Bioresource Technology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced current production by Desulfovibrio desulfuricans biofilm in a mediator-less microbial fuel cell Christina S. Kang a,1, Numfon Eaktasang a,b,1, Dae-Young Kwon c, Han S. Kim a,⇑ a
Department of Environmental Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea Department of Advanced Technology Fusion, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea c Department of Civil and Urban Engineering, Inje University, 607 Eobang-dong, Gimhae, Gyeongnam 621-749, Republic of Korea b
g r a p h i c a l a b s t r a c t
H2SO4/HNO3 3:1
Modified electrode
O
Electrode
electrochemical oxidation. The treated anode facilitated formation of electrogenic biofilm. Strong hydrogen and/or peptide bonds contributed to bacterial attachment. Cytochrome c of D. desulfuricans was efficiently bound to the electrode and transferred electrons directly.
O OH
O OH
i n f o
Article history: Received 26 December 2013 Received in revised form 26 March 2014 Accepted 27 March 2014 Available online xxxx Keywords: Electrode surface modification Biofilm Cytochrome c Microbial fuel cell Sulfate-reducing bacteria
NH2
O
O
N H N H N H
a b s t r a c t In this study, a mediator-less microbial fuel cell (MFC) inoculated with a sulfate-reducing bacterium (SBR), Desulfovibrio desulfuricans, was equipped with bare and surface-treated graphite felt electrodes. Electrochemical treatment of the anode surface facilitated biofilm formation on the electrode, resulting in rapid and enhanced current production. The maximum current density of the treated anode was 233 ± 24.2 mA/m2, which was 41% higher than that of the untreated anode. The electron transfer rate also increased from 2.45 ± 0.04 to 3.0 ± 0.02 lmol of electrons/mg of proteinmin. Biofilm formation on the treated anode was mainly due to the strong hydrogen or peptide bonds between the amide groups of bacterial materials (including cytochrome c) and carboxyl groups formed on the electrodes. These results provide useful information on direct electron transfer by SRB in a mediator-less MFC through cytochrome c and the effects of the electrochemical treatment of electrodes on MFC performance. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Electrogenic bacteria convert organic or inorganic substances into electricity by transferring free electrons obtained from oxidation of fuel compounds to an electrode in a microbial fuel cell (MFC). Numerous investigations have been directed to improve performance of an MFC by changing/modifying the following com⇑ Corresponding author. Tel.: +82 2 450 4092; fax: +82 2 450 3542. 1
NH2
O OH
a r t i c l e
NH2
Electrode
Graphite felt electrode was treated by
Modified electrode
h i g h l i g h t s
E-mail address: [email protected] (H.S. Kim). Christina S. Kang and Numfon Eaktasang equally contributed to this paper.
ponents: MFC design/architecture/configuration, electrolytes in solutions, organic fuels, materials and surfaces of electrodes, and electrogenic biofilm on the anode (Logan et al., 2006). In particular, the efficiency of electron transfer from electrogens to the anode has been noted to be the most critical factor controlling the overall circuit of an MFC for current production (Crittenden et al., 2006; Feng et al., 2010). The following mechanisms of electron transfer to the anode have been proposed for dissimilatory metal-reducing bacteria: (1) direct electron transfer via bacterial membrane-bound c-type cytochrome (Chaudhuri and Lovley, 2003) or bacterial nanowires
http://dx.doi.org/10.1016/j.biortech.2014.03.148 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kang, C.S., et al. Enhanced current production by Desulfovibrio desulfuricans biofilm in a mediator-less microbial fuel cell. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.148
2
C.S. Kang et al. / Bioresource Technology xxx (2014) xxx–xxx
(Reguera et al., 2005) and (2) indirect electron transfer via extracellular electron shuttles (e.g., manganese, iron, humate, phenazine, and quinones; Crittenden et al., 2006; Newman and Kolter, 2000; Rabaey et al., 2006). Meanwhile, a number of previous studies have demonstrated that sulfate-reducing bacteria (SRBs), including Desulfovibrio desulfuricans, utilize sulfate/sulfide as an electron mediator to generate electricity in an MFC (Eaktasang et al., 2013; Rabaey et al., 2006). However, it is worth noting that D. desulfuricans also possesses several low potential cytochrome c proteins, all of which are membrane-bound and presumably function in direct extracellular electron transfer (Fritz et al., 2001). Electrode material is an important factor that primarily controls energy conversion, particularly in the case of direct electron transfer from electrogens to the anode. Currently, carbon-based materials, such as graphite fiber brush, graphite rod, graphite felt, graphite plate, carbon paper, and carbon cloth, are the most widely used anode materials due to their high electrical conductivity, strong biocompatibility, and low cost (Logan et al., 2006). Furthermore, the modification of electrode surfaces has proven to be an effective way to improve the performance of an MFC because it changes the physicochemical properties of electrodes to facilitate microbial attachment and electron transfer (Table S1). In this study, it was hypothesized that SRBs convert organic fuels to electricity in a mediator-less MFC. D. desulfuricans, a predominant SRB in the subsurface environment, was inoculated and graphite felt was used for electrodes. The surface of the anode was modified by electrochemical oxidation, and the improvement in current production and the efficiency of electron transfer were assessed. To further investigate the direct electron transfer, a membrane-bound cytochrome c was cloned from D. desulfuricans, and then its adsorption onto the anode and electron transfer capability were evaluated. 2. Methods
solids were then freeze-dried overnight. The protein concentrations in the supernatant and washing solutions were measured to determine the amount of bound proteins. 2.4. Electron transfer test for cytochrome c The oxidized and reduced forms of cytochrome c were characterized by a spectrophotometric assay using a UV–visible spectrophotometer (Optizen, Mecasys, Korea). The responses of protein were scanned from 275 to 650 nm. The purified cytochrome c (55.4 lg) was anaerobically reduced with 5 lL of 100-mM dithiothreitol after dissolution in 0.9 mL of Tris–HCl (10 mM and pH 7.0). Fe(III) oxide (a-Fe2O3, 10 mg) was added to the buffer solution containing cytochrome c as a solid electron acceptor. Changes in the redox potential of cytochrome c and the production of Fe2+ were monitored, and the electron transfer rate was determined by the rate of Fe2+ generation. 2.5. Analytical procedures Biomass attached to the electrode was extracted and quantified by Bradford analysis with a bovine serum albumin standard (Quick Start™ Bradford Protein Assay, Bio-Rad, USA). To extract protein, the electrode (1 cm2) was washed with Milli-Q water and the protein was then extracted with 0.2-N NaOH at 100 °C for 10 min (Tang et al., 2011). Organic levels of the anodic chamber were analyzed as chemical oxygen demand (COD). The graphite felt electrode was ground and mixed with KBr for functional group analysis using a Nicolet 6700 Fourier transform infrared (FT-IR) spectrometer (Thermo, USA). The surface area of the graphite felt was measured based on a Brunauer–Emmett–Teller adsorption isotherm (TriStar II 3020 V1.05, Micromeritic Instrument, USA). The electrode surfaces and biofilm were examined using scanning electron microscopy (SEM) and detailed procedures for the SEM analysis are provided in the Supplementary data.
2.1. Microbial fuel cell 3. Results and discussion
2.2. Electrochemical oxidation treatment of electrodes The graphite felt was cleaned with acetone overnight, rinsed thoroughly with Milli-Q water, and then oven-dried at 100 °C for 4 h. It was subsequently soaked for 6 h in a sonication bath (50 Hz) and filled with a mixture of 1-M H2SO4 and concentrated HNO3 (3:1, v/v) in a constant current density of 30 mA/cm2. Then, the graphite felt was washed with Milli-Q water (pH of 7.0) and was oven-dried at 100 °C for 4 h. 2.3. Attachment of cytochrome c onto electrodes Recombinant cytochrome c was obtained from D. desulfuricans and the procedure for its cloning, overexpression, and purification is described in the Supplementary data. Treated graphite felt (1 mg) was added to 1 mL of a 50-mM phosphate buffer solution (pH 7) containing a pre-determined amount of cytochrome c. The mixture was homogenized on a rotating shaker (50 rpm) for 30 min at 25 °C followed by centrifugation (13,000g) using a microcentrifuge (Centrifuge 5424, Eppendorf, Germany). The precipitate (cytochrome c bound to graphite felt) was collected and washed 3 times with a 50-mM phosphate buffer solution to remove any unbound or loosely bound cytochrome c. The washed
3.1. Current production Current production was initiated almost immediately after circuit connection was established (Fig. 1). The current density was low for the first day (lag period) and then increased rapidly, reaching a maximum level at day 3 in the case of unmodified anode. Then, the current dropped almost instantly to a background level as the electron donor was depleted. In the case of the treated anode, the initial lag period and time to reach the maximum current
400 Treated graphite felt Current density (mA/m2)
As presented in Fig. S1, a dual-chamber MFC reactor equipped with cathodic and anodic electrodes made of graphite felt was used in this study. Detailed procedures for the MFC preparation and operation are provided in the Supplementary data.
Untreated graphite felt
300
200
100
0 0
2
4
6
8
10
12
14
16
18
20
Time (d) Fig. 1. Current production by D. desulfuricans in MFCs equipped with untreated and treated anodes. Medium in the anode chamber was replaced when the current dropped below 0.03 mA to start the new cycles of batch operation.
Please cite this article in press as: Kang, C.S., et al. Enhanced current production by Desulfovibrio desulfuricans biofilm in a mediator-less microbial fuel cell. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.148
3
C.S. Kang et al. / Bioresource Technology xxx (2014) xxx–xxx Table 1 Performance of MFC operation. Cycle
1 2 3 4 5 a
Maximum current production (mA/m2)
Time to reach maximum current production (h)
Untreated
Treated
Untreated
Treated
Untreated
Treated
Untreated
Treated
92.0 ± 8.9a 121.0 ± 20.1 142.0 ± 25.2 152.0 ± 35.1 165.0 ± 16.8
103.0 ± 15.3 154.9 ± 24.3 178.0 ± 32.4 217.0 ± 10.1 233.0 ± 24.2
64 126 181 253 340
32 83 151 237 328
75.0 ± 5.0 79.0 ± 4.5 82.0 ± 3.5 85.0 ± 3.0 90.0 ± 4.0
85.0 ± 4.0 90.0 ± 4.5 92.0 ± 3.2 95.0 ± 4.0 97.0 ± 2.5
75.0 ± 5.0 79.0 ± 4.0 83.0 ± 3.0 87.0 ± 5.0 90.0 ± 3.0
89.0 ± 5.0 92.0 ± 3.0 93.0 ± 6.0 95.0 ± 4.0 98.0 ± 2.0
Coulombic efficiency, CE (%)
COD removal (%)
s.d. (n = 3).
density became evidently shorter than those of the untreated anode (Table 1 and Fig. 1). Both MFCs exhibited a growth state, which was supported by gradual increases in maximum current levels. However, the increase in the maximum current level became insignificant (
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced current production by Desulfovibrio desulfuricans biofilm in a mediator-less microbial fuel cell Christina S. Kang a,1, Numfon Eaktasang a,b,1, Dae-Young Kwon c, Han S. Kim a,⇑ a
Department of Environmental Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea Department of Advanced Technology Fusion, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea c Department of Civil and Urban Engineering, Inje University, 607 Eobang-dong, Gimhae, Gyeongnam 621-749, Republic of Korea b
g r a p h i c a l a b s t r a c t
H2SO4/HNO3 3:1
Modified electrode
O
Electrode
electrochemical oxidation. The treated anode facilitated formation of electrogenic biofilm. Strong hydrogen and/or peptide bonds contributed to bacterial attachment. Cytochrome c of D. desulfuricans was efficiently bound to the electrode and transferred electrons directly.
O OH
O OH
i n f o
Article history: Received 26 December 2013 Received in revised form 26 March 2014 Accepted 27 March 2014 Available online xxxx Keywords: Electrode surface modification Biofilm Cytochrome c Microbial fuel cell Sulfate-reducing bacteria
NH2
O
O
N H N H N H
a b s t r a c t In this study, a mediator-less microbial fuel cell (MFC) inoculated with a sulfate-reducing bacterium (SBR), Desulfovibrio desulfuricans, was equipped with bare and surface-treated graphite felt electrodes. Electrochemical treatment of the anode surface facilitated biofilm formation on the electrode, resulting in rapid and enhanced current production. The maximum current density of the treated anode was 233 ± 24.2 mA/m2, which was 41% higher than that of the untreated anode. The electron transfer rate also increased from 2.45 ± 0.04 to 3.0 ± 0.02 lmol of electrons/mg of proteinmin. Biofilm formation on the treated anode was mainly due to the strong hydrogen or peptide bonds between the amide groups of bacterial materials (including cytochrome c) and carboxyl groups formed on the electrodes. These results provide useful information on direct electron transfer by SRB in a mediator-less MFC through cytochrome c and the effects of the electrochemical treatment of electrodes on MFC performance. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Electrogenic bacteria convert organic or inorganic substances into electricity by transferring free electrons obtained from oxidation of fuel compounds to an electrode in a microbial fuel cell (MFC). Numerous investigations have been directed to improve performance of an MFC by changing/modifying the following com⇑ Corresponding author. Tel.: +82 2 450 4092; fax: +82 2 450 3542. 1
NH2
O OH
a r t i c l e
NH2
Electrode
Graphite felt electrode was treated by
Modified electrode
h i g h l i g h t s
E-mail address: [email protected] (H.S. Kim). Christina S. Kang and Numfon Eaktasang equally contributed to this paper.
ponents: MFC design/architecture/configuration, electrolytes in solutions, organic fuels, materials and surfaces of electrodes, and electrogenic biofilm on the anode (Logan et al., 2006). In particular, the efficiency of electron transfer from electrogens to the anode has been noted to be the most critical factor controlling the overall circuit of an MFC for current production (Crittenden et al., 2006; Feng et al., 2010). The following mechanisms of electron transfer to the anode have been proposed for dissimilatory metal-reducing bacteria: (1) direct electron transfer via bacterial membrane-bound c-type cytochrome (Chaudhuri and Lovley, 2003) or bacterial nanowires
http://dx.doi.org/10.1016/j.biortech.2014.03.148 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kang, C.S., et al. Enhanced current production by Desulfovibrio desulfuricans biofilm in a mediator-less microbial fuel cell. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.148
2
C.S. Kang et al. / Bioresource Technology xxx (2014) xxx–xxx
(Reguera et al., 2005) and (2) indirect electron transfer via extracellular electron shuttles (e.g., manganese, iron, humate, phenazine, and quinones; Crittenden et al., 2006; Newman and Kolter, 2000; Rabaey et al., 2006). Meanwhile, a number of previous studies have demonstrated that sulfate-reducing bacteria (SRBs), including Desulfovibrio desulfuricans, utilize sulfate/sulfide as an electron mediator to generate electricity in an MFC (Eaktasang et al., 2013; Rabaey et al., 2006). However, it is worth noting that D. desulfuricans also possesses several low potential cytochrome c proteins, all of which are membrane-bound and presumably function in direct extracellular electron transfer (Fritz et al., 2001). Electrode material is an important factor that primarily controls energy conversion, particularly in the case of direct electron transfer from electrogens to the anode. Currently, carbon-based materials, such as graphite fiber brush, graphite rod, graphite felt, graphite plate, carbon paper, and carbon cloth, are the most widely used anode materials due to their high electrical conductivity, strong biocompatibility, and low cost (Logan et al., 2006). Furthermore, the modification of electrode surfaces has proven to be an effective way to improve the performance of an MFC because it changes the physicochemical properties of electrodes to facilitate microbial attachment and electron transfer (Table S1). In this study, it was hypothesized that SRBs convert organic fuels to electricity in a mediator-less MFC. D. desulfuricans, a predominant SRB in the subsurface environment, was inoculated and graphite felt was used for electrodes. The surface of the anode was modified by electrochemical oxidation, and the improvement in current production and the efficiency of electron transfer were assessed. To further investigate the direct electron transfer, a membrane-bound cytochrome c was cloned from D. desulfuricans, and then its adsorption onto the anode and electron transfer capability were evaluated. 2. Methods
solids were then freeze-dried overnight. The protein concentrations in the supernatant and washing solutions were measured to determine the amount of bound proteins. 2.4. Electron transfer test for cytochrome c The oxidized and reduced forms of cytochrome c were characterized by a spectrophotometric assay using a UV–visible spectrophotometer (Optizen, Mecasys, Korea). The responses of protein were scanned from 275 to 650 nm. The purified cytochrome c (55.4 lg) was anaerobically reduced with 5 lL of 100-mM dithiothreitol after dissolution in 0.9 mL of Tris–HCl (10 mM and pH 7.0). Fe(III) oxide (a-Fe2O3, 10 mg) was added to the buffer solution containing cytochrome c as a solid electron acceptor. Changes in the redox potential of cytochrome c and the production of Fe2+ were monitored, and the electron transfer rate was determined by the rate of Fe2+ generation. 2.5. Analytical procedures Biomass attached to the electrode was extracted and quantified by Bradford analysis with a bovine serum albumin standard (Quick Start™ Bradford Protein Assay, Bio-Rad, USA). To extract protein, the electrode (1 cm2) was washed with Milli-Q water and the protein was then extracted with 0.2-N NaOH at 100 °C for 10 min (Tang et al., 2011). Organic levels of the anodic chamber were analyzed as chemical oxygen demand (COD). The graphite felt electrode was ground and mixed with KBr for functional group analysis using a Nicolet 6700 Fourier transform infrared (FT-IR) spectrometer (Thermo, USA). The surface area of the graphite felt was measured based on a Brunauer–Emmett–Teller adsorption isotherm (TriStar II 3020 V1.05, Micromeritic Instrument, USA). The electrode surfaces and biofilm were examined using scanning electron microscopy (SEM) and detailed procedures for the SEM analysis are provided in the Supplementary data.
2.1. Microbial fuel cell 3. Results and discussion
2.2. Electrochemical oxidation treatment of electrodes The graphite felt was cleaned with acetone overnight, rinsed thoroughly with Milli-Q water, and then oven-dried at 100 °C for 4 h. It was subsequently soaked for 6 h in a sonication bath (50 Hz) and filled with a mixture of 1-M H2SO4 and concentrated HNO3 (3:1, v/v) in a constant current density of 30 mA/cm2. Then, the graphite felt was washed with Milli-Q water (pH of 7.0) and was oven-dried at 100 °C for 4 h. 2.3. Attachment of cytochrome c onto electrodes Recombinant cytochrome c was obtained from D. desulfuricans and the procedure for its cloning, overexpression, and purification is described in the Supplementary data. Treated graphite felt (1 mg) was added to 1 mL of a 50-mM phosphate buffer solution (pH 7) containing a pre-determined amount of cytochrome c. The mixture was homogenized on a rotating shaker (50 rpm) for 30 min at 25 °C followed by centrifugation (13,000g) using a microcentrifuge (Centrifuge 5424, Eppendorf, Germany). The precipitate (cytochrome c bound to graphite felt) was collected and washed 3 times with a 50-mM phosphate buffer solution to remove any unbound or loosely bound cytochrome c. The washed
3.1. Current production Current production was initiated almost immediately after circuit connection was established (Fig. 1). The current density was low for the first day (lag period) and then increased rapidly, reaching a maximum level at day 3 in the case of unmodified anode. Then, the current dropped almost instantly to a background level as the electron donor was depleted. In the case of the treated anode, the initial lag period and time to reach the maximum current
400 Treated graphite felt Current density (mA/m2)
As presented in Fig. S1, a dual-chamber MFC reactor equipped with cathodic and anodic electrodes made of graphite felt was used in this study. Detailed procedures for the MFC preparation and operation are provided in the Supplementary data.
Untreated graphite felt
300
200
100
0 0
2
4
6
8
10
12
14
16
18
20
Time (d) Fig. 1. Current production by D. desulfuricans in MFCs equipped with untreated and treated anodes. Medium in the anode chamber was replaced when the current dropped below 0.03 mA to start the new cycles of batch operation.
Please cite this article in press as: Kang, C.S., et al. Enhanced current production by Desulfovibrio desulfuricans biofilm in a mediator-less microbial fuel cell. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.148
3
C.S. Kang et al. / Bioresource Technology xxx (2014) xxx–xxx Table 1 Performance of MFC operation. Cycle
1 2 3 4 5 a
Maximum current production (mA/m2)
Time to reach maximum current production (h)
Untreated
Treated
Untreated
Treated
Untreated
Treated
Untreated
Treated
92.0 ± 8.9a 121.0 ± 20.1 142.0 ± 25.2 152.0 ± 35.1 165.0 ± 16.8
103.0 ± 15.3 154.9 ± 24.3 178.0 ± 32.4 217.0 ± 10.1 233.0 ± 24.2
64 126 181 253 340
32 83 151 237 328
75.0 ± 5.0 79.0 ± 4.5 82.0 ± 3.5 85.0 ± 3.0 90.0 ± 4.0
85.0 ± 4.0 90.0 ± 4.5 92.0 ± 3.2 95.0 ± 4.0 97.0 ± 2.5
75.0 ± 5.0 79.0 ± 4.0 83.0 ± 3.0 87.0 ± 5.0 90.0 ± 3.0
89.0 ± 5.0 92.0 ± 3.0 93.0 ± 6.0 95.0 ± 4.0 98.0 ± 2.0
Coulombic efficiency, CE (%)
COD removal (%)
s.d. (n = 3).
density became evidently shorter than those of the untreated anode (Table 1 and Fig. 1). Both MFCs exhibited a growth state, which was supported by gradual increases in maximum current levels. However, the increase in the maximum current level became insignificant (
