S1
Supplementary Information
Simultaneous, high-precision measurements of
δ
2
H and
δ
13
C in nanomole
quantities of acetate using ESI-Quadrupole-Orbitrap MS
Elliott P. Mueller
1*
Alex L. Sessions
1
Peter E. Sauer
2
Gabriella M. Weiss
1,3
John M. Eiler
1
1
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA
91125
2
Department of Earth and Atmospheric Sciences, Indiana University, Bloomington, IN, USA 47405
3
Astrobiology Center for Isotopologue Research, Department of Geosciences, Pennsylvania State
University, State College, PA, USA 16802
*Corresponding Author: emueller@caltech.edu
Table of Contents:
Description of EA-IRMS measurements of acetate standards
Table S1: Acetate working standards measured by EA-IRMS
Figure S1: Effect of concentration on isotopic accuracy
Figure S2: Ionization yield of 50μM acetate in pure methanol versus in 1 mM solutions of salts
Table S2: Bacterial strains grown in this study
Table S3: Growth medium composition for cultures analyzed in this study.
Culture conditions of
Acetobacterium woodii, Sporomusa ovata,
and
Clostridium pasteurianum
S2
Supplementary Information
13
C and
2
H values of acetate measured by EA-IRMS:
Carbon isotope compositions of three acetate working standards were measured on a Thermo-
Fisher Flash combustion Elemental Analyzer (EA) coupled to a Thermo Scientific Delta-V
isotope-ratio mass spectrometer (IRMS) at Caltech. Carbon isotope ratios of combusted CO
2
were calibrated to the VPDB scale using a single CO
2
reference gas (-12.04‰) measured before
and after sample peaks. International reference materials valine (USGS74, 9.3‰), glycine
(USGS64, -40.81‰), sucrose (NIST8542, -10.47‰), and caffeine (IAEA600, -27.77‰) were
used to correct for further instrumental fractionations associated with combustion.
Hydrogen isotope compositions were measured on a Thermo thermal-conversion (TC) EA
coupled to a Thermo Finnigan Delta Plus XP IRMS at Indiana University, Bloomington.
Samples were weighed into silver capsules and dried in a glass vacuum desiccator for 6 hours.
The desiccator was flushed with dry nitrogen gas before being opened. Silver capsules were
quickly crushed and sealed in a Costech Zero-Blank autosampler, which was flushed with
helium. The TC/EA was set to a furnace temperature of 1100
C, a GC oven temperature of 40
C,
and a flow rate of 70 mL/min. Samples were calibrated to the VSMOW scale by analyzing
USGS77 (polyethylene powder) and C
36
n-alkane #2 provided by Arndt Schimmelman (Indiana
University).
Description of working standards:
Table S1: Acetate working standards measured by EA-IRMS
Standard
13
C (VPDB)
2
H (VSMOW)
Source
AcSt (Sodium acetate)
-19.2
0.1‰
-127
1.9‰
Malinckrodt
AcA (Sodium acetate)
-26.5
0.1‰
-153
2.0‰
Fisher Scientific
AcB (Sodium acetate)
-34.6
0.1‰
-95
1.1‰
Allied Chemicals
S3
Effect of concentration on isotopic accuracy
Figure S1:
An acetate standard (AcB) was measured against working standard AcSt across a
range of concentrations. AcSt was either kept at a constant 50
μM
concentration (blue) or was
diluted to match the concentration of AcB (black). Accuracy decreased when the solutions were
< 30
μM,
potentially due to the larger influence of background acetate at low concentrations.
Figure S2:
Ionization yield of 50μM acetate in pure methanol versus in 1 mM solutions of NaCl,
KCl and NH
4
Cl. Addition of inorganic salts reduces the ionization of acetate by more than 10-
fold.
S4
Bacterial Cultures:
Table S2
: Bacterial strains grown in this study
*Leibniz Insitute Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)
Table S3:
Growth medium composition for cultures analyzed in this study.
Component
A. woodii & S. ovata
C. pasteurianum
units
KH2PO4
0.2
2.15
g/L
NH
4
Cl
0.25
--
g/L
Na
2
HPO
4
•
7H
2
O
--
9.16
g/L
NH
4
SO
4
--
3
g/L
NaCl
1.16
--
mg/L
MgSO
4
•
7H
2
O
1.45
--
mg/L
CaCl
2
•
2H
2
O
0.11
0.013
g/L
KCl
0.5
--
g/L
KHCO
3
6
--
g/L
Na
2
S •
9H
2
O
0.3
--
g/L
Na
2
SeO
3
17
--
μg/L
Na
2
WO
4
•
2H
2
O
33
--
μg/L
FeSO
4
•
7H
2
O
2
--
mg/L
FeCl
3
⋅
6H
2
O
2027
2027
μg/L
H
3
BO
3
30
30
μg/L
MnCl
2
⋅
4H
2
O
100
100
μg/L
CoCl
2
⋅
6H
2
O
190
190
μg/L
NiCl
2
⋅
6H
2
O
24
24
μg/L
CuCl
2
⋅
2H
2
O
2
2
μg/L
ZnCl
2
68
68
μg/L
Riboflavin
100
100
μg/L
Biotin
30
30
μg/L
Thiamine HCl
100
100
μg/L
L-Ascorbic acid
100
100
μg/L
d-Ca-pantothenate
100
100
μg/L
Species
Strain
Source
Source Location
Sporomusa ovata
DSM 2662
Volker Müller
Goethe University
Frankfurt, Germany
Acetobacterium woodii
DSM 1030
Volker Müller
Goethe University
Frankfurt, Germany
Clostridium pasteurianum
LMG 3285
DSMZ
Brunswick, Germany
S5
Folic acid
100
100
μg/L
Nicotinic acid
100
100
μg/L
4-aminobenzoic acid
100
100
μg/L
pyridoxine HCl
100
100
μg/L
Lipoic acid
100
100
μg/L
Thiamine
pyrophosphate
100
100
μg/L
Cyanocobalamin
10
10
μg/L
Culture Conditions:
Acetobacterium woodii
and
Sporomusa ovata
were both grown in 25 mL balch tubes with 10 mL
of media under 20 psi 80:20 H
2
:CO
2
at pH 7 and 30ºC. Cultures were shaken at 180 rpm and
grew to completion over one day. Media aliquots were taken in stationary phase, filtered through
a 0.2
m filter, and frozen.
Clostridium pasteurianum
was grown in 1L sealed culture bottles with 100 mL of media under
an N
2
atmosphere at 37ºC. Cultures were not shaken. Media aliquots were taken in end-log
phase, filtered through a 0.2
m filter, and frozen.