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Decolorization of azo dye and generation of electricity by microbial fuel cell with laccase-producing white-rot fungus on cathode
Applied Energy 188 (2017) 392–398
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Decolorization of azo dye and generation of electricity by microbial fuel cell with laccase-producing white-rot fungus on cathode Chi-Yung Lai a, Chih-Hung Wu b, Chui-Ting Meng b, Chi-Wen Lin b,⇑ a b
Department of Biology, National Changhua University of Education, Changhua 510, Taiwan, ROC Department of Safety, Health and Environmental Engineering, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan, ROC
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A laccase-producing fungus on
1 0 -1 Current (mA)
cathode of MFC was used to enhance degradation of azo dye. Laccase-producing fungal cathodes performed better than laccase-free control cathodes. A maximum power density of 13.38 mW/m2 and an >90% decolorization of acid orange 7 were obtained. Growing a fungal culture with continuous laccase production improved MFC’s electricity generation.
-2 -3 -4 -5 -6 -7 -1.0 -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 Potential vs. Ag/AgCl (V)
a r t i c l e
i n f o
Article history: Received 29 August 2016 Received in revised form 9 December 2016 Accepted 9 December 2016
Keywords: Laccase-catalyzed cathode White-rot fungi Electricity generation Azo dye Decolorization
a b s t r a c t Wood-degrading white-rot fungi produce many extracellular enzymes, including the multi-copper oxidative enzyme laccase (EC 1.10.3.2). Laccase uses atmospheric oxygen as the electron acceptor to catalyze a one-electron oxidation reaction of phenolic compounds and therefore has the potential to simultaneously act as a cathode catalyst in a microbial fuel cell (MFC) and degrade azo dye pollutants. In this study, the laccase-producing white-rot fungus Ganoderma lucidum BCRC 36123 was planted on the cathode surface of a single-chamber MFC to degrade the azo dye acid orange 7 (AO7) synergistically with an anaerobic microbial community in the anode chamber. In a batch culture, the fungus used AO7 as the sole carbon source and produced laccase continuously, reaching a maximum activity of 20.3 ± 0.3 U/L on day 19 with a 77% decolorization of the dye (50 mg/L). During MFC operations, AO7 in the anolyte diffused across a layer of polyvinyl alcohol-hydrogel that separated the cathode membrane from the anode chamber, and served as a carbon source to support the growth of, and production of laccase by, the fungal mycelium that was planted on the cathode. In such MFCs, laccase-producing fungal cathodes outperformed laccase-free controls, yielding a maximum open-circuit voltage of 821 mV, a closed-circuit voltage of 394 mV with an external resistance of 1000 X, a maximum power density of 13.38 mW/m2, a maximum current density of 33 mA/m2, and a >90% decolorization of AO7. This study demonstrates the feasibility of
⇑ Corresponding author at: Department of Safety, Health and Environmental Engineering, National Yunlin University of Science and Technology, 123 University Rd. Sec. 3, Douliou, Yunlin 64002, Taiwan, ROC. E-mail address: [email protected] (C.-W. Lin). http://dx.doi.org/10.1016/j.apenergy.2016.12.044 0306-2619/Ó 2016 Elsevier Ltd. All rights reserved.
C.-Y. Lai et al. / Applied Energy 188 (2017) 392–398
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growing a white-rot fungal culture with continuous laccase production on the cathode of MFCs to improve their electricity generation and azo dye removal efficiency. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Azo dyes are a group of poorly biodegradable synthetic colorants that are often found in waste water that is produced by the textile industry [1]. These compounds are highly resistant to degradation by aerobic bacteria but can be easily reduced by anaerobic bacteria to form aromatic amines, which are a group of carcinogens that are stable under anaerobic conditions and must be returned to aerobic conditions before they can be further degraded and mineralized [2,3]. Therefore, a combination of aerobic and anaerobic treatments have been suggested to be required for the effective mineralization of these pollutants [4]. Pollutants in waste water from various sources can be removed by a microbial fuel cell (MFC), in which microorganisms break down organic compounds and convert their chemical energy into electrical energy [5,6]. An MFC typically consists of an anaerobic anode chamber and an aerobic cathode chamber that are separated by a proton exchange membrane. In the anode chamber is a microbial community that often comprises a large number of both fermenting and respiring bacteria that contribute to the versatility of MFCs in degrading pollutants. When waste water enters the anode chamber, fermenting bacteria firstly convert large organic molecules into smaller fermentation products, such as lactate, which are then oxidized by anaerobically respiring bacteria to produce CO2, protons, and electrons. In the absence of a suitable electron acceptor in the anolyte, electrons pass to the anode interface and are transferred through an external wire to the cathode, where they join oxygen molecules (O2) and protons to form water molecules, thus completing a circuit [7]. Azo dyes can be degraded in either the anode or cathode chamber of an MFC. When introduced into the catholyte, azo dyes can act as electron acceptors and be decolorized in reductive cathode reactions. In most cases, these reactions transform azo dyes into less colorful compounds, such as aromatic amines and hydrazines, but fail to degrade and mineralize them completely [8]. The dyereducing reactions proceed better under anaerobic conditions as oxygen competes for electrons from the cathode, and the rate of the reaction depends heavily on the pH of the catholyte [9], the structure of the dye molecules [10], and the use of catalysts such as noble metals. Ding et al. demonstrated that photocatalysis with visible light on a rutile-modified cathode greatly increases the rate of dye reduction [11]. Azo dyes can be introduced into the anolyte, as in an MFC that forms the first part of a two-stage system in which azo dyecontaining waste water firstly enters the anode chamber, where dye molecules are reduced by anaerobic bacteria to form aromatic amines, which were then transferred to the second stage, an aerobic bioreactor, for further degradation into smaller compounds by aerobic microorganisms [4,8]. Although these two-in-one systems can effectively remove dye, they are structurally complex and expensive to construct and operate. In theory, azo dyes can be completely degraded in a single MFC if they are firstly introduced into the anode chamber for reductive transformation by anaerobic bacteria and then transferred to the cathode chamber for aerobic degradation by a second group of aerobic microbes. However, in most MFCs, the anode and cathode chambers are separated by a proton exchange membrane, which prevents movement of azo dyes and their transformed products between the chambers. Furthermore, growing aerobic microbes
in the cathode chamber inevitably reduces the level of dissolved oxygen there, and so reduces the cathode potential and the power output of the MFC. This study involves testing the feasibility of growing a laccasesecreting white-rot fungus on the cathode surface of a singlechamber MFC, in which the proton exchange membrane was replaced with a layer of polyvinyl alcohol-hydrogel (PVA-H), allowing the pollutant acid orange 7 (AO7, an azo dye) to diffuse from the anode chamber to the cathode. White-rot fungi are the only group of organisms that can completely degrade azo dyes. Laccase is one of the enzymes that they produce when they degrade lignin, which refers to a group of highly heterogeneous aromatic polymers that are abundant in the natural habitats of white-rot fungi, and is also used by these fungi to degrade azo dyes and many other aromatic compounds [12]. Additionally, laccase has been used as an enzyme catalyst on the cathode of many MFCs, replacing noble metals in catalyzing the reduction of oxygen [13]. In the singlechamber configuration, the MFCs herein have no cathode chamber and their cathodes are exposed directly to the air to increase oxygen availability [14,15]. Our results demonstrate that such fungal MFCs outperformed control MFCs that are equipped with abiotic or enzyme-free cathodes in both the decolorization of dye and the generation of electrical power.
2. Materials and methods 2.1. Microbes Anaerobic sludge was collected from an oil-cracking wastewater treatment plant of Nan-Ya Plastics Co., Ltd. (at the Six Naphtha Cracking Industry Site at Mailiao in Yunlin County). The sludge microbial community was acclimated for an extended period in the laboratory and used for the degradation of aromatic compounds; it tested positive for AO7 decolorization. The fungal strain that was planted on the cathode was obtained from a local mushroom grower and was identical to the Ganoderma lucidum BCRC 36123 strain from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan, ROC. The fungal strain was maintained on potato dextrose agar (PDA) plates. 2.2. Fungal culture on cathode Mycelium of Ganoderma lucidum BCRC 36123 was inoculated onto a 2 mm-thick layer of potato dextrose agar (PDA) medium and incubated to cover fully the surface of the medium. A 1.4 cm 1.4 cm square of the mycelium-covered medium was then cut from the culture plate to be placed on a cathode. 2.3. MFC setup and operation Fig. 1 schematically depicts the single-chamber MFC. The anode chamber was a 350 cm3 container at the bottom of which was placed a 70 cm2 carbon felt anode. The anolyte (of which each liter contained 1.75 g K2HPO4, 2.145 g KH2PO4, 10 mg NH4Cl, 100 mg MgCl26H2O, 45 mg CaCl2, 1 mg FeCl36H2O, 0.25 mg CuCl22H2O, 0.25 mg CoCl26H2O, 1 mg ZnCl2, 1 mg MnCl24H2O, 0.1 mg Na2MoO42H2O, and 0.02 mg NiCl26H2O) was inoculated with 403.29 mg/L of sludge. The air cathode was made from a 6 cm
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Fig. 1. Schematic of a fungal MFC.
(height) 5 cm (diameter) garden pot. A 10% PVA-H gel solution that contained 2.4% potato dextrose broth (PDB) medium was poured into the pot and kept frozen at 30 °C for 12 h. The solidified gel was warmed to room temperature and then covered with a 28.27 cm2 piece of carbon cloth. The carbon cloth was connected with electrical wire, wetted with 6 ml of PDB, and overlaid with a 1.4 cm 1.4 cm square of fungal mycelium-covered medium. The top of the pot was sealed with a parafilm membrane and incubated for 14 days to allow mycelial growth into the carbon cloth. The cathode assembly was then fitted onto the top of the anode chamber such that the underside of the PVA-H layer faced the carbon felt anode at a distance of 10 cm. The parafilm membrane was then replaced with a perforated petri-dish cover to increase ventilation during MFC operations. 2.4. Determination of laccase activity Laccase activity in the cathode assembly was assayed using the substrate 2 20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Absorbance of the green reaction product at 420 nm was determined using a spectrophotometer [16]. The reaction was carried out in 3 ml of a 0.1 M sodium acetate buffer (pH 4.5) that contained 1 mM of ABTS. One unit (U) of laccase is defined as the amount of activity that converts 1 lmole of the substrate in one minute, and is calculated from absorbance readings, using Eq. (1), which is based on Beer’s law:
U¼
Ak V 10 ek l t
(b) Laccase activity in the PVA-H layer: After the myceliumcovered carbon cloth was removed, the remaining PVA-H layer was soaked in 100 ml of a 0.1 M sodium acetate buffer (pH 4.5) with agitation for four hours. The buffer was then clarified by centrifugation (4500 rpm) and its laccase activity was determined. (c) Laccase activity in heat-inactivated fungal cathode: A fungal cathode was incubated for 14 days after mycelial transplantation and soaked three times in sterile distilled water for two hours each time to remove PDB ingredients. The fungal mycelium/carbon cloth/PVA-H assemblies were then baked at 80 °C for one hour to inactivate laccase [17,18]. The assembly was then soaked in 20 ml of the 0.1 M sodium acetate buffer (pH 4.5) with agitation for six hours. The buffer was then clarified by centrifugation (4500 rpm) and its laccase activity was determined. 2.5. Detection of laccase production by fungal mycelia To detect the production of laccase by a fungal culture, guaiacol (0.01%) was added to the PDA medium as an indicator. Laccase production is revealed by a zone of red precipitation around the fungal colony, and the size and shade of the red zone can be used to estimate the amount of laccase produced [18]. 2.6. AO7 decolorization
6
ð1Þ
where Ak is the change in absorbance at wavelength k; V is reaction volume (L), ek is the extinction coefficient of ABTS at wavelength k (e420 = 36,000 M1 cm1); l is the length of the light path (cm), and t is the reaction time (min). Laccase activity on the following samples was evaluated. (a) Laccase activity on a fresh fungal cathode: The fungal cathode was incubated for 14 days after mycelial transplantation and was tested for laccase activity. The mycelium-covered carbon cloth cathode was peeled from the PVA-H layer and soaked in 20 ml of 0.1 M sodium acetate buffer (pH 4.5) with agitation for six hours. The buffer was then clarified by centrifugation (4500 rpm) and its laccase activity was determined.
The absorption spectrum of AO7 in the visible light range peaks at a wavelength of 485 nm. Absorption at 485 nm was therefore used to calculate AO7 from Eq. (2) where A0 and A are the initial and final absorptions, respectively.
Decolorization ð%Þ ¼
A0 A 100 A0
ð2Þ
2.7. Electrochemical analyses A multimeter (CHY 48R, Taiwan) was used to measure the voltage (V) of the MFC. Current (I) was calculated from the external resistance (R) using Eq. (3).
I¼
V R
ð3Þ
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Power (P) was calculated from voltage and current using Eq. (4) and divided by the anode surface area to obtain power density.
P ¼IV
ð4Þ
The internal resistance of each MFC was determined using the polarization slope method and the power density peak method [7]. MFCs were operated to establish a constant voltage and then disconnected for six to eight hours to maximize their open circuit voltage. Voltage was recorded using a range of external resistors and a multimeter. Current and power that were calculated from Eqs. (3) and (4) are used to plot polarization and power density curves. The electrochemical properties of the fungal cathode were investigated using cyclic voltammetry in a three-electrode setup with an Ag/AgCl reference electrode (201 mV vs. NHE) and a Pt counter electrode that were connected to a Bio-Logic SP-150 potentiostat (Bio-Logic, France). The electrodes were submerged in a 0.1 M acetate buffer (pH 4.5) that contained 100 mg/L of AO7 at 25 °C and scanned over the range 1 V to 1 V at a speed of 20 mV/s for three cycles.
Fig. 3. Laccase production and AO7 decolorization of a Ganoderma lucidum BCRC 36123 batch culture in a basal salt medium containing 50 mg/L of AO7 as the sole carbon source; laccase activity ( ), decolorization ( ).
3. Results and discussion 3.1. Production of laccase by fungal mycelia To test the production of laccase by the fungal strain Ganoderma lucidum BCRC 36123, the substrate compound guaiacol was added to PDA medium plates. Dark red precipitation indicated the presence of laccase within and around a colony (Fig. 2) [19]. The red zone expanded as the mycelium grew but faded after day five when growth stopped. 3.2. Decolonization of AO7 by laccase in batch culture Liquid cultures were used to test whether the fungus Ganoderma lucidum BCRC 36123 can use AO7 as a sole carbon source to produce laccase. Such an ability would eliminate the need for
the addition of a co-substrate, such as glucose, to the electrolyte during MFC operations. Fig. 3 presents a typical growth/decolorization cycle of the fungus in a basal salt medium that contained 50 mg/L of AO7. The growth of the mycelium began six days following inoculation, as indicated by a sharp increase of laccase activity in the medium. The activity was 7.5 U/L on day 6, reaching a plateau of 20.3 ± 0.2 U/L on days 8–19. At the same time, the laccase-catalyzed oxidation of AO7 decolorized the medium. The decolorization rate peaked on days 8–19 with 5, 8, and 58% decolorization on days 7, 8, and 20, respectively. The culture aged and the laccase activity declined rapidly after day 20, and decolorization gradually reached a plateau of 77% on day 50. These results clearly demonstrate that Ganoderma lucidum BCRC 36123 grew with AO7 as the sole carbon source and produced laccase during the phase of active growth.
Day 0
Day 1
Day 2
Day 5
Day 6
Day 7
Fig. 2. Growth of the fungal strain Ganoderma lucidum BCRC 36123 on laccase detection medium containing 0.01% guaiacol. The presence of laccase was indicated by red precipitation around fungal mycelium. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.3. Laccase activity of the fungal cathode Laccase production by mycelium that grew on the cathode membrane was tested by soaking the mycelium-covered carbon cloth (cathode membrane) and the underlying PVA-H gel in an acetate buffer for 4–6 h and assayed for laccase activity in the buffer using the substrate ABTS. After 14 days of incubation, the mycelium-covered carbon cloth contained 1.88 ± 0.22 U/L of laccase. The PVA-H gel contained 2.28 ± 0.71 U/L of laccase. No laccase activity was detected on control electrodes without mycelium or with heat-inactivated fungal mycelium. 3.4. Generation of electricity by MFC with fungal cathode Although the anodic reaction in an MFC is always catalyzed by microbes, the reduction of oxygen at the cathode of most MFCs is catalyzed by non-biological catalysts such as platinum [10]. Oxidative enzymes including laccase are sometimes immobilized on the surface of the cathode to catalyze the reaction [13]. However, noble metal and enzyme catalysts are expensive and can deteriorate rapidly under the moist and often corrosive conditions in which most MFCs operate [20]. Instead of applying a purified enzyme to the cathode, Wu et al. demonstrated that growing a laccasesecreting white-rot fungus in the cathode chamber increased the power density of a two-chamber MFC sevenfold. However, such a setup requires the continuous addition of nutrients and mediator compounds into the cathode chamber [21]; this process not only increases cost but also encourages the growth of contaminating microorganisms to become another source of pollution. In this work a white-rot fungus was planted on the surface of the cathode in a single-chamber MFC to serve as both a continuous source of fresh laccase and an aerobic dye-degrading microbe. Single-chamber MFCs, shown in Fig. 1, with a fungal cathode (fMFC), a fungus-free cathode (a-MFC) or a heat-inactivated fungal cathode (if-MFC), were set up and stabilized in a 20 day pre-run. Their close- and open-circuit voltages were monitored and compared in repeated cycles of dye addition in 30 days of continuous operation. Fig. 4 shows that in the early stage, between days 3 and 16, the f-MFC produced higher open circuit voltages than the two control MFCs, reaching a maximum of 817 mV on day 10, an 18% increase over the 692 mV that was produced by the a-MFC. After day 10, the voltage of the f-MFC slowly declined, eventually reaching the same level as that of the a-MFC on day 17. In the late stage, between days 17 and 30, the two cells exhibited the same
voltage, under both the open circuit condition and the closed circuit condition with a 1000 X external resistance, as shown in Fig. 4. This loss of increase in potential by the f-MFC in the late stage was probably caused by excessive growth of fungal mycelium on the cathode membrane. The greater thickness of the fungal layer was responsible for reduced availability of oxygen to the cathode, and the greater distance from the most active laccaseproducing cells on the growing tips of the mycelium to the electrode was responsible for reduced laccase activity on the electrode surface. The heat-inactivated if-MFC produced lower voltages than either the a-MFC or the f-MFC throughout the operation, probably because the inactivated mycelium on the cathode membrane acted as a barrier to oxygen transport. The increased open circuit voltage of the f-MFC on days 10–17 suggests a reduced internal resistance, and laccase-modified electrodes have been shown to have lower activation over voltages than unmodified ones [22]. The internal resistance of MFCs was examined herein using the polarization slope method and the power density peak method [7]. Figs. 5 and 6 plot the polarization curves and the power density curves of the three MFCs that were recorded on day 17 of continuous operation. The maximum power densities that were produced by the f-MFC, a-MFC, and if-MFC were 13.38, 10.41, and 9.04 mW/m2 at current densities of 33, 27.6, and 23.3 mA/m2, respectively (Table 1). The internal resistances of the f-MFC, a-MFC, and if-MFC that were calculated using the polarization slope method were 1574, 1680, and 1859 X, respectively. Whereas the internal resistances that were calculated
Fig. 5. Polarization curves of f-MFC ( ), a-MFC ( ), and if-MFC (d).
Fig. 4. Voltage-time plots of f-MFC ( ), a-MFC ( ), and if-MFC (d). Open circuit voltages are shown for days 1–16 and 24–30 while closed circuit voltages with a 1000 X external resistance are shown for days 17–23.
Fig. 6. Power density curves of f-MFC ( ), a-MFC (s), and if-MFC (d).
C.-Y. Lai et al. / Applied Energy 188 (2017) 392–398 Table 1 Electrochemical properties of f-MFC, a-MFC, and if-MFC.
a b
Items
f-MFC
a-MFC
if-MFC
Maximum open-circuit potential (mV) Comparison of open-circuit potential efficiency (%) Closed-circuit potential (mV)a Comparison of closed-circuit potential efficiency (%) Maximum power density Based on the anode surface (mW/m2) Based on the anode chamber volume (mW/m3) Maximum current density Based on the anode surface (mA/m2) Based on the anode chamber volume (mA/m3) Internal resistance (X)b
821 18
695 –
655 6
394 4
378 –
375 1
13.38 267.60
10.41 208.30
9.04 180.70
33 659.10 1574
27.60 551 1680
23.30 465.80 1859
With 1000 X of external resistance. Determined using the polarization slope method.
using the power density peak method were 1760, 1960, and 2380 X. Since the three cells shared the same configuration and operating conditions, except for the presence of laccase in the fMFC, the reduction of more than 100 X in the internal resistance of the f-MFC can be attributed to a reduction in the activation loss owing to laccase activity. Although thickened with prolonged operation, the fungal mycelium did not increase internal resistance because it was planted on the outer surface of the cathode and continuously grew away from the cathode into the air. The electrochemical properties of freshly prepared cathodes of the f-MFC, a-MFC, and if-MFC were examined using cyclic voltammetry (CV). Each cathode was scanned from 1.0 V to 1.0 V vs. an Ag/AgCl electrode at a rate of 20 mV/s for three cycles. Fig. 7 presents the results of the second cycle. The f-MFC cathode exhibited an increase in reduction current that peaked at 0.73 V (vs. Ag/ AgCl). No increase in oxidation current was detected during the forward scan. Many studies of laccase-modified electrodes have yielded the same results [22] as, under aerobic conditions, laccase molecules that are reduced during the backward scan are rapidly oxidized again by binding to dissolved oxygen; they therefore do not contribute to the oxidation current during the subsequent forward scan. Increases in reduction currents in many laccasemodified carbon electrodes have been attributed to two mechanisms, which are (1) direct electron transfer from the electrode to the T1 copper ion in the enzyme; (2) mediated electron transfer between the electrode and the enzyme [22]. In the latter mechanism, free radicals that are generated by laccase-catalyzed oneelectron oxidation of phenolic substrates frequently act as media-
Fig. 7. Cyclic voltammograms of the cathodes of f-MFC ( MFC ().
), a-MFC ( ), and if-
397
tors. Direct electron transfer from electrode to laccase can be observed using CV under aerobic conditions as an enzymedependent reduction current, whose magnitude depends on the cathode material, the method of preparation of the cathode, and the substrate concentration. As no faradaic current was detected on the fungal cathodes by CV when AO7 was omitted from the anolyte, a mediated mechanism with either AO7 or its oxidized products as mediators was more likely to be involved. However, CV is not sufficiently sensitive to detect small reduction currents, so the involvement of direct electron transfer cannot be completely excluded.
3.5. Decolorization of AO7 by MFC with mycelium-covered cathode During operation of the MFC, the decolorization of the anolyte usually reached 90% in four to five days, as determined using a spectrophotometer. Fresh AO7 solution was therefore added periodically to restore the substrate concentration. Fig. 8 compares the decolorization of the anolyte in the f-MFC with that in the aMFC. In the early stage of the operation before day 10, the f-MFC decolorized AO7 faster than did the a-MFC. From day 10 to day 17, the two cells decolorized at similar rates. This trend paralleled the changes in the voltages of the MFCs over the same period. Fungal mycelium on the cathode increased the decolorization and removal of dye by several mechanisms. First, fungal mycelium functions as an oxygen barrier, keeping the anaerobic conditions of the anode chamber, which favor dye reduction. Second, laccase that is produced by the mycelium increases the rate of reduction of the dye on the cathode. On abiotic cathodes, the rate of reduction of dye depends heavily on its molecular structure [10]. As many azo dyes are substrates of laccase, the use of laccase as a cathode catalyst greatly increases the range of dyes that can be effectively reduced and the rate of their reduction. Third, white-rot fungi are effective azo dye degraders, and laccase is one of the dyedegrading enzymes that they produce in a study to determine the products of azo dye degradation, Zille et al. concluded that laccase is not suitable for removing azo dyes because the enzyme initiates a series of free radical-mediated coupling and polymerization reactions that lead to a variety of end products, many of which are large azo compounds that are recalcitrant to further degradation [23]. However, this conclusion is accurate only when laccase is the only enzyme that acts on the dye. In the presence of living fungal cells and their complete metabolic network,
Fig. 8. Decoloration of AO7 in f-MFC ( ), a-MFC ( ), and if-MFC (d). Fresh solution of AO7 was added to anolyte to the concentration of 50 mg/L on days 4, 10, 12, 17, and 23.
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most azo dyes and their degradation intermediates can be absorbed and assimilated. After day 17, the f-MFC surpassed the a-MFC in dye decolorization despite of the similarity between their voltages, probably because of the overgrowth of fungal mycelium in the f-MFC, which oxidizes and uses AO7 as its carbon source, again indicating that fungus on the cathode increased the decolorization rate by improving the electrochemical performance of the MFC in the early stage and by directly removing the dye pollutant from the anolyte in both the early and the late stages. The fungal colony thus served as a continuous pollutant sink made possible by the use of PVAH gel, which allows AO7 and its degradation intermediates to move freely between the electrodes. The PVA-H gel might additionally help to prevent acidification of the anolyte during prolonged operations. Recent studies showed that the use of proton exchange membranes in most MFCs causes accumulation of cations on the anode side because these selective materials favor the passage of proton. Non-proton cations that migrate toward the cathode tend to be stopped at the anolytemembrane interface. During prolonged operations, the build-up of cation concentration near the membrane surface in turn produces a large resistance to the passage of proton, and thus the flow of current, leading to a rapid acidification of the anolyte [24]. By replacing the proton exchange membrane, the PVA-H gel therefore served the triple role of lowering the cost, enabling a direct participation of the fungus in pollutant removal, and mitigating acidification during prolonged operations. 4. Conclusions The f-MFC with a fungal cathode achieved 90% decolorization of the azo dye AO7 in five days of continuous operation and outperformed control MFCs with respect to electrochemical parameters such as open-circuit voltage, closed circuit voltages with a fixed external resistance, current density, and power density. Unlike two-chamber MFCs in which fungus grows in the cathode chamber, the single-chamber f-MFC did not require the continuous addition of substrate and mediator to the catholyte, because the proton exchange membrane that is used in most fuel cells was replaced with a layer of PVA-H, allowing AO7 molecules to diffuse to the cathode, to serve as a substrate, and to be removed by the fungal mycelium. However, thickening of the fungal cathode during prolonged operations appeared to inhibit oxygen transport to the cathode surface and thereby reduce power generation without significantly affecting the decolorization rate. These results demonstrate that the fungal cathode is feasible for removing azo dyes, but further modifications of MFC design and operating conditions must be made to prevent fungal overgrowth and improve longterm effectiveness. Acknowledgements The authors would like to thank the Ministry of Science and Technology of the Republic of China, Taiwan, for financially supporting this research under Contract No. MOST 105-2221-E-224003. Ted Knoy is appreciated for his editorial assistance.
References [1] Cai M-Q, Wei X-Q, Du C-H, Ma X-M, Jin M-C. Novel amphiphilic polymeric ionic liquid-solid phase micro-extraction membrane for the preconcentration of aniline as degradation product of azo dye Orange G under sonication by liquid chromatography–tandem mass spectrometry. J Chromatogr A 2014;1349:24–9. [2] van der Zee FP, Villaverde S. Combined anaerobic–aerobic treatment of azo dyes—a short review of bioreactor studies. Water Res 2005;39:1425–40. [3] García-Martínez Y, Bengoa C, Stüber F, Fortuny A, Font J, Fabregat A. Biodegradation of acid orange 7 in an anaerobic–aerobic sequential treatment system. Chem Eng Process 2015;94:99–104. [4] Fernando E, Keshavarz T, Kyazze G. Complete degradation of the azo dye acid orange-7 and bioelectricity generation in an integrated microbial fuel cell, aerobic two-stage bioreactor system in continuous flow mode at ambient temperature. Bioresour Technol 2014;156:155–62. [5] Du Z, Li H, Gu T. A state of the art review on microbial fuel cells: a promising technology for wastewater treatment and bioenergy. Biotechnol Adv 2007;25:464–82. [6] Pant D, Van Bogaert G, Diels L, Vanbroekhoven K. A review of the substrates used in microbial fuel cells (MFCs) for sustainable energy production. Bioresour Technol 2010;101:1533–43. [7] Logan BE. Microbial fuel cells. Hoboken, New Jersey: John Wiley & Sons; 2008. [8] Savizi IS, Kariminia HR, Bakhshian S. Simultaneous decolorization and bioelectricity generation in a dual chamber microbial fuel cell using electropolymerized-enzymatic cathode. Environ Sci Technol 2012;46:6584–93. [9] Mani P, Keshavarz T, Chandra TS, Kyazze G. Decolourisation of acid orange 7 in a microbial fuel cell with a laccase-based biocathode: influence of mitigating pH changes in the cathode chamber. Enzyme Microbial Technol 2017;96:170–6. [10] Liu L, Li FB, Feng CH, Li XZ. Microbial fuel cell with an azo-dye-feeding cathode. Appl Microbiol Biotechnol 2009;85:175–83. [11] Ding H, Li Y, Lu A, Jin S, Quan C, Wang C, et al. Photo catalytically improved azo dye reduction in a microbial fuel cell with rutile-cathode. Bioresour Technol 2010;101:3500–5. [12] Spadaro JT, Gold MH, Renganathan V. Degradation of azo dyes by the lignindegrading fungus Phanerochaete chrysosporium. Appl Environ Microbiol 1992;58:2397–401. [13] Luo H, Jin S, Fallgren PH, Park HJ, Johnson PA. A novel laccase-catalyzed cathode for microbial fuel cells. Chem Eng J 2010;165:524–8. [14] Fan Y, Hu H, Liu H. Enhanced Coulombic efficiency and power density of aircathode microbial fuel cells with an improved cell configuration. J Power Sources 2007;171:348–54. [15] Vogl A, Bischof F, Wichern M. Increase life time and performance of microbial fuel cells by limiting excess oxygen to the cathodes. Biochem Eng J 2016;106:139–46. [16] Niku-Paavola ML, Karhunen E, Salola P, Raunio V. Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem J 1988;254:877–83. [17] Chefetz B, Chen Y, Hadar Y. Purification and characterization of laccase from Chaetomium thermophilum and its role in humification. Appl Environ Microbiol 1998;64:3175–9. [18] Sharma KK, Shrivastava B, Sastry VR, Sehgal N, Kuhad RC. Middle-redox potential laccase from Ganoderma sp.: its application in improvement of feed for monogastric animals. Sci Rep 2013;3:1299. [19] Nishida T, Kashino Y, Mimura A, Takahara Y. Lignin biodegradation by woodrotting fungi, 1: Screening of lignin-degrading fungi. J Jpn Wood Res Soc 1998:34. [20] Harnisch F, Schroder U. From MFC to MXC: chemical and biological cathodes and their potential for microbial bioelectrochemical systems. Chem Soc Rev 2010;39:4433–48. [21] Wu C, Liu X-W, Li W-W, Sheng G-P, Zang G-L, Cheng Y-Y, et al. A white-rot fungus is used as a biocathode to improve electricity production of a microbial fuel cell. Appl Energy 2012;98:594–6. [22] Shleev S, Jarosz-Wilkolazka A, Khalunina A, Morozova O, Yaropolov A, Ruzgas T, et al. Direct electron transfer reactions of laccases from different origins on carbon electrodes. Bioelectrochemistry 2005;67:115–24. [23] Zille A, Gornacka B, Rehorek A, Cavaco-Paulo A. Degradation of azo dyes by Trametes villosa laccase over long periods of oxidative conditions. Appl Environ Microbiol 2005;71:6711–8. [24] Jannellia N, Nastrob RA, Cigolottic V, Minutillob M, Falcucci G. Low pH, high salinity: Too much for microbial fuel cells? Appl Energy 2016. doi: http://dx. doi.org/10.1016/j.apenergy.2016.07.079.
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Decolorization of azo dye and generation of electricity by microbial fuel cell with laccase-producing white-rot fungus on cathode Chi-Yung Lai a, Chih-Hung Wu b, Chui-Ting Meng b, Chi-Wen Lin b,⇑ a b
Department of Biology, National Changhua University of Education, Changhua 510, Taiwan, ROC Department of Safety, Health and Environmental Engineering, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan, ROC
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A laccase-producing fungus on
1 0 -1 Current (mA)
cathode of MFC was used to enhance degradation of azo dye. Laccase-producing fungal cathodes performed better than laccase-free control cathodes. A maximum power density of 13.38 mW/m2 and an >90% decolorization of acid orange 7 were obtained. Growing a fungal culture with continuous laccase production improved MFC’s electricity generation.
-2 -3 -4 -5 -6 -7 -1.0 -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 Potential vs. Ag/AgCl (V)
a r t i c l e
i n f o
Article history: Received 29 August 2016 Received in revised form 9 December 2016 Accepted 9 December 2016
Keywords: Laccase-catalyzed cathode White-rot fungi Electricity generation Azo dye Decolorization
a b s t r a c t Wood-degrading white-rot fungi produce many extracellular enzymes, including the multi-copper oxidative enzyme laccase (EC 1.10.3.2). Laccase uses atmospheric oxygen as the electron acceptor to catalyze a one-electron oxidation reaction of phenolic compounds and therefore has the potential to simultaneously act as a cathode catalyst in a microbial fuel cell (MFC) and degrade azo dye pollutants. In this study, the laccase-producing white-rot fungus Ganoderma lucidum BCRC 36123 was planted on the cathode surface of a single-chamber MFC to degrade the azo dye acid orange 7 (AO7) synergistically with an anaerobic microbial community in the anode chamber. In a batch culture, the fungus used AO7 as the sole carbon source and produced laccase continuously, reaching a maximum activity of 20.3 ± 0.3 U/L on day 19 with a 77% decolorization of the dye (50 mg/L). During MFC operations, AO7 in the anolyte diffused across a layer of polyvinyl alcohol-hydrogel that separated the cathode membrane from the anode chamber, and served as a carbon source to support the growth of, and production of laccase by, the fungal mycelium that was planted on the cathode. In such MFCs, laccase-producing fungal cathodes outperformed laccase-free controls, yielding a maximum open-circuit voltage of 821 mV, a closed-circuit voltage of 394 mV with an external resistance of 1000 X, a maximum power density of 13.38 mW/m2, a maximum current density of 33 mA/m2, and a >90% decolorization of AO7. This study demonstrates the feasibility of
⇑ Corresponding author at: Department of Safety, Health and Environmental Engineering, National Yunlin University of Science and Technology, 123 University Rd. Sec. 3, Douliou, Yunlin 64002, Taiwan, ROC. E-mail address: [email protected] (C.-W. Lin). http://dx.doi.org/10.1016/j.apenergy.2016.12.044 0306-2619/Ó 2016 Elsevier Ltd. All rights reserved.
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growing a white-rot fungal culture with continuous laccase production on the cathode of MFCs to improve their electricity generation and azo dye removal efficiency. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Azo dyes are a group of poorly biodegradable synthetic colorants that are often found in waste water that is produced by the textile industry [1]. These compounds are highly resistant to degradation by aerobic bacteria but can be easily reduced by anaerobic bacteria to form aromatic amines, which are a group of carcinogens that are stable under anaerobic conditions and must be returned to aerobic conditions before they can be further degraded and mineralized [2,3]. Therefore, a combination of aerobic and anaerobic treatments have been suggested to be required for the effective mineralization of these pollutants [4]. Pollutants in waste water from various sources can be removed by a microbial fuel cell (MFC), in which microorganisms break down organic compounds and convert their chemical energy into electrical energy [5,6]. An MFC typically consists of an anaerobic anode chamber and an aerobic cathode chamber that are separated by a proton exchange membrane. In the anode chamber is a microbial community that often comprises a large number of both fermenting and respiring bacteria that contribute to the versatility of MFCs in degrading pollutants. When waste water enters the anode chamber, fermenting bacteria firstly convert large organic molecules into smaller fermentation products, such as lactate, which are then oxidized by anaerobically respiring bacteria to produce CO2, protons, and electrons. In the absence of a suitable electron acceptor in the anolyte, electrons pass to the anode interface and are transferred through an external wire to the cathode, where they join oxygen molecules (O2) and protons to form water molecules, thus completing a circuit [7]. Azo dyes can be degraded in either the anode or cathode chamber of an MFC. When introduced into the catholyte, azo dyes can act as electron acceptors and be decolorized in reductive cathode reactions. In most cases, these reactions transform azo dyes into less colorful compounds, such as aromatic amines and hydrazines, but fail to degrade and mineralize them completely [8]. The dyereducing reactions proceed better under anaerobic conditions as oxygen competes for electrons from the cathode, and the rate of the reaction depends heavily on the pH of the catholyte [9], the structure of the dye molecules [10], and the use of catalysts such as noble metals. Ding et al. demonstrated that photocatalysis with visible light on a rutile-modified cathode greatly increases the rate of dye reduction [11]. Azo dyes can be introduced into the anolyte, as in an MFC that forms the first part of a two-stage system in which azo dyecontaining waste water firstly enters the anode chamber, where dye molecules are reduced by anaerobic bacteria to form aromatic amines, which were then transferred to the second stage, an aerobic bioreactor, for further degradation into smaller compounds by aerobic microorganisms [4,8]. Although these two-in-one systems can effectively remove dye, they are structurally complex and expensive to construct and operate. In theory, azo dyes can be completely degraded in a single MFC if they are firstly introduced into the anode chamber for reductive transformation by anaerobic bacteria and then transferred to the cathode chamber for aerobic degradation by a second group of aerobic microbes. However, in most MFCs, the anode and cathode chambers are separated by a proton exchange membrane, which prevents movement of azo dyes and their transformed products between the chambers. Furthermore, growing aerobic microbes
in the cathode chamber inevitably reduces the level of dissolved oxygen there, and so reduces the cathode potential and the power output of the MFC. This study involves testing the feasibility of growing a laccasesecreting white-rot fungus on the cathode surface of a singlechamber MFC, in which the proton exchange membrane was replaced with a layer of polyvinyl alcohol-hydrogel (PVA-H), allowing the pollutant acid orange 7 (AO7, an azo dye) to diffuse from the anode chamber to the cathode. White-rot fungi are the only group of organisms that can completely degrade azo dyes. Laccase is one of the enzymes that they produce when they degrade lignin, which refers to a group of highly heterogeneous aromatic polymers that are abundant in the natural habitats of white-rot fungi, and is also used by these fungi to degrade azo dyes and many other aromatic compounds [12]. Additionally, laccase has been used as an enzyme catalyst on the cathode of many MFCs, replacing noble metals in catalyzing the reduction of oxygen [13]. In the singlechamber configuration, the MFCs herein have no cathode chamber and their cathodes are exposed directly to the air to increase oxygen availability [14,15]. Our results demonstrate that such fungal MFCs outperformed control MFCs that are equipped with abiotic or enzyme-free cathodes in both the decolorization of dye and the generation of electrical power.
2. Materials and methods 2.1. Microbes Anaerobic sludge was collected from an oil-cracking wastewater treatment plant of Nan-Ya Plastics Co., Ltd. (at the Six Naphtha Cracking Industry Site at Mailiao in Yunlin County). The sludge microbial community was acclimated for an extended period in the laboratory and used for the degradation of aromatic compounds; it tested positive for AO7 decolorization. The fungal strain that was planted on the cathode was obtained from a local mushroom grower and was identical to the Ganoderma lucidum BCRC 36123 strain from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan, ROC. The fungal strain was maintained on potato dextrose agar (PDA) plates. 2.2. Fungal culture on cathode Mycelium of Ganoderma lucidum BCRC 36123 was inoculated onto a 2 mm-thick layer of potato dextrose agar (PDA) medium and incubated to cover fully the surface of the medium. A 1.4 cm 1.4 cm square of the mycelium-covered medium was then cut from the culture plate to be placed on a cathode. 2.3. MFC setup and operation Fig. 1 schematically depicts the single-chamber MFC. The anode chamber was a 350 cm3 container at the bottom of which was placed a 70 cm2 carbon felt anode. The anolyte (of which each liter contained 1.75 g K2HPO4, 2.145 g KH2PO4, 10 mg NH4Cl, 100 mg MgCl26H2O, 45 mg CaCl2, 1 mg FeCl36H2O, 0.25 mg CuCl22H2O, 0.25 mg CoCl26H2O, 1 mg ZnCl2, 1 mg MnCl24H2O, 0.1 mg Na2MoO42H2O, and 0.02 mg NiCl26H2O) was inoculated with 403.29 mg/L of sludge. The air cathode was made from a 6 cm
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Fig. 1. Schematic of a fungal MFC.
(height) 5 cm (diameter) garden pot. A 10% PVA-H gel solution that contained 2.4% potato dextrose broth (PDB) medium was poured into the pot and kept frozen at 30 °C for 12 h. The solidified gel was warmed to room temperature and then covered with a 28.27 cm2 piece of carbon cloth. The carbon cloth was connected with electrical wire, wetted with 6 ml of PDB, and overlaid with a 1.4 cm 1.4 cm square of fungal mycelium-covered medium. The top of the pot was sealed with a parafilm membrane and incubated for 14 days to allow mycelial growth into the carbon cloth. The cathode assembly was then fitted onto the top of the anode chamber such that the underside of the PVA-H layer faced the carbon felt anode at a distance of 10 cm. The parafilm membrane was then replaced with a perforated petri-dish cover to increase ventilation during MFC operations. 2.4. Determination of laccase activity Laccase activity in the cathode assembly was assayed using the substrate 2 20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Absorbance of the green reaction product at 420 nm was determined using a spectrophotometer [16]. The reaction was carried out in 3 ml of a 0.1 M sodium acetate buffer (pH 4.5) that contained 1 mM of ABTS. One unit (U) of laccase is defined as the amount of activity that converts 1 lmole of the substrate in one minute, and is calculated from absorbance readings, using Eq. (1), which is based on Beer’s law:
U¼
Ak V 10 ek l t
(b) Laccase activity in the PVA-H layer: After the myceliumcovered carbon cloth was removed, the remaining PVA-H layer was soaked in 100 ml of a 0.1 M sodium acetate buffer (pH 4.5) with agitation for four hours. The buffer was then clarified by centrifugation (4500 rpm) and its laccase activity was determined. (c) Laccase activity in heat-inactivated fungal cathode: A fungal cathode was incubated for 14 days after mycelial transplantation and soaked three times in sterile distilled water for two hours each time to remove PDB ingredients. The fungal mycelium/carbon cloth/PVA-H assemblies were then baked at 80 °C for one hour to inactivate laccase [17,18]. The assembly was then soaked in 20 ml of the 0.1 M sodium acetate buffer (pH 4.5) with agitation for six hours. The buffer was then clarified by centrifugation (4500 rpm) and its laccase activity was determined. 2.5. Detection of laccase production by fungal mycelia To detect the production of laccase by a fungal culture, guaiacol (0.01%) was added to the PDA medium as an indicator. Laccase production is revealed by a zone of red precipitation around the fungal colony, and the size and shade of the red zone can be used to estimate the amount of laccase produced [18]. 2.6. AO7 decolorization
6
ð1Þ
where Ak is the change in absorbance at wavelength k; V is reaction volume (L), ek is the extinction coefficient of ABTS at wavelength k (e420 = 36,000 M1 cm1); l is the length of the light path (cm), and t is the reaction time (min). Laccase activity on the following samples was evaluated. (a) Laccase activity on a fresh fungal cathode: The fungal cathode was incubated for 14 days after mycelial transplantation and was tested for laccase activity. The mycelium-covered carbon cloth cathode was peeled from the PVA-H layer and soaked in 20 ml of 0.1 M sodium acetate buffer (pH 4.5) with agitation for six hours. The buffer was then clarified by centrifugation (4500 rpm) and its laccase activity was determined.
The absorption spectrum of AO7 in the visible light range peaks at a wavelength of 485 nm. Absorption at 485 nm was therefore used to calculate AO7 from Eq. (2) where A0 and A are the initial and final absorptions, respectively.
Decolorization ð%Þ ¼
A0 A 100 A0
ð2Þ
2.7. Electrochemical analyses A multimeter (CHY 48R, Taiwan) was used to measure the voltage (V) of the MFC. Current (I) was calculated from the external resistance (R) using Eq. (3).
I¼
V R
ð3Þ
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Power (P) was calculated from voltage and current using Eq. (4) and divided by the anode surface area to obtain power density.
P ¼IV
ð4Þ
The internal resistance of each MFC was determined using the polarization slope method and the power density peak method [7]. MFCs were operated to establish a constant voltage and then disconnected for six to eight hours to maximize their open circuit voltage. Voltage was recorded using a range of external resistors and a multimeter. Current and power that were calculated from Eqs. (3) and (4) are used to plot polarization and power density curves. The electrochemical properties of the fungal cathode were investigated using cyclic voltammetry in a three-electrode setup with an Ag/AgCl reference electrode (201 mV vs. NHE) and a Pt counter electrode that were connected to a Bio-Logic SP-150 potentiostat (Bio-Logic, France). The electrodes were submerged in a 0.1 M acetate buffer (pH 4.5) that contained 100 mg/L of AO7 at 25 °C and scanned over the range 1 V to 1 V at a speed of 20 mV/s for three cycles.
Fig. 3. Laccase production and AO7 decolorization of a Ganoderma lucidum BCRC 36123 batch culture in a basal salt medium containing 50 mg/L of AO7 as the sole carbon source; laccase activity ( ), decolorization ( ).
3. Results and discussion 3.1. Production of laccase by fungal mycelia To test the production of laccase by the fungal strain Ganoderma lucidum BCRC 36123, the substrate compound guaiacol was added to PDA medium plates. Dark red precipitation indicated the presence of laccase within and around a colony (Fig. 2) [19]. The red zone expanded as the mycelium grew but faded after day five when growth stopped. 3.2. Decolonization of AO7 by laccase in batch culture Liquid cultures were used to test whether the fungus Ganoderma lucidum BCRC 36123 can use AO7 as a sole carbon source to produce laccase. Such an ability would eliminate the need for
the addition of a co-substrate, such as glucose, to the electrolyte during MFC operations. Fig. 3 presents a typical growth/decolorization cycle of the fungus in a basal salt medium that contained 50 mg/L of AO7. The growth of the mycelium began six days following inoculation, as indicated by a sharp increase of laccase activity in the medium. The activity was 7.5 U/L on day 6, reaching a plateau of 20.3 ± 0.2 U/L on days 8–19. At the same time, the laccase-catalyzed oxidation of AO7 decolorized the medium. The decolorization rate peaked on days 8–19 with 5, 8, and 58% decolorization on days 7, 8, and 20, respectively. The culture aged and the laccase activity declined rapidly after day 20, and decolorization gradually reached a plateau of 77% on day 50. These results clearly demonstrate that Ganoderma lucidum BCRC 36123 grew with AO7 as the sole carbon source and produced laccase during the phase of active growth.
Day 0
Day 1
Day 2
Day 5
Day 6
Day 7
Fig. 2. Growth of the fungal strain Ganoderma lucidum BCRC 36123 on laccase detection medium containing 0.01% guaiacol. The presence of laccase was indicated by red precipitation around fungal mycelium. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.3. Laccase activity of the fungal cathode Laccase production by mycelium that grew on the cathode membrane was tested by soaking the mycelium-covered carbon cloth (cathode membrane) and the underlying PVA-H gel in an acetate buffer for 4–6 h and assayed for laccase activity in the buffer using the substrate ABTS. After 14 days of incubation, the mycelium-covered carbon cloth contained 1.88 ± 0.22 U/L of laccase. The PVA-H gel contained 2.28 ± 0.71 U/L of laccase. No laccase activity was detected on control electrodes without mycelium or with heat-inactivated fungal mycelium. 3.4. Generation of electricity by MFC with fungal cathode Although the anodic reaction in an MFC is always catalyzed by microbes, the reduction of oxygen at the cathode of most MFCs is catalyzed by non-biological catalysts such as platinum [10]. Oxidative enzymes including laccase are sometimes immobilized on the surface of the cathode to catalyze the reaction [13]. However, noble metal and enzyme catalysts are expensive and can deteriorate rapidly under the moist and often corrosive conditions in which most MFCs operate [20]. Instead of applying a purified enzyme to the cathode, Wu et al. demonstrated that growing a laccasesecreting white-rot fungus in the cathode chamber increased the power density of a two-chamber MFC sevenfold. However, such a setup requires the continuous addition of nutrients and mediator compounds into the cathode chamber [21]; this process not only increases cost but also encourages the growth of contaminating microorganisms to become another source of pollution. In this work a white-rot fungus was planted on the surface of the cathode in a single-chamber MFC to serve as both a continuous source of fresh laccase and an aerobic dye-degrading microbe. Single-chamber MFCs, shown in Fig. 1, with a fungal cathode (fMFC), a fungus-free cathode (a-MFC) or a heat-inactivated fungal cathode (if-MFC), were set up and stabilized in a 20 day pre-run. Their close- and open-circuit voltages were monitored and compared in repeated cycles of dye addition in 30 days of continuous operation. Fig. 4 shows that in the early stage, between days 3 and 16, the f-MFC produced higher open circuit voltages than the two control MFCs, reaching a maximum of 817 mV on day 10, an 18% increase over the 692 mV that was produced by the a-MFC. After day 10, the voltage of the f-MFC slowly declined, eventually reaching the same level as that of the a-MFC on day 17. In the late stage, between days 17 and 30, the two cells exhibited the same
voltage, under both the open circuit condition and the closed circuit condition with a 1000 X external resistance, as shown in Fig. 4. This loss of increase in potential by the f-MFC in the late stage was probably caused by excessive growth of fungal mycelium on the cathode membrane. The greater thickness of the fungal layer was responsible for reduced availability of oxygen to the cathode, and the greater distance from the most active laccaseproducing cells on the growing tips of the mycelium to the electrode was responsible for reduced laccase activity on the electrode surface. The heat-inactivated if-MFC produced lower voltages than either the a-MFC or the f-MFC throughout the operation, probably because the inactivated mycelium on the cathode membrane acted as a barrier to oxygen transport. The increased open circuit voltage of the f-MFC on days 10–17 suggests a reduced internal resistance, and laccase-modified electrodes have been shown to have lower activation over voltages than unmodified ones [22]. The internal resistance of MFCs was examined herein using the polarization slope method and the power density peak method [7]. Figs. 5 and 6 plot the polarization curves and the power density curves of the three MFCs that were recorded on day 17 of continuous operation. The maximum power densities that were produced by the f-MFC, a-MFC, and if-MFC were 13.38, 10.41, and 9.04 mW/m2 at current densities of 33, 27.6, and 23.3 mA/m2, respectively (Table 1). The internal resistances of the f-MFC, a-MFC, and if-MFC that were calculated using the polarization slope method were 1574, 1680, and 1859 X, respectively. Whereas the internal resistances that were calculated
Fig. 5. Polarization curves of f-MFC ( ), a-MFC ( ), and if-MFC (d).
Fig. 4. Voltage-time plots of f-MFC ( ), a-MFC ( ), and if-MFC (d). Open circuit voltages are shown for days 1–16 and 24–30 while closed circuit voltages with a 1000 X external resistance are shown for days 17–23.
Fig. 6. Power density curves of f-MFC ( ), a-MFC (s), and if-MFC (d).
C.-Y. Lai et al. / Applied Energy 188 (2017) 392–398 Table 1 Electrochemical properties of f-MFC, a-MFC, and if-MFC.
a b
Items
f-MFC
a-MFC
if-MFC
Maximum open-circuit potential (mV) Comparison of open-circuit potential efficiency (%) Closed-circuit potential (mV)a Comparison of closed-circuit potential efficiency (%) Maximum power density Based on the anode surface (mW/m2) Based on the anode chamber volume (mW/m3) Maximum current density Based on the anode surface (mA/m2) Based on the anode chamber volume (mA/m3) Internal resistance (X)b
821 18
695 –
655 6
394 4
378 –
375 1
13.38 267.60
10.41 208.30
9.04 180.70
33 659.10 1574
27.60 551 1680
23.30 465.80 1859
With 1000 X of external resistance. Determined using the polarization slope method.
using the power density peak method were 1760, 1960, and 2380 X. Since the three cells shared the same configuration and operating conditions, except for the presence of laccase in the fMFC, the reduction of more than 100 X in the internal resistance of the f-MFC can be attributed to a reduction in the activation loss owing to laccase activity. Although thickened with prolonged operation, the fungal mycelium did not increase internal resistance because it was planted on the outer surface of the cathode and continuously grew away from the cathode into the air. The electrochemical properties of freshly prepared cathodes of the f-MFC, a-MFC, and if-MFC were examined using cyclic voltammetry (CV). Each cathode was scanned from 1.0 V to 1.0 V vs. an Ag/AgCl electrode at a rate of 20 mV/s for three cycles. Fig. 7 presents the results of the second cycle. The f-MFC cathode exhibited an increase in reduction current that peaked at 0.73 V (vs. Ag/ AgCl). No increase in oxidation current was detected during the forward scan. Many studies of laccase-modified electrodes have yielded the same results [22] as, under aerobic conditions, laccase molecules that are reduced during the backward scan are rapidly oxidized again by binding to dissolved oxygen; they therefore do not contribute to the oxidation current during the subsequent forward scan. Increases in reduction currents in many laccasemodified carbon electrodes have been attributed to two mechanisms, which are (1) direct electron transfer from the electrode to the T1 copper ion in the enzyme; (2) mediated electron transfer between the electrode and the enzyme [22]. In the latter mechanism, free radicals that are generated by laccase-catalyzed oneelectron oxidation of phenolic substrates frequently act as media-
Fig. 7. Cyclic voltammograms of the cathodes of f-MFC ( MFC ().
), a-MFC ( ), and if-
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tors. Direct electron transfer from electrode to laccase can be observed using CV under aerobic conditions as an enzymedependent reduction current, whose magnitude depends on the cathode material, the method of preparation of the cathode, and the substrate concentration. As no faradaic current was detected on the fungal cathodes by CV when AO7 was omitted from the anolyte, a mediated mechanism with either AO7 or its oxidized products as mediators was more likely to be involved. However, CV is not sufficiently sensitive to detect small reduction currents, so the involvement of direct electron transfer cannot be completely excluded.
3.5. Decolorization of AO7 by MFC with mycelium-covered cathode During operation of the MFC, the decolorization of the anolyte usually reached 90% in four to five days, as determined using a spectrophotometer. Fresh AO7 solution was therefore added periodically to restore the substrate concentration. Fig. 8 compares the decolorization of the anolyte in the f-MFC with that in the aMFC. In the early stage of the operation before day 10, the f-MFC decolorized AO7 faster than did the a-MFC. From day 10 to day 17, the two cells decolorized at similar rates. This trend paralleled the changes in the voltages of the MFCs over the same period. Fungal mycelium on the cathode increased the decolorization and removal of dye by several mechanisms. First, fungal mycelium functions as an oxygen barrier, keeping the anaerobic conditions of the anode chamber, which favor dye reduction. Second, laccase that is produced by the mycelium increases the rate of reduction of the dye on the cathode. On abiotic cathodes, the rate of reduction of dye depends heavily on its molecular structure [10]. As many azo dyes are substrates of laccase, the use of laccase as a cathode catalyst greatly increases the range of dyes that can be effectively reduced and the rate of their reduction. Third, white-rot fungi are effective azo dye degraders, and laccase is one of the dyedegrading enzymes that they produce in a study to determine the products of azo dye degradation, Zille et al. concluded that laccase is not suitable for removing azo dyes because the enzyme initiates a series of free radical-mediated coupling and polymerization reactions that lead to a variety of end products, many of which are large azo compounds that are recalcitrant to further degradation [23]. However, this conclusion is accurate only when laccase is the only enzyme that acts on the dye. In the presence of living fungal cells and their complete metabolic network,
Fig. 8. Decoloration of AO7 in f-MFC ( ), a-MFC ( ), and if-MFC (d). Fresh solution of AO7 was added to anolyte to the concentration of 50 mg/L on days 4, 10, 12, 17, and 23.
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most azo dyes and their degradation intermediates can be absorbed and assimilated. After day 17, the f-MFC surpassed the a-MFC in dye decolorization despite of the similarity between their voltages, probably because of the overgrowth of fungal mycelium in the f-MFC, which oxidizes and uses AO7 as its carbon source, again indicating that fungus on the cathode increased the decolorization rate by improving the electrochemical performance of the MFC in the early stage and by directly removing the dye pollutant from the anolyte in both the early and the late stages. The fungal colony thus served as a continuous pollutant sink made possible by the use of PVAH gel, which allows AO7 and its degradation intermediates to move freely between the electrodes. The PVA-H gel might additionally help to prevent acidification of the anolyte during prolonged operations. Recent studies showed that the use of proton exchange membranes in most MFCs causes accumulation of cations on the anode side because these selective materials favor the passage of proton. Non-proton cations that migrate toward the cathode tend to be stopped at the anolytemembrane interface. During prolonged operations, the build-up of cation concentration near the membrane surface in turn produces a large resistance to the passage of proton, and thus the flow of current, leading to a rapid acidification of the anolyte [24]. By replacing the proton exchange membrane, the PVA-H gel therefore served the triple role of lowering the cost, enabling a direct participation of the fungus in pollutant removal, and mitigating acidification during prolonged operations. 4. Conclusions The f-MFC with a fungal cathode achieved 90% decolorization of the azo dye AO7 in five days of continuous operation and outperformed control MFCs with respect to electrochemical parameters such as open-circuit voltage, closed circuit voltages with a fixed external resistance, current density, and power density. Unlike two-chamber MFCs in which fungus grows in the cathode chamber, the single-chamber f-MFC did not require the continuous addition of substrate and mediator to the catholyte, because the proton exchange membrane that is used in most fuel cells was replaced with a layer of PVA-H, allowing AO7 molecules to diffuse to the cathode, to serve as a substrate, and to be removed by the fungal mycelium. However, thickening of the fungal cathode during prolonged operations appeared to inhibit oxygen transport to the cathode surface and thereby reduce power generation without significantly affecting the decolorization rate. These results demonstrate that the fungal cathode is feasible for removing azo dyes, but further modifications of MFC design and operating conditions must be made to prevent fungal overgrowth and improve longterm effectiveness. Acknowledgements The authors would like to thank the Ministry of Science and Technology of the Republic of China, Taiwan, for financially supporting this research under Contract No. MOST 105-2221-E-224003. Ted Knoy is appreciated for his editorial assistance.
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