MEC, can both successfully remove color and produce high levels of hydrogen.

Fig. 2.

Photograph of decolorization from the 0th hour to the forty-eighth hour when the dye is added to the MEC. 0 h (a),12 h (b), 36 h (c), 48 h (d)

Dye solutions at different concentrations (40 mg L⁻¹, 20 mg L⁻¹, 10 mg L⁻¹, and 5 mg L⁻¹) were treated with ECV 304 cells for 24 h with Ponceau S dye. According to the MTT assay results, Ponceau S dye did not show a toxic effect on the ECV 304 cell line. Compared to the control group, the 10 mg L⁻¹ dose cell viability was 105%, and the 40 mg L⁻¹ dose cell viability was 118% (Fig. 3). The highest cell viability was 124% at 20 mg L⁻¹ dose. All doses applied ECV 304 showed the same proliferation compared to the control group. COD analysis was performed on samples collected from MEC reactors. The percentage of COD removal was 94.78% in the experimental group in which 0.7 V electricity was applied in a 40 mg L⁻¹ concentration in Ponceau S. In future, other types of azo dyes such as monoazo, diazo and triazo would be investigated as a sole contaminant or in various combinations using well-defined media or real wastewater samples for hydrogen production in MECs.

Fig. 3.

Cytotoxicity results of Ponceau S (0–40 mg L⁻¹) on the healthy cell line ECV 304 using MTT assay

Conclusions

Hydrogen was generated using Ponceau S dyes at the concentration levels (10–40 mg L⁻¹) in single chamber-membrane-free MECs. Maximum hydrogen production was calculated as 18 mL in the dye solution prepared at a concentration of 20 mg L⁻¹. Decolorization of Ponceau S was 100% at the concentration level of 10 mg L⁻¹. The examined concentrations of the Ponceau S were not significantly affected ECV cells. In conclusion, hydrogen gas can be produced in the presence of Ponceau S found in various industrial wastewaters such as textile industry, and simultaneous decolorization of Ponceau S can be achieved in MECs. This technology could also help decreasing wastewater treatment plant operational costs in the treatment of wastewaters coming from textile industry.

Authors’ contributions

RC and DA performed the investigations of the study. RC and TC analyzed the results and wrote the manuscript.

Funding

Not applicable.

Data availability

All the data used in the current study are available from the corresponding author on reasonable request.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.