Bioelectrochemical decolorization of a reactive diazo dye: Kinetics, optimization with a response surface methodology, and proposed degradation pathway

Bioelectrochemistry 128 (2019) 9–16

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Bioelectrochemical decolorization of a reactive diazo dye: Kinetics, optimization with a response surface methodology, and proposed degradation pathway Hou-Yun Yang a,b, Jing Liu b, Yi-Xuan Wang b, Chuan-Shu He b,⁎, Li-Shan Zhang c,⁎, Yang Mu b, Wei-Hua Li a a

School of Environment and Energy Engineering, Key Laboratory of Anhui Province of Water Pollution Control and Wastewater Reuse, Anhui Jianzhu University, Hefei, China CAS Key Laboratory for Urban Pollutant Conversion, Collaborative Innovation Centre of Suzhou Nano Science and Technology, Department of Applied Chemistry, University of Science & Technology of China, Hefei, China c College of Environment and Resources, Guangxi Normal University, Guilin, China b

a r t i c l e

i n f o

Article history: Received 19 November 2018 Received in revised form 15 February 2019 Accepted 16 February 2019 Available online 9 March 2019 Keywords: Bioelectrochemical systems Diazo dye Kinetics Response surface methodology Decolorization pathway

a b s t r a c t Bioelectrochemical systems (BESs) have shown great potential for azo dye removal. However, comprehensive evaluation of the bioelectrochemical decolorization performance for reactive diazo dyes remains limited, particularly the kinetics and operation parameter optimization. This study evaluated the decolorization of the diazo dye Reactive Black 5 (RB5) in BESs, particularly with regard to kinetics, parameter optimization using response surface methodology (RSM), and the degradation pathway. The results indicated that the pseudo–first–order kinetic rate constant of RB5 decolorization increased from 0.023 ± 0.001 to 0.146 ± 0.008 h−1 with a decrease in cathode potential from −400 mV to −500 mV. RSM optimization suggested that the linear effects of RB5 concentration, cathode potential and hydraulic retention time (HRT), interaction of RB5 concentration with cathodic HRT, and the quadratic effect of cathodic HRT were most influential on the bioelectrochemical decolorization of RB5. Further, the decolorized RB5 products in the BESs were characterized by ultraviolet–visible spectrophotometry, Fourier-transform infrared spectroscopy, and liquid chromatograph–mass spectrometry. From this, a potential decolorization mechanism is proposed based on cleavage of azo bonds. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Bioelectrochemical systems (BESs), which represent an emerging technology, are designed and developed to remove wastewater pollutants by using bioelectrochemical or oxidization–redox processes. These pollutants include nitrophenols, nitro-aromatics, perchlorate, hexavalent chromium, chloramphenicols, and azo dyes [1–11]. However, research has only focused on the effect of several key factors on the removal of recalcitrant contaminants from wastewater in BESs. Among these factors are cathode potential [12,13], initial compound concentration [8,12], cathodic pH [14,15], and so on. Mu et al. [8] and Shen et al. [11] demonstrated that reduced initial concentration and enhanced cathode potential contribute to degradation efficiency. Meanwhile, comprehensive evaluation of the kinetics and operation parameter optimization of contaminant removal in BESs remains inadequate. Azo dyes in wastewater from textile, paper, and dyestuff industries comprise 70% of all known commercial dyes and are the topically ⁎ Corresponding authors. E-mail addresses: [email protected] (C.-S. He), [email protected] (L.-S. Zhang).

https://doi.org/10.1016/j.bioelechem.2019.02.008 1567-5394/© 2019 Elsevier B.V. All rights reserved.

common chromophores in reactive dyes [16]. The discharge of diazo dyes not only severely threatens lives and the environment but also affects the aesthetics of nature. Large quantities of diazo dyes are produced, subsequently generating intermediate substances that are toxic and mutagenic [13]. Most reactive diazo dyes exhibit poorer decolorization owing to their more complex structures and number of azo bonds; moreover, functional groups and the arrangement on the compounds of diazo reactive dyes can considerably affect their decolorization performance [17]. Physicochemical and biological methods have been developed to remove these reactive diazo dyes from wastewater; however, they use significant quantities of chemicals and/or produce large amounts of sludge, requiring safe disposal [18]. Reactive dyes can be degraded or transformed into degradable products through biological and physicochemical processes in BESs [13,15,19–24]; however, the decolorization of diazo dyes in BESs is scarcely reported [25–27]. Studies have rarely considered the degradation kinetics of azo dyes under various conditions, which is required to achieve optimal design and operation of BESs. Conventional methods to evaluate the factors affecting a process often select one factor as a variable and other factors fixed to a certain extent, failing to depict the combined effects of all factors involved. This technique is also time-consuming and lacks reliability because it

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requires a number of experiments to determine optimal levels [28]. Response surface methodology (RSM) is a set of statistical methods for experimental design, generating enhanced and pragmatic predictions for near-optimal process factors in the prescribed operability region [29]. RSM has been successfully applied in several complex systems, such as biological hydrogen production [30], enzyme synthesis [31], bacterial growth [32], acetic acid production in acidogenesis of swine wastewater [33], as well as azo dye decolorization, by using biological and physicochemical methods [34,35]. However, RSM has rarely been adopted to optimize azo dye decolorization in BESs. The present study aimed to confirm the feasibility of diazo dye decolorization in BESs and analyze the degradation kinetics. Reactive Black 5 (RB5), one of the widely used reactive diazo dyes for textile finishing, was used as a model because it has the largest consumption, among all reactive dyes, for cotton and other cellulose fibers in textile industries [36]. Subsequently, RSM was performed to determine the optimal operation parameters for diazo dye decolorization. Dehalogenation intermediates and products were identified by ultraviolet–visible spectrophotometry (UV–vis), Fourier-transform infrared spectroscopy (FTIR), and high-performance liquid chromatography–mass spectrometry to propose a possible RB5 degradation pathway in BESs. 2. Materials and methods 2.1. Chemical reagents The diazo dye RB5 was purchased from Sigma–Aldrich Co. (USA); its characteristic structure is shown in the Supplementary material (Fig. S1). Na2SO4 and CH3COONa of analytical grade were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were used without further purification. 2.2. Evaluation of RB5 decolorization kinetics with batch experiments BES was constructed as described by Aelterman et al. [37]. Anodic and cathodic compartments were separated by a cation exchange membrane (Membranes International Inc., USA), each with an empty volume of 650 cm3. Carbon felt (25.2 cm × 5.0 cm) in a roll form was used as the anode material so that the working volume of anode was 550 cm3. Before use, all carbon electrode materials were washed in 32% HCl for 24 h to remove any potentially catalytic foreign compound from the carbon materials. The anodic compartment of the BES was inoculated with a microbial consortium that had been enriched in BESs, with acetate as the carbon source, and continuously fed with a modified M9 medium as described in a previous study [38]. The inoculum, which was enriched in BESs, was collected from a full-size, enlarged granular sludge-bed reactor treating starch wastewater located in Shandong Province, China. The anolyte was recirculated at a rate of approximately 200 mL·min−1 to maintain well-mixed conditions and to prevent concentration gradient. The carbon felt (6.5 cm × 7.0 cm) was used as the cathode material, reaching an effective cathode volume of 600 cm3. The catholyte of the BES contained RB5 at desired concentrations, with a 50 mM phosphate buffer to maintain the solution pH at 7.0. The startup and operation of the reactor are discussed in the study by Freguia et al. [39]. To maintain well-mixed conditions and avoid concentration gradient, the cathode chamber was stirred with a magnetic stirrer during batch experiments. The graphite rod contacts of both the anodic and cathodic electrodes were connected to a potentiostat (VMP3, Bio-Logic Science Instruments, France) for cathode potential control and online measurement. The Ag/AgCl reference electrode (+0.197 V vs. standard hydrogen electrode or SHE) was placed close to the cathodic electrode for potential measurement; the potential reported refers to the SHE in this study. All batch experiments were repeated at least twice and performed at 25 °C ± 1 °C.

2.3. RSM optimization of RB5 decolorization with continuous experiments The anode of the BES, as well as the electrochemical monitoring for continuous experiments, was the same as the aforementioned. Granular graphite with diameters ranging from 2 mm to 6 mm was used as the electrode in the cathode compartment, reducing the compartment volume to 330 cm3 (net cathodic compartment or NCC). The cathode influent of the BES contained RB5 and sulfate at desired concentrations, with a 50 mM phosphate buffer (pH at 7.0), and the catholyte was also recirculated at approximately 200 mL·min−1 to maintain well-mixed conditions and to avoid concentration gradients. Before changing to the next condition, each experiment lasted 1–2 weeks to ensure that the reactor reaches the pseudo–steady-state, as determined by the constant RB5 decolorization rate (DR) and the anode and cathode potentials. Each trial was replicated at least two times. Only the results obtained under pseudo–steady-state conditions are reported in this study. The most popular class of second-order designs called central composite design (CCD) was used for RSM in the experimental design. The CCD is suitable for fitting a quadratic surface, which is usually efficiently for process optimization [29]. The range and levels of the variables investigated, as well as the experimental setup, are given in the Supplementary material (Tables S1 and S2). Influent RB5 concentration (X1), cathode potential (X2), cathodic HRT (X3), and influent sulfate concentration (X4) were selected as independent input variables. The reason sulfate is selected as an input variable is that textile-processing wastewaters usually contain moderate to high sulfate concentrations [40]. The following were the central values chosen for the experimental design: RB5 concentration, 0.12 mM; cathodic electrode potential, −400 mV; HRT, 2.0 h; and sulfate concentration, 50 mM. The variables Xi were codes as xi according to Eq. (1). xi ¼

X i −X 0 δX

ð1Þ

where X0 is the value of Xi at the central point, and δX represents the step change. The RB5 DR was chosen as the dependent output variable. The response variable of the RB5 DR was fitted using a predictive polynomial quadratic equation to correlate the response variable to the independent variables. The general form of the predictive polynomial quadratic equation is Y ¼ A0 þ

k X i¼1

Ai xi þ

k X i¼1

Aii x2i þ

k X k X i

Aij xi x j

ð2Þ

j

where xi are the input variables, which influence the response variable Y; A0 is the offset term; Ai is the ith linear coefficient; Aii is the quadratic coefficient, and Aij is the ijth interaction coefficient. Eq. (2) can be used to locate the optimum for the set of independent variables by the partial derivatives of the model response with respect to the individual independent variables is equal to zero. Experimental data were analyzed by response surface regression of a statistical analysis system (Minitab, Version 14, Minitab Inc., USA) and fitted to a second-order polynomial model. The corresponding analyses of the variations were assessed using MATLAB 7.0 (MathWorks Inc., USA). Subsequently, three-dimensional response surfaces were constructed to provide a visual insight into the effects of RB5 concentration, cathode potential, HRT, and sulfate concentration on the RB5 DR. Two additional experiments were conducted to verify the validity of the statistical experimental methods. 2.4. Identification of RB5 decolorization products The decolorization of RB5 was monitored using a UV–visible spectrophotometer (Cary 60, Agilent Inc., USA) over a wavelength range

H.-Y. Yang et al. / Bioelectrochemistry 128 (2019) 9–16

from 200 nm to 800 nm. Subsequently, the products of RB5 degradation were characterized by FTIR (FTIR, Nicolet 6700, Thermo Fisher, USA) in the mid-IR region of 400–4000 cm−1. Moreover, the final products of RB5 decolorization in BESs were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LTQ-Orbitrap XL, ThermoFisher Co., USA). The mobile phase was composed of methanol and H2O with a volume ratio of 4:1 at a constant flow rate of 0.2 mL·min−1. Electrospray ionization MS was conducted in a negative mode at 4 kV. The vaporizer temperature was set at 80 °C, and the capillary temperature was 275 °C. 2.5. Chemical analysis and calculation Samples taken from the reactor were immediately filtered through a 0.22 μm membrane. The RB5 concentration was measured using a UV–visible spectrophotometer (Cary 60, Agilent Inc., USA) at its characteristic absorption wavelength of 597 nm. For acetate analysis, 1.0 mL sample was added to 1.0 mL of 3% formic acid solution and then analyzed by gas chromatography (model 7890A, Agilent Inc., USA), using the DB-FFAP polar capillary column at 140 °C and a flame ionization detector at 250 °C. Sulfate concentration was determined using an ion chromatography system (ICS-1100, Dionex, Sunnyvale, CA, USA) with a 4.0 mm × 250 mm analytical column (IonPac AS19, Dionex) at 30 °C and a UV detector. KOH gradient elution ranged from 10 mM to 40 mM, and the flow rate was 1.0 mL·min−1. The bacterial morphologies on the graphite felt were analyzed by scanning electron microscopy (SEM, SIRION 200, FEI, USA). The graphite felt was cut from the anode of the reactor. The specimens were first immersed in 5% glutaraldehyde at 4 °C for 12 h and then gradually dehydrated using ethanol solution at different concentrations (30%, 50%, 70%, 80%, 95%, and 100%) for 15 min each. The electrode pieces were finally dried and coated with gold before micrographs were taken. Batch RB5 decolorization in the BES was described by a pseudo–firstorder kinetic model according to Mu et al. [41]: dC ¼ k1  C dt

3. Results and discussion 3.1. Typical RB5 decolorization in the BES As shown in Fig. 1A, SEM images reveal that a biofilm is formed on the surface of the anode, and the abiotic anode is smooth without microorganism attachment (Fig. 1B). The DE and DR of RB5 were 74.74% ± 0.17% and 4.84 ± 0.01 mol m3-NCC d−1 in the BES with a controlled cathode potential of −400 mV under HRT of 2.2 h, respectively. The acetate DE was 39.33% ± 6.70% in the anode chamber of the BES. The CE for RB5 decolorization at the cathode was up to 96.58% ± 0.22%, indicating that the majority of electrons from the cathode was used for RB5 reduction. However, the CE for acetate oxidation was only 21.76% ± 0.06% at the bioanode of the BES in which aceticlastic methanogens and bacterial growth could be responsible for electron loss [8]. Earlier studies have suggested various techniques to achieve a high CE for acetate oxidation. These methods include inhibiting the bioactivity of methanogenesis and improving the relative abundance of exoelectrogens [42–44]. Chae et al. (2010) indicated that with 2-bromoethanesulfonate injection, the CE of acetate oxidation increased from 35% (non-injected) to 70% (injected); in addition, they proposed cross-inhibition, such as combining air exposure and injection. With this combination, methanogen is suppressed, but exoelectrogen remains unchanged, which would be desirable to improve the CE for acetate oxidation and improve microbial fuel cell performance [45]. Chen et al. (2019) combined electro-autotroph and acetate enrichment processes to selectively eliminate non-bioelectrochemical microorganisms and increase the amount of exoelectrogens on the electrode, thereby doubling the CE for acetate oxidation after five batches [46]. However, further research is necessary to elucidate the nature of anode processes.

ð3Þ

where C (mM) is the concentration of RB5, t (h) is the reaction time, and k1 (h−1) represents the apparent kinetic rate constant. The RB5 decolorization efficiency (DE) and DR for continuous experiments were calculated as follows: DE ¼

C in−RB5 −C ef −RB5  100% C in−RB5

ð4Þ

DR ¼

  C in−RB5 −C ef −RB5  ν cathode −1  103 mol m−3 −NCC d NCC

ð5Þ

The Coulombic efficiencies for acetate oxidation (CEacetate) and RB5 decolorization (CERB5) during continuous experiments were evaluated in accordance with Li et al. [15]: CEacetate ¼

CERB5 ¼

I 8  ΔC acetate  F 

νcathode  100% 24  3600

  8  C in−RB5 −C ef −RB5  I

νcathode F 24  3600  100%

ð6Þ

ð7Þ

where Cin-RB5 is the influent RB5 concentration (mM), Cef-RB5 is the effluent RB5 concentration (mM), I is the current (mA), F represents the Faraday constant (96,485C mol−1 e−), ΔCacetate is the change in acetate concentration in the anode (mM), 8 represents the number of electrons exchanged per mole of acetate oxidation and RB5 reduction, and νanode and νcathode are the anode and cathode flow rates, respectively (L d−1).

11

Fig. 1. SEM images of anode (A) with biofilm and (B) without biofilm in BES.

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3.2. RB5 decolorization kinetics in the BES Fig. 2A illustrates the kinetics of RB5 decolorization in the BES. The RB5 concentration almost remained at the initial value with reaction time in the open circuit, indicating insignificant RB5 adsorption on the reactor and electrode materials. After the circuit was closed, RB5 concentration rapidly decreased from 0.503 ± 0.002 mM to 0.124 ± 0.007 mM after operating for 10 h when cathode potential was controlled at −500 mV in the BES. Moreover, the RB5 decolorization in the BES was well fit to the pseudo–first-order kinetic model with a high correlation coefficient R2, (Fig. 2A). This result is consistent with that obtained in the study by Kong et al., who also found that the reduction of the diazo dye Congo red fitted well with the first-order kinetic model in BES [25]. When the cathode potential was decreased from −400 mV to −500 mV, the reaction rate constant (k1) for RB5 decolorization increased from 0.023 ± 0.0002 h−1 to 0.146 ± 0.008 h−1 (Fig. 2B). This result is consistent with those in several previous studies regarding mono-azo dye decolorization kinetics in BESs. Sun et al. observed that the reaction rate constant of Alizarin Yellow R decolorization significantly improved with an increase in applied voltage in the BES [47]. Yang et al. also reported that the reaction rate constant of Methyl Orange decolorization increased from 0 to 0.503 ± 0.001 h−1 with a decrease in cathode potential from 0 to −600 mV [13]. A more negative cathode potential indicates a better reducing environment for electron transfer from the electrode to the azo dye for its removal in the BES. However, the k1 value of RB5 decolorization was generally lower than those of mono-azo dye reduction in the BES. Such a

difference suggests that the decolorization of diazo dyes in the BES can be limited by several factors, such as their complex structures, number of azo bond, and functional groups [48]. 3.3. Optimization of RB5 decolorization in the BES The regression coefficient values, standard deviation, texp, and significance level for the optimization of RB5 decolorization in the BES by using RSM are listed in Table 1. A1, A2, and A3 as well as the interaction coefficient A13, are significant at a level below 5%. The quadratic coefficient A33 is also significant at the same level. Therefore, the linear effects of the influent RB5 concentration, cathode potential and HRT (coefficients A1, A2, and A3), interaction of influent RB5 concentration with HRT (coefficient A13), and quadratic effect of cathodic HRT (coefficient A33) are the parameters with the most influence. The significance of these quadratic and interaction effects between the variables would have been lost if the experiments were conducted using the conventional single-variable method. On the basis of parameter estimation (Table 1), the following empirical relationship between the RB5 DR and independent variables was obtained. DR ¼ 1:2080 þ 0:8767x1 −0:1767x2 −1:1233x3 −0:0017x4 þ0:0003x21 −0:0347x22 þ 0:7753x23 −0:0647x24 −0:0300x1 x2 −0:7900x1 x3 −0:0050x1 x4 þ 0:0150x2 x3 −0:0000x2 x4 −0:0000x3 x4

ð8Þ

The measured and predicted RB5 DRs under different conditions are presented in the Supplementary material for comparison (Table S2). The ANOVA results in Table 2 show that the predictive model ensures high representativeness of the experimental data because the significance level calculated from the ratio of the mean square attributed to regression was b0.01. In addition, the regression model had a high coefficient of determination (R2 = 0.9867), further suggesting good agreement between the measured and predicted RB5 DRs in the BES. Table 1 Estimated regression coefficients and corresponding texp. and significance level. Coefficient

Value

Standard deviation

texp.

Significance level/%

A0 A1 A2 A3 A4 A11 A22 A33 A44 A12 A13 A14 A23 A24 A34

1.2080 0.8767 −0.1767 −1.1233 −0.0017 0.0003 −0.0347 0.7753 −0.0647 −0.0300 −0.7900 −0.0050 0.0150 −0.0000 −0.0000

0.0524 0.0478 0.0478 0.0478 0.0478 0.0920 0.0920 0.0920 0.0920 0.1170 0.1170 0.1170 0.1170 0.1170 0.1170

23.070 18.340 −3.700 −23.500 −0.030 0.000 −0.380 8.430 −0.700 −0.260 −6.750 0.040 0.130 0.000 −0.000

b0.01a b0.01a 0.2a b0.01a 97.3 99.7 71.2 b0.01a 49.3 80.2 b0.01a 96.7 90.0 100 100

texp. value was obtained from the t-test, which indicates the significance of the regression coefficients. a Means highly significant.

Table 2 ANOVA analysis.

Fig. 2. The kinetic of RB5 decolorization: (A) at open (black square: □) and closed (red circle: ) circuit at batch mode in BES (red dash line represents regression equation: , k1 = 0.146 ± 0.008 h−1, R2 = 0.987) and (B) under various cathode potentials in the BES (pH 7.0).

Source

Degree of freedom

Sum of square

Mean square

Ratio of mean square

Significance level/%

Regression Residues Total

14 14 28

14.0977 0.1920 14.2897

1.0070 0.0137

73.44

b0.01a

R2 = 0.9867. a Means highly significant.

H.-Y. Yang et al. / Bioelectrochemistry 128 (2019) 9–16

Moreover, the residual plots for the model and data set on the DR of RB5 were randomly distributed (shown in the Supplementary material, Fig. S2). The assumption of constant variance could be verified because a random plot of residuals meant homogeneous error variances across

13

the observed values [33]. Improved prediction of maximum responses, along with constant variance in residual plots, suggests the high adequacy of the predictive quadratic model for evaluating the RB5 DR in the BES.

Fig. 3. Three-dimensional graphs of the quadratic model for RB5 DR within orthogonal design: (A) influent RB5 concentration and cathode electrode potential; (B) cathodic HRT and cathode electrode potential; (C) influent RB5 concentration and cathodic HRT; (D) cathode electrode potential and influent sulfate concentration; (E) cathodic HRT and influent sulfate concentration; (F) influent RB5 concentration and influent sulfate concentration.

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Three-dimensional response surface plots for estimating the decolorization surface over independent variables are presented in Fig. 3, which indicates the relative effects of two variables on the DR while the other two variables remain constant in their central values. Fig. 3A shows the effects of the influent RB5 concentration and cathode potential on RB5 decolorization in the BES. The response surface of the plot implied that the high RB5 DR was achieved at a high influent RB5 concentration and more negative potential. The Acid Orange 7 (AO7) DR also increased from 2.48 ± 0.02 mol m−3 NCC d−1 to 4.08 ± 0.24 mol m−3 NCC d−1 as the influent AO7 concentration increased from 0.19 mM to 0.70 mM. Moreover, AO7 DR increased with a decrease in cathodic electrode potential under the continuous mode [8]. Fig. 3A also suggested that the influent RB5 concentration in the 0.04–0.20 mM range markedly affected its DR in the BES, whereas the variation in cathode potential was less important. The RB5 DR increased when the cathodic HRT decreased from 3.8 h to 0.2 h and then slightly increased when the cathode potential decreased from −300 mV to −500 mV (Fig. 3B). Moreover, the effect of the cathodic HRT on the RB5 DR was more important than that of the cathode potential in the BES. The contour plot suggested the presence of an apparent interactive effect between the influent RB5 concentration and the cathodic HRT on RB5 DR. This effect is confirmed by an obvious peak on the response surface, with the optimal conditions being exactly located in the experimental range. Consequently, the variations in influent RB5 concentration and cathodic HRT exerted the same significant effect on the DR. Figs. 3D–F present the interactive effects of the cathode potential, cathodic HRT, and influent RB5 concentration with influent sulfate concentration on RB5 DR. Further, cathode potential, cathodic HRT, and influent RB5 concentration were identified as the three key factors controlling the RB5 DR. Sulfate concentration only slightly affected RB5 decolorization in the BES, as indicated by considerably higher linear, quadratic, and interaction significance levels of sulfate concentration (Table 1). Sulfate concentration changed slightly even when the cathode potential was −500 mV. Although the electrochemical reduction of sulfate in aqueous solution is thermodynamically possible, sulfate reduction is apparently kinetically inhibited and requires considerably high overvoltage particularly when using a graphite cathode [49]. 3.4. RB5 degradation pathway in the BES Fig. 4 illustrates the UV–visible absorption spectra of catholyte at different reaction times with a controlled cathode potential of −500 mV in

Fig. 4. UV–visible absorption spectra during RB5 decolorization at batch mode in BES (black dash dot (-•-•-): PBS; red line ( ): 0 h; blue dash ( ): 2 h; pink dash ( ): 4 h; green dash ( ): 6 h; black dash (——): 8 h; purple dash ( ): 10 h) (cathode potential −500 mV, pH 7.0).

Fig. 5. FTIR spectrum of RB5: (A) before (blue line: ) and (B) after (red line: decolorization at cathode potential of −500 mV, respectively.

)

the BES. The absorption bands of RB5 at 597 and 310 nm were attributed to the azo bands and naphthalene moieties, respectively [50]. The characteristic absorbance peak of RB5 at 597 nm decreased and shifted to a shorter wavelength; meanwhile, an absorbance peak at 260 nm was simultaneously observed with prolonged reaction time. This result indicated that the molecular structure of RB5 changed evidently and that corresponding products were generated after decolorization because the primary chromophore was destroyed [51]. The FTIR spectra of RB5 before and after decolorization in the BES are presented in Fig. 5. The expected absorption levels are summarized in the Supplementary material (Table S3), as indicated in the more or less classic studies [52]. Significant

Fig. 6. LC-MS chromatogram of catholyte after decolorization at cathode potential of −500 mV in BES.

H.-Y. Yang et al. / Bioelectrochemistry 128 (2019) 9–16

15

Scheme 1. Tentative mechanism for RB5 decolorization in the BES.

changes were observed in the 1800–1400 and 1200–600 cm−1 ranges. The bands located at 1497 and 1402 cm−1 were attributed to azo linkages \\N_N\\ on the aromatic structures and to \\N_N\\ stretching in substituted compounds, respectively [53]. These peaks decreased after RB5 decolorization, confirming the cleavage of the azo bands in RB5 [54]. After the azo linkage peak was reduced, a band at about 1633 cm−1 was generated after operation, which was consistent with \\NH2 bending derived from secondary amines. Meanwhile, the peak represented the stretching vibrations of N\\H at 3377 cm−1; the intense peak indicating an N\\H stretch (primary and secondary amines) at 619 cm−1 appeared and was significantly strengthened, respectively (Fig. 5B). These results suggested that the products were generated because of the reductive cleavage of the\\N_N\\bond, which is in agreement with the disappearance of the band at 597 nm in Fig. 4. LC–MS was further employed to identify the products of RB5 degradation in the BES. The peak at m/z = 279.99 was identified as one of the reductive products of RB5 (i.e., 2-(4-aminobenzene-sulphonyl)-ethoxi-sulphonate (Fig. 6)). However, another reductive product of RB5, 1,2,7-triamino-8hydroxynaphthalene-3,6-disulfonate (TAHNDS), was not identified in this study probably because TAHNDS is unstable and can be autoxidized immediately even in the presence of trace oxygen in the solution [55]. Moreover, the total organic carbon concentration of the cathode solution was almost the same before and after BES operation (data not shown), indicating that no mineralization of RB5 occurred. On the basis of the results of UV–visible absorption spectroscopy, FTIR, and LC–MS, it could be concluded that RB5 decolorization in the BES occurred via cleavage of azo bonds (Scheme 1). 4. Conclusions RB5 decolorization in BES was successfully achieved in this study. The kinetics of RB5 decolorization in BES were well fitted with the pseudo–first-order model, and the reaction rate constant increased

from 0.023 ± 0.0002 h−1 to 0.146 ± 0.008 h−1 as the cathode potential decreased from −400 mV to −500 mV. RSM optimization showed that the linear effects of RB5 concentration, cathode potential and HRT, the interaction of RB5 concentration with cathodic HRT, and the quadratic effect of cathodic HRT were the most important factors in RB5 decolorization in BESs. The results obtained from UV–visible spectrophotometer, FTIR, and LC–MS also suggested that the RB5 decolorization mechanism in BESs was through cleavage of azo bonds. Acknowledgements The authors wish to thank the Natural Science Foundation of China (51538012, 51478446, and 51469006), the Fundamental Research Funds for the Central Universities and the Startup Fund for Advanced Talents of AHJZU (2018QD05) for financially supporting this study. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bioelechem.2019.02.008.

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