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Mediating effects of ferric chelate compound in microbial fuel cells
Bioelecrrochemisrry and Bioenergetics, 11 (1983) 135-143 A section of J. Elecmanal. Chem., and constituting Vol 156 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
591 -MEDIATING EFFECTS OF FERRIC CHELATE MICROBIAL FUEL CELLS
KAZUKO
TANAKA,
Inorganic Chemistty
CARMEN Laboratory,
A. VEGA
l
135
COMPOUNDS
IN
and REITA TAMAMUSHI
The Institute of Physical and Chemical Research, Wako-shi,
Saitama 351
(Japan) (Manuscript
received June 23rd 1983)
SUMMARY The performance of b&fuel cells containing Escherichia coli, glucose and a series of ferric chelate reagents was studied. The measured coulombic outputs indicate that most of the compounds work effectively as electron-transfer mediators in the fuel cells. These outputs were compared with measured rate constants for the reduction of ferric chelates by E. co/i and the electrochemical reaction of these compounds at a carbon electrode. The results suggest that a good mediator for microbial fuel cells is one which shows fast reduction by E. co/i. together with a fast electrode reaction. In regenerative fuel cells which were run for five days, coulombic yields over 70 % were obtained on the basis of complete oxidation of added glucose.
INTRODUCTION
In a microbial fuel cell, electrical energy is obtained from oxidizable substrate through the catalytic ,action of microorganisms [l]. The functioning of such direct bio-fuel cells requires the presence of a mediator, which facilitates the transfer of electrons from the microorganisms to the anode [2]. Previous studies have shown that many redox dyes, such as thionine and new methylene blue, are effective mediators, as indicated from spectrophotometric measurements of their reduction rates by microorganisms [3,4], and from preliminary estimates of the coulombic outputs of fuel eells [2]. The development of effective mediators or mediator systems is clearly important for improving the efficiency of this type of electrochemical cell, and the requirements for a suitable mediator may be detailed as follows: (a) The mediator should not be toxic to microorganisms. This requirement is not fulfilled by many of the redox dyes employed so far, especially at higher concentrations. (b) The reagent should be able to penetrate the cell wall of the microorganism to l
On leave (1982) from University of Puerto Rico, Mayaguez, Puerto Rico.
0302-4598/83/$03.00
0 1983 Elsevier Sequoia S.A.
136
react with the source of electrons. Although details of this process remain to be clarified, some importance is attached to the lipophilicity and structure of the mediator [3]. (c) It should be electrochemically active at the electrode surface. (d) The formal redox potential should be near to that of the redox couple providing the reducing action within microorganism, which is likely in many cases [l] to be NAD+/NADH. (e) For practical application over long periods of time, the reagent should be chemically stable in solution. This is often not the case for organic dyes, especially when they are in the reduced state, at elevated temperature, or continuously exposed to light. (f) The mediator must be reasonably soluble in buffer solutions, which are usually near pH 7. The use of metallic chelate compounds as mediators has not yet been explored, but many of them have the potential to satisfy these requirements for a suitable mediator. By using different chelating agents together with a given metal, it is possible to vary the redox potential, solubility, lipophilicity, stability, etc. The purpose of the present study was to examine the mediating effects of a series of ferric chelate compounds in fuel cells containing Escherichia coli. The ferric complexes of ethylenediaminetetraacetic acid (EDTA) and five of its variants were chosen, together with nitrilotriacetic acid. The coulombic outputs from the fuel cells were measured as described in previous papers [2]. Rates of reduction of these ferric chelates by E. coli together with electrode reaction rates of the complexes were determined by electrochemical techniques. These rates were compared with the coulombic ouputs from the fuel cells measured under comparable conditions. Fe(III)TTHA, Fe(III}DTPA and Fe(III)EDADPA complexes were further investigated in longer term experiments during which the fuel cells were operated regeneratively over a five day period. Coulombic yields were compared with the theoretical yield calculated on the basis of complete oxidation of glucose to CO, and water. EXPERIMENTAL
Materials Escherichia coli KI2 was grown aerobically in nutrient broth (Difco Laboratories, U.S.A.) for 24 hours. It was harvested by centrifugation at 4 “C and washed three times with 0.1 mol dme3 phosphate buffer solutions of pH 7.0. In each fuel cell experiment the anode compartment contained 6 X 10” cells (approx. 0.05 g, dry weight). All chemicals were used without further purification. The chelating reagents (Dojin Kagaku) were complexed by mixing equivalent amounts with ferric ammonium sulphate in aqueous solution. Names and structures of the chelating compounds are given in Table 1. For comparison some experiments were performed using thionine (Eastman Kodak) as a mediator.
137
TABLE
1
Chelating
reagents
used as mediators
Name
Structure
(1) EDTA
HooCCH~
\
N -
/
HOOCCH~
/
CH2-CH2--N
\
Ethylenediaminetetraacetic
CH2COOH
CH2COOH
acid
,CH2COOH
(2) CyDTA
N\ a
CH&OOH CH$OOH
N”
‘CHzCOOH
1,2-Cyclohexanediamine
tetraacetic
acid
HOOCCHZ
(3) DTPA
/
\ /
HOOCCHz
\ CH$OOH
Diethylenetriamine (4) TTHA
pentaacetic
,CH2COOH \ /
N’--CHp2-~-c~,c~,-
HOOCCH2CH2
\
Ethyknediamine HOOCCH,
HOOCCH2
\
CH2CCOH
acid
/ \
diaceticacid
CH2 CKIH
dipropionic
acid
NCH2CH2-0-CH2CH2--0-CH2CH2-N
/ \
/
N(CH,COOH), Nitrilotriacetic
\
CHzCH$OOH NCH2CH2N’
Glycoletherdiaminetetraacetic (7) NTA
CH2COOH
hexaacetic
/
HOOCCH2
(6) GEDTA
N-CH~CH~-N”
HOOCH&
Triethylenetetramine (5) EDADPA
CHzCOOH
acid
HooCCH* HOOCCHz
CHzCOOH
N’-CH2-CH2-N-CH2-CH2--N’
CH$OOH
CH,COOH
acid
acid
Microbial fuel cells The composition of the microbial fuel cells is represented below: Carbon cloth (cathode) (10 cm2)
K3[Fe(CN)a] (0.1 mol dmm3) in phosphate buffer, pH 7 (15 cm3)
/ ; ) ; 1
E. coli (6 X 10” cells); glucose (lO.pmoI); mediator (1 x low2 or 1 x low3 mol dm-3) in phosphate buffer, pH 7 (15 cm3)
Carbon cloth (anode) (10 cm2 >
138
The cells were constructed of polyacrylic material and were similar to those used previously [2]. Carbon cloth (Nihon Carbon) electrodes were connected with Pt-wire. The anode and cathode compartments were separated by a cation-permeable ion-exchange membrane (Asahi Glass). Nitrogen was bubbled through both compartments to maintain anaerobic conditions. All the measurements were carried out at 30 “C using a digital voltmeter and a chart recorder (Riken Denshi SP-6 36 Speedex), and the procedure for the measurements of the cell performance was as follows. After the cells had been assembled and glucose added to the anode compartments, they were left on open circuit for 1-2 hours, after which time the voltage was around -0.5 V. The cells were allowed to discharge through a known resistance (usually 500-600 52) and the cell voltages were recorded over a 24 hour period, which was sufficient to extract most of the electric charge available from the oxidation of glucose. In regenerative-cell experiments the load was removed after 24 hours and a further 10 pmol of glucose was added to the anode compartment. The cells were allowed to recover for a period of one hour, and the discharge procedure was then repeated. This regeneration was carried out for five consecutive experiments.
Polarography
and linear sweep voltammetry
Reduction rates of ferric chelate compounds by E. coli in phosphate buffer solutions at 30 “C were measured polarographically using a potentiostat/galvanostat system (Princeton Applied Research Model 173) with a chart recorder (Riken Denshi). In order to follow the changes in concentration of ferric or ferrous chelates by measuring the current at the dropping mercury electrode (d.m.e.) in the limiting current region, applied electrode potentials of - 0.4 V and + 0.05 V versus s.c.e. were switched, in turn, at 50 second intervals with an external trigger mode. All measurements were carried out in deaerated phosphate buffer solutions containing about 1Or2 cells of E. coli and 1 X 10m3 mol dmW3 ferric chelate. Linear sweep voltammetric measurements for the determination of the electrode reaction rate constants were carried out at 25 “C in a three-electrode cell using a conventional reversal technique. The apparatus consisted of a potentiostat (Fuso Seisakusho Model 361), a potential scanning unit (Fuso Seisakusho Model 321-Sl), a transient converter (Riken Denshi Co Ltd. Model TCA-lOOO), and an X-Y recorder. The transient converter was used to record voltammograms at scanning rates higher than 0.2 V s-‘. The working electrode was a pyrolytic graphite rod (diameter 5 mm) whose surface was renewed for each experimental run, and its electrode potential was varied from +0.5 V to -0.5 V against a AgJAgCIlsat. KC1 reference electrode with a scan rate ranging between 0.01 and 10 V s-l. All measurements were made in deaerated phosphate buffer solutions containing lop2 mol dm-’ ferric chelate compound.
139 RESULTS AND DISCUSSIONS
The typical voltage W.SW time curves for microbial fuel cells using Fe(III)EDTA as mediator are shown in Fig. 1, together with an experiment using thionine. Thionine is an effective mediator for E. coli fuel cells [2,4] and was used as a reference. As the figure shows, dilute Fe(III)EDTA (curve B) has a limited mediating effect compared with the cell without a mediator (curve D), but a more concentrated solution of Fe(III)EDTA gives a good mediating effect (curve A) comparable with that of thionine (curve C). Coulombic outputs (Q) from the fuel cells were obtained from integration of the current uersta time plots. The coulombic outputs for the first 24 hours of E. coli fuel cells for several ferric chelate mediators are presented in Table 2. The figures give evidence that the chelates are good mediators in comparison with thionine, particularly at the higher concentration of 0.01 mol dme3. The Fe(III)GEDTA complex is an exception; the coulombic output of the cell using this mediator was similar to that of the blank cell without a mediator. Fe(II1) chelates and their reduced forms (Fe(II)chelates) gave well-defined polarographic waves at the d.m.e. in phosphate buffer solutions of pH 7.0, and their concentrations were estimated from the wave heights in the limiting current plateau region. The changes in concentrations of Fe(II1) chelates and Fe(I1) chelates were determined from the changes in wave heights at - 0.4 V and + 0.05 V versus s.c.e., respectively. The plots of log(h, - h,) uersur time, where h, is the wave height at time t, and h, is the wave height when the reaction is completed, gave straight lines, 0 O-
4
I
8
12
16
20
24
1 I ' ;.' .....'.'.........--.._...~.._.~...__._.__...._........_....... D c__-_---- _____------7= I I
' t(hr)
Fig. 1. Cell voltage versus time curves for microbial fuel cells. Cells were loaded (510 51) after two hours. (A) 1 x lo-* mol drne3 Fe(III)EDTA; (B) 1 X lo-’ mol dm-’ Fe(III)EDTA; (C) 1 X 10m3 mol dmm3 tbionine; (D) no mediator.
140
TABLE
2
Coulombic outputs of E. coli fuel cells, rate constants electrode reaction rate constants of these compounds
of ferric chelates
by E. co/i, and
QW
Mediator
Fe(III)EDTA Fe(III)CyDTA Fe(III)DTPA Fe(III)TTHA Fe(III)EDADPA Fe(III)GEDTA Fe(III)NTA Thionine
1x10-2 mol dm-’
1x10-s mol drne3
31 25 24 22 26 8
13 8 15 11 11 -
20
13
-
31
104k( E. co/i) (s-l) 1.1 0.54 0.26 0.17 100”
lO*k(EIectrode) (cm s-‘) 1.5 1.8 1.2 0.22 1.1 slow 0.07 -
5
No mediator a Obtained
of reduction
spectrophotometrically
under comparable
conditions.
as shown in Fig. 2. The first order reduction rate constants of ferric chelate compounds by E.coli [k(E.con)] were estimated from the slopes of these lines, and are listed in the third column of Table 2. The figures appear to indicate that the smaller the size of the mediator, the faster is its reduction rate, which suggests that
0
10
20
3Q
40
Fig. 2. First order plots for the reduction
50
60
of ferric chelate
compounds
by E. co/i. (h expressed
in cm.)
141
the smaller mediator molecules penetrate the cell wall to accept electrons at the cell membrane more easily. All the ferric chelate compounds studied gave well-defined cyclic voltammograms at a pyrolytic graphite electrode in 0.1 mol dmV3 phosphate buffer solutions; the voltammogram exhibited the quasi-reversible behaviour corresponding to a one-electron redox reaction between Fe(II1) and Fe(I1). The electrochemical rate constant [k(Electrode)] for the redox reaction was estimated by determining the separation between the cathodic and anodic peak potentials at different scan rates [5]. Approximate [k(Electrode)] values, which were obtained under the assumption that the transfer coefficient is equal to 0.5 and that the diffusion coefficient of the reacting species is 1 X lop5 cm* s-l, are listed in the fourth column of Table 2. It was not possible to measure the electrochemical rate constant of thionine under the same conditions because of its strong adsorption on the pyrolytic graphite electrode surface. The electrode reaction rate constant of Fe(III)GEDTA was too small to be measured by this method, and this is considered to be a reason why Fe(III)GEDTA did not work as an effective mediator. Figure 3 shows the relationship between the coulombic outputs of the fuel cells, the reduction rate constants by E. coli [k( E. cofi)], and the electrochemical rate constants [( k(Electrode)] for the ferric chelate compounds. It is apparent from this figure that the coulombic output of the cell is higher when the mediator has high [ k( E. cofi)] and k(Electrode)]. The coulombic outputs of the fuel cells containing Fe(III)TTHA, Fe(III)DTPA and Fe(III)EDADPA as mediators are tabulated in Table 3. The higher coulombic
J 15 Fig. 3. Relationship between coulombic outputs of fuel cells, rate constants of reduction of ferric chelates by E. cofi [k(E. co/i)] and electrochemical rate constants of these compounds [ k(Electrode)]. (0) k(E. cob); (0) k(Electrode).
TABLE
3
Coulombic
outputs
and percent
Mediator day Fe(III)DTPA Fe(III)TTHA FE(III)EDADPA
yields recovered
from regenerative
fuel cells
Q CC)
Yield
1
2
3
4
5
2-5
(%)
24 22 26
22 23 19
17 28 14
17 22 16
18 19 15
74 92 64
80 99 70
outputs observed for the first 24 hours in the case of cells with Fe(III)DTPA and Fe(III)EDADPA as mediators are attributed to the contribution of endogenous metabolism. Subtraction of this contribution from the total coulombic output is necessary in order to calculate the true yields for a given amount of added glucose, but in view of the uncertainties in estimating the endogenous component, calculations for the recovery over four days were made by omitting the coulombic output for the first 24 hours. This procedure appears to be justified by the levelling off in the coulombic yields after the first day. The estimation of the recovery of electric charge from the fuel cell supplied with a given amount of glucose can be obtained by comparing with the theoretical yield for the complete oxidation of substrate, i.e. 23.2 coulombs per 10 pmol of added glucose. The recoveries of the cells over days 2-5 so obtained are listed in the last column of Table 3. These results are important in suggesting that in the presence of a suitable mediator, glucose can be converted to give electric energy by the microorganisms in the anode compartment, with a better than 70 % recovery. CONCLUSION
The results are conclusive in showing the effectiveness of a number of Fe(II1) chelate compounds as mediators in microbial fuel cells. Higher concentrations are required than in the case of the most effective dye-mediators such as thionine, but this presents no problems in view of the ready solubility of these and many other metal chelates. At the levels of concentration used in this work, the ferric chelates are probably less toxic to microorganisms than, e.g., thionine. Evidence to support this came from observations that E. coli can be grown in the presence of 10m3 mol dmp3 Fe(III)CyDTA without any change in number of cells yielded per unit volume of culture medium, whereas the same organisms failed to grow in the presence of low4 mol dmV3 thionine. Although the reduction rates of ferric chelate compounds by E. coli were much smaller than for thionine (Table 2), the comparable coulombic yields were obtained from fuel cells using ferric chelates or thionine as mediators. The effectiveness of these ferric chelates may be attributed mainly to the efficiency of the reoxidation of the reduced mediator at the anode. It is evident that mixed-mediator systems having coupled redox reactions for efficient scavenging and delivery of electrons have many advantages, and investigations of this sort will be reported in later papers.
143
ACKNOWLEDGEMENTS
We thank Miss T. Akahori for her help with preparation of E. coli, Drs. K. Nozaki and H. Kaneko for providing materials and their helpful discussions, and Dr. H.P. Bennett0 for discussions and help in preparing this manuscript. One of us enjoyed a fellowship from the Japan Society for Promotion of Science. REFERENCES 1 L.B. Wingard, Jr., C.H. Shaw and J.F. Castner, Enzyme Microb. Technol., 4 (1982) May 137. 2 H.P. Bennetto, J.L. Stirling, K. Tanaka and C.A. Vega, Sot. Gen. Microb. Quart., 8 (1980) 37; Biotecbnol. Bioeng., 25 (1983) 559. 3 H.P. Bennetto, M.E. Dew, J.L. Stirling and K. Tanaka, Chem. Ind., (1981) 776. 4 H.P. Bennetto, G.M. Delaney, J.R. Mason, S.D. Roller, J.L. Stirling and C.F. Thurston, J. Chem. Tech. Biotech., in press. 5 A.J. Bard and L.R. Faulkner, Electrochemical Methods, Wiley, New York, 1980, Ch. 6.5.2.
591 -MEDIATING EFFECTS OF FERRIC CHELATE MICROBIAL FUEL CELLS
KAZUKO
TANAKA,
Inorganic Chemistty
CARMEN Laboratory,
A. VEGA
l
135
COMPOUNDS
IN
and REITA TAMAMUSHI
The Institute of Physical and Chemical Research, Wako-shi,
Saitama 351
(Japan) (Manuscript
received June 23rd 1983)
SUMMARY The performance of b&fuel cells containing Escherichia coli, glucose and a series of ferric chelate reagents was studied. The measured coulombic outputs indicate that most of the compounds work effectively as electron-transfer mediators in the fuel cells. These outputs were compared with measured rate constants for the reduction of ferric chelates by E. co/i and the electrochemical reaction of these compounds at a carbon electrode. The results suggest that a good mediator for microbial fuel cells is one which shows fast reduction by E. co/i. together with a fast electrode reaction. In regenerative fuel cells which were run for five days, coulombic yields over 70 % were obtained on the basis of complete oxidation of added glucose.
INTRODUCTION
In a microbial fuel cell, electrical energy is obtained from oxidizable substrate through the catalytic ,action of microorganisms [l]. The functioning of such direct bio-fuel cells requires the presence of a mediator, which facilitates the transfer of electrons from the microorganisms to the anode [2]. Previous studies have shown that many redox dyes, such as thionine and new methylene blue, are effective mediators, as indicated from spectrophotometric measurements of their reduction rates by microorganisms [3,4], and from preliminary estimates of the coulombic outputs of fuel eells [2]. The development of effective mediators or mediator systems is clearly important for improving the efficiency of this type of electrochemical cell, and the requirements for a suitable mediator may be detailed as follows: (a) The mediator should not be toxic to microorganisms. This requirement is not fulfilled by many of the redox dyes employed so far, especially at higher concentrations. (b) The reagent should be able to penetrate the cell wall of the microorganism to l
On leave (1982) from University of Puerto Rico, Mayaguez, Puerto Rico.
0302-4598/83/$03.00
0 1983 Elsevier Sequoia S.A.
136
react with the source of electrons. Although details of this process remain to be clarified, some importance is attached to the lipophilicity and structure of the mediator [3]. (c) It should be electrochemically active at the electrode surface. (d) The formal redox potential should be near to that of the redox couple providing the reducing action within microorganism, which is likely in many cases [l] to be NAD+/NADH. (e) For practical application over long periods of time, the reagent should be chemically stable in solution. This is often not the case for organic dyes, especially when they are in the reduced state, at elevated temperature, or continuously exposed to light. (f) The mediator must be reasonably soluble in buffer solutions, which are usually near pH 7. The use of metallic chelate compounds as mediators has not yet been explored, but many of them have the potential to satisfy these requirements for a suitable mediator. By using different chelating agents together with a given metal, it is possible to vary the redox potential, solubility, lipophilicity, stability, etc. The purpose of the present study was to examine the mediating effects of a series of ferric chelate compounds in fuel cells containing Escherichia coli. The ferric complexes of ethylenediaminetetraacetic acid (EDTA) and five of its variants were chosen, together with nitrilotriacetic acid. The coulombic outputs from the fuel cells were measured as described in previous papers [2]. Rates of reduction of these ferric chelates by E. coli together with electrode reaction rates of the complexes were determined by electrochemical techniques. These rates were compared with the coulombic ouputs from the fuel cells measured under comparable conditions. Fe(III)TTHA, Fe(III}DTPA and Fe(III)EDADPA complexes were further investigated in longer term experiments during which the fuel cells were operated regeneratively over a five day period. Coulombic yields were compared with the theoretical yield calculated on the basis of complete oxidation of glucose to CO, and water. EXPERIMENTAL
Materials Escherichia coli KI2 was grown aerobically in nutrient broth (Difco Laboratories, U.S.A.) for 24 hours. It was harvested by centrifugation at 4 “C and washed three times with 0.1 mol dme3 phosphate buffer solutions of pH 7.0. In each fuel cell experiment the anode compartment contained 6 X 10” cells (approx. 0.05 g, dry weight). All chemicals were used without further purification. The chelating reagents (Dojin Kagaku) were complexed by mixing equivalent amounts with ferric ammonium sulphate in aqueous solution. Names and structures of the chelating compounds are given in Table 1. For comparison some experiments were performed using thionine (Eastman Kodak) as a mediator.
137
TABLE
1
Chelating
reagents
used as mediators
Name
Structure
(1) EDTA
HooCCH~
\
N -
/
HOOCCH~
/
CH2-CH2--N
\
Ethylenediaminetetraacetic
CH2COOH
CH2COOH
acid
,CH2COOH
(2) CyDTA
N\ a
CH&OOH CH$OOH
N”
‘CHzCOOH
1,2-Cyclohexanediamine
tetraacetic
acid
HOOCCHZ
(3) DTPA
/
\ /
HOOCCHz
\ CH$OOH
Diethylenetriamine (4) TTHA
pentaacetic
,CH2COOH \ /
N’--CHp2-~-c~,c~,-
HOOCCH2CH2
\
Ethyknediamine HOOCCH,
HOOCCH2
\
CH2CCOH
acid
/ \
diaceticacid
CH2 CKIH
dipropionic
acid
NCH2CH2-0-CH2CH2--0-CH2CH2-N
/ \
/
N(CH,COOH), Nitrilotriacetic
\
CHzCH$OOH NCH2CH2N’
Glycoletherdiaminetetraacetic (7) NTA
CH2COOH
hexaacetic
/
HOOCCH2
(6) GEDTA
N-CH~CH~-N”
HOOCH&
Triethylenetetramine (5) EDADPA
CHzCOOH
acid
HooCCH* HOOCCHz
CHzCOOH
N’-CH2-CH2-N-CH2-CH2--N’
CH$OOH
CH,COOH
acid
acid
Microbial fuel cells The composition of the microbial fuel cells is represented below: Carbon cloth (cathode) (10 cm2)
K3[Fe(CN)a] (0.1 mol dmm3) in phosphate buffer, pH 7 (15 cm3)
/ ; ) ; 1
E. coli (6 X 10” cells); glucose (lO.pmoI); mediator (1 x low2 or 1 x low3 mol dm-3) in phosphate buffer, pH 7 (15 cm3)
Carbon cloth (anode) (10 cm2 >
138
The cells were constructed of polyacrylic material and were similar to those used previously [2]. Carbon cloth (Nihon Carbon) electrodes were connected with Pt-wire. The anode and cathode compartments were separated by a cation-permeable ion-exchange membrane (Asahi Glass). Nitrogen was bubbled through both compartments to maintain anaerobic conditions. All the measurements were carried out at 30 “C using a digital voltmeter and a chart recorder (Riken Denshi SP-6 36 Speedex), and the procedure for the measurements of the cell performance was as follows. After the cells had been assembled and glucose added to the anode compartments, they were left on open circuit for 1-2 hours, after which time the voltage was around -0.5 V. The cells were allowed to discharge through a known resistance (usually 500-600 52) and the cell voltages were recorded over a 24 hour period, which was sufficient to extract most of the electric charge available from the oxidation of glucose. In regenerative-cell experiments the load was removed after 24 hours and a further 10 pmol of glucose was added to the anode compartment. The cells were allowed to recover for a period of one hour, and the discharge procedure was then repeated. This regeneration was carried out for five consecutive experiments.
Polarography
and linear sweep voltammetry
Reduction rates of ferric chelate compounds by E. coli in phosphate buffer solutions at 30 “C were measured polarographically using a potentiostat/galvanostat system (Princeton Applied Research Model 173) with a chart recorder (Riken Denshi). In order to follow the changes in concentration of ferric or ferrous chelates by measuring the current at the dropping mercury electrode (d.m.e.) in the limiting current region, applied electrode potentials of - 0.4 V and + 0.05 V versus s.c.e. were switched, in turn, at 50 second intervals with an external trigger mode. All measurements were carried out in deaerated phosphate buffer solutions containing about 1Or2 cells of E. coli and 1 X 10m3 mol dmW3 ferric chelate. Linear sweep voltammetric measurements for the determination of the electrode reaction rate constants were carried out at 25 “C in a three-electrode cell using a conventional reversal technique. The apparatus consisted of a potentiostat (Fuso Seisakusho Model 361), a potential scanning unit (Fuso Seisakusho Model 321-Sl), a transient converter (Riken Denshi Co Ltd. Model TCA-lOOO), and an X-Y recorder. The transient converter was used to record voltammograms at scanning rates higher than 0.2 V s-‘. The working electrode was a pyrolytic graphite rod (diameter 5 mm) whose surface was renewed for each experimental run, and its electrode potential was varied from +0.5 V to -0.5 V against a AgJAgCIlsat. KC1 reference electrode with a scan rate ranging between 0.01 and 10 V s-l. All measurements were made in deaerated phosphate buffer solutions containing lop2 mol dm-’ ferric chelate compound.
139 RESULTS AND DISCUSSIONS
The typical voltage W.SW time curves for microbial fuel cells using Fe(III)EDTA as mediator are shown in Fig. 1, together with an experiment using thionine. Thionine is an effective mediator for E. coli fuel cells [2,4] and was used as a reference. As the figure shows, dilute Fe(III)EDTA (curve B) has a limited mediating effect compared with the cell without a mediator (curve D), but a more concentrated solution of Fe(III)EDTA gives a good mediating effect (curve A) comparable with that of thionine (curve C). Coulombic outputs (Q) from the fuel cells were obtained from integration of the current uersta time plots. The coulombic outputs for the first 24 hours of E. coli fuel cells for several ferric chelate mediators are presented in Table 2. The figures give evidence that the chelates are good mediators in comparison with thionine, particularly at the higher concentration of 0.01 mol dme3. The Fe(III)GEDTA complex is an exception; the coulombic output of the cell using this mediator was similar to that of the blank cell without a mediator. Fe(II1) chelates and their reduced forms (Fe(II)chelates) gave well-defined polarographic waves at the d.m.e. in phosphate buffer solutions of pH 7.0, and their concentrations were estimated from the wave heights in the limiting current plateau region. The changes in concentrations of Fe(II1) chelates and Fe(I1) chelates were determined from the changes in wave heights at - 0.4 V and + 0.05 V versus s.c.e., respectively. The plots of log(h, - h,) uersur time, where h, is the wave height at time t, and h, is the wave height when the reaction is completed, gave straight lines, 0 O-
4
I
8
12
16
20
24
1 I ' ;.' .....'.'.........--.._...~.._.~...__._.__...._........_....... D c__-_---- _____------7= I I
' t(hr)
Fig. 1. Cell voltage versus time curves for microbial fuel cells. Cells were loaded (510 51) after two hours. (A) 1 x lo-* mol drne3 Fe(III)EDTA; (B) 1 X lo-’ mol dm-’ Fe(III)EDTA; (C) 1 X 10m3 mol dmm3 tbionine; (D) no mediator.
140
TABLE
2
Coulombic outputs of E. coli fuel cells, rate constants electrode reaction rate constants of these compounds
of ferric chelates
by E. co/i, and
QW
Mediator
Fe(III)EDTA Fe(III)CyDTA Fe(III)DTPA Fe(III)TTHA Fe(III)EDADPA Fe(III)GEDTA Fe(III)NTA Thionine
1x10-2 mol dm-’
1x10-s mol drne3
31 25 24 22 26 8
13 8 15 11 11 -
20
13
-
31
104k( E. co/i) (s-l) 1.1 0.54 0.26 0.17 100”
lO*k(EIectrode) (cm s-‘) 1.5 1.8 1.2 0.22 1.1 slow 0.07 -
5
No mediator a Obtained
of reduction
spectrophotometrically
under comparable
conditions.
as shown in Fig. 2. The first order reduction rate constants of ferric chelate compounds by E.coli [k(E.con)] were estimated from the slopes of these lines, and are listed in the third column of Table 2. The figures appear to indicate that the smaller the size of the mediator, the faster is its reduction rate, which suggests that
0
10
20
3Q
40
Fig. 2. First order plots for the reduction
50
60
of ferric chelate
compounds
by E. co/i. (h expressed
in cm.)
141
the smaller mediator molecules penetrate the cell wall to accept electrons at the cell membrane more easily. All the ferric chelate compounds studied gave well-defined cyclic voltammograms at a pyrolytic graphite electrode in 0.1 mol dmV3 phosphate buffer solutions; the voltammogram exhibited the quasi-reversible behaviour corresponding to a one-electron redox reaction between Fe(II1) and Fe(I1). The electrochemical rate constant [k(Electrode)] for the redox reaction was estimated by determining the separation between the cathodic and anodic peak potentials at different scan rates [5]. Approximate [k(Electrode)] values, which were obtained under the assumption that the transfer coefficient is equal to 0.5 and that the diffusion coefficient of the reacting species is 1 X lop5 cm* s-l, are listed in the fourth column of Table 2. It was not possible to measure the electrochemical rate constant of thionine under the same conditions because of its strong adsorption on the pyrolytic graphite electrode surface. The electrode reaction rate constant of Fe(III)GEDTA was too small to be measured by this method, and this is considered to be a reason why Fe(III)GEDTA did not work as an effective mediator. Figure 3 shows the relationship between the coulombic outputs of the fuel cells, the reduction rate constants by E. coli [k( E. cofi)], and the electrochemical rate constants [( k(Electrode)] for the ferric chelate compounds. It is apparent from this figure that the coulombic output of the cell is higher when the mediator has high [ k( E. cofi)] and k(Electrode)]. The coulombic outputs of the fuel cells containing Fe(III)TTHA, Fe(III)DTPA and Fe(III)EDADPA as mediators are tabulated in Table 3. The higher coulombic
J 15 Fig. 3. Relationship between coulombic outputs of fuel cells, rate constants of reduction of ferric chelates by E. cofi [k(E. co/i)] and electrochemical rate constants of these compounds [ k(Electrode)]. (0) k(E. cob); (0) k(Electrode).
TABLE
3
Coulombic
outputs
and percent
Mediator day Fe(III)DTPA Fe(III)TTHA FE(III)EDADPA
yields recovered
from regenerative
fuel cells
Q CC)
Yield
1
2
3
4
5
2-5
(%)
24 22 26
22 23 19
17 28 14
17 22 16
18 19 15
74 92 64
80 99 70
outputs observed for the first 24 hours in the case of cells with Fe(III)DTPA and Fe(III)EDADPA as mediators are attributed to the contribution of endogenous metabolism. Subtraction of this contribution from the total coulombic output is necessary in order to calculate the true yields for a given amount of added glucose, but in view of the uncertainties in estimating the endogenous component, calculations for the recovery over four days were made by omitting the coulombic output for the first 24 hours. This procedure appears to be justified by the levelling off in the coulombic yields after the first day. The estimation of the recovery of electric charge from the fuel cell supplied with a given amount of glucose can be obtained by comparing with the theoretical yield for the complete oxidation of substrate, i.e. 23.2 coulombs per 10 pmol of added glucose. The recoveries of the cells over days 2-5 so obtained are listed in the last column of Table 3. These results are important in suggesting that in the presence of a suitable mediator, glucose can be converted to give electric energy by the microorganisms in the anode compartment, with a better than 70 % recovery. CONCLUSION
The results are conclusive in showing the effectiveness of a number of Fe(II1) chelate compounds as mediators in microbial fuel cells. Higher concentrations are required than in the case of the most effective dye-mediators such as thionine, but this presents no problems in view of the ready solubility of these and many other metal chelates. At the levels of concentration used in this work, the ferric chelates are probably less toxic to microorganisms than, e.g., thionine. Evidence to support this came from observations that E. coli can be grown in the presence of 10m3 mol dmp3 Fe(III)CyDTA without any change in number of cells yielded per unit volume of culture medium, whereas the same organisms failed to grow in the presence of low4 mol dmV3 thionine. Although the reduction rates of ferric chelate compounds by E. coli were much smaller than for thionine (Table 2), the comparable coulombic yields were obtained from fuel cells using ferric chelates or thionine as mediators. The effectiveness of these ferric chelates may be attributed mainly to the efficiency of the reoxidation of the reduced mediator at the anode. It is evident that mixed-mediator systems having coupled redox reactions for efficient scavenging and delivery of electrons have many advantages, and investigations of this sort will be reported in later papers.
143
ACKNOWLEDGEMENTS
We thank Miss T. Akahori for her help with preparation of E. coli, Drs. K. Nozaki and H. Kaneko for providing materials and their helpful discussions, and Dr. H.P. Bennett0 for discussions and help in preparing this manuscript. One of us enjoyed a fellowship from the Japan Society for Promotion of Science. REFERENCES 1 L.B. Wingard, Jr., C.H. Shaw and J.F. Castner, Enzyme Microb. Technol., 4 (1982) May 137. 2 H.P. Bennetto, J.L. Stirling, K. Tanaka and C.A. Vega, Sot. Gen. Microb. Quart., 8 (1980) 37; Biotecbnol. Bioeng., 25 (1983) 559. 3 H.P. Bennetto, M.E. Dew, J.L. Stirling and K. Tanaka, Chem. Ind., (1981) 776. 4 H.P. Bennetto, G.M. Delaney, J.R. Mason, S.D. Roller, J.L. Stirling and C.F. Thurston, J. Chem. Tech. Biotech., in press. 5 A.J. Bard and L.R. Faulkner, Electrochemical Methods, Wiley, New York, 1980, Ch. 6.5.2.