Drinking water disinfection by hemin-modified graphite felt and electrogenerated reactive oxygen species

Electrochimica Acta 56 (2011) 8278–8284

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Drinking water disinfection by hemin-modified graphite felt and electrogenerated reactive oxygen species Qiang Ma a , Tao Liu a , Tiantian Tang b , Huanshun Yin a , Shiyun Ai a,∗ a b

College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong, PR China College of Resources and Environment, Shandong Agricultural University, Taian 271018, Shandong, PR China

a r t i c l e

i n f o

Article history: Received 24 February 2011 Received in revised form 20 June 2011 Accepted 27 June 2011 Available online 28 July 2011 Keywords: Drinking water disinfection Hemin Graphite felt Electrochemistry

a b s t r a c t The electrochemical inactivation of microorganisms by a hemin/graphite felt (GF) composite electrode was investigated, and Escherichia coli was treated as the testing species. The composite electrode was constructed by chemically bonding hemin molecules onto an amino-mineralized GF (AGF) surface. Then, the electrode was characterized systematically by electrochemical methods, and the kinetic parameters of the modified electrode were investigated. The hemin molecules on the surface of the composite electrode have high activity for the reduction of O2 . When the composite electrode was applied with negative potentials, the dissolved oxygen was electrochemically reduced to reactive oxygen species (ROS, such as H2 O2 and • OH) at the cathode surface. The ROS can cause biological damage and can eventually result in the death of bacteria. A sterilizing rate up to 99.9% could be obtained after 60 min of inactivation. Thus, this composite electrode could be applied to disinfect drinking water efficiently at a low potential (−0.6 V vs. SCE) without any addition of chloride. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction In a large number of countries, especially developing ones, water-borne diseases (e.g., diarrhea, cholera, typhoid, amoebiasis, and schistosomiasis) and their associated deaths remain problems. These diseases contribute considerably to high morbidity and mortality rates [1]. The inactivation of these pathogenic microorganisms is the most important step in producing safe drinking water. Therefore, the effectiveness of inactivation is a crucial measure to ensure food hygiene and human health [2]. The most popular method for drinking water disinfection is the addition of chlorine and/or chlorine by-products, which are able to eliminate most harmful microorganisms. Despite the effectiveness of chlorination as a water disinfection method, its disadvantages include unfavorable taste and odor, its ineffectiveness when used alone against some resistant microorganisms, and the generation of potentially toxic or mutagenic products, such as chloroform (the most common chemical by-product of water disinfection) [3]. These disadvantages motivated the search for alternative disinfection methods. Numerous alternatives to chlorination for drinking water disinfection have been proposed. Electrochemical disinfection has emerged as one of the most promising alternatives to chlorination, providing both primary and residual disinfection. Recently, a significant amount of research has focused on elec-

∗ Corresponding author. Tel.: +86 538 8247660; fax: +86 538 8242251. E-mail address: [email protected] (S. Ai). 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.06.088

trochemical disinfection because of its high treatment efficiency, low cost, convenience, and environmental compatibility [4]. Electrochemical disinfection has become an alternative process for water treatment. The mechanism of electrochemical disinfection using chloride-containing solutions, termed electrochlorination, has been mainly attributed to the action of electrogenerated active chlorine. Research examining the generation of other disinfecting agents in water treated with these systems has been considered. The debate as to whether electrochemical systems can replace chlorination remains open [5]. Fortunately, new evidence on the oxidizing action of reactive oxygen species (ROS; e.g., • OH, • O2 − , H2 O2 , O3 ) in electrochemical disinfection with a boron-doped diamond (BDD) electrode [6] and a RuO2 /Ti anode [7] has been obtained by electrolyzing chlorine-free waters [8]. These strong oxidants, especially • OH, can attack the cell membrane and wall, disrupt membrane integrity, or electrolyze the molecules at the cell surface, which induces massive cell death and lysis [4]. Disinfection is aimed at all types of microorganisms without generating secondary pollutants [6,7]. Most of these experiments were conducted using anodes operating at high voltages. Electrodes fabricated by depositing films on a basement usually develop cracks, which can cause detachment of the films during long-term electrolysis [1]. Recently, enzymatic films prepared by immobilizing horseradish peroxidase (HRP) [9] or hemoglobin (Hb) [10] onto carbon electrodes, which has been used as the cathode for the degradation of organic pollutants with the help of in situ generated H2 O2 , has obtained excellent results.

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Scheme 1. Idealized scheme of the procedure for the stepwise preparation of hemin-modified GF electrodes.

The active center of the heme protein family, such as b-type cytochromes, peroxidase, myoglobin, and hemoglobin, is hemin (iron protoporphyrin IX, a well-known natural porphyrinatoiron) [11]. The antimicrobial activity induced by hemin can inhibit cell viability in the dark and induce the development of an irregular cell wall [12]. This property is ascribed to the strong pro-oxidant activity of ferric porphyrin (FeIII -P). Ferric porphyrin can catalyze various oxidative and per-oxidative reactions, which results in the observed antimicrobial activity [13]. Hemin has excellent electrocatalytic properties towards the detection of many important analytes, such as oxygen [14], hydrogen peroxide [15], and antioxidants [16]. Hemin has higher activity than Hb towards the reduction of oxygen and hydrogen peroxide [16]. However, few investigations on the application of hemin’s catalytic property to electrochemically inactivate microorganisms have been reported. Due to its high stability and low cost, hemin is a suitable candidate for the fabrication of electrochemical electrodes [11]. In this work, hemin was covalently immobilized onto the surface of graphite felt (GF) (Scheme 1). This composite material, hemin–AGF, was used as the cathode for the electrochemical disinfection of Escherichia coli. The objectives of our research were (1) to verify the effectiveness of this electrochemical water treatment system on bacterial inactivation and (2) to show the influence of processing factors, such as working electrode potential and detention time on E. coli viability.

2. Experimental 2.1. Materials and reagents Graphite felt (GF) was purchased from Hunan Jiuhua Carbon Company, Ltd. (China). Hemin chloride was purchased from Yiwei Biochemical Reagent Co., Ltd. (Yiyang, China) and was used as received. N,N -dicyclohexylcarbodiimide (DCC) was obtained from China National Medicines Co., Ltd. Nutrient broth (NB) and nutrient agar (NA) were obtained from Hope Bio-Technology Co., Ltd. (Qingdao, China). 2,9-Dimethyl-1,10-phenanthroline (DMP) was purchased from Aladdin Chemistry Co., Ltd. E. coli was supplied by the College of Animal Science and Technology, Shandong Agri-

cultural University (Taian, China). Phosphate buffer solution (PBS; 0.2 M, pH 7.0) was prepared by mixing stock solutions of Na2 HPO4 and NaH2 PO4 . All of the other chemicals were of analytical reagent grade and were used as received without further purification. 2.2. Immobilization of hemin on graphite felt The graphite felt was cut into 2 cm × 5 cm pieces and was airoxidized at 500 ◦ C for 8 h. Carbonylic and carboxylic groups formed on the surface of the GF [17]. Afterwards, the oxidized GF (OGF) was immersed in a 1 M hydrazine aqueous solution for 3 days to form an amino-coated GF (AGF). The excess hydrazine was removed by washing with deionized water (Scheme 1). After drying, ten pieces of the AGF were immersed in 200 mL of anhydrous tetrahydrofuran in the presence of 0.2 g of hemin chloride and 0.3 g of DCC for two days. The hemin-modified AGF (hemin–AGF) was thoroughly washed with PBS prior to use. 2.3. Cyclic voltammetry The cyclic voltammetry (CV) experiments were performed with a CHI 660C electrochemical workstation (CH Instruments, Shanghai, China). A conventional three-electrode system was used in the measurements with a saturated calomel electrode (SCE) as the reference, a Pt wire as the auxiliary electrode, and either GF or modified GF (cut into 1.0 cm × 0.5 cm pieces) as the working electrode. The CV measurements were conducted in PBS. The blank solutions were purged with nitrogen for at least 30 min to remove oxygen prior to starting a series of experiments. 2.4. Culture of E. coli One hundred microliters of the E. coli suspension, stored in glycerin, was inoculated into 5 mL of NB and cultured aerobically for 12 h at 37 ◦ C with constant agitation. Then, 50 ␮L of the anabiotic E. coli suspension was inoculated into 5 mL of NB at 37 ◦ C for 4 h to reach the late logarithmic phase. The bacterial cells were collected by centrifuging at 4000 rpm for 5 min, washing twice, and resuspending in 5 mL of sterilized PBS. The cell concentration was

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Fig. 1. (A) Cyclic voltammograms of GF (a), AGF (b), and hemin–AGF (c) electrodes in deaerated 0.2 M PBS, pH 7.0. (B) Cyclic voltammograms of the hemin-modified GF electrode in deaerated 0.2 M PBS, pH 7.0, at different scan rates: (a) 100 mV s−1 , (b) 200 mV s−1 , (c) 300 mV s−1 , (d) 400 mV s−1 , (e) 500 mV s−1 , (f) 600 mV s−1 , (g) 700 mV s−1 , (h) 800 mV s−1 , (i) 900 mV s−1 , and (j) 1000 mV s−1 . (C) The plots of the anodic and cathodic peak currents vs. the scan rate. (D) The relationship of the peak potential (Ep ) vs. the logarithm of the scan rate (log ). Inset: plots of the peak potential (Ep ) vs. the scan rate ( ≥ 0.6 V s−1 ).

determined by using the plate counting method. The stock suspension of E. coli, containing approximately 107 CFU/mL, was prepared by diluting the counted E. coli suspension with PBS.

2.5. Electrochemical inactivation procedure The inactivation experiments were performed in a 25-mL beaker containing 20 mL of the E. coli suspension. A conventional threeelectrode system was used in the procedure with a saturated calomel electrode (SCE) as the reference, a Pt sheet (0.5 cm × 0.5 cm, single side) as the counter electrode, and either GF or modified GF (2 cm × 5 cm pieces) as the working electrode. The three electrodes were installed and spaced 2 cm apart by a Teflon top. An electrochemical working station, which was obtained from Ingsens Instruments Co., Ltd. (Ingsens-1010, Guangzhou, China), was used to provide a constant potential. During the inactivation reaction, the suspension was magnetically stirred to ensure efficient mass transfer. All of the materials used in the experiments were autoclaved at 121 ◦ C for 15 min. During the experiments, 100 ␮L of the suspension with a defined concentration was withdrawn at each sampling time. Then, the sample was serially diluted with a 0.9% saline solution. Three replicates of the diluted samples were used for counting by the spread plate method with NA at 37 ◦ C for 12 h. The sterilizing rate was defined as follows. Sterilizing rate (%) = [(the number of viable cells when no potential was applied, N0 ) − (the number of viable cells after applied potential, N)]/N0 × 100%. The procedure was repeated three times to examine the level of reproducibility and stability of the experimental data for the selected experiments.

2.6. H2 O2 analysis The formation of H2 O2 was determined by a colorimetric using copper(II) ions and 2,9-dimethyl-1,10method phenanthroline (DMP) at a wavelength of 454 nm in a UV-5301 visible spectrophotometer (Shimadzu, Japan) [18]. The literature and our preliminary tests confirmed that these analytical methods do not significantly interfere with each other under the experimental conditions in this study. For the analysis, an appropriate amount of sample taken from the electrolytic solution was injected into the analytical reagents as quickly as possible to minimize the decay of the oxidants. The concentration was measured while varying the electrode potential.

3. Results and discussion 3.1. Electrochemical characterization The electrochemical behavior of hemin immobilized on the GF electrode was investigated in PBS by CV. No peaks were observed for GF (curve a, Fig. 1A) or AGF (curve b, Fig. 1A). The background current of GF (curve a, Fig. 1A) was slightly higher than AGF (curve b, Fig. 1A). This difference is ascribed to the active surface groups of AGF that decrease the capacity of the electrode double layer and decrease the charging current [19]. Curve c (Fig. 1A) shows the distinct electrochemical response of hemin that is attributed to the redox process of FeII /FeIII at an anodic peak potential (Epa ) of −0.123 V and cathodic peak potential (Epc ) of −0.370 V. The formal potential (E◦ , defined as the average value of the anodic and cathodic peak potentials) is −0.246 V, and the peak potential sepa-

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Scheme 2. Reduction processes at hemin–AGF electrodes in the presence of O2 and H2 O2 .

ration (Ep ) is 0.247 V. The peak currents (Ip ) of the anode (Ipa ) and the cathode (Ipc ) are 0.558 mA and 0.618 mA, respectively. The welldefined and quasi-reversible redox peaks suggest favorable direct electron transfer between the electrode and the redox centers of the hemin molecules. The influence of the scan rate on the CV performance of the hemin–AGF was investigated (Fig. 1B). The hemin redox processes gave roughly symmetric anodic and cathodic peaks at relatively slow scan rates. When the scan rate increased, the redox potentials (Epa and Epc ) of hemin shifted slightly, which resulted in Ep increasing (Fig. 1D). At the same time, the redox peak current increased linearly with the scan rate (Fig. 1C; linear regression equations: Ipa = 0.00555 + 0.889, R = 0.997; Ipc = −0.00540 − 1.535, R = 0.994) in accordance with the equation Ip = nFQ/4RT. Integration of the area under the reduction peaks gave nearly constant charge (Q) values, independent of the scan rate. All of the characteristics suggest that the redox reaction of hemin on the AGF electrode is a quasi-reversible, surface-controlled electrochemical process [11]. The heterogeneous electron transfer rate (ks ) of adsorbed hemin was estimated from the CV experiments at a higher scan rate using Laviron’s treatment [20]. The plots of Epa and Epc versus the logarithm of the scan rate produce two straight lines with slopes of 2.3RT/(1 − ˛)nF and −2.3RT/˛nF [11] when  ≥ 0.6 V s−1 (Fig. 1D). From the slopes, the charge transfer coefficient (˛) was estimated to be 0.82. The average electrode transfer rate constant, ks = 3.4 s−1 , was obtained according to Eq. (1). log ks = ˛ log(1 − ˛) + (1 − ˛) log ˛ − log

 −

˛(1 − ˛)nFEp 2.3RT

 RT 



nF (1)

This ks value is higher than that of hemin immobilized on multi-walled carbon nanotubes (MWCNT; 2.9 s−1 ) [14], but it is lower than other hemin-modified electrodes, such as a hemin/single-walled carbon nanotube (SWCNT)/Nafion/graphite (11 s−1 ) electrode [21] and a hemin/polyamidoamine (PAMAM)/MWCNT/glassy carbon electrode (GCE) (3.8 s−1 ) [11]. These differences in the ks value can be ascribed to the different electrode materials as well as the orientation and aggregation of hemin affecting the electrode transfer rate [22]. The solution pH can cause changes in the iron ligation of the protoporphyrin macrocycle [23]. Accordingly, the pH effect of the supporting electrolyte on the E◦ of the immobilized hemin was investigated from pH 5.0 to pH 10.0 (Fig. 2). E◦ decreased by 57.2 mV per unit pH (R = 0.973, n = 6). This value is close to the theoretical value (59 mV pH−1 ) for the number of electrons transferred accompanied by an equal number of protons in the electrode reaction. These results support the 1e− /1H+ process (reaction b, Scheme 2) [21,23]. 3.2. Electrocatalytic reduction of O2 and H2 O2 Hemin adsorbed on the electrode surface can catalyze the reduction of oxygen and hydrogen peroxide [14,16,24,25]. To assess the electrocatalytic properties of hemin immobilized to the GF surface,

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the electrocatalytic activity in terms of the reduction of O2 and H2 O2 was examined. In deaerated PBS, no peaks were observed for the GF electrode (curve a, Fig. 3A). A pair of reversible and well-defined peaks, which was attributable to the redox process of FeII /FeIII , was observed with the hemin–AGF electrode (curve a, Fig. 3B). In air-saturated PBS, the O2 reduction current started at −200 mV and reached its peak at approximately −650 mV on the GF electrode (curve b, Fig. 3A). In comparison, the peak current for the reduction of FeIII was enhanced by the hemin–AGF electrode (curve b, Fig. 3B). The magnitude of the reductive current was more than 1.4 times greater than the current observed using the GF electrode without the immobilization of hemin. A positive shift of the potential from −0.65 V to −0.4 V was also observed. These results can be interpreted as a catalytic mechanism, in which O2 reacts with FeII to form FeIII and then in which FeIII can be electrochemically reduced. This mechanism recycles the catalytic process at the hemin–AGF electrode surface (reaction a, Scheme 2) [16]. Upon addition of H2 O2 , the hemin–AGF electrode (curve c, Fig. 3B) has higher activity than the GF electrode (curve c, Fig. 3A) towards H2 O2 reduction, and the reduction current peaks, starting at −0.13 V. The increment of the reductive current is more than 2.1 times larger than that observed with the GF electrode. This enhancement is interpreted as the same aforementioned catalytic mechanism in that H2 O2 reacts with FeII and then electrochemically reduces FeIII (reaction c, Scheme 2). 3.3. E. coli inactivation by different electrodes Fig. 4A shows the electrochemical inactivation of E. coli by different GF electrodes in the chloride-free PBS at a potential of −0.6 V. The initial population of E. coli was 107 CFU/mL. A successful but weak inactivation of E. coli was observed (the sterilizing rate was 97.0% after 60 min) by the unmodified GF electrode. When GF was replaced by AGF, which was oxidized and treated with hydrazine, the sterilization efficiency (the sterilizing rate was 98.6%) was enhanced slightly. This enhancement can be attributed to the amino groups on the AGF surface facilitating the access of bacterial cells to the electrode [26]. More than 99.9% of the initial E. coli population was killed as a result of 60 min inactivation at the hemin–AGF electrode, indicating that this type of GF cathode can be effectively applied to the electrochemically inactivate E. coli. To facilitate the comparison of the performances by the different electrochemical disinfection methods, the electrochemical cell, working electrode, effluent, electrolysis conditions, inactivated microorganisms and sterilization rate are listed in Table 1. Table 1 shows that the greatly enhanced sterilization efficiency of the hemin–AGF electrode could be ascribed to the hemin molecules and the amino-rich electrode surface, which directly catalyzed the formation of absorbable H2 O2 for the inactivation of E. coli. According to Fig. 3 and previous research [9], the cathodic potential is an essential factor controlling the electrochemical generation of H2 O2 . To optimize the potential for hydrogen peroxide generation and bacterial inactivation, electrochemical disinfection experiments were carried out under the conditions as shown in Fig. 4. Fig. 4B shows that, when the voltage increases from −0.4 V to −1.0 V, the sterilizing rate increases initially, reaching a maximum value at −0.6 V and then decreases at higher negative voltages. Hence, the optimal voltage was fixed at −0.6 V. This result should be in accordance with the current efficiency for accumulating of H2 O2 [10]. This value is very similar to the results obtained by Lee et al. (−0.6 V vs. Ag/AgCl) [9] and Hsiao (−0.55 V vs. SCE) [32]. To substantiate this inference, the formation of H2 O2 , whose presence is widely accepted as evidence for the production of • OH [6,33], was measured while varying the electrode potential. Fig. 5 shows the trend of the H2 O2 concentration in the electrolyte solution during electrolysis in PBS at different electrode potentials. The

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Fig. 2. (A) Cyclic voltammograms of hemin-modified GF electrodes at different pHs in deaerated solutions (from right to left, pH 5–pH 10). The scan rate is 0.1 V s−1 . (B) Plots of the anode potential (, Epa ), formal potential (, E◦  ), and cathode potential (䊉, Epc ) vs. pH values.

Fig. 3. Cyclic voltammetry measurements of a GF electrode (A) and a hemin–AGF electrode (B) in a deaerated solution using nitrogen (see Fig. 1A) (a), in air-saturated solution (b), and with 100 ␮M H2 O2 in a deaerated solution (c). Conditions: PBS buffer (pH 7.0); scan rate, 100 mV s−1 and potential/V vs. SCE.

concentration of H2 O2 due to oxygen reduction increases with time until reaching a limiting value after approximately 60 min, implying that H2 O2 is electrogenerated and simultaneously degraded in the system at the same rate [10,34]. The limiting concentration follows almost the same trend as the sterilizing rate with the applied potential, and it is higher than that obtained with other electrode materials, such as boron-doped diamond (BDD) [6,8,33] or RuO2 [8].

3.4. Stability and reusability To demonstrate the reusability of hemin–AGF electrodes, the electrode was recovered after each batch and rinsed thoroughly with deionized water for the subsequent batch. For each batch, the electrolysis was allowed to proceed for 300 min. The sterilizing rate at the sampling time of 60 min decreased with each subsequent cycle of the electrode. After six cycles, the sterilizing rate, in order,

Fig. 4. (A) The effect of GF (a), AGF (b) and hemin–AGF (c) electrodes on E. coli inactivation at a potential of −0.6 V in pH 7.0 PBS. (B) The effect of the potential on E. coli inactivation by a hemin–AGF electrode. Inset: a plot of the sterilizing rate following 60 min of inactivation by a hemin–AGF electrode vs. potential (E).

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Table 1 A comparison of the performances for different electrochemical disinfection methods. Working electrode

Effluent

Electrolysis conditions

Inactivated microorganisms

Sterilization rate (%)

References

Cylinder-shaped carbon fiber (34 mm diameter, 100 mm length, 9 mm thickness)

Tap water with a suspension of 2.3 × 103 cells/mL; continuous flow at 300 mL/min Tap water with a suspension of 73 cells/mL; continuous flow at 15 mL/min Tap water with a suspension of 102 cells/mL; continuous flow Drinking water with 22 cells/mL; continuous flow at 2 mL/min (12 h); after an interruption for 24 h, it was started again at 1 mL/min (6 h) 10 L of contaminated 0.030 M Na2 SO4 or 0.036 M NaH2 PO4 ; batch treatment at 6 L/min 50 mL of 0.1 M PBS (pH 7.1) with a suspension of 2 × 106 CFU/mL 1 mM Na2 SO4 with a suspension of 6.4 × 102 CFU/mL; batch flow up to 100 mL/min 50 mL 0.1 M pH 7.1 PBS with a suspension of 1 × 107 CFU/mL 20 mL 0.2 M pH 7.0 PBS with a suspension of 1 × 107 CFU/mL

Flow-through cell; 1.0 V vs. SCE for 20 min and cycling from 0.2 to −0.8 V for 10 min

E. coli

99.10

[27]

Flow-through cell; 1.2 V vs. Ag/AgCl for 60 min and then −0.6 V for 30 min

E. coli, Klebsiella pneumoniae, etc.

93.10

[28]

Flow-through cell; 0.7 V vs. SCE with a residence time more than 10 min Flow-through cell; 0.8 V vs. SCE.

E. coli

99.90

[29]

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99.70

[30]

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E. coli, bacteriophage MS2

Above 99.00

[5]

Stirred tank reactor; 1 A

Saccharomyces cerevisiae

About 96.80

[31]

Stirred tank reactor; 2.8–3.1 V vs. SCE for about 1 min

E. coli

99.80

[2]

Stirred tank reactor; 10 mA/cm2 for 60 min

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99.99

[4]

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99.90

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TiN mesh (2 cm2 )

Carbon-cloth sheet (1170 cm2 ) Carbon fiber (18 mm diameter, 100 mm length, 5 mm thickness)

Pt–Nb mesh (522 cm2 )

Pt sheet (4.6 cm2 )

Si/BDD plate (30 cm2 )

Ti/PbO2 sheet (6 cm2 )

Hemin–graphite felt (10 cm2 )

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