Supplementary Information
Pre-enrichment procedure for enhanced start-up of anaerobic facultatively
autotrophic biocathodes in bioelectrochemical systems
Zehra Zaybak1, John M. Pisciotta1, 2 , Justin C. Tokash1 , and Bruce E. Logan1*
1
Department of Civil and Environmental Engineering, The Pennsylvania State University, 212
Sackett Building, University Park, PA 16802, USA
2
Department of Biology, West Chester University of Pennsylvania, West Chester, PA
19383,USA
*Corresponding author. Tel 1-814-963-7908, Fax 1-814-863-7304, E-mail [email protected]
1
a
b
Pre-enrichment (with
e- donor/ substrate
in batch culture)
Pre-enrichment with
glucose in batch
culture (2 weeks)
Inoculation of BES
cathode chamber +
acclimation (with edonor/substrate)
Inoculation of BES
cathode chamber +
acclimation with
glucose (2 weeks)
Cathode is sole
electron source
(no external e- donor)
Cathode is sole
electron source and
CO2 sole carbon
source
Fig. S1 Steps for (a) general pre-enrichment procedure for development of anaerobic
biocathodes and (b) pre-enrichment for development of facultatively autotrophic biocathodes.
2
Fig. S2 BES biocathode start-up experiment without pre-enrichment step. Current density
produced by two replicate reactors (B1 and B2) that were directly inoculated with bog sediment
(3g) and operated at a cathode set potential of −0.4 V (vs. SHE).
3
Fig. S3 HPLC chromatograms obtained from analysis of cathode chamber filtrates from
(a, b) biotic replicates and (c) control reactors to examine excreted metabolites. Red
arrows show to peak of acetic acid; blue arrows show to peak of propionic acid; black
arrows show to unknown peaks.
4
Fig. S4 GC chromatograms obtained from analysis of cathode chamber filtrates from (a,
b) duplicate reactors to examine excreted volatile fatty acids (VFAs). Green arrows show
to peak of ethanol; pink arrows show to peak of butanol; purple arrows show peak of
butyrate.
5
Tab. S1 Number of clones belonging to major bacterial groups in duplicate reactors (n
=93 and n =87).
# of clones
# of clones
(reactor 1)
(reactor 2)
Trichococcus palustris
44
54
Oscillibacter sp.
22
9
Anaerofilum sp.
2
7
Clostridium sp.
20
4
Other
7
13
Total (n)
93
87
Genus/ Species
6
1. Mass Balance Analysis of Organic and Inorganic Carbon in Biotic BESs
1.1 Analysis of total carbon in glucose added to cathode chamber of BESs
Glucose added to serum bottles for pre-enrichment (Step 1): 28 mM
Glucose added to BESs during start-up operation (Step 2):
42 mM
Conversion of these concentrations into mass in units of moles gives:
Glucose (Step 1): 28 mM * 0.05 L = 1.4 mmol
Glucose (Step 2):
42 mM * 0.26 L = 10.92 mmol
Total Glucose:
Sum = 12.32 mmol
(Volume of inoculum for BESs: 0.05 L; Final working volume of BESs cathode chamber:
0.26 L)
The mole fraction of carbon in total glucose is:
Total Organic Carbon (Glucose): 4.93 mmol
1.2 Analysis of total carbon in supplied inorganic carbon to cathode chamber of BESs
Sources of inorganic carbon at time point zero of BES operation are from degassing of
medium and reactor headspace with 20% CO2 and from sodium bicarbonate (NaHCO3)
added to the medium. We assume equilibrium at pH 7.3 (final pH) for following
calculations of inorganic carbon.
We use corrected Henry’s constant kH,pc(30 °C) as described before [1, 2],
k
,
30°C
33.59
(1)
Using Henry’s law [3], (cCO2(aq): aqueous molar concentration of CO2; pCO2: partial
pressure of CO2)
k
(2)
,
we calculate concentration of dissolved CO2 for pCO2 = 0.2 atm (20% CO2):
c
,
.
.
0.00595
(3)
We can now calculate moles of CO2(aq)
7
n
0.00595 ∙ 250 ∙ 10
1.488mmol
(4)
Given pH 7.3, we can calculate each species of the carbonic acid system in equilibrium.
Since about 99% of carbonic acid (H2CO3) is dissolved in water in form of CO2(aq) [4],
we expect according to above equation (3)
CO
H CO∗
5.95 mM (5)
Moles of carbonic acid species: nH2CO3* = 1.488 mmol
Calculation of bicarbonate concentration [4]:
K
,
∗
.
HCO
∙ .
.
(6)
= 59.5 mM
(7)
Moles of bicarbonate species: nHCO3- = 14.875 mmol
Calculation of carbonate species concentration [4]:
K
CO
(8)
,
.
∙
. .
= 0.0595 mM
(9)
Moles of carbonate species: nCO32- = 0.015 mmol
The mole fraction of carbon in each species is:
Carbonic acid species: 0.40 mmol
Bicarbonate species:
2.98 mmol
Carbonate species:
0.003 mmol
Total Inorganic Carbon from degassing:
3.38 mmol
In addition to carbon from degassing, the medium in BESs contained 8g/L NaHCO3.
Conversion into mass in units of moles gives NaHCO3: 23.81 mmol
The mole fraction of carbon is
NaHCO3: 3.33 mmol
Therefore, moles of total dissolved inorganic carbon is
Total Inorganic Carbon: 6.71 mmol
1.3 Analysis of total carbons in detected organic metabolites
Acetic acid:
1.90 ± 0.73 g/L
8
Propionic acid:
Butyric acid:
Ethanol:
Butanol:
2.09 ± 0.56 g/L
2.25 ± 0.20 g/L
16.04 ± 0.01 mg/L
26.82 ± 0.00 mg/L
Conversion of these concentrations into mass in units of moles gives:
Acetic acid:
8.23 ± 3.15 mmol
Propionic acid: 7.32 ± 1.96 mmol
Butyric acid:
6.62 ± 0.60 mmol
Ethanol:
0.09 ± 0.00 mmol
Butanol:
0.09 ± 0.00 mmol
The mole fraction of carbon in each substrate is:
Acetic acid:
3.29 ± 1.26 mmol
Propionic acid: 3.59 ± 0.96 mmol
Butyric acid:
3.64 ± 0.33 mmol
Ethanol:
0.05 ± 0.00 mmol
Butanol:
0.06 ± 0.00 mmol
The summation of all carbon fractions in detected organic metabolites gives us:
Total Organic Carbon (Metabolites): 10.63 ± 2.55 mmol
Conclusions:
Comparison of total organic carbon in metabolites and glucose shows that only 49.2 ±
11.8% of carbons in metabolites could have been provided by added glucose. Therefore,
more than 50% of the remaining carbon assimilated in form of detected organic
metabolites could have been provided by added inorganic carbon. Mass analysis of
inorganic carbon added to cathode chamber BESs, showed that there was sufficient
inorganic carbon available for biosynthesis of remaining detected organic metabolites.
The added amount of total inorganic carbon could have provided carbon for up to 67.0 ±
16.1% of detected organic metabolites if it was completely consumed.
2. Stoichiometric Analysis
Calculation of electrons consumed (Q: electric charge):
Q
1 mol e186 C
1 mol e= =1.93 mmol e-
96485 C/F
1F
96485 C/F
1F
Moles of electrons consumed:
1.93 ± 0.2 mmol
9
Cathodic half-reaction reaction for Acetic acid:
2 CO2(aq) + 8 H+ + 8 e- → CH3COOH + 2 H2O
1.93 ± 0.2 mmol e-
1 mol CH3COOH
8 mol e-
= 0.24 ± 0.03 mmol
Cathodic half-reaction reaction for Propionic acid:
3 CO2(aq) + 14 H+ + 14 e- → CH3CH2COOH + 4 H2O
1 mol CH3CH2COOH
14 mol e-
1.93 ± 0.2 mmol e-
= 0.14 ± 0.01 mmol
Cathodic half-reaction reaction for Butyric acid:
4 CO2(aq) + 20 H+ + 20 e- → CH3(CH2)2COOH + 6 H2O
1.93 ± 0.2 mmol e-
1 mol CH3(CH2)2COOH
20 mol e-
= 0.10 ± 0.01 mmol
Cathodic half-reaction reaction for Ethanol:
2 CO2(aq) + 12 H+ + 12 e- → CH3CH2OH + 3 H2O
1.93 ± 0.2 mmol e-
1 mol CH3CH2OH
12 mol e-
= 0.16 ± 0.02 mmol
Cathodic half-reaction reaction for Butanol:
4 CO2(aq) + 24 H+ + 24 e- → CH3(CH2)3OH + 7 H2O
1.93 ± 0.2 mmol e-
1 mol CH3(CH2)3OH
24 mol e-
= 0.08 ± 0.01 mmol
10
The mol relation of detected organic metabolites is
Acetic acid : Propionic acid : Butyric acid : Ethanol : Butanol
0.368 :
0.328
:
0.296
: 0.004 : 0.004
Calculation of moles metabolites produced electrotrophically,
Acetic acid:
88.32 ± 11.04 µmol
Propionic acid: 45.92 ± 3.28 µmol
Butyric acid:
29.60 ± 2.96 µmol
Ethanol:
0.64 ± 0.08 µmol
Butanol:
0.32 ± 0.04 µmol
Calculation of metabolites produced electrotrophically into mg/L gives,
Acetic acid:
20.34 ± 2.55 mg/L
Propionic acid: 13.08 ± 0.93 mg/L
Butyric acid:
10.03 ± 1.00 mg/L
Ethanol:
0.11 ± 0.01 mg/L
Butanol:
0.09 ± 0.01 mg/L
Conclusions:
Stoichiometric analysis of consumed electrons shows that 20.34 ± 2.55 mg/L acetic acid,
13.08 ± 0.93 mg/L propionic acid, 10.03 ± 1.00 mg/L butyric acid, 0.11 ± 0.01 mg/L
ethanol and 0.09 ± 0.01 mg/L butanol were theoretically produced by microbial
electrosynthesis.
References:
[1] Pisciotta, J.M., Zaybak, Z., Call, D.F., Nam, J.-Y., Logan, B.E., 2012. Enrichment of microbial
electrolysis cell (MEC) biocathodes from sediment microbial fuel cell (sMFC) bioanodes. Appl.
Environ. Microbiol. 78, 5212-5219.
[2] CRC Handbook of Chemistry and Physics, 76th Edition, D.R. Lide and H.P.R. Frederikse, ed(s),
Inc., Boca Raton, FL, 1995
[3] Physical Chemistry, P.W. Atkins, Oxford University Press, 2006
[4] Aquatic Chemistry, 3rd Edition, W. Stumm and J.J. Morgan, Wiley-Interscience, Inc. ,NY, 1996
11