SUPPORTING INFORMATION
Comparison of non-precious metal cathode materials for methane
production by electromethanogenesis
Michael Siegert1, Matthew D. Yates1, Douglas F. Call1,2, Xiuping Zhu1, Alfred Spormann3,
Bruce E. Logan1*
1
Department of Civil and Environmental Engineering, The Pennsylvania State University,
University Park, PA, USA
2
Syracuse University, Department of Civil and Environmental Engineering, Syracuse, NY, USA
3
Department of Civil and Environmental Engineering and Department of Chemical Engineering,
Stanford University, Stanford, CA, USA
*Corresponding author: Bruce E. Logan, 231Q Sackett Bldg, Department of Civil and
Environmental Engineering, The Pennsylvania State University, University Park, PA 16802,
Email: [email protected]; Phone: +1 814-863-7908, Fax: +1 814-863-7304
S1
Medium. The vitamins solution contained (100× stock concentration in mg L–1): pyridoxine
HCl, 10; thiamin HCl, 5; riboflavin, 5; nicotinic acid, 5; calcium pantothenate, 5; vitamin B12, 5;
p-aminobenzoic acid, 5; lipoic (thioctic) acid, 5; biotin, 2; folic acid, 2.1 The trace element
solution contained (100× stock concentration in g L–1): nitrilotriacetic acid, 1.5; MgSO4·7H2O, 3;
NaCl, 1; MnSO4·H2O, 0.5; NiCl2·6H2O, 0.2; FeSO4·7H2O, 0.1; CoCl2, 0.1; CaCl2·2H2O, 0.1;
ZnSO4, 0.1; CuSO4·5H2O, 0.01; AlK(SO4)2, 0.01; H3BO3, 0.01; Na2MoO4·2H2O, 0.01.1 All
chemicals were purchased from VWR (Radnor, PA, USA) or Sigma-Aldrich (St. Louis, MO,
USA) in the highest available purity.
Additional Details of Reactor Materials Preparation. Nafion® membranes were pretreated by boiling them successively for 1 h each in a 4% H2O2 in de-ionized water, 1 M H2SO4,
and again in de-ionized water. Butyl rubber stoppers were used to prevent loss of gas from the
system, and allow samples to be extracted using a gas-tight syringe and needle. The stoppers
(6.4 mm thick and 43 mm diameter) were cut from large butyl rubber sheets (McMaster-Carr,
Cleveland, OH, USA) Before being used, stoppers were cleaned by boiling them for 1 h in soap
(Alconox® powder VWR, Radnor, PA, USA), rinsing with de-ionized water, sitting overnight in
0.6 M HCl, autoclaving (121˚C for 20 minutes), flushing again with de-ionized water, and finally
by cleaning with 96% ethanol and paper tissue (KimWipe, VWR, Radnor, PA, USA).
Linear sweep voltammetry (LSV) experiments were conducted once under abiotic
conditions before the first abiotic batch was started and at the beginning and the end of every
consecutive cycle. The consecutive scans were carried out as cyclic voltammetry (CV) with two
full cycles between –700 and 0 mV vs. SHE and the last part of the second CV cycle between 0
and –700 mV is shown as LSV. The scan rate was 1 mV s–1.
S2
Gas Chromatography (GC) Analyses. Weekly methane and hydrogen measurements were
conducted, with samples analyzed using a gas chromatograph equipped with a 6-foot long
molsieve-column (SRI 310C, SRI Instruments, Torrance, CA, USA) at an oven temperature of
80˚C.2 In abiotic tests, hydrogen gas concentrations were measured every two days during a 10 d
period.
High Pressure Liquid Chromatography (HPLC) Analyses. Concentrations of formic,
acetic, propionic and butyric acids were determined only during the final cycle (split potentials)
using an HPLC (CTO-20A UFLC; Shimadzu, Columbia, MD, USA) equipped with an
autosampler (model SIL-20A HT, Shimadzu, Columbia, MD, USA).2 The mobile phase was
50 mM KH2PO4 adjusted to a pH of 2.3 using H3PO4. The column (250 × 4.6 mm “Allure
Organic Acids”, 5 μm particle size; Restek, Bellefonte, PA, USA) oven temperature was fixed at
40°C, and each run lasted 50 min. Samples and standards were filtered using syringe filters
(polytetrafluoroethylene, 13 mm Acrodisc® CR, 0.2 μm pore size, PALL Life Sciences, NY,
USA) prior to analyses.
Environmental scanning electron microscope ESEM. Electrodes were removed and fixed
overnight at 4°C in approximately 5 mL of a phosphate buffer solution (pH 7.2) containing 2.5%
glutaraldehyde and 1.5% paraformaldehyde. Subsequently, electrodes were sequentially dried in
ethanol-water solution of 70% and 90% ethanol and stored at 4°C in 100% ethanol in the dark.
Prior to electron microscopy, electrodes were critical point CO2-dried until all traces of water
were removed. Electrodes were stored in a desiccator until analyzed with an environmental
scanning electron microscope (E-SEM; FEI Quanta 200 instrument, FEI company, Hillsboro,
OR, USA) equipped with an electron dispersive X-ray spectroscopy (EDX).
S3
References
(1)
Wolin, E. A.; Wolfe, R. S.; Wolin, M. J., Viologen dye inhibition of methane formation
by Methanobacillus omelianskii. J. Bacteriol. 1964, 87, (5), 993-998.
(2)
Pisciotta, J. M.; Zaybak, Z.; Call, D. F.; Nam, J.-Y.; Logan, B. E., Enrichment of
microbial electrolysis cell biocathodes from sediment microbial fuel cell bioanodes. Appl.
Environ. Microbiol. 2012, 78, (15), 5212-5219.
(3)
Thauer, R. K.; Kaster, A.-K.; Seedorf, H.; Buckel, W.; Hedderich, R., Methanogenic
archaea: ecologically relevant differences in energy conservation. Nat. Rev. Microbiol. 2008, 6,
(8), 579-591.
(4)
Lide, D. R., CRC handbook of chemistry and physics. 89 ed.; CRC Press, Inc.: Boca
Raton, 2008.
S4
Table S 1: Batch cycle times in days. Batch cycle 6 was the potential split cycle. Asterisks (*)
denote actual batch cycles 7 because of oxygen intrusion into 1 reactor with an iron sulfide
cathode and 2 reactors with graphite electrodes. Plus (+) indicates graphite reactors that ran over
116 days during cycle 2. All results displayed in the article were shifted accordingly, i.e. failed
cycles (cycle 2 of iron sulfide and graphite and cycle 6 of steel) are never shown because no
methane was produced. The reference electrodes failed during cycle 6 of one steel reactor and
the entire cycle was repeated.
Material
Platinum
Steel
Nickel
Ferrihydrite
Magnetite
Iron sulfide
MoS2
C-brush
C-black
Graphite
inoculation
24
24
23
24
24
24
24
24
24
24
Batch Cycle Times in days
batch 2
batch 3
batch 4
batch 5
16
32
22
27
57
34
32
28
60
34
32
40
71
27
40
34
57
34
32
28
49
43
32
40
63
46
63
48+
34
22
27
33
40
41
32
34
S5
34
33
34
28
batch6
25
21*
27
34
33
34*
34
20
41
27*
Table S 2: Potential intermediate reactions which can cause methanogenic cathode corrosion and their respective energy requirements
ΔG° (standard conditions) and ΔGMMC° (approximated standard conditions in MMC reactors) a.
reaction
reaction
2 C0 + 3 H2O
1
0
2
C + 3 H2O
0
3
C + 2 H2
2+3
5
10 + 11

+
-50
-
+
34
8 C + 4 H2 + 9 H2O
17
84
CH4 + HCO3 + H
HCOOH + H2
4 C6H6 + 27 H2O
42
-

6 C + 3 H2
113
-
+
-62
-
+
51
CH4 + 3 HCO3 + 3 H

CH4 + 3 HCO3 + 3 H + 4 H2

C6H6


-47
-22
-34
-56
-25
CH4 + HCO3 + H
0
17
+


∆GMMC°
[kJ mol 1]a
-
0
4 C + 9 H2O
0
CH4
0
0
11

H3C-COOH
4 HCOOH + H2O
10
HCO3 + H + 2 H2

C + 2 H2O
7+8

CH4 + HCO3 + H
2 C + 3 H2O
8
+

H3C-COOH + H2O
7
-
0
2 C + 2 H2O
4+5
-
CH4 + HCO3 + H+
0
2 C + 3 H2O
4

∆G°
[kJ mol 1]
125
-
15 CH4 + 9 HCO3 + 9 H
-
5 CH4 + 3 HCO3 + 3 H
+
-123
+
0
normalized to
per mol carbon
per mol hydrogen
per mol hydrogen
per mol hydrogen (2)
-132 per mol acetate
-99 per mol acetate
-231 per mol acetate (1)
16
per mole formate
-76 per mole formate
-43 per mol hydrogen (4)
124
per mol benzene
-412 per mol benzene
-96 per mol hydrogen (4)
Hydrogen (or e- + H+) is needed to make methanogenesis from elemental carbon (C0, graphite) thermodynamically feasible (reaction
3). However, the near infinite abundance of water (1/[H+]under MMC reactor conditions makes all reactions involving water and
protons thermodynamically feasible.
a
Reaction condition ΔGMMC°: [methane] = 98 Pa (GC detection limit), [hydrogen] = 10 Pa (limit for cytochrome methanogenesis),3
[formate] = 1 mM, [acetate] = 1 mM, [bicarbonate] = 30 mM, [H+] = 0.1 μM at pH 7, [H2O] = 1/[H+] all other concentrations 1 M
S6
Table S 3: Correlation factors R2 of methane production rates over poised potentials. The poised
potentials were -550 mV and -650 mV during cycle 6 and -600 mV during cycle 5.
Material
Pt
Steel
Ni
ferrihydrite
magnetite
FeS
MoS2
C-brush
C-black
graphite
R2
-0.93
-0.96
-0.75
-0.83
-0.94
-0.19
-0.86
-0.95
-0.59
-0.83
S7
Table S 4: List of materials used in the experiment and their respective energies of formation
ΔGf° taken from reference 4. Coulombic recoveries of cycle 2 were selected to show that ΔGf° of
0 tend to be closer to 100% than negative or unspecified ΔGf°.
0
unspecified
0
Coulombic
recovery
(cycle 2)
93
115
105
rate in nmol ml 1 d 1
(95% CI)
251
26
25
Fe3O4
indeterminate
FeS
-1015.4
unspecified
-100.4
807
721
265
30
21
41
MoS2
indeterminate
indeterminate
C
-225.9
unspecified
unspecified
0
395
587
1226
71
30
100
34
34
Material
Platinum
Steel
Nickel
Chemical
formula
Pt
alloy
Ni
∆Gf° in
kJ mol 1
Magnetite
Ferrihydrite
Iron sulfide
Molybedenum disulfide
Carbon brush
Carbon black
Graphite
CI = confidence interval
S8
-
-
CH4 produced in µmol CH4 produced in µmol
Inoculation cycle
200
Cycle 2
100
100
50
0
0
100
0
25
Cycle 4
Cycle 3
Pt
steel
Ni
ferrihydrite
magnetite
0
25
Cycle 5
FeS
MoS2
C-brush
C-black
graphite
0
25
50
Cycle 6
50
0
0
0
25
0
25
50
25
Cycle time in days Cycle time in days Cycle time in days
Figure S 1: Methanogenesis in open circuit controls. Note that cycle 2 lasted longer than depicted
but no methanogenesis was observed in any of the reactors until the cycle ended.
S9
Steel
Nickel
0
I in mA
-0.5
-1.0
-1.5
-2.0
nabiotic= 3
-2.5
nabiotic= 3
0
I in mA
-0.5
-1.0
-1.5
Platinum
nabiotic= 3
nabiotic= 3
Carbon brush
-2.0
-2.5
0
I in mA
-0.5
-1.0
-1.5
-2.0
nabiotic= 5
Abiotic (+/- errorn)
Inoculation cycle
nabiotic= 3
Last cycle
Graphite
Carbon black
-2.5
0
I in mA
-0.5
-1.0
-1.5
-2.0
nabiotic= 4
Ferrihydrite
nabiotic= 3
Magnetite
-2.5
0
I in mA
-0.5
-1.0
-1.5
-2.0
nabiotic= 3
nabiotic= 4
MoS2
Iron sulfide
-2.5
-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1
E vs. SHE in V
E vs. SHE in V
Figure S 2: Linear sweep voltammograms of the cathodes at different stages of the experiment:
before the abiotic cycle 0 (bold continuous lines with error margins as fine lines), right after
inoculation (dashed lines) and right before the final cycle (fine dashed lines). Abiotic
volatmmograms show the average value of n reactors whereas the bold is the average and the
fine lines are the error margins (standard deviation of n reactors).
S10
A
B
C
10 μm
Fe
A-EDX
C
O
Cr
Fe
NiNa
Zn S
SiP
1
2
5 μm
10 μm
Mo&S
B-EDX
C F Zn
Ca
Ni
O
Mo
Cr
4
5
S
P
Cr
3
C-EDX
6
FeNi
7
C
NiZn Zn
8
9 keV
F
0.5
1.0
1.5
Mo
Mo
S
2.0
2.5
Al
Mo
keV
Ca
1
2
3
4
5
6
Zn
Ni Cu CuZn
7
8
9 keV
Figure S 3: ESEM micrographs taken of particles found on different carbon black electrodes and
their respective EDX scans: A, steel particle, B, MoS2 particle; note that the molybdenum and
the sulfur EDX bands overlap, C, carbon black-only with a presumably precipitated particle. All
particles were overgrown by microbes indicating that they were present before colonization.
S11
10000
1000
100
graphite
C-black
C-brush
MoS2
FeS
magnetite
ferrihydrite
Ni
1
steel
10
Pt
Coulombic recovery in %
Cycles 2-5
Cycles 3-5
Figure S 4: Coulombic recoveries for duplicate reactors over several cycles illustrating the
decrease of the errors. Mean values are shown for cycles 2-5 in dark blue and for cycles 3-5 in
light green. Errors are standard deviations of the mean.
S12
1000
100
graphite
C-black
C-brush
MoS2
FeS
magnetite
ferrihydrite
Ni
1
steel
10
Pt
Gas produced nmol cm–3 d–1
¼H2
CH4
Figure S 5: Gas production rates for hydrogen (mean) and methane (confidence intervals). The
difference to the figure in the main text is that here, 95% confidence intervals over all cycles and
duplicate reactors were calculated for methanogenesis. Shown are the mean values with their
corresponding standard deviations within these confidence intervals.
S13